AN INVESTIGATION INTO THE MECHANISMS OF FORMATION AND THE DEVELOPMENT OF IMPROVED TECHNIQUES FOR ITS QUANTITATION.

Submitted for the degree of Doctor of Philosophy

M arch 1996

by: Mark Pearson. B.Sc., A.R.C.S.

Department of Physiology & Biophysics Imperial College of Science, Technology & Medicine St. Mary's Hospital Medical School Norfolk Place Paddington London W 2 IPG To Leonard, my mentor and friend ABSTRACT

Rouleaux formation (RF) influences a variety of rheological properties of blood (eg shear thinning, thixotropy) and is believed to have effects in-vivo. However, the actual process of RF is little understood, and the primary aim here was to investigate factors that may be involved. There are currently two proposed hypotheses for describing the mechanisms involved in RF, one associated with macromolecular bridging between RBC's, and the second with osmotic forces bringing and holding RBC's together. OifFerentiatwj these two hypotheses, and gm/v/Kj further insight into the mechanisms of RF, was attempted here using two approaches:

The first approach involved making measurements, before and after enzyme modification, of RBC aggregation and cellular factors (size, deformability and surface charge), so any changes in the latter could be related to the former. However, many of the enzyme modified RBC's showed extremely high levels of aggregation, which caused problems in obtaining, viscosity and optical measures of aggregation. Hence new experimental procedures and analytical methods had to be developed to deal with this. With these new methods it was possible to show large differences in the aggregating potential of RBC's modified with various enzymes and, as far as dextran-induced-aggregation was concerned, good correlation was found with changes in cellular charge. However, -induced- aggregation followed a different pattern, suggesting that the physico-chemical factors involved differed to dextran.

The second approach involved a more direct method for investigating fibrinogen interactions with the RBC surface. Radioactively- and fluorescently-labelled fibrinogen were used in this work in an attempt to directly observe adsorption to the RBC surface, which is an essential feature of the cross-bridging hypothesis. Despite many experimental problems with this approach it was conclusively shown that fibrinogen adsorbed to the RBC surface and was associated with RF. CONTENTS OF THESIS

• A B ST R A C T 3

• LIST OF SYMBOLS AND ABBREVIATIONS 5

• LIST OF EQUATIONS 7

• L IST OF T A B L E S 11

• L IST OF FIG U R E S 13

• LIST OF IMAGES (PICTURE SETS) 19

CHAPTER 1. INTRODUCTION 21

CHAPTER 2. METHODS, MATERIALS AND INSTRUMENTATION 61

CHAPTER 3. A STUDY OF THE CONTRAVES LS30 VISCOMETER 85

CHAPTER 4. A STUDY OF THE MYRENNE ERYTHROCYTE

AGGREGOMETER (MEA), THE SYLLECTOGRAM

AND METHODS OF ITS ANALYSIS 113

CHAPTER 5. HAEMATOCRIT EFFECTS ON VARIOUS BLOOD

SUSPENSIONS 151

C H A PT E R 6 . USING ENZYMES TO DEGRADE THE RBC SURFACE TO

INVESTIGATE CELLULAR FACTORS OF IMPORTANCE

TO ROULEAUX FORMATION / AGGREGATION. 173

CHAPTER 7. A METHOD FOR DIRECTLY INVESTIGATING THE

MECHANISMS OF ROULEAUX FORMATION. 210

CHAPTERS. DISCUSSION 231 •

• R E FE R E N C E S 237

• ACKNOWLEDGEMENTS 249

• IMAGES (PICTURE SETS) - in sleeve of back cover 250

4 SYMBOLS AND ABBREVIATIONS

Y Shear R ate V iscosity

H data Viscosity data Hr Relative viscosity 128.5 _ ‘ 1 r Relative viscosity at a high shear rate of 128.5s*1 027, Tir Relative viscosity at a low shear rate of 0.277s*1

A t | Change in viscosity

M an(0, 3) a -^S v l Area under aggregating phase of the MEA syllectogram calculated

by the MEA at either 0 or 3 s *1 C om pC Y )^^ ^ Area under aggregating phase of the MEA syllectogram calculated by computer analysis program at a shear rate y .< <1

vi Change in area of the syllectogram

^ 1 1 2 5 Iodine (radioactively) labelled fibrinogen

^ R h -la b Rhodamine (fluorescently) labelled fibrinogen

^ U n la b Unlabelled fibrinogen B Brom elain CT a-Chymotrypsin CV Coefficient of variation ds deci seconds ( 10*1 s) ESR Erythrocyte Sedimentation Rate F Formaldehyde G Glutaraldehyde H ct Haematocrit (or Packed Cell Volume) HSR High Shear Rate LSR Low Shear Rate LT Light Transmission MCV Mean cell volume MEA Myrenne Erythrocyte Aggregometer MSM The microscope slide method of fixation MW Mean weight molecular weight

5 M n Mean number molecular weight NA Neuraminidase P Paraformaldehyde MS PerioJ/c fe.i&£cktf RBC (erythrocyte) R®CSusp Red blood cell suspension RF Rouleaux formation SD Standard deviation T Trypsin T h Throm bin TTM The test tube method of fixation WBC White blood cell (leukocyte)

6 LIST OF EQUATIONS:

Eqn 1.1: Smoluchowski's equation. UE . C (-) 30 n

Eqn 1.2: Relation of electrophoretic mobility ( UE) to Debye-Hiickel length (k 1) and surface charge UE - (—r\K ) 30 density ( a).

Eqn 1.3: Describes velocity profile of Newtonian liquid — ■ 1 - ( — )2 31 flow through a tube. v max r max dv Eqn 1.4: Definition of shear rate ( y). Y ■ —dr . 31

Ft Eqn 1.5: Definition of shear stress (r). T - ----A 31

X E q n 1 . 6 : Definition of viscosity ( 77). v - — 32 Y

Eqn 1.7: Darcy's Law. 0 « AP - R — 32

Eqn 1.8: Poiseuille-Hagen equation. Q - LF 11 r< 32 8 /

Eqn 1.9: Combination of equations 1.7 and 1.8. K . 8 7 ’l- 32 3i r 4

Eqn 1.10: Einsteins equation for rigid particles. * 1 ♦ a.C 35

Eqn 1.11: Definition of apparent relative T\g of the blood sample ^ or ' of the suspending phase 35 viscosity (q j

Eqn 1.12: Change in r|a with hct (H) between log T\a - k1 * k"*H 39 30-60% at a fixed y.

Eqn 1.13: rj changes with temperature (T) for C r 40 Newtonian fluids and blood )-Ae T

7 Eqn 1.14: Forces involved in the aggregation F.'Fh-Ft -F,- Fn 51 o f RBC's.

Eqn 2.1: Calculation of the concentration (C) of a Wi 63 dialysed substance. FIN

E q n 2 .2 : Calculation of fibrinogen concentration [

Eqn 2.3: Loge-linear relationship between r) of Ln (?\susp) ■ 65 RBCSusp and polymer concentration [P].

Eqn 2.4: Calculation of the volume of enzyme 5 H stock solution needed to give 5mg/ml v ■ V' 68 EN2' ^ c STOCK 'm 'rsAS{P of RBC's at a hct H.

Eqn 2.5 : Calculation of the volume of stock aggregating agent solution needed to / AGG H . y 69 produce a specified concentration per AGG U C STOCK C 1UUIOC) ^ ml of supernatant.

Eq" 2.6 : Calculation of the supernatant volume change aAV y SUSP . AC -yv SOL 70 needed to correct a blood sample to 45% hct. 45

H l Eqn 2.7: Calculation for RBC size (MCV). MCI' - too N, 72

Eqn 2.8: Relation of torque (T) and shear stress (x) in the 2 2 T rl*r2 gap between the bob (radius r l5 height h) and T - — .[— 81 A *h rl 2 -r2 2J cup (radius r2) of the Viscometer.

Eq" 3.1 : Conversion equation of torque t) - EXP [-4.5627 .(R. 1.6095 ) 91 readings to r\ values. -Ln (y)] . Torque Reading

8 Eq" 3.2 : Power law relationship between q and y valid for blood in an aggregation phase (Phase-A) ti - c.yG 91 and in a deformation phase (Phase-D).

Eqn 3.3: Equation 3.2 manipulated to give the equation of a straight line representing shear thinning ^ Ln(x ir) - G A'L n (y ) ♦ C A g \ characteristics of LSR q ^ - ie aggregation phase (A-Phase).

Eqn 3.4: As for equation 3.3, but for HSR q ^ - ie L n 0lr) - G D«Ln(y) ♦ CD 91 deformation phase (D-Phase).

Eqn 3.5: Definition of 0-277q r; an index of • RBC Susp Viscosity at 0.277 s A 92 aggregation Suspending Phase Viscosity

RBC Viscosity at j Eqn 3.6: Definition of 1285qr; an index of 128.5 _ Susp______' ______128 .5 '1 g j 'If* ** deformation. Suspending Phase Viscosity

Eqn 4.1: Monoexponential curve fitting equation for fitting to the aggregation phase of LT - K m - b m,ExP [-

Eq" 4.2 : As for equation 4.1, but the curve fitting l t - K b- a b.Exp [-&fc»(r-'min)] 117 equation is biexponential. ’ c^Exp

Eq" 5.1 : Modified form of equation 1.12 that assumes Ln(T\J - mx • H 157 k' equals logc(supernatant q) at all y's.

Eq" 5.2: Relationship between nq and y for whole (mj) - -0.171 .Ln(y) - 2.79 158 blood between y's 0.277-27.7s*1.

Eq" 5.3 : As for equation 5.2 but between y's L n (m j) - -0.0895 *Ln{ y) - 3.13 158 69.5 and 128.5s'1.

9 Eq" 5.4: Authors' equation relating r|p H and y. In(T,r) - H.iA'X x 158

E q n 5.5: Whittington et al's equation linking q p H i"(n P - H.Ln(.— ) and y based on a Weavers et al's linear-log Ay) 170 * relationship between ml and y . Ay)

E q n 5.6: Matrais' equation derived from removing irij 45 , \H~ 170 from equation 5.1. ^ r 45 ’ f ^ r not*

10 LIST OF TABLES:

T ab 1.1: Some blood vessels with their average diameter and wall thickness. 25 T ab 1.2: List of various cells found in blood, with approximate adult human values for diameter, abundance and volume. 26 T ab 1.3: Reference list of approximate specifications for some 'artificial' and 'natural' macromolecules, most of which induce RBC aggregation. 50

T ab 2.1: List of coefficients of equation 2.3 for various molecular weight dextran fractions or fibrinogen. 67 T ab 2.2: Reference table for Contraves LS30 Viscometer - y relationship. 79 —— ■**!* •*!» T ab 5.1: List o f changes in M-( ° ‘"d 3)Asy) and Comp<0’6’ 12and24)A SyI (A A Syl) betw een the hcts 43 to 47%. Also listed is the hct at which the MEA gives the peak response. 154 T ab 5.2: List of coefficients to equation 1.12 (ie Ln(r|r)=k'+k"*H) and equation 5.1 (Ln(r|r)=m]*H) derived from whole blood data. 158 T ab 5.3: Coefficients of equation 5.4 (ie Ln(rir)=H .yA.K1) derived from whole blood data. 160 T ab 5.4: Coefficients of equation 5.4 obtained from various enzyme treated RBC's (W, CT or T) with 15g/l dextran 70 present. 161 T ab 5.5: Coefficients of equation 5.4 obtained from various non-aggregating enzyme treated RBC's (W, CT, T, T+CT or CT+T). 164 T ab 5.6: Comparison of Matrais' equation (equation 5.6) and the authors'

equation (equation 5.4) for correcting hct differences of t\ measurements of whole blood. 171

Tab 6.1: Lists °'277r|r and 128'5r)r data for untreated (W) and treated RBC's (CT, T, T+CT or CT+T) with either 15g/l dextran 70 or 10g/l dextran 40 present to induce aggregation. 178 Tab 6.2: As table 6.1 but for the parameters GA and GD. 178 Tab 6.3: As table 6.1 but for the parameter Comp{12)ASvl. 179

11 Tab 6.4: As table 6.1 but for the parameter Comp(0)ASvI and only for 10g/l dextran 40. 179

Tab 6.5: Lists measurements of RBC Size (MCV), size distributions and deformability (rFR(O)) for untreated (W) or treated (CT, T, T+CT, CT+T) RBC's. 182

Tab 6.6: Lists measurements of UE at the ionic strengths 0.172 and 0.0043 for untreated (W) or treated (CT, T, T+CT or CT+T) RBC's. 184

Tab 6.7: Lists 0-277r|r, 128'5r|n GA and GD for non-aggregating untreated (W) or enzyme modified (CT, T, T+CT, CT+T) RBC's. 184

Tab 6.8: Repeats the results of table 6.1, and shows additional results from the various enzyme treated RBC's in the presence of either 6 or 10g/l of fibrinogen. Compares the actions of dextran and fribrinogen. 186

Tab 6.9: As table 6.8 but for the parameters GA and GD. 187

Tab 6.10: As table 6.8 but for the parameter Compn2)ASvl. 188

Tab 6.1 l:Lists °'277r|r, 1285r|r, GA and GD for untreated (W) or thrombin (Th) treatred, density (age) fractionated (ie young, middle and old), RBC's. 189

Tab 6.12:Lists a277r)r and GA for enzyme modified (T, B or NA) RBC's with either 10g/l dextran 40, 15g/l dextran 70 or 6g/l fibrinogen present to induce aggregation. 190

Tab 6.13:As table 6.12 but for the parameters 1285r|r and GD. 190

Tab 6.14:A s table 6.12 but for Comp(12)A Svl. and Comp(24)A SvI. 192

Tab 7.1: Lists measures of 4>1125 fixed to the RBC surface using the TTM with various concentrations of the fixatives formaldehyde, paraformaldehyde or glutaraldehyde. 220

12 LIST OF FIGURES:

Fig 1.1: Schematic representation of the cardiovascular system. 24 Fig 1.2: Various forms of "the red blood cell" under different conditions. 28 Fig 1.3: (a) Laminae telescopically moving across each other when liquid flows through circular tubes. (b) Parabolic change in velocity of laminae across a tube. (c) Two adjacent laminae with velocity and geometry parameters. 32 Fig 1.4: Changes of rj with y for non-aggregating, aggregating or fixed RBC's. 35 Fig 1.5: (a) Plot of r\ against y for a sample of normal human whole blood. (b) Data in (a) plotted on log-log axes. (c) Schematic representation of the shear thinning of blood. 37 Fig 1.6: Schematic representation of thixotropy. 38 Fig 1.7: Effects of haematocrit on whole human blood at four different y's, 0.052, 0.52, 5.2 and 52s'1. 40 Fig 1.8: Flow profile of Newtonian fluids and blood. 43 Fig 1.9: (a) Schematic representation of the cross bridging hypothesis (CBH). (b) Schematic representation of the depletion layer hypothesis (DLH). 49 Fig 1.10: Electron microscopy measurements of changes in intercellular distance against molecular weight dextran fractions. Also shown is the dextran concentration of peak aggregation against molecular weight dextran fraction. 53 Fig 1.11: Effects of increasing concentration of different molecular weight dextran fractions on aggregation. 53 Fig 1.12: Effects of increasing dextran 80 concentration on the aggregation of normal or neuraminidase treatedRBC's. 56 Fig 1.13: Adsorption isotherms of dextran 70 on the surface of normal or neauraminidase treated RBC's. 56

■ t f Fig 2.1: Log-linear relationship between r| and dextran 70 concentration. 67 Fig 2.2: Schematic representation of the Coulter Counter Zbi, a device for measuring cell size. 71

13 Fig 2.3: Size distribution curves obtained from the Coulter Channelyzer. 71 Fig 2.4: Schematic representation of the St. George's Filtrometer, a device for measuring deformation. 74 Fig 2.5: Demonstrates how the index of deformation 'relative filtration rate at time zero (rFR(O))' is determined. 74 Fig 2.6: Schematic representation of the Malvern Zetasizer III, a device for measuring electrophoretic mobility (UE). 77 Fig 2.7: Shows how UE changes across the cell chamber of the Malvern Zetasizer III, if aligned correctly. 77 Fig 2.8: Schematic representation of the Contraves LS30, bob-in-cup style, Viscometer. 80 Fig 2.9: Schematic representation of the Myrenne Erythrocyte Aggregometer (MEA), an optical device for measuring aggregation. 80

Fig 3.1: The response of the Viscometer with time after applying a low shear rate to: (a) a Newtonian liquid, (b) a normally aggregating RBCSusp, (c) a non-aggregating RBCSusp, or (d) a hyperaggregating R B C Susp. 88 Fig 3.2: (a) Shows change in r|r against y for RBC's with zero, normal or hyper- aggregating tendencies. (b) Data from (a) plotted on log-log axes for the purpose of analysis.. 90 Fig 3.3: Some potentially useful parameters associated with r)^. 92 Fig 3.4: Demonstrates how computer program (AnalEta) allows ri^ to be investigated by allowing user to select limits where points can be ignored from the linear regressions. 92 Fig 3.5: Changes in r|r with y for various T, B or NA modified RBC's with 15g/l dextran 70 present; ie aggregating RBCSusp's. Demonstrates (a) the plateau effect, and (b) how this problem may be avoided. 95

F ig 3.6: HjLjuj fro™ three RBCSusp's with very different levels of aggregation. Demonstrates how the transition effect increases with increasing levels of aggregation. 97 Fig 3.7: Changes in intercept point (Iy) and HSR gradient (GD) with increasing dextran 70 concentration. 100 Fig 3.8: Changes in °'277r|r and °‘277*n/l28'5r|r for normal RBC's suspended in increasing concentrations of dextran 70. 100 Fig 3.9: Shows how Logc(r|)-loge(Y) curves are shifted down with increasing dextran concentration, and thus supernatant r\. 101 Fig 3.10: Changes in 0 277r|r and a277r|I/128-5r|r for trypsin treated RBC's suspended in increasing concentrations of dextran 70. 101 Fig 3.11: Shows how GA changes with increasing concentrations of (a) dextran 70 or (b) dextran 110, for normal, CT or T modified RBC's. 103 Fig 3.12: Changes in (a) a277r|/128-5r|n and (b) GA, for normal RBC's suspended in various concentrations of fibrinogen (3-40g/l). 105 Fig 3.13: Changes in Man(0 and 3 >a Sv, for normal RBC's suspended in various concentrations of fibrinogen (3-40g/l). 106 Fig 3.14: Shows how transition effect is also seen with’whole blood. 106 Fig 3.15: Shows two sets of t) ^ (a and b) from a paper by Bernasconi, that has points of transition. Also shown are predictions of the r\ at 200s’1. 110 -»■ **!*«." Fig 4.1: The different response of the indices of aggregation from theViscometer ( a 277 T lr ) and the M EA (Man(3)ASvI) for washed normal RBC's suspended in various concentrations of fibrinogen (0-12g/l). 115 Fig 4.2: Changes in the syllectogram for increasing levels of aggregation. 118 Fig 4.3: Demonstrates how well monoexponential and biexponential curves fit to the aggregation phase of a syllectogram. 118 Fig 4.4: Some potential parameters associated with the syllectogram. 119 Fig 4.5: Problems associated with the syllectogram, in the form of (a) 'Noise' and (b) regular dips. 122 Fig 4.6: Demonstrates the occurrence of regular dips at the beginning of the aggregation phase of the syllectogram, which makes analysis difficult. 123 Fig 4.7: Examples of "blips" that occur occasionally in the syllectogram. 124 Fig 4.8: Demonstrates the method for extending the lower limits of sensitivity of the MEA. 126 Fig 4.9: Shows the different behaviour of syllectograms obtained at 6s’1 from normal aggregating blood samples. 126 Fig 4.10: The occurrence of oscillations in the syllectogram obtained at HSR's. 127 15 Fig 4.11: Compares the syllectograms from computer runs of two different aliquots of a blood sample where very different results were obtained. 130 Fig 4.12: Demonstrates the "crenation problem" for washed RBC's suspended in various concentrations of fibrinogen (0-12g/l). 130 Fig 4.13: Shows the normal behaviour of syllectograms obtained from RBC's suspended in PBS where the "crenation problem" was found. 132 Fig 4.14: Shows "settling" in syllectograms obtained from normal washed RBC's in the presence of 15 or 25g/l dextran 500. 132 Fig 4.15: Shows the large variation in the fast decay constant (db) of syllectograms. 134 Fig 4.16: Demonstrates changes in the syllectogram with time. 135 Fig 4.17: Man(0and3>ASyI measurements for whole blood at various hcts (20-60%). 137 Fig 4.18: As figure 4.17 but for Comp(12)ASyl. 137 Fig 4.19: Demonstrates good correlation of Klan(3)ASyI with (a) °‘277T|r and (b) GA for washed RBC's from five subjects with 25g/l dextran 70 present. 139 Fig 4.20: Comp(Y)ASyl at various y's for washed RBC's from five subjects with 25g/l dextran 70 present. 140 Fig 4.21: Shows good correlation between Comp(Y)ASyl and °’277r)r for washed RBC's from five subjects with 25g/l dextran 70 present. 140 Fig 4.22: Comp(0-3-6-9-12>18 “ d 24)ASyI for B treated RBC's with 10g/l dextran 40, 15g/l dextran 70 or 6g/l fibrinogen. Shows how insensitive this parameter is on these RBCSusp's at LSR's, but'not for higher y's. 142 Fig 4.23: Shows good correlation between Coinp{12’ 18and24)ASv) and (a) °‘277r)r or (b) Ga, demonstrating the upper limit of the MEA has been extended. 143

F ig 5.1: P lots o f (a) Man(0 and 3)A Syl and (b) Comp(0’6’ 12and24)A Syl against hct (30-51% ) for normal washed RBC's with 15g/l dextran 70. 155

Fig 5.2: Plots of (a) Man(0 and 3)ASyl and (b) c™** 6. 12-nd 24)^ against hct (30-51%) for trypsin treated RBC's with 15g/l dextran 70. 156 Fig 5.3: Logc-linear plot of T|r against hct for whole blood with linear regressions of the forms of equations 1.12 and 5.1. 159 Fig 5.4 : Shows Logc-Logc relationship between the coefficient m, (from equation 5.1) and y, for data shown in figure 5.3. 159 Fig 5.5 : As for figure 5.4 but irij derived from various RBCSusp's. 161

16 Fig 5.6: As for figure 5.4 but m, derived from trypsin treated RBC's with 15g/l dextran 70 present to induce aggregation. 162 Fig 5.7: As for figure 5.3, but for trypsin treated RBC's. 162 Fig 5.8: As for figure 5.4, but for non-aggregating RBCSusp's. 164 Fig 5.9: Compares changes of0-277r|r and GA between the hcts 30-50%. 164 Fig 5.10: (a - c) Demonstrates steady improvement of correlation between Man(3)ASyl and a277r|r as small corrections are made to each parameter for small changes in hct. 165 Fig 5.11: Shows poor linear-log relationship between m, and .y for whole blood data. Hence, Weaver et al's earlier work appears to be wrong. 170

Fig 6.1: UE and 0277rir data taken from a paper from Ertan et al which suggests trypsin modification of RBC's is complete after ~30 minutes. 177 Fig 6.2: a277r|r data for untreated (W) or enzyme treated RBC's (CT, T, T+CT, CT+T) with either 15g/l dextran 70 or 10g/I dextran 40 present to induce aggregation. 177 Fig 6.3: ESR data for some RBCSusp's showing agreement with °'277r|r for changes in aggregating tendencies of W, CT or T treated RBC's. 180 Fig 6.4: Shows exponential decrease in UE for increasing ionic strength of the suspending phase for normal RBC's. 183 Fig 6.5: Measurements of UE for untreated (W) or enzyme modified (CT, T, T+CT or CT+T) RBC's in suspending phase solution of ionic strengths 0.172 or 0.0043. 183 Fig 6.6: Loge-loge plot of r)r against y for non-aggregating untreated (W) or enzyme treated (CT, T, T+CT or CT+T) RBC's. 185 Fig 6.7 : Same as for figure 6.2, but also showing data for 6g/l or 10g/l fibrinogen. 185 Fig 6.8: (a & b) Combines the results of r| indices of aggregation (°'277r|r and Ga) for all treatments of RBC (W, CT, T, T+CT, CT+T, B or NA) and with all the aggregating agents used (dextran 70, dextran 40 and fibrinogen). 191

17 Fig 6.9: Compares °- 277r|r to RBC surface charge, as modified by the enzymes (CT, T, T+CT, CT+T, B orNA), suspended in solutions of ionic strength 0.172 (a), or 0.0043 (b). 193

F ig 6 . 1 0 : As for 6.9, but with GA. 194

F ig 6 . 1 1 : ri^ for trypsin modified RBC's suspended in various concentrations of albumin (15-75g/l). 196

Fig 6.12: As figure 6.11 but showing Man( 0)ASyI data. 196

F ig 6.13: S h ow s °' 277r|p (a), and GA, (b), for time incubation studies of CT or T modification of RBC's with 10g/l dextran 40. 199

Fig 6.14: As figure 6.13 but showing (a) Comp( 12)A s>.|, and (b) Comp( 0)ASyl data. 200

F ig 6.15: 0 277r)n (a), GA, (b), and °- 277r|r/ 128-5r|r, (c), for time incubation studies

o f CT. 2 0 1

Fig 6.16: As figure 6.13 but showing Comp( 12)ASyI data. • 202

Fig 6.17: As figure 6.13 but showing (a) GD and (b) 128'5r)r data. 203

Fig 7.1: Schematic representation of (a) the test tube method (TTM) and (b) the microscope slide method of fixation. 216 Fig 7.2: Schematic representation of the imaging equipment. 216

F ig 7.3: (|)I125 fixed to the RBC surface using the TTM with either 0.5 or 1.0% P, or 1.0% F or G fixatives. 218

F ig 7.4: 4 >I125 fixed to the RBC surface using the TTM with either 2.5% G or 4.0% P fixatives. 218

Fig 7.5: (a) Fixation time course study (l-5min) of 4 )I125 fixed to the RBC

surface using 1 % G as the fixative. (b) Fixative concentration study for F (0.1 or 1.0%) or G (0.1, 0.5

or 1 .0 %) for fixing (J)I125 to the surface of the RBC. 219 Fig 7.6: Absorption curves for equal concentrations of 4>Un]ab or

passed through chromotography equipment. 2 2 1 Fig 7.7: Schematic representation of fibrinogen clustering and the optical trapping method. 229

18 LIST OF IMAGES f PICTURE SETSI:

All obtained with the MSM as described in figure 7. lb.

Pic. Set 7.1: Shows autofluorescence of RBC's caused by glutaraldehyde fixation. 250 Pic. Set 7.2: Compares fluorescence at a point of contact between a single RBC and a rouleaux when (a)

These picture sets are found in the sleeve of the back cover of this thesis.

19 20 CONTENTS OF CHAPTER 1: INTRODUCTION

1.1 THE CARDIOVASCULAR SYSTEM 23 1.2 BLOOD COMPOSITION AND FUNCTION 25 1.2.1 WHITE BLOOD CELLS (WBC'S) AND PROTECTION 26 1.2.2 , HAEMOSTASIS AND COAGULATION 26 1.2.3 RED BLOOD CELLS (RBC's) 27 • GENESIS OF THE RBC 27

• TRANSPORT OF 0 2 A N D C 0 2 27 • THE STRUCTURE OF THE RBC’ 28 • THE 'MANY' RBC'S IN THE CIRCULATION 30 1.3 RHEOLOGICAL PRINCIPLES 31 1.3.1 DEFINING VISCOSITY 31 1.3.2 LIQUID FLOW THROUGH A TUBE 33 1.4 BLOOD VISCOSITY AND NON-NEWTONIANISM 34 1.5 CAUSES OF BLOOD NON-NEWTONIANISM 34 1.5.1 THE IMPORTANCE OF RBC AGGREGATION 36 1.5.2 FACTORS AFFECTING RBC AGGREGATION 39 • H A E M A T O C R IT 39 • TEMPERATURE 40 • PLASMA PROTEINS AND VISCOSITY 41 1.5.2 RBC DEFORMABELITY AND BLOOD NON-NEWTONIANISM 41

1 .6 BLOOD FLOW IN THE CIRCULATION 42 1.7 BLOOD FLOW IN THE MACROCIRCULATION 43

1 .8 BLOOD FLOW IN THE MICROCIRCULATION 44 1.8.1 WBC'S AND THE MICROCIRCULATION 44 1.8.2 RBC'S AND THE MICROCIRCULATION 45 • RBC AGGREGATION 45 • RBC DEFORMATION 46 • HAEMATOCRIT 46 1.8.3 PLASMA VISCOSITY IN THE MICROCIRCULATION 47

21 1.9 ROULEAUX FORMATION (RF) / AGGREGATION 47 1.9.1 HOW DOES RF/AGGREGATION OCCUR? 48 • THE CROSS BRIDGING HYPOTHESIS (CBH) 48 • THE DEPLETION LAYER HYPOTHESIS (DLH) 48 • COMPARING THE CBH AND DLH 51 1.9.2 CONDITIONS NECESSARY FOR RF/AGGREGATION 51 1.9.3 INTRINSIC AND EXTRINSIC FACTORS AFFECTING RF/AGGREGATION 52 • EXTRINSIC FACTORS AFFECTING RF/AGGREGATION 54 • INTRINSIC FACTORS AFFECTING RF/AGGREGATION 58 1.10 WHAT IS TO FOLLOW ...... 59

2 2 CHAPTER 1: INTRODUCTION

Haemorheology is the study of flow, deformation and vessel wall interactions of blood, and is the field in which this thesis is based. It seems usual to start a thesis with some historical facts that lead up to modem day understanding, but for a number of reasons no attempt at giving this historical account is made. Instead the author refers the reader to several good historical reviews that are already available11*41. Also available are several good books and reviews on various aspects of the field of Haemorheology15*81, and chapters from these will be referred to at numerous times in this thesis.

This introduction starts by looking at the large scale anatomical and functional aspects of the cardiovascular system and some properties of'blood' that flows through this vasculature. Then some general principles of liquid flow through tubes are given and extended to look more specifically at blood flow in the circulation. The central issue of this thesis is the mechanisms behind the important property of blood ’’rouleaux formation" (RF), the loose sticking together of red blood cells to form linear networks like piles of coins191. This introduction concludes conventionally with work that will follow in the rest of the thesis.

1.1 THE CARDIOVASCULAR SYSTEM

The heart is an organ, comprising four chambers, that mechanically act as two serial pumps that drive blood around a complex network of parallel and serially connected tubular vessels. One pump drives blood through the lungs (Pulmonary Circulation) and the other pump drives blood around the remainder of the vasculature (Systemic Circulation) (figure 1.1). At the time of the ejection phase of the heart, blood pressure leaving the heart is about 120 and 25 mmHg (16 and 3.3 kPa) above atmospheric pressure in the systemic and pulmonary circulations respectively, dropping to 80 and 10 mmHg (10.7 and 1.3 kPa) in between each ejection phase.

23 flow through the left and right lungs, and the systemic (oxygenated) blood flow through the remainder of the body. Figure Schematic1.1: representation of the cardiovascular system showing the pulmonary(deoxygenated) blood SYSTEMIC CIRCULATION PULMONARY CIRCULATION SYSTEMIC CIRCULATION Capillaries Veins 24 a n r s arm and ead H Less Arteries

INTERNAL WALL BLOOD VESSEL DIAMETER THICKNESS

AORTA 25m m 2 mm

ARTERIOLE 30p m 2 0 pm

CAPILLARY 4 -7 pm 1 pm

VENULE 50-200p m 2 pm V E N A C A V A 30m m 1.5m m Table 1.1: Some blood vessels with their average diameter and wall thicknesses.

The various vessels differ enormously in internal (lumen) size, wall thickness and structure and play very different roles (table 1.1). The arteries (supply vessels) deAver blood to various parts of the body, varying in size to form a continuum from the aorta to the arterioles. The arterioles (resistance vessels) control local blood flow to local need by changing the vessel diameter through constricting and as necessary. They pr&v/tle much of the circulation resistance and pressure drop by being relatively few in number, compared to the capillaries, and having a small lumen diameter. The capillaries (exchange vessels) are large in number, thus having a large total cross sectional area which acts to slow blood flow down. This, coupled with their thin walls, make them highly suited for their job of allowing 0 2, metabolites etc to the tissues, and removing waste products. The veins (capacity vessels) act as a reservoir for blood returning to the heart, storing -60% of total blood volume. As for arteries, these vary in size to form a continuum from venules to the vena cava.

1.2 BLOOD COMPOSITION AND FUNCTION

The normal human body has a total blood volume of -5% 1 for males and -4% 1 for females of which 48±7% and 42±6% are cells respectively. There are three types of cell: red blood cells (erythrocytes), white blood cells (leucocytes) and platelets (thrombocytes) (table 1 .2 ). These cells are suspended in a straw yellow coloured fluid called plasma that can be split into two parts; the clottable protein fibrinogen and the liquid serum. Serum contains proteins (albumins and globulins), nutrients and metabolic waste products, mineral electrolytes.

25 CELL TYPE DIAMETER ABUNDANCE VOLUME Oim) (10’/1 blood) (fl or fim3) Mature Erythrocytes 6.5-8.5 male: 4500-6500 . 80-100 female: 3800-5800 -10.0 10-100 300 Platelets 2.5-3.5 150-400 15 Leucocytes -7.0 3-10 -200 Table 1.2: List of various cells found in blood, with approximate values for size, abundance and volume corresponding to adult humans.

1.2.1 WHITE BLOOD CELLS (WBC's) AND PROTECTION

The white blood cells (WBC's) main function is to fight and cancer. There are three groups of leucocyte: (, and ), M onocytes and Lymphocytes. Granulocytes and protect the body against invading microorganisms, toxins etc by ingesting and destroying them, and lymphocytes give the body specific immunity to recognised invading microorganisms through synthesis of antibodies.

1.2.2 PLATELETS, HAEMOSTASIS AND COAGULATION

Platelets are smaller than erythrocytes and leucocytes and play a major role in haemostasis, the process of stopping blood loss. When vascular damage occurs, platelets aggregate and plug the hole. If the damage is severe enough then blood coagulation, the precipitation of the fibrinogen intorfibrin threads, is also triggered that acts to fill the hole more rapidly by trapping RBC's. The fibrinogen to fibrin conversion occurs through the action of the proteolytic enzyme thrombin that is not normally present in blood, but is cleaved from its precursor prothrombin when blood loss occurs. Also if blood is removed from the circulation, then it is able to detect a foreign environment which causes it to coagulate. There are two types of anticoagulant that are used to prevent this from happening: heparin, which inhibits enzymes that induce coagulation, and a group of anticoagulants that reduces Ca2+ ions needed in coagulation (eg. sodium citrate, EDTA, salts).

26 1.2.3 RED BLOOD CELLS (RBC’s)

Red blood cells (RBC's) constitute the majority of cells found in blood, and it is these RBC's that are most relevant to this thesis and so will be covered in some detail. More is known about the human RBC than any other cell, because it is a relatively simple cell lacking a nucleus, and is easily obtainable in small or large volumes.

GENESIS OF THE RBC

The production of RBC's in the adult is stimulated by the hormone erythropoietin found in circulating blood. Adult RBC's are non-nucleated cells, produced in bone marrow and released into the circulation as reticulocytes which survive one to two days before becoming mature RBC's. It is known that mature adult RBC's survive in the circulation for ~ 121 days before being removed by the phagocytic cells mainly of the reticuloendothelial system primarily in the spleen, but also in the liver and bone marrow. As yet how senescent RBC's are recognised is unknown, although it is believed to be related to the structural changes that take place with the RBC as it ages as will be described below.

TRANSPORT OF P2 AND CO:

Simplistically RBC's are just membraneous sacks ftllaJ with a fluid containing the red pigment of blood, haemoglobin (normally at whole blood concentrations of 13-18g/dl for males and 11.5-16.5g/dl for females), a complex protein that binds 0 2 during their transport to and from the lungs as follows. 0 2 depleted (but C 0 2 rich) blood passes through the right atrium to the right ventricle, and is pumped around the pulmonary circulation where the C 0 2 is lost for exhalation and 0 2 absorbed. This 0 2 rich blood is passed through the left atrium to the left ventricle and pumped around the systemic circulation, feeding the tissues with 0 2 and absorbing the C 0 2 before returning to the left atrium via the venous reservoir. The RBC's large surface area (~ 135pm2) to volume (~90pm 3) ratio (~ 1 .5) m akes them particularly suited to act as carriers, since 0 2 and C 0 2 diffusion is facilitated. The capacity of blood to transport 0 2 to, and remove C0 2 from, the tissues is of obvious importance and depends on haemoglobin concentration (with associated regulating factors) and blood flow to the tissues.

27 Figure 1.2: Various forms of "the red blood cell" under different conditions: unstressed (biconcave disc), deforming to pass through narrow vessels (parabaloid) and under high flow conditions (fluid drop). THE STRUCTURE OF THE RBC

The adult human RBC is a biconcave disk shaped cell with an average diameter of ~ 7.5pm, which has the remarkable ability to deform to a fluid drop shape when under high stresses,

or to deform to a parabaloid to pass through capillaries as small as 2 .9pm diameter (figure 1.2). Much is now known about the general structure of the RBC membrane, which will now be covered in some detail.

The RBC membrane is composed of a lipid bilayer with eight associated major polypeptide chains (glycoproteins), when analysed by SDS gels, which have been split into two categories depending how easily they can be extracted from the membrane. Polypeptides easily extracted, and not destroying membrane (ie only requiring manipulation of pH or ionic strength), have been considered to be peripheral to the membrane and thus referred to as extrinsic membrane proteins. Conversely those not so easily removed, needing complete membrane destruction (ie requiring detergents, etc), have been considered to be more intimately connected to the membrane and thus referred to as intrinsic membrane proteins[10]. These extrinsic and intrinsic proteins make up the channels, the supporting structure (cytoskeleton), etc of the RBC and have been further split up into bands depending 28 of their molecular weight and locations; eg band 1 is spectrin a, band 3 the anion transporter, band 5 is actin, etc. The composition of certain polypeptides is known to differ between individuals presumably accounting for some of the differences in RBC's as will be discussed in the next section.

O f most relevance to this thesis is the major integral glycoprotein 'band 3', of which much is now known. It has a molecular weight (MW) of ~90kD, a concentration of ~ 1.2* 10 6 per RBC[11], and as already mentioned acts as the RBC anion transporter^91. It has been thought for some time that the glycoproteins laterally diffuse through the membrane112,13]. More recently it has been found that band 3 laterally diffuses through the membrane forming enriched and diminished areas, domains, of band 3 and lipids1141. These domains depend on the ionic strength, but not temperature, of the suspending phase.

Much of the information available about the RBC membrane structure has come from work using enzymes to degrade intact RBC's or ghost RBC membranes. Thus what was cleaved from the cytoplasmic or external side of the membrane is known. Identification of what is removed is made using SDS gel electrophoresis and staining chemicals specific to proteins, sialoglycoproteins or lipids115*211. Since enzyme modification of the RBC surface is a major ia 1^3 concern of this thesis, this work is of obvious importance and will helpAunderstand^findings as will be discussed further in chapter 6 .

RBC's carry a net negative charge of which -90% is due to different sialic acids (PAS 1-4, the glycophorins), and the remaining - 1 0 % due to the amino acids of the glycoproteins protruding from the cell surface (glycocalyx). It is interesting that despite all of the different normal human RBC's, the surface charge density has been shown to be the same for all individuals (age, sex, race and blood groups (ABO, MMS, etc) and for different aged cells*221. However, more recently this has been put in doubt by a finding that RBC's of the AB group have a surface charge density greater than the other groups (A, B and O) that show no difference between each other*231. The surface charge is measured by means of cell electrophoresis that measures particle movement, electrophoretic mobility (UE - units pm .s'1.V" 1.cm), under an applied electric field. UE per unit applied electric field is related to surface charge or zeta potential (Q by Smoluchowski's equation (valid for particles greater than 2 0 nm) as follows: 29 UE = « - ) (1.1) 11 where e is the permittivity of the sample and r| is the viscosity, a quantity that is defined in the next section, of the suspending phase. The protruding glycoproteins carry a net negative charge that, in an ionic solution, will attract counter-ions and repel co-ions causing a charge bi-layer to be formed around the cells whose thickness k*1 (Debye-Hiickel thickness) is related to U E as follows: 1

UE = (— ) (1.2) T|K ds^ihj o is the surface charge and is assumed to be uniform.

THE 'MANY' RBC'S IN THE CIRCIJLA TION

'The RBC' is a misnomer as there are a wide range of RBC's in the circulation, having the general features described above, but that vary both mechanically and structurally. Scanning electron microscopy studies have shown that one individuals RBC's, whilst having the same biconcave feature, vary considerably in size and shape1241. Size differences are known to be caused by the loss of membrane and sialic acid moieties as they age. Another variation is known to come from the different blood groups, such as the ABO group where the RBC's differ in what A.B.antigens (agglutinogens) they have on their surfaces. The picture is further complicated by RBC's differing in various pathologies such as diabetes125,261, sickle cell disease 1271 etc., from neonates 1281 and finally between species1291. As if this was not enough variation, RBC's are known to change between different normal subjects, between different diabetic subjects etc, as assessed by changes in their aggregating tendencies; ie subject variability1301. So there are a wide variety of RBC's, making the overall picture very com plex.

30 1.3 RHEOLOGICAL PRINCIPLES

1.3.1 DEFINING VISCOSITY

By definition the viscosity (r|) of a fluid is a measure of its intrinsic resistance to flow. This is easily demonstrated by comparing water with treacle. Water flows much easier than treacle because treacle has a much higher r| than water. Quantification is a little more difficult, and it is simpler to start by looking at tubular flow of simple "single-phase" liquids, such as water where r) is constant.

The flow behaviour of water through a tube consists of theoretical finite fluid layers (laminae) that telescopically slide across each other (figure 1.3a). The velocity profile of water can be shown, by simple algebra, to vary parabolically across the tube (figure 1.3b) as described by the equation:

= i - ( ^ f (1.3) where v is the lamina velocity of radius r, and vmax and rmax are the maximum velocity and radius respectively. Consider two adjacent laminae (figure 1.3c). Lamina / moves with a velocity v, and lamina /+ / moves with a greater velocity v,+; separated by a finite distance (dr). This velocity difference (dv) allows the important quantity, shear rate (y) with units of inverse seconds (s*1), to be defined: dv dr (1.4)

To extend this a little further, molecules from lamina i will diffuse to lamina i+J slowing it down, and molecules from lamina i+J will diffuse to lamina i speeding it up. These interactions induce a tangential frictional force (Fr) between the laminae. This leads to another important quantity, the shear stress (r) with units Pascal (Pa), and is defined as the tangential force per unit area (A):

T A (1.5)

31 Figure 1.3: (a) Shows laminae telescopically moving across each other when liquid flows through circular tubes, (b) 2 dimensional section (A'-A*) through (a) showing change in velocity of laminae across tube, (c) Two adjacent laminae with velocity and geometry parameters.

32 It is the ratio of the quantities t and y that gives the important quantitative definition of viscosity ( r\); ie:

’ = 7 (!•« )

t j has the units Pa.s, or more usually mPa.s because of its normal small value for every day liquids.

1.3.2 LIQUID FLOW THROUGH A TUBE

Before considering the far more complex issue of blood flow in the circulation, it is better to stay with the simpler system of water and build up to this. Consider water flowing through a straight, cylindrical, rigid tube, ignoring the initial acceleration effect a liquid experiences when first entering a narrower tube. It is known, from Darcy's Law, that the rate of passage of water ( Q) is proportional to the driving pressure gradient ( AP) as follows:

AP Q « &P R (1.7) where the constant of proportionality is defined as the conductivity, or its reciprocal resistance (R), of water through the tube. This equation shows that there are only two ways flow can be controlled; by pressure or resistance.

To extend this a little, consider a tube of length / and radius r, with water of viscosity rj flowing through it. The Poiseuille-Hagen equation relates these quantities as follows:

A P 7i r4 Q = (1.8 ) 8 / T[

Combining this with equation 1.2 allows us to see what quantities affect resistance:

8 / T| R = (1.9) 7 t r

33 This shows that any small variation in r drastically affects R making it a highly influential factor, and is a parameter for investigation in Microcirculation. Here the interest is with the r\ parameter, where it too will be seen to be of significant influence, particularly in the macrocirculation.

Combining the rheological contents of this section with the haematology of §1.2 allows some haemorheological principles to be looked at next, followed by the influences of blood flow in the circulation.

1.4 BLOOD VISCOSITY AND NON-NEWTONIANISM

Water was chosen above because it is an example of the simplest of liquids from a rheological point of view, such that r a y (ie t j= constant). Such fluids are referred to as linear or Newtonian. Other examples include plasma, alcohol, mercury etc. This is all very good, but blood, and some other liquids, are not homogeneous and their r\ depends on y (ie tj constant). Such liquids are referred to as nonlinear or non-Newtonian. Other examples of these are oil emulsions, water based paint and mayonnaise.

Many non-Newtonian fluids are multiphase; eg blood has cells suspended in plasma. This makes defining absolute quantities such as r, y and rj a little more difficult. When whole blood flows, cells tend to disturb the streamlines (velocity flow lines) of plasma such that they become more complicated around the cells. This means that y and t are not evenly distributed throughout the sample, yet rj is still calculated from equation 1 .6 as if they were. Normally these complex flow patterns are ignored and to avoid confusion the quantities are referred to as apparent shear rate (ya), apparent shear stress (t ) and apparent viscosity

( ii, ) 15".

1.5 CAUSES OF BLOOD NON-NEWTONIANISM

There are a number of contributing factors to the non-Newtonian behaviour of blood, but they fall into two distinct parts: RBC deformability and aggregation. To show this, making the RBC's stiff by fixing should remove the non-Newtonian characteristics of blood. Chien confirmed this by fixing canine and human RBC's 132,331 with acetaldehyde (ethanal) for one 34 month. Most of the non-Newtonianism was removed as shown in figure 1.4. As an approximation, Chien also related this to Einstein's equation for rigid particles in a dilute solution, relating apparent relative viscosity ( rjar) to cell concentration (C):

V = 1 + a-C - , (1.10)

The constant a depends on the particle shape and is equal to 2.5 for spherical particles, and larger than this value for non-spherical particles due to larger volumes being swept out by the surface, r\u was used because this quantity allows the r| characteristics of RBC's suspended in different media to be compared and is defined as follows:

ri of the blood sample r\ar = ------(1.11) r| of the suspending phase

RBC aggregation is a phenomenon that is very important and central to this thesis, and the technical details are covered in §1.9. Here, the importance of RBC aggregation is demonstrated by discussing its influence vn a number of phenomena, and then the effects of , „ , . . MaAM//uf/K€ a number of characteristics / aggregation are discussed.

Figure 1.4: Logarithmic relationship between T |„ and y, for non-aggregating and aggregating RBC's. Also shown is the q,, behaviour of fixed RBC's which does not change with y t. Modified from a paper by Chien1311. 35 1.5.1 THE IMPORTANCE OF RBC AGGREGATION

The non-Newtonian behaviour of blood is shown in figure 1.5a, where rja can be seen to drop exponentia/as y, increases. In fact to a good approximation the drop is bi-exponential; as shown by plotting the same data on log-log axes (figure 1.5b). Furthermore, with the aid of power law equations, two straight lines can be regressed through points of the aggregation and deformation phases which has useful analytical properties as is discussed and extended in chapter 3. This bi-exponential drop in r)a, nonlinear behaviour, is caused

by RBC's and is known as the shear thinning phenomenon 18,31,341 and is easily explained as follows. When forces are small RBC's are relatively free to aggregate, but as these forces increase, RBC's find aggregation more difficult because of a build up of forces between the RBC's. Eventually, if the force is increased enough, RBC's can no longer aggregate, and the RBC's are monodispersed throughout the sample. Increasing the force beyond this point causes the RBC's to deform (figure 1.5c). To emphasize this, at low shear rates (LSR's) RBC's aggregate and at high shear rates (HSR's) they deform. Another example of the shear thinning effect is found with non drip paint. In the pot it is highly viscous preventing it from being spilt. As it is applied to the walls with a roller, the forces are such that its r| drops and if* spreads across the wall.

When blood is allowed to come to rest, the RBC's are relatively free to aggregate and form rouleaux as already mentioned. These rouleaux would then act as a logjam against blood starting to flow again and it is believed, and seems logical, that a certain minimum force, the yield stress, is needed to make blood flow again19,35'371. Although the value of yield stress for normal whole blood is small, it is believed that it may still be of some physiological influence which would increase in pathological conditions where levels of aggregation increase.

If a constant, but low, ya is applied to blood then r|a can be seen to continuously change with

time as shown in figure 1 .6 First r|a increases until a peak is reached and then it decreases. This change in r|a over time is referred to as Thixotropy and is associated with RBC aggregation because the phenomenon disappears at HSR's or in non aggregating blood137'391.

36 Figure 1.5: (a) Plot of Ha against y, for a sample of normal human whole blood, (b) Data in (a) plotted on Log-log axes, (c) Schematic representation of the shear thinning of blood.

37 Y-

Figure 1.6: Schematic representation of thixotropy. When a constant, but low, y. is applied to whole blood, then rja can be seen to change with time.

Another phenomenon that aggregation contributes to is viscoelasticity 131,39*421 that becomes manifest in oscillatory flow, as found in arteries for example. Under a continuously changing force the RBC aggregates exhibit dual viscous and elastic properties. The viscous property limits the rate of deformation of the aggregate and depends on cytoplasmic, external and membrane viscosities of the RBC's making up the aggregate. The elastic if is property comes entirely from the membrane of the RBC's forming the aggregate,^believed to be

38 1.5.2 FACTORS AFFECTING RBC AGGREGATION

RBC aggregation is known to be the largest contributing factor to the non-Newtonianism of blood, and it is affected by many different factors. These factors have some effect at HSR's, but these are always much smaller in comparison to those at LSR because of the occurrence of RBC aggregation. Hence, here HSR effects will be largely ignored,

HAEMA TOCRJT

The haematocrit (hct), or packed cell volume (PCV) as it is sometimes called, is the percentage of RBC's to total blood volume. This quantity varies considerably between individuals, but has a normal range of between 40-54% for males, and 35-47% for females. Raised hcts occur at high altitudes (60%), after a heavy drinking session due to dehydration

(55%) or in many blood disorders 17,31,43*451 such as polycythemia rubra vera (overproduction of RBC's), where hcts may rise to 75% or more. Also neonates can have hcts of 50-60% and premature infants of 65-75%. Lowered hcts, anaemias17,31,43*451, occur at times such as in pregnancy, or in disorders such as thalassaemia where hct can drop to 2 0 % or less.

Hct has a large affect on r)a at LSR's, that is directly related to the RBC's increase in aggregation; for hct changes of 35 - 55% at a LSR of 0.3s*1, r|a increases by -500% (at a

HSR of 100s *1 the increase is much less at almost 100%)[9]. These large effects at LSR's makes hct the most influential of blood characteristics.

Chien et al have looked at changes over a wide range of hcts and some of their findings are presented in figure 1.7129,461. It can be seen that there is a good semi-logarithmic relationship between r|a and hct (at fixed shear rates), between the range of 30-60% as described by the following equation:

log T)a = k' + k"*H (1.12) w here H is hct and k' and k" are shear and sample dependant constants.

39 Figure 1.7: Shows the logarithmic relationship between r| and haematocrit for whole human blood at four different y,'s, 0.052,0.52, 5.2 and 52 s'1. Taken from a paper by Chien et al|441.

TEMPERATURE

Another factor that normally has a small contribution towards t| is temperature. For Newtonian liquids it has been shown that rj of simple liquids change inversely with temperature as follows:

_c r\-Ae T (1.13)

where A is the constant of proportionality, Tis temperature (K) and c=E/k; E is activation energy for viscous flow and k is Boltzmanns constant. \

The t| of water is about 0.7 mPa.s at 37°C and changes by 2.4%/°C between 20 and 40°C[47]. Rampling and Whittingstall demonstrated that equation 1.13 also describes ri^. 40 measurements of the more complex liquid blood1481. They looked at the effects of temperature on RBC's suspended in various media (plasma, serum, dextran etc) and found

this equation worked, but in two distinct regions above ( 0= 0^ and below (c ^ ) 22°C where c^Ch. This they concluded to be caused by changes in the deformability of the membrane that occurs at ~22°C. They also found distinct differences: Prohto sufppmm h&ve higher

adivAliort energies dexfaw solutions abL$l\ aAJ d r hwpZrahsrtS btkn-> VL °C.

Rampling and Whittingstall showed that aggregation decreases with increasing temperature, but work by Neumann et al[491 found the opposite of this.

Temperature can become more significant in certain disorders such as when cryoglobulins are produced (cryoglobunaemia) that precipitate at low temperatures. This may also lead to other complications such as secondary Raynaud's Syndrome where circulation is impaired in certain bodily extremities such as fingers1501.

PLASMA PROTEINS AND VISCOSITY

Plasma r| has an adult normal range 1.16-1.33 mPa.s at 37°C with no sex variation1471. Plasma is very heterogeneous in proteins that are known to induce RF during times of low shear,* in their absence there is no RF (figure 1.4). The most influential protein Ifl aggregation is fibrinogen, but other large proteins such as the serum globulins (IgG, IgM etc) can also induce RF1511 (§1.8). In plasma disorders, such as hyperproteinaemia or Waldenstrom's macroglobulinaemia, blood ri, is known to be affected considerably at LSR's due to a large increase in RF and can lead to severe hyperviscosity syndromes143"451. Smaller proteins such as Albumin, do not induce RF, but may still affect blood r|a by influencing larger protein interactions with RBC's138,52, 531. Finally, in grossly abnormal disorders levels of plasma viscosity may rise to 20mPa.s or more and would place severe strain on the circulation.

1.5.2 RBC DEFORMABILITY AND BLOOD NON-NEWTONIANISM

Deformability of the RBC depends on the cytoplasmic r| (haemoglobin concentration, etc) and on the membrane viscoelasticity. The ability of a RBC to deform is generally affected 41 little by external factors, and there are very few circumstances where deformability changes enough to significantly affect blood r|a. One example is sickle cell anaemia127,43*45, However, deformability is also known to be of influence towards RF. Chien showed, with some early scanning electron microscopy work, that RBC's deform to lose their bi-concave disk shape when they form rouleaux1551. It was also shown that if RBC's lose some of their deformability, accomplished by heating RBC's at 50°C, then they find it harder to form rouleaux156-581.

1.6 BLOOD FLOW IN THE CIRCULATION

In the circulatior^blood experiences drastic changes in hydrodynamic conditions, "pie cardiovascular system controls local blood flow to local need through resistance, the majority of which takes place by vasodilation and vasoconstriction. Also, since the vessel radius varies considerably in-vivo, going from 12.5mm in the aorta down to as little as 1.5pm in capillaries, from equation 1.4 we see the resistance in individual vessels will vary enormously.

The importance of RF to in-vivo blood flow remains a controversial issue as was highlighted at the recent Clinical Haemorheology meeting in Big Sky, Montana, when Johnson and Gaehtgens debated the "pro"1591 and "con"1601 views. Johnson believes that vascular resistance increases with increasing RF. However Gaehtgens believes that the in-vivo effective rj, and thus resistance, should remain unchanged (or even decrease) because of the increasing 'lubricating' plasmatic layer with increased RF. Understanding blood flow in the circulation is obviously an important aim and important to this is a belief that much in-vitro work can not be extrapolated to the in-vivo situation. For instance, r|a measurements in- vivo show that hct influences are only minorAwhich is very different to the in-vitro results shown above (figure 1.7). The in-vivo vessel structure and geometry is so very different from the in-vitro setup in that vessels are not smooth or rigid, they are permeable with bifurcations, valves etc. Flow behaviour of blood in the large vessels that make up the macrocirculation (>300pm diameter) will now be discussed followed by the behaviour of blood in the small vessels that make up the microcirculation (<300pm diameter).

42 Figure 1.8: Flow profiles of Newtonian fluids and blood. Note the blunted flow profile of blood compared to the parabolic profile of Newtonian fluids.

1.7 BLOOD FLOW IN THE MACROCIRCULATION

When looking at blood flow in the macrocirculation, it is only really necessary to consider blood as a continuum with a bulk blood r\. At steady flow, the flow profile for blood is not parabolic, as found with Newtonian fluids because r| is not constant (§1.4), but instead is more blunted at the centre and steeper at the walls (figure 1.8). This is a direct consequence CojI&hjh of preferentially flowing down the central region of the vessel, where shear is less, and the smaller components of blood (WBC's[611 and platelets1621) migrating to the outer region of the vessel by interaction with the aggregates and also by hydrodynamical forces. This very important phenomenon is known as margination, and has important contributions towards the defence mechanism of the body, ie WBC's and platelets are closer to the vessel wall, then they can act faster during or damage. In addition to the profile change, there is a cell free region (plasmatic layer), next to the vessel wall that acts as a lubricant that reduces resistance1361. As aggregation and/or blood velocity increases, so the thickness of the plasmatic layer increases. More recently Thurston, who is interested in linking the microstructure of blood to the blood rj, found that flowing blood cells orient themselves into 43 "cell layers" depending on yv separated by "plasma layers" along which they slide. At LSR's rouleaux form large cell layers with few plasma layers "..like a traditional logjam", and at HSR's the deforming cells form compressed cell layers, ie have many plasma layers "...like crowded traffic on a highway"1631.

Pyital-iJikj Blood flow from the heart through the arteries is pulsatile*64,651 and is removed by the 'arterioles' along with much of the high pressure. It is believed that whole blood viscoelastic properties would be of some influence during pulsatile flow, due to the continually cf.&£cetif changing shape. Another type of flow is turbulence, that occurs in large arteries at high pressure*651, at arterial bifurcations where it is thought to act to increase the occurrence of atherosclerosis*661, at the valves of veins, etc.

Understanding blood flow in veins is of particular interest because veins accommodate -60% of total blood volume, and venular flow is sluggish which means that RF can occur. Sluggish flow is enhanced at times when muscle pumps, that help blood flow through legs, are not operational such as with patients confined to bed, people on long flights, etc where the onset of deep vein thrombosis occurs. In clinical disorders inducing hyperviscosity syndromes (eg Waldenstrom's macroglobulaemia, polycytaemia etc), flow becomes even more sluggish and the above mentioned phenomena become even more influential.

1.8 BLOOD FLOW IN THE MICROCIRCULATION

In the microcirculation everything becomes more complicated as one can no longer consider blood as homogeneous, because the micro-vessels are comparable in size to the cells flowing through them. Hence, considering blood rj as one entity is no use as the different subfactors of blood become much more influential to flow, and it is necessary to look at the various characteristics of WBC's, RBC's and plasma.

1.8.1 WBC’S IN THE MICROCIRCULATION

The WBC concentration (or leucocrit) is normally much smaller than RBC's (table 1.1) by a factor o f-1000, but V&r are -1000 times less deformable which makes their effect in the capillaries probably at least as influential as RBC's*671. They pass through small 44 capillaries relatively slowly, because of this lack of deformability, and clog the vessel. Such slower passage of a WBC through a small vessel would lead to a greater concentration of cells passing through other adjoining vessels1441. Also because WBC's are frequently found close to vessel walls, due to margination (§1.6), and have a tendency to role along and stick to vessel walls, this would certainly offer further hindrance to flow. In disorders, such as leukaemias1681, when their numbers are elevated, their effect will obviously be even greater. Also if marginatiofi were increased by increased RBC aggregation, then this would affect resistance as already mentioned.

1.8.2 RBC'S IN THE MICROCIRCULATION

R B C AG G REG ATIO N

The shears in the microcirculation vary considerably from the highest in the whole circulation, at the walls of certain capillaries, to some of the lowest in the venules. At times of low shear, RBC aggregation can which is believed to have certain influences in the microcirculation. For instance, flow could be affected by the failure of an aggregate breaking up at the vessel entrance, thus obstructing (plugging) the vessel depending on its size and shape.

Also of importance is when flow is close to stasis for a time as occurs in vasomotion, the contraction and relaxation of the arterioles. This would effectively cause a log jam and require a certain minimum force, the yield stress, to make blood flow again as already mentioned. The yield stress for blood may therefore contribute to the resistance of flow in the microcirculation even though the value is very small. During times of hyperviscosity syndrome, where RF is greatly increased, yield stress would rise due to the highly elevated degree of aggregation and may become a more influential factor.

Another way RF is thought to be influential in the microcirculation is when blood is slowly passing through horizontal or inclined vessels. Here gravity is more influential and could induce settling that in itself would increase with increasing degrees of RF. In extreme cases this would lead to blood sludging*371, which in turn would lead to uneven spread of hcts in the different vessels. RF is also believed to have an effect in vertical microvessels where RF 45 reduces resistance (ie the reverse to in-vitro measurements) because of its influence towards the cell free zone at vessel walls. The effects of such phenomena remain unclear in vivo.

RBC DEFORMATION

The RBC's ability to deform, possible because of the large surface area to volume ratio, is necessary for them to pass through the very small capillaries less than half their diameter. Gregersen et al have shown that RBC's can deform down to as little as 2.9pm in diameter1691. Small changes in deformability will have more effect at this level, but again only a few disorders will severely affect the flexibility of the membrane or cytoplasmic r| enough to cause a large increase in resistance.

HAEMATOCRIT

It has been shown experimentally that the r| of blood in vessels of the microcirculation is less than the feeding vessels of the macrocirculation170’721. This drop in rj is known to be caused by a drop in hct from the feeding reservoir, occurring when blood enters narrow vessels (<300pm), a phenomenon called the Fahraeus Effect19,70'72]. The hct difference increases as microvessel diameter decreases, such that the hct in 3 pm vessels can be as little as 20% of the feeding reservoir. This is an important physiological phenomenon because the lowering of the hct will greatly reduce resistance by lowering aggregation levels and increasing the cell free zone.

There are a number of in-vivo phenomenon that can lead to local hct rising, some of which have already been mentioned. A further way local hct can rise is by increased plasma diffusion across the microvessel wall as occurs in inflammation etc. If plasma loss is elevated microvascular occlusion can occur when the local hct rises to almost 100%; a phenomenon called compaction stasis136,731. It is also now believed that aggregation- dependant settling in horizontal tubes may play an important part in producing this elevated haemoconcentration1731.

46 1.8.3 PLASMA VISCOSITY IN THE MICROCIRCULATION

It has been shown that in capillaries, there is a thin layer of plasma separating the cells from the wall. As mentioned above, there is also the complex flow pattern of plasma surrounding the cells that is generally ignored (§1.6). In the microcirculation these complex flow patterns are more influential towards flow, resistance etc and so can not so readily be ignored, especially considering disorders that dramatically affect plasma r| as in paraproteinaemias1681.

This concludes the general background material that has shown many ways aggregation can influence the in-vivo system. A more complete account of RBC aggregation, or more precisely the previously mentioned linear form of aggregation termed rouleaux formation (RF), is now given with hypotheses for the mechanisms that leads to RF.

1.9 ROULEAUX FORMATION (RF) / AGGREGATION

Above the term RBC aggregation was used and has a general definition of the weak (easily reversible) sticking together of RBC's. There are however various forms of aggregation from the linear face-to-face loose sticking together of RBC's (RF), to an extreme case of the tight sticking together of RBC's to form a complex clump (clumping)174,75]. Moving from one form of aggregation to the other can be invoked by changing the RBC environment (eg temperature, protein concentration, etc) which is why the term "aggregation" is generally used. It may be useful to know what form of aggregation is occurring in a blood sample, because of the potential different behaviour of the various forms of aggregate, and also the potential different, or additional, factors involved in the aggregation process. It appears that the present consensus is that clumping is just an extreme case of RF where the same mechanisms are at work, but this may not be true.

Another process aggregation can easily be confused with is agglutination174,76-781 which involves stronger (covalent) bonds which are known to cross-link (bridge) RBC's together. This can be induced in-vivo by antibodies or artificially by lectins and differs from aggregation by the difficult or impossible (irreversible) separation of the RBC's.

47 1.9.1 HOW DOES RF/AGGREGATION OCCUR?

One of the first things the author found out in the field of Haemorheology was that the mechanism explaining the fundamental phenomenon “RF” remains unknown. Currently, there are two hypotheses that explain how RBC's aggregate as follows.

THE CROSS BRIDGING HYPOTHESIS (CBH)

The first more simplistic hypothesis was proposed by Chien and Jan in 1973l55] after extensive studies using dextran. Tfoy proposed that the aggregating agents present are adsorbed over the whole surface of the RBC, and when two RBC's come close to each other they are bridged and held together by this aggregating agent. As the RBC's move about each other, with the aid of rotating membranes etc, more bridges form until the two cells form a rouleau (figure 1.9a). Hence this was called the "Cross Bridging Hypothesis" (CBH). Due to the large number of different polymers (table 1.3) that induce aggregation adsorption to the RBC surface is considered to be non-specific. The thermodynamics of this system describes how free energy reduction associated with adsorption is available to do work required to aggregate the RBC's.

THE DEPLETION 1A YER HYPO THESIS (DLH)

The second hypothesis is far more complex, evolved more slowly and there currently appears to be much confusion surrounding it. Work supporting this hypothesis, appears to go back to the 1954 when Asakura & Oosawa proposed some theoretical work that showed aggregation of parallel plates does not require adsorption or requires non-adsorption1791. This work stems from the thought that close to a surface the Brownian motion of the aggregating polymers is restricted, and in the absence of energetic interactions with the plates' surface^ their configurational entropy is decreased. Such a decrease in polymer entropy near to a surface, leads to a decrease in concentration in this region; ie depletion. When the depleted region between two parallel plates overlap, the osmotic pressure in this region is much less than the bulk phase, and thus the osmotic force difference acts to push the plates together. Hence, aggregation by this hypothesis is considered an entropic rather than an enthalpic event and is referred to as the "Depletion Layer Hypothesis" (DLH). 48 (a) The Cross Bridging Hypothesis

Overlapping regions of depletion Osmotic forces

/ \

(b) The Depletion Layer Hypothesis

Figure 1.9: Schematic representations of the cross bridging hypothesis (CBH) (a), taken from a paper by Chien and Jan1”1, and the depletion layer hypothesis (DLH) (b). See text for details. It can be seen for the CBH the RBC's are held together by macromolecular bridges, and the lower panel shows an enlarged view of the regions bridged together. For the DLH a region of depletion is shown around the RBC's, and the arrows represent the external osmotic forces that cause the RBC’s to aggregate. 49 Evans and Needham added further foundation to the DLH by extending this colloidal work to plasma proteins. They have shown with experiments, and have developed supporting theoretical work, that in the presence of large molecular weight proteins, but in the absence of adsorption, strong attraction between vesicles occurs. They explained their findings in term s o f the D L H 180, 811.

However, the above work was not on RBC's, and it was Janzen and Brooks who applied the colloidal theories, and Evans and Needhams work, to RBC's and RBC aggregation (figure 1.9b),82]. Janzen carried out a number of experiments on protein adsorption to the RBC surface and concluded there was very little adsorption, and certainly not enough to induce aggregation by the CBH: "to promote aggregation surface adsorption would ideally be zero"1821. Janzen argued that most of the evidence supporting adsorption was artifactual, and much of the work explained by the CBH could be easily explained by the DLH.

Aggregating A v erag e M W LENGTH DIMENSIONS CHARGE A g en t (kD ) (n m ) (nm x n m )

ARTIFICIAL:

‘Dextran 20 20 24 N eutral TDextran 40 42 36 N eutral D extran 70 53 N eutral D extran 80 74 55 N eutral Dextran 150 140 75 N eutral Dextran 500 450 150 N eutral Dextran 2000 2000 29 0 N eutral Polylysine 9 P ositive Poly-l-Glutamic Acid 50, 66, ‘20 N egative PolyVinylPyrrolidone 360 N eutral H eparin 17 N egative PROTEINS: Fibrinogen 340 47.7 5 .0 x 4 5 .0 N egative IgG 150 20 N egative ‘Album in 66 15 3 .8 x 15.0 N egative Table 1.3: Reference list of approximate specifications for some macromolecules, artificial and natural, which generally induce RBC aggregation, ‘indicates macromolecules do not induce aggregation on normal RBC's. Indicates macromolecules induce a little aggregation.

50 COMPARING THE CBH AND DLH

What is of fundamental importance to both theories is the length of the macromolecule. The CBH requires the length to be sufficient to bridge the cells together at a distance where electrostatic repulsion effects are small. The DLH requires a minimum sized macromolecule to form a depletion layer at the surface of two cells, which is large enough to overlap and thus induce aggregation. Dextran, a neutral aggregating agent, has been the main aggregating agent used in studying aggregation, and it is known that dextran fractions have to be of molecular weight 40,000 or greater to induce aggregation1551.

One way of directly assessing if the CBH were true, would be to see if the distance between aggregating RBC's increases with aggregant length; Chien et al used transmission electron microscopy to measure the distance between cells aggregated by different dextran fractions and showed a linear increase in distance with dextran size up to about 150,000 daltons (150kD), although the distance plateaus off at 2000kD for some reason (figure 1.10)I55). The distances measured was less than aggregant length, and was explained by parts of each end of the aggregant adsorbing to the RBC surface. The DLH can not explain this hypothesis other than its being artifactual.

If the CBH were true a question is raised as to where the aggregating agents bind to the RBC. Maeda et al has looked at the possible binding sites of fibrinogen by looking at the effectiveness of different fibrinogen fragments in inducing RF1831. Based upon this they proposed a binding site on the fibrinogen protein, but the assumptions appear dubious.

1.9.2 CONDITIONS NECESSARY FOR RF/AGGREGATION

Chien summarised the forces acting towards aggregation by the following equation131,551:

F a = Fb - F* - F, ~ Fm (1.14)

Aggregating force (Fa) equals the bridging force (F ) less the disaggregating effects of electrostatic repulsion due to the RBC's net negative charge (Fe), mechanical shearing on the cells (F,) and the membrane bending forces (FJ. For the DLH, Fb could be simply replaced with a force relating the overall osmotic attractive force (F0), as the other forces

51 still apply.

The electrostatic repulsion acting between the RBC's increases exponentially as they get closer together. If there is no aggregating agent present (or it is not long enough), then Van der Waals force of attraction or hydrogen bending alone ^ not enough to overcome the electrostatic repulsion.

For RF to occur a number of criteria have to be satisfied regarding the environment and, of more interest here, the aggregating agents. Firstly, that concentration exceeds a certain minimum threshold necessary to induce RF1351. By the CBH this is because there is a minimum number of bridges necessary to link the RBC's. The DLH needs a minimum concentration to induce enough osmotic difference when the depletion layers between RBC's overlap. The secondly criteria is that the aggregating agent has to be over a certain molecular weight and hence length131,33J as briefly mentioned above (table 1.3).

Chien used electron microscopy studies to show that when RBC's aggregate they deform and lose their biconcave shape becoming flat against each other1331 separated by an apparent regular distance. Hence, the reason for the membrane bending force term Fm in equation 1.14.

There does not have to be one definitive way in which RBC's come together to form rouleaux; both the DLH and the CBH may be true with certain aggregating agents, in certain circumstances or they may even work together in a combined theory.

1.9.3 INTRINSIC AND EXTRINSIC FACTORS AFFECTING RF/AGGREGATION

In the past research has been mainly based on looking at how extrinsic factors (eg environment, different aggregating agents, etc) affect aggregation, but very little work has looked at what intrinsic factors (ie RBC properties) affect aggregation. These two categories will now be looked at in turn.

52 Figure 1.10: Changes in intercellular distance and dextran concentration of peak aggregation (ESR) against molecular weight of dextran fraction. Data taken from a paper by Chien and Jant55].

MOLAR CONCENTRATION OF DEXTRAN Figure 1.11: Effects of increasing dextran concentrations, of various dextran fraction molecular weights (20- 2000kD), on aggregation. Aggregation was assessed by a microscopic aggregation index (MAI) for RBC suspensions at 1% haematocrit. Taken from a paper by Chien et al131*.

53 EXTRINSIC FACTORS AFFECTING RF/AGGREGA TION

The environment in which the RBC's are suspended is highly influential to how they form rouleaux, and as such much work has been carried out to look at the different factors including temperature148, 49, 141 (§ 1.5.1), ionic strength185, osmolality*841, pH1841 These factors influence or affect the RBC properties which in turn changes their aggregating tendencies. Findings from these studies provide more insight into the behaviour of aggregation, but are not relevant to the rest of this thesis as they are kept constant in the experimental studies reported by the author.

There are known to be a wide range of macromolecules that induce aggregation, some of which are shown in table 1.3, whose characteristics differ considerably. probably interact with the RBC surface in different ways. For instance polylysine is a positively charged polymer that will be attracted to the surface of the RBC, whereas dextran is neutral and so will not have any attraction, polyglutamic acid is negatively charged (as are all serum proteins) and so will have a tendency to be repelled. All of these different aggregating agents, from studies carried out so far, appear to induce aggregation in exactly the same wayP».

Another extrinsic effect that is important here, is the concentration of aggregating agent. Chien has looked extensively at the effect of varying dextran concentrations on aggregation1551. To summarise, over a certain sized dextran fraction (~40kD), increasing dextran concentration caused RBC aggregation to increase until a peak at a dextran concentration of -3.5%, and Me/* // a disaggregating phase beyond this point. Chien obtained these results from three independent methods; one method was a microscopic aggregation index (MAI) and the results are shown in figure 1.11. This aggregation/disaggregation behaviour has been confirmed several times since, and more recently Whittingstall et al looked at several different aggregating agents, and showed the peak occurred at different points (dextran 70=3%, heparin=6%, PVP=0.5%, PGA=0.6% )[921.

Previously the disaggregation phase, seen at high concentrations of dextran in figure 1.11, has been explained in two ways:

54 1. increasing zeta potential with increasing dextran concentration causing greater electrostatic repulsion 155,93'961 2. greater adsorbed dextran layer than free dextran layer (depletion stabilization )1971

The first explanation has recently been put in serious doubt by BSumler et al 198,991 who showed that measures of RBC UE in different dextran concentrations did not increase substantially as explanation T requires. Also values obtained were less than those predicted by the Smoluchowski's equation (equation 1.1) as expected by the DLH. Using the same principles of measuring UE, similar UE work was carried out using PEG instead of dextran and has produced similar findings11001. A theory has been proposed by Donath et al, based on this work, which suggests "....adsorbed dextran creates conditions favouring the development of a depletion layer. The available adsorption sites are already occupied and the polymer near the adsorbed dextran has to lose some conformational entropy "11011 - ie depletion stabilization. Hence, in contrast to what earlier workers thought, as mentioned above, this theory suggests adsorption can occur. To enable the two possible mechanisms for the DLH to be discussed, they will be referred to as the adsorption DLH, and the non- adsorption DLH.

Also Chien and coworkers looked at the effects of increasing dextran concentration on neuraminidase treated RBC's where surface charge is much reduced (-90%). Here the disaggregating phase was removed, and instead the cells remain aggregating at the same maximal level (figure 1.12)11021. This was believed to be caused by the surface saturation not being reached and Brooks has shown the plateau remains up to 150g/l dextran 80 on normal RBC's195]; higher concentrations are not possible because of the insolubility of dextran.

Chien also looked at the surface adsorption of dextran to normal and neuraminidase treated RBC's and the results are shown for dextran 80 in figure 1.13,103); similar curves were obtained with dextran 500. Adsorption to both normal and neuraminidase treated RBC's, with dextran 80 and 500, showed a two-step behaviour with secondary adsorption occurring at a dextran concentration of ~5g/l, that is possibly caused by increased number of available adsorption sites. Also seen u/M /iewro/mW/reefedHOc'/ it &rphkn plaf-Qov/ /owe- Mar, un/'recif-sJ A&c}. This is believed to be because the disaggregation process, found with normal, but not neuraminidase treated RBCs, allows more dextran adsorption. 55 T

------1------1 I------1------1------1 I 0 2 4 6 8 10 12 Dextran concentration ( g B/o) Figure 1.12: Effects of increasing dextran concentration on the aggregation of normal (dotted line) and neuraminidase treated (solid line) RBC's. The latter shows no disaggregation phase at higher concentrations of dextran. Taken from a paper by Jan and Chien,lo:1.

Figure 1.13: Adsorption isotherms of dextran 70 to the surface of normal (solid lines) and neuraminidase treated (dashed line) RBC's. Taken from a paper by Jan and Chien'1011.

56 Rampling and Whittingstall found that aggregation increased linearly with increasing fibrinogen concentration from ~ 1.5g/l up to 13g/l11041. Aggregation effects may plateau at higher concentrations of fibrinogen, but technical difficulties in obtaining higher concentrations of fibrinogen have prevented this from being investigated.

Maeda et al has carried out extensive work looking at how extrinsic factors affect RF, and these are all summarised in a recent review paper*511. Much of his work has repeated earlier work looking at these extrinsic factors, but he has also extended many observations. He has set up his own video analysing system based around the rheoscope1105, 1061 that allows measurements of the velocity of aggregation to be made. The velocity of aggregation froM his work appears to directly relate to measures of aggregation using other techniques. A few of his findings are as follows: • Fibrinogen is more effective than IgG on normal RBC's but the reverse is true on iieuraminidase treated RBC's11071. • Increasing albumin concentrations inhibits IgG based aggregation but accelerates fibrinogen based aggregation1521. Also 8 kD and 20kD MW polyglutamic acid, known not to induce aggregation themselves, inhibits IgG aggregation but leaves fibrinogen aggregation unchanged1891. • Fibrinogen induces 1-dimensional rouleaux but IgG preferentially induces 3-dimensional globular aggregates11081. Maeda explains all of his results with the CBH giving no acknowledgement or account in terms of the DLH. With recent from work ffom Baumler, and others researchers, the DLH has been gaining more support recently, and so ignoring it from analysis (discussion) seems unwise!

57 INTRINSIC FACTORS AFFECTING RFMGGREGA TION

In looking at the intrinsic factors responsible in aggregation, it is useful to start by listing the factors that may influence aggregation:

• Size • Deformability • Surface properties (charge etc.) • Adsorption sites

Knowing what factors are responsible for changes in RF can contribute valuable insight into understanding the mechanisms of aggregation.

Meiselman, and other researchers in his laboratory, have looked at intrinsic properties of RBC's and has shown subject variability, as was assessed by measuring aggregation for different RBC's under the same conditions11091. RBC's of different ages were also looked at, and again each age population showed a similar degree of subject variability11101. Another approach was to look at subject variability in disorders where RBC's were known to have raised tendencies towards aggregation (eg Diabetes and Hansens's disease); again a similar degree of subject variability was found in these disorders11111. Also looked at was lowered aggregation levels, as found with full term neonates, and again similar degrees of subject variability were found1281. These variabilities were related to cellular factors (cell size1921, deformability1109,1101, surface charge 11101 or ABO group or Rh factor1921), but none explained the subject variability. Also, corrections were made for different RBC numbers but this did not significantly change the findings. Such work has clearly shown the importance of cellular factors to RF, and understanding these factors better can indirectly lead to greater understanding of the mechanisms of RF.

58 1.10 WHAT IS TO FOLLOW

There has been much work carried out looking at different aspects of potential mechanisms for RBC-RBC interactions but presently there is much confusion and uncertainty. The original aim of this thesis was to specifically look at the affects of different enzymes on the aggregating tendencies of RBC's. It soon became apparent that this provided new information about the aggregation process, and about the CBH and DLH hypotheses. erqy*#. tfjech rtAUtin Although A . an important part of this thesis, there is a second equally important theme included here. This is referred to by the second part of the thesis title; ie that of improving techniques for the quantitation of RF. To appreciate where the necessary twist towards this direction took place consider the original project.

The main approach to investigating the mechanism(s) of RF in this thesis was to enzymatically degrade the RBC surface and measure RF and cellular factors (size, deformability and surface charge) before and after this enzyme modification (chapter 6). Changes in the RF could then be related to the cellular factors. It was the measuring of RF where problems started to occur, mostly due to the extremely high level of RF caused by the modified RBC's.

Two well established instruments were used for measuring RF, one based on T], and the other on optical measurements, and both initially failed in some way to accurately measure the levels of aggregation (hyperaggregation) found with the enzyme modified RBC's. What soon became apparent, was that making accurate measurements of hyperaggregating blood suspensions, with these two techniques, was something that appears not to have specifically been investigated. This not only posed a very interesting challenge, but also has some potentially clinical relevance when investigating abnormal hyperaggregating blood samples. It was obvious that the data from both instruments had potential for yielding much more information. This was particularly true in the case of the optical method which also had many failings, many of which are documented here along with some ways of avoiding or recognising the failings (chapter 4). Chapter 3 looks in more detail at methods of measuring r|„ and the different possibilities for what information the data may provide.

59 A number of researchers have used equation 1.12 as the starting point to derive other equations for predicting whole blood rj,'s at a certain hct and also correcting r|a for changes in hct in whole blood. Chapter 5 contains the authors attempts at manipulating this equation and also looks at how hct affects hyperaggregating RBC suspensions. It is known that small changes in hct for normal aggregating suspensions can have significant affects on r\M, but surprisingly this had never been looked at for hyperaggregating suspensions. Preparing results for chapter 6 included correcting tj, of the various RBC suspensions for small changes in hct. How small hct changes affects optical measures of RF have been fairly much ignored previously, and this was reassessed here. Chapter 5 was yet another chapter necessary for data presented in chapter 6.

The final results chapter of this thesis, chapter 7, uses a completely different approach for investigating the mechanisms behind RF. Interactions of fibrinogen with the RBC surface were looked at using radiolabelled and fluorescently labelled fibrinogen. The main method used for this was extended to allow an assessment to be made, as to whether the distance between RBC's increases with increasing dextran length as the CBH would suggest, and as Chien et al found (§1.9.1).

Note, throughout all of the chapters that follow, when the term aggregation is used, this refers specifically to RBC's aggregating in linear networks, ie 'rouleaux formation' unless otherwise stated. Also, for simplicity the term apparent viscosity and apparent shear rate will be referred to as simply viscosity and shear rate respectively for the remainder of this thesis.

60 CONTENTS OF CHAPTER 2: METHODS, MATERIALS AND INSTRUMENTATION

GENERAL PREPARATIONS:

2.1 PREPARING STOCK SOLUTIONS 62 2.1.1 RBC WASHING AND SUSPENDING SOLUTION 62 2.1.2 FILTERING SOLUTIONS 62 2.1.3 ENZYMES 63 2.1.4 FIBRINOGEN 63 2.1.5 DEXTRAN FRACTIONS 65 2.1.6 VISCOSITY MEASUREMENTS OF FIBRINOGEN AND DEXTRAN STOCK SOLUTIONS 65 2.1.7 ALBUMIN 66 2.2 BLOOD SAMPLING 66 2.3 PREPARING RBCS 66 2.3.4 NORMAL RBCS 66 2.3.1 ENZYME DIGESTION OF THE RBC SURFACE 68 2.3.3 DENSITY FRACTIONATING RBCS 68 2.4 PREPARING RBC SUSPENSIONS (RBCSusp) 69

EQUIPMENT USED AND MEASUREMENTS MADE:

2.5 MEASURING AND ADJUSTING HAEMATOCRIT 69 2.6 MEASURING CELLULAR FACTORS OF THE RBC 70 2.6.1 SIZE AND DISTRIBUTION 70 2.6.2 DEFORM ABILITY 73 2.6.3 SURFACE CHARGE 76 2.7 MEASURING LEVELS OF AGGREGATION 78 2.7.1 VISCOSITY (r|) MEASUREMENTS 79 2.7.2 AUTOMATED OPTICAL MEASUREMENTS OF AGGREGATION 82 2.7.3 ERYTHROCYTE SEDIMENTATION RATE 84

61 CHAPTER 2: METHODS, MATERIALS AND INSTRUMENTATION

The purpose of this chapter is to describe the various techniques and devices used to obtain the results presented in this thesis. Details are given of how stock solutions were prepared and used, how blood was collected and prepared and how the general measurements were carried out on the various devices used. Also a brief description of some of the main devices used is given. Methods, materials and instrumentation specific to only one chapter are given at the beginning of that chapter.

GENERAL PREPARATIONS

2.1 PREPARING STOCK SOLUTIONS

2.1.1 RBC WASHING AND SUSPENDING SOLUTIONS

For the majority of work carried out in this thesis, the RBC's were washed in an isotonic (pH 7.4±.01) physiological buffer, phosphate buffered saline (PBS) with osmolarity 293±5 mOsm. There are various different recipes for PBS solutions, but for work carried out in this thesis the following was usedIII2J: 0.121 MNaCl 0.030 M Sorensens A (81%, Na 2HP04) + B (19% KH2P04) 0.1% NaN3 NaN3 was added to prevent biological growth in the PBS.

2.1.2 FILTERING SOLUTIONS

It was sometimes necessary to work with filtered distilled water or filtered PBS as will be seen later. Filtering involved simply passing the solution through a Millipore filter of 0.22pm diameter pore size.

62 2.1.3 ENZYMES

All enzymes used were obtained from Sigma (Sigma Chemical Co, St. Louis, Mo. USA). The serine proteases trypsin and a-chymotrypsin and the proteolytic enzyme bromelain, were prepared by dissolving lg of enzyme into 10ml PBS. The proteolytic enzyme neuraminidase, was dissolved in PBS to give lOOU/ml1. These were dialysed overnight against PBS at room temperature to remove any impurities that may interfere with the RBC's, centrifuged at 3300 rpm for five minutes and the pure enzyme solutions collected free of any precipitates. The final volume (Vnh) was measured and concentration (C) was given by the following equation:

where Wt is the initial weight, or number of enzyme units, dissolved. This assumes that the concentration of impurities is small.

These were stored in small aliquots (~lml) at -20°C until needed, and were then defrosted at room temperature or at 37°C, used that day and kept at 4°C in between subsequent experiments until finished.

2.1.4 FIBRINOGEN

Human fibrinogen was obtained in lyophilized powder form from Chromogenix (Chromogenix AB, Taljegardsgatan 3, S-431 53 Molndal, Sweden) and contained 42% 4> and 58% salts by weight. This powder was added to PBS (~0.25g : 5ml) and left undisturbed over a heat bath of 37°C until completely dissolved. Once dissolved it was dialysed against PBS overnight at room temperature to remove unwanted impurities, centrifuged on a Centaur II MSE centrifuge at 3300rpm for five minutes and the homogeneous purified fibrinogen solution collected free of any precipitates. These stock *

*Trypsin (MW=24 kD : activity 12,900 U/mg protein), a-chymotrypsin (MW=22.5 kD : activity 52 U/mg protein), bromelain (MW=33 kD, 2480 U/g protein) and neuraminidase (l.lU/mg protein). (NB: 1 enzyme unit (U) catalyses the transformation of lpM substrate per minute under standard conditions.) 63 solutions were stored in small aliquots ( 2ml) at -20°C, defrosted at 37°C when needed and returned to -20°C after use. It was also found that if fibrinogen was stored at -20°C for long periods of time (generally greater than a month), it would to some extent degrade. Hence, concentrations of fibrinogen stock solutions stored for long periods were re-measured.

Fibrinogen is well known to be a fairly unstable protein that sporadically precipitates at room temperature for no apparent reason. This has been assumed to be caused by some impurity introduced into the system, and so attempts were made to avoid this by rinsing all containers with filtered distilled water before use.

MEASURING FIBRINOGEN CONCENTRA TION:

The concentration of the stock solution of fibrinogen was measured using the standard Rampling/Gaffney thrombin clotting technique*1131. Briefly, Thrombin Topical' (MW 33,580) obtained from Armour Pharmaceutical Co (Kankaka, Illinois, 60901, USA) was diluted down to 200 U/ml with PBS. lOOpl of thrombin stock solution was added to a mixture of200pl PBS with either 200jj1, lOOpl or 50pl of fibrinogen solution for estimated fibrinogen concentrations < 5mg/ml, ~5-12mg/ml or > 12mg/ml respectively. After leaving this for at least 30 minutes for all active fibrinogen to form a fibrin clot, the clot was collected with a wooden applicator stick, washed in PBS and added to 5mJ of dissolving solution (4M Urea + 0.1M NaOH) taking care not to dip the stick into the solution. The control (background) was just 5ml of dissolving solution. Both were covered with a marble and left for at least 15 minutes at 80°C for the fibrin clot to dissolve, and absorptions were measured at 280nm (A2g0) using a Philips SP8-500 Spectrophotometer, with the Autocell Z64 attachment that allowed automatic infusion and measurement of the sample. First the control was measured and the Spectrophotometer zeroed. Then the dissolved fibrinogen solution was mixed and measured. This was repeated three times and the average taken. The fibrinogen stock solution concentration [

15.3 . 0.2 [4>] 280 (2.2) where Vs is the volume of fibrinogen suspension used in ml. 64 2.1.5 DEXTRAN FRACTIONS

Dextran fractions used in this study were dextran 10, dextran 20, dextran 40, dextran 70, dextran 110, dextran 500, dextran 2000 of number averaged molecular weight distributions (N't) 5.7, 15.0, 28.9, 43.4, 76.0, 177.2 and unknown kD respectively. Also used was FITC labelled dextran 40 (M„ 32.2 kD). These were obtained commercially from Pharmacia (Pharmacia Fine Chemicals AB, Uppsala, Sweden) except dextran 500 that was from Sigma.

All stock solutions were made by dissolving 3g of dextran in 20ml PBS, except for dextran 2000 which, due to its solubility, was 0.3g in 5ml. These were all dissolved in PBS and dialysed overnight against PBS to remove any impurities that may affect the RBCs. The final volume ( V of each was measured and concentration (C) given by equation 2.1, again with the assumption of negligible impurities.

All of these dextran stock solutions were stored at -20°C in small aliquots of about 3 ml until needed for experiments. When required a dextran aliquot was defrosted at room temperature (or 37°C) used for that days experiments, and stored at 4°C in between subsequent experiments (up to a few weeks). Dextran was found to be highly stable over very long periods, as assessed by its viscosity measurement and by the induced level of aggregation of the authors RBCs.

2.1.6 VISCOSITY MEASUREMENTS OF FIBRINOGEN AND DEXTRAN STOCK SOLUTIONS

It was necessary to make viscosity (q) measurements (§2.7.1) of various concentrations of fibrinogen and dextran fractions, at 37°C, in order to calculate the relative q's (qr) of RBCs suspended in different media as described in §1.5. Over the range used here, there is a log- linear relationship between suspension viscosity ( rjSUSP) and polymer concentration (JPJ) and results were combined to fit the following equation:

LnCnsusp) • w-lpl ♦ ^(’1®) (2.3) where m is the gradient and rjPBS the PBS viscosity that was found to be ~0.7mPas in accordance with standard value for water. 65 Obtaining standardised equations of this form are beneficial in two ways. Firstly, as a way of checking the dextran concentration and secondly, to obtain rj's of different dextran concentrations quickly and easily. Figure 2.1 shows an example of the linear relationship between ln(r|) and dextran concentration (JDxJ) for dextran 70. The coefficient 'm' describing fits through fibrinogen and various dextran fractions are summarised in table 2.1.

2.1.7 ALBUMIN

Human albumin was obtained from AB KABI (Stockholm, Sweden). Solutions were prepared by dissolving 0.5 g in 5ml PBS, dialysing overnight, centrifuging to remove precipitates, and the final volume measured ( VF]N). Assuming negligible impurities and loss, the approximate concentration (C) was given by equation 2.1.

2.2 BLOOD SAMPLING

Heparin was always used as the anticoagulant, but it should be noted that RBC's were isolated and washed making the anticoagulant used irrelevant. Blood was collected intravenously from a number of healthy, normal human volunteers, put into heparin coated containers (12.5 I.U./ml) and mixed by inverting several times to prevent coagulation. RBC's were nearly always prepared and used immediately after collection, and experiments completed within twelve hours.

2.3 PREPARING RBC'S

To avoid confusion, the terms "whole-blood samples" and "RBCSusp's" are used here to distinguish between the whole blood samples collected from the various subjects and the various "artificial" RBC suspensions respectively.

2.3.1 NORMAL RBC'S

Blood samples were centrifuged on a Centaur II centrifuge MSE at 3300 rpm for five minutes, the plasma and bufly coat (WBC's and 's) were removed using a pasteur pipette and discarded. The remaining cells were re-suspended in PBS, vigorously agitated

66 Number of Total number of Gradient stock solutions T | measurements (m) Dextran 10 1 4 0.0076 Dextran 20 1 4 0.0113 Dextran 40 2 10 0.0153 Dextran 70 6 37 0.0187 Dextran 110 1 13 0.0211 ‘Fibrinogen 3 8 0.0427 Table 2.1: Coefficient, m, of equation 2.3 for various dextran fractions and also for fibrinogen. Derived from a varying number of r| measurements from a varying number of stock solutions. Note: coefficients derived from dextran concentrations up to ~ 100g/l, and from fibrinogen concentrations up to - 12g/l. Linear regression were carried out at a fixed constant (c) of ln(0.7).

Figure 2.1: Log* - linear plot of n against dextran 70 concentration. Also showing a linear regression line calculated through the data points.

67 with a vortex mixer to remove any bound proteins, centrifuged and the supernatant and buffy coat removed again. This constituted one wash, and was performed another twice. After the third wash no buffy coat was present and the RBC's, free of any plasma proteins, were suspended in PBS and referred to as 'Normal' RBC's.

2.3.2 ENZYME DIGESTION OF THE RBC SURFACE

The enzymes trypsin and a-chymotrypsin were the main enzymes used in this study to degrade the surface of the RBC. This work consisted of comparing five different samples: untreated normal washed RBC's (W), trypsinised RBC's (T), chymotrypsinised RBC's (CT) and cocktails of the two where washed trypsinised cells were treated with chymotrypsin (T+CT) and washed chymotrypsinised cells were treated with trypsin (CT+T).

Due to a previous report11141, 5mg of enzyme /ml of RBC's was used for all incubations mentioned above. The volume of enzyme ( Vf;vz) needed to constitute 5mg of enzyme /ml cells in the sample of volume (V ^ / p) was calculated from the following equation:

. 5 H y C ' 100 SAXtP (2.4) '-STOCK IUU whereCmx:K is the concentration of the enzyme stock solution and H is the hct of the RBC suspension. Each sample was mixed with a vortex mixer, and left at 37°C for various times. All incubations were followed by removing the supernatant, and washing the remaining RBC's two to three times in PBS in order to remove the enzyme. No substantial haemolysis occurred during the incubation.

Briefly the enzymes bromelain (B) (5mg/ml of RBC's) and neuraminidase (NA) (33U/ml of RBC's) were also looked at using the same protocol as above.

2.3.3 DENSITY FRACTIONATED RBC'S

Normal washed RBC's were transferred to Dupont ultracentrifuge tubes, centrifuged and the supernatant removed. These cells were density separated by the ultracentrifugation method of Murphy11151. Briefly this involved centrifuging the RBC's, using a Sorvall SA

68 600/RC6S (Dupont) centrifuge with the 30° angled SS34 rotor, at 15,000 rpm for one hour at 15 °C (±5°C). After centrifugation the RBC's were packed to a hct of -98%. This method assumes that the RBC's have been sorted by density corresponding to large, young RBC's at the top and small older RBC's at the bottom. -10% of the top, middle and bottom portions were carefully extracted using a pasteur pipette and the rest discarded. The collected RBC's were again washed in PBS and resuspended at 45% hct.

2.4 PREPARING RBC SUSPENSIONS (RBC^’s)

RBCSusp's consisted of RBC's suspended in PBS with and without aggregating agents present. The calculation of the volume of aggregant solution needing to be added ( VAGG) to give the required concentration of aggregant per ml of supernatant (CAGG\ was made using the following equation:

'AGO AGO •0 - (2.5) 'STOCK whereCSPXK is the aggregant stock concentration, H is the % hct of the sample with volume V ^ p . After the aggregant was added, the sample was mixed with a vortex mixer.

EQUIPMENT USED AND MEASUREMENTS MADE

2.5 MEASURING AND ADJUSTING HAEMATOCRIT

MEASUREMENTS

A small aliquot of homogeneous blood sample, was drawn into a glass capillary tube by capillary attraction and one end sealed by heating for a few seconds. This was performed in triplicate for each blood sample, because occasionally capillaries leak. These were then spun at high speed (15,000g) for five minutes on a Hawksley Microhaematocrit Centrifuge. Measurements were made on each capillary using a calibrated sliding scale device, and the average of the three used. They were nearly always identical to within ±0.25%, but if one tube was grossly different, due to a slow leak, this was excluded..

69 ADJUSTMENTS

In many of the studies the parameters investigated are very haematocrit dependent, and thus to remove this variability hct was usually adjusted to a standard of 45%. The suspending phase volume change (AVSUSP) needed to adjust the hct (H) of a RBCSutp of volume V^ to 45% was calculated from the following equation:

A v, H-AS .V SUSP 45 SOL (2.6)

A positive AVSUSP value represents adding that volume of PBS and a negative value represents removing that volume of supernatant. The hct of all RBCSusp's were re-measured after being corrected because there was a ± 2.0% hct variability due to experimental error.

2.6 MEASURING CELLULAR FACTORS

2.6.1 RBC SIZE AND DISTRIBUTION

MEAN CELL VOLUME (MCV)

The MCV was calculated from the hct and the number of RBC's /I blood. The instrument used to count the number of cells was the Coulter Counter Zbi (Coulter Electronics Ltd, Northwell Drive, Luton, Bedfordshire. LU3 3RH. UK). The Coulter Counter required a 1:50,000 dilution of the RBCSujp in Isoton II2, which was achieved by pipetting lOpl of blood sample into 10ml Isoton II, mixing and adding 1ml of this to 49ml Isoton II. The diluted sample was transferred to an Acouvette container (Coulter) for the purpose of measurement.

The Coulter Counter is schematically represented in figure 2.2. The Coulter was switched on for ~ 15 minutes so that a partial vacuum can build up and then the Acouvette containing the sample, was positioned on the Coulter as shown. When a tap is opened, the cell

2Isoton II is a commercially available, azide free, isotonic diluent based in saline. It is available only from Coulter (or registered retailers) and the formula is unavailable. 70 Figure 2.2: Schematic representation of the Coulter Counter, showing positions of the RBC suspension, orifice and electrodes.

Figure 2.3: Size distribution curves obtained from the Coulter Channelyzer. Three consecutive distributions from the same dilution of RBC's are shown, demonstrating good reproducibility. All points above bascline+45 units were used to give a measure of RBC size spread (o) and mean position (M) as shown. 71 JJfo*, A) « /"* «/trt'JtforHerpJvrfaJ-/U & be *^4-4 V*Mf H* ,nj.Li«a Of, yl«w M t u cnpce, IAiMCj/MjMfArtJj M i W U «■**> hAtyiMhceMsrcJm** Cv\lbr&hi>n Ivklt. '1't'^rtftsrZj fiQCr C^iH, ctlrnQfnuA

With an estimate of the number of cells (Nc) known, and the hct of the sample measured (//) as described above in §2.6, MCV of units fl, was calculated from the following equation:

u c v - JL . _L 100 Nc (2.7)

RBC SIZE DISTRIBUTION

A Coulter Channelyzer (Coulter Electronics Ltd) was attached to the Coulter Counter, and is designed to collect cell size information during the counting process. Frequency distributions were given of amplitudes of the pulses, that change with cell size, produced by changes in conductivity as mentioned above. A test cable was obtained from Coulter Counter Electronics, that allowed the Channelyzer to be calibrated to the Coulter such that there was a linear relationship between pulse amplitude and the distribution position as described in the manual.

Previously a Commodore Pet computer was attached to the Channelyzer and collected and analysed the frequency distribution curves. However, due to technical difficulties the author devised another system as follows. An IBM compatible computer was connected to the Channelyzer via a parallel port with an 8 bit, 0 - 5 V, analogue to digital converter (ADC 10) (Pico Technology Ltd, Broadway House, 149-151 St Neots Road, Hardwick, Cambridge, CB3 7QJ, UK). The computer was programmed (PASCAL) to receive distribution curves and export these to Microsoft Excel for analysis. Figure 2.3 shows the 72 frequency distributions from different dilutions of a sample were highly reproducible, and have the potential to offer information on the overall population of the RBC's as well as the MCV. Shown are limits over which analysis was performed, and the parameters 'M', which is equivalent to MCV, and 'o', a measure of spread or overall population, were simply obtained from measuring off the graphs in Microsoft Excel.

2.6.2 DEFORMABILITY

Deformability measurements were made using the St. George's Filtrometer (Carri-med Ltd, Glebelands Centre, Vincent Lane, Dorking, Surrey. RH4 3YX. UK) connected to a BBC model B computer that calculated and printed out results. This instrument was developed in 1985, and was described in detail by Dormandy et al at that time11161. For measurements made on the filtrometer it was important that the RBC's were washed 2-3 times in filtered PBS and the top 5% of the RBC's were discarded to minimise WBC's. Each RBCSujp was split into three parts of different hct, and the hct of each were corrected to 10±1%. With this method experimental error had a CV o f-5%. Samples were at 37°C before running on the filtrometer at room temperature.

The St George's Filtrometer is diagrammatically represented in figure 2.4. A glass capillary

( 1 .6 mm internal diameter), into which the samples were injected, was separated from a reservoir by a removable filter. The reservoir applies a pressure of ~4cm H20 across the filter. In all experiments carried out, 5pm pore diameter Nuclepore Polycarbonate filters (batch no. 110424) were used (Hemaphil, Costar Corporation, 10 The Valley Centre,

Gordon Road, High Wycombe, Bucks, HP 13 6 EQ . U K ).

After a filter was placed into the filtrometer, it needed to be calibrated for the suspending solution (PBS). Filtered PBS was injected into the capillary to a marked level, and the tap was opened allowing the PBS to pass through the filter. Four detectors were used to obtain three transit times, based on the time it takes the fluid to pass between two adjacent detectors. After the sample had passed the fourth detector the tap was closed. This was repeated for a second time, and if the two consecutive runs did not agree to within 1% o f each other, data was cleared and the calibration started again.

73 SAMPLE IN

Figure 2.4: Schematic representation of the St. George's Filtrometer.

Figure 2.5: Plot of relative filtration rate (rFR) against volume for transit times 1, 2 and 3. Demonstrates how rFR at lime zero (rFR(O)) is calculated by linear extrapolation back to time (volume) zero. 74 Once standardised for the suspending solution, a homogenous 1 0 % RBCSusp was injected into the capillary and passed through the same filter. RBC's have a slower transit time than PBS. depend on their deformability. Transit times^were measured between detectors, and because the volumes involved were the same, the relative flow rate of the RBCSu>p could be calculated by dividing the transit times of the suspending phase with the respective transit times of the RBCSusp. With transit times known, and assuming a linear clogging rate, linear extrapolation back to time zero was calculated as shown in figure 2.5; hence the initial relative filtration rate, rFR(O), could be calculated and was used here as the index of deformability.

Due to the fact that filters vary from batch to batch, the same batch of filters had to be used for one experiment. This posed a problem in that there was only a limited number of filters in a batch ( 10 0). A method was proposed by Bilto & Stuart 1" ! 1where used filters could be cleaned. Briefly, after use filters were suspended in l%^>D$)until cleaning was required. Then the used filters were washed in water and ultrasonicated for one minute (Sonicleaner 6440 - Dawe Instruments Ltd) to remove any rubbish from the filter. Filters were then re-suspended in SDS and sonication repeated three times. Filters were finally washed a few times in PBS to remove any trace of SDS, and stored at 4 °C until needed for re-use.

Problems could occur with protein build up on the capillary walls that greatly hindered, and often prevented, filters from being calibrated. Protein build up was found to be greatly reduced if the capillary and outflow system was thoroughly cleaned after a days experiment and left to soak in 1% SDS until next used. Before being used for experiments, the system was again thoroughly cleaned with filtered PBS to remove SDS.

75 2.6.3 SURFACE CHARGE

For electrophoretic mobility (UE) measurements, RBC's were suspended in buffers of various ionic strengths. These buffers were made up using different combinations of the following base solutions11181:

SOLUTION A: 9 parts 1% NaCl, 0.2 parts M/15 KH^O, and 0.8 parts M/15 Na 2H P 0 4 (pH of 7.32) SOLUTION B: 5.4% glucose

Buffers of ionic strengths (7) 0.172, 0.086, 0.043, 0.017, 0.0086, 0.0069, 0.0043 were made by diluting solution A with 0, 50, 75, 90, 95, 96 ,97.5 parts of solution B respectively. All solutions were degassed (deionizing) to help prevent small bubbles forming in the capillary and interfering with results. Suspensions were prepared by initially washing the RBC's with 1% NaCl and then diluting 5-1 Opl of packed RBC's in 50ml of one of the above degassed ionic strength solutions.

The Malvern Zetasizer 3, based on a 5mW (633nm) HeNe laser, was used to measure UE. It utilizes some specialised techniques for simplifying, speeding up and improving measurements of either cell size or UE. The Zetasizer was connected to and controlled by an IBM compatible PC, where all of the results were stored and allowed temperature, timing, etc to be controlled.

A diagrammatic representation of the Zetasizer is shown in figure 2.6. First, before any measurements could be made, the Zetasizer had to know the position of the left and right walls of the capillary. This alignment was performed manually by positioning the converging point of two laser beams on the left then right walls of the capillary as shown in figure 2.6. With these positions set, the Zetasizer would know the position of the central region of the capillary where measurements were made. Accurately knowing the location of the central region was important because of artifacts associated with electro-osmosis, caused by the negative charge of the glass capillary. Only a small region in the centre of the capillary is free of electo-osmosis, and hence the Zetasizer needs to know the position of this region. Alignment was checked each time the Zetasizer was used by putting a dilute sample

76 BUTFTR WTO SAMPLE WTO

Figure 2.6: Schematic representation of the Malvern Zetasizer III.

0 10 20 30 40 50 60 70 80 90 100

Figure 2.7: AutoScan of what apparent electrophoretic mobility (Ug) across the electrophoresis chamber should look like if Malvern Zetasizer is aligned correctly; ie minimum close to centre. Measurements of latex spheres (3mg/ml PG Monomcrmin in 0.1M NaCl pH 7.0) in chamber.

77 ^ f\s RQc's ftovC /Vi

After making sure the alignment and temperature were correct measurements could be made. The automatic plumbing of the system was bypassed due to problems associated with using it (dirt, leaksj and because better control of volume used was obtained manually. The RBCSusp was injected into the capillary tube and the suspending phase injected into the electrodes. The two solutions were separated by a membrane. The computer was then set up to initiate three consecutive runs separated by a 60 second delay, to allow cooling of the chamber, and UE was measured over a 25 second duration. Briefly, the Zetasizer works by passing a laser beam through a beam splitter, and then converging it to a point in the centre of the capillary. At this point interference fringes occur and any particles present interfere with these fringes causing scattering. This scattering is detected by a 64 channel photomultiplier and as the particles move under the influence of a known constant DC current applied across the chamber, the signals picked up by the photomultiplier changes. From this information the UE could be calculated.

Problems were encountered in obtaining data from this system. One particular problem was that once blood was in the system for a few runs, the capillary would become contaminated with protein deposits, and flow would be affected. If this became too problematic, ethanol was flushed through the system to clean it.

2.7 MEASURING LEVELS OF AGGREGATION

The viscosity, or relative viscosity (r|r) can be used as an index of RBC aggregation in a number of ways that will be looked at more fully in chapter 3. There are problems associated with this method that are based around it being an indirect way of measuring aggregation, and as such care must be taken in interpreting results. Also aggregation was measured more directly using an optical method which reinforced many of the findings.

7 8 Both of these methods for measuring the level of aggregation will now be briefly considered.

2.7.1 VISCOSITY (rj) MEASUREMENTS

Measurements of t| for the prepared RBCSufp's (§2.4), supernatants etc were carried out using a Contraves Low Shear 30 (LS30) Viscometer (Contraves AG, Zurich, Switzerland), based on a 1+ 1 bob in cup type configuration of the original design of Gilinson et al’s11191, and is schematically shown in figure 2.8. The Viscometer was equipped with a wind shield and a heat bath, always set to 37±0.5°C, to keep the cup and contents at a constant temperature. The Viscometer was controlled by an electronic device that allows it to be driven at 30 different speeds, corresponding to different y's, and displayed a value that is related to r\ of the suspension being measured. Only speeds between 10-30 were used that corresponded to y's 0.277-128.5s’1 as shown in table 2.2. The control device had five different ranges of sensitivity; the range used depended on the r| of the sample and the speed it was being run at. (ie. blood might need range 2 at LSR's and range 4 at HSR's). A wind shield was used at all times to prevent the influence of draughts.

S p eed y (s'1) S p eed Y (s ') S p eed Y (s'1) 10 0.277 17 2.37 24 20.4 11 0.376 18 3.23 25 27.7 12 0.512 19 4.39 26 37.6 13 0.695 20 5.96 27 51.2 14 0.945 21 8.11 28 69.5 15 1.285 22 11.02 29 94.5 16 1.747 23 14.98 30 128.5 Table 2.2: Reference table relating Contraves LS30 Viscometer speeds to y's.

79 Tweldw Mra

Figure 2.8: Schematic representation of a Contraves LS30 (bob-in-cup style) Viscometer. Features are exaggerated for clarity.

APPLE IIGS DISPLAY COMPUTER IBM PC

Figure 2.9: Schematic representation of the Myrenne Erythrocyte Aggregometer, an optical device designed for measuring aggregation.

80 Before the start of a days experiments, the Viscometer had to be zeroed and centred. To do this, in the absence of a sample, the bob was lowered into the cup and the reading on the control device (range 1) zeroed using an adjustment located on the Viscometer. The bob was then centred in the cup using the balance adjustments located on the bottom of the Viscometer. If the bob was not centred properly a continuously rising reading, after a short plateau, was found at speed 30. Having made these adjustments, the Viscometer was left to reach 37°C before being used.

Solutions (eg PBS) or RBCSusp's (~lm l), at 37±0.5°C, were transferred to the cup with a pasteur pipette and the bob was lowered. The sample was left for ~5-10s to stabilise temperature to 37±0.5°C. If blood was being measured, aggregates were dispersed by moving the bob vigorously up and down during this time, thus inducing a y of -300-400s_1 on the sample, taking care not to let the bob leave the sample as this introduced air bubbles. This caused any RBC aggregates to be broken up and sample history removed and was proposed by Matrai et al as a way of improving Viscometry accuracy*1201. This vigorous bob movement was not necessary for newtonian fluids like PBS, plasma etc. The speed was selected on the control device and the motor turned on, which rotated the cup thus applying a constant y across the sample to the bob, suspended by a torsion wire, which twisted under the force; ie a torque is applied. A mirror, located at the top of the wire, reflects a measure of the torque to a detector, that via a feedback system electrically removes the torque, and the measure of such removal is transduced into a measure of torque. This torque (7) is related to the mean shear stress (r) in the gap between the bob and cup by the following equation:

2 2 r \ * r 2 . X (2.8 ) rx.r22 2 w here r, and r 2 are the radii of the bob and cup respectively, and h is the height of the bob. With y and t known, using equation 1.8 gives the t|, and the control device continually displays a calibrated value related to the q being measured by a calibration table supplied by Contraves. Measurements were carried out for as many different speeds as required. Also, two different sets of bob and cup were used alternately, being warmed on the heater between runs, for different samples to minimise temperature changes.

81 priiK ipk o f cpvJic'X <-b A te McA is />~jeJ c/> Hie ihotcvi m /fejeg m of- A j / H W v Mi /ruviy^A U>Ui intri(i.(hlpt t-'iCttcScd( /An/At cunc^A^efc u h q ^ a !-■i f ffMJLf/JMJL bih-r bzhs& i file'sftfiC'j/a^ri^eshu WitciejCjn\crt&it{; iqhl'/" har>iwSsio\ IncrtoJCi'i isitiiuiHi //KKAr/^ iA(r<6si/\(i fr& (r&t Xpzce. heJ-u^t^ heJ-u^t^ ^(JC'j/A^it^cd^i, ^(JC'j/AA^it^cd^i, There weree many developments made to making measurements and analysing data with the

ViscometerM M that4 t* Aare » A the 4 l* A subject M l • tft I A m A ofa ^ chapter ^ L A M A A M3. O

2.7.2 AUTOMATED OPTICAL MEASUREMENTS OF AGGREGATION

The Myrenne Erythrocyte Aggregometer (MEA) (model MA-1, Myrenne GmbH, Roetgen, ah men fa^fOn/vA and UUS Germany) is an optical device for specifically measuring aggregation. It worksA the cone on plate type configuration and has a distinct advantage over most other methods because it works on extremely small quantities of blood sample or RBCSlttp (~25pl). A large part of this thesis is based on work carried out on the MEA and the data it produces, and so a full discussion will be given in chapter 4. However, here the general principles of the MEA will be given.

The MEA is schematically represented in figure 2.9. Initially the MEA needs to be calibrated by closing the lid, pressing the button followed by the 'A' button and waiting until 'O' is displayed. A small volume of homogeneous blood sample, or RBCSusp, is pipetted onto a transparent cone and the lid of the MEA is closed bringing a glass slide down onto the sample, thus forming the cone on plate arrangement. There is an infrared light source under the cone which illuminates the sample, and light is picked up by a detector •k located in the lid. Then there are two ways measurements can be made corresponding to two modes of operation: manual mode or computer mode, Tfeswill now be covered in turn.

In manual mode either M or M l can be selected by pressing the appropriate button on the front of the MEA. In both cases a HSR (600s*1) is then applied across the sample for 10s to disperse any aggregates and mix the sample. The MEA then automatically switches to apply a y of 0 or 3s *1 across the sample depending on whether M or M l was selected respectively. The RBC's reorientate themselves and if the conditions are right, they aggregate. Figure 2.10 shows how the light transmission changes: A-B is the HSR disaggregation phase, B-C the reorientation phase and C-D the aggregation phase. As an index of aggregation, the MEA simply measures the area under the curve over 5 s from point C and displays a value proportional to this area on the front of the device. The curve from B - D is knowji as the syllectogram. Tfe shape. / / fyjicdpnjpar-U&s Ctfjaciekd tJilh fhstsc^ple Such a4 Mt vtloc/iucy . Cf^^cjcJe Auxffh<.L^ cifA&a ^vtM - 82 A B C D

Figure 2.10: Changes in light transmission through a sample of whole human blood during 10s (600s*1) disaggregation and 10s aggregation phases. LT data taken from MEA, and the curve from phase B-D is called the syllectogram.

In computer mode, the MEA was controlled by software 3 running on an Apple IIGS computer which allowed the user to choose to apply any y (0-600S*1) on the sample during phase B-D. Up to ten cycles (A-D) could be defined to run one after another. Data was captured from each cycle over the whole period from A-D (20s) and stored for later analysis. The author decided not to develop analysis software on the Apple IIGS computer as it was dated and non-compatible with modem computers that have many advantages. Instead a communications link between the Apple IIGS, running AppleWorks communications software package, and an IBM PC, running communications software written by the author, was devised. With the data on the IBM compatible computer, an analysis system was developed and an extensive study carried out on various aspects of the MEA and the syllectogram, and is the topic of chapter 4.

3Written by: Guillet, R. and Tourain, C. Laboratoire de Biophysique Appliquee. Universite Rene Descartes, 45 rue des Saints-Peres, 75006 Paris, France (1993) 83 The protocol used for obtaining most MEA data was as follows. The MEA was zeroed in manual mode, and then switched to computer mode where software was set up to perform 10 cycles, with y's 0, 3,6 , 9, 12, 12, 9, 6, 3, Os*1 for phase B-D. Following this, the MEA was switcl^to manual mode and three 'MO' and three *M1' readings were taken. Then the above was repeated on a second aliquot, from the sample, but with the software set to apply y's of 0, 6, 12, 18, 24, 24, 18, 12, 6, Os*1. If the two sets of manual readings differed substantially, a further run was carried out and the odd one out ignored. ^ vduet

2.7.3 ERYTHROCYTE SEDIMENTATION RATE

The erythrocyte sedimentation rate (ESR) is a very simple technique for measuring the degree of aggregation of a sample11211. The technique involves sucking 1ml of the homogeneous blood into ESR tube (200mm long) that is then sealed, placed upright and left. Under the influence of gravity, cell-cell interactions etc RBC's aggregate and begin to settle; the more or larger the aggregates the faster the sedimentation. The amount of settling that occurs in 1 hour is defined as the ESR measurement (units - mm)11221; longer settling times were looked at here to attempt to exaggerate differences. This is a very sensitive technique, but is highly dependent on hct, especially with hyperaggregating samples. However, hct was corrected to 45±2% hct which mostly removed this problem.

84 CONTENTS OF CHAPTER 3: A STUDY OF THE CONTRAVES LS30 VISCOMETER

A IM 86

FOREWORD 86

S T U D Y : 87 3.1 VISCOSITY (rj) MEASUREMENTS 87 3.1.1 DEVELOPED TECHNIQUES FOR MAKING ti MEASUREMENTS 87 • LOW t| NEWTONIAN LIQUIDS 87 • NON-AGGREGATING RBCSlup'S 88 • NORMAL AGGREGATING RBCSufp’S 89 • HYPERAGGREGATING RBCSlup’S 89 3.2 T| DATA (ri^u) 89 3.3 CALCULATING r] 91 3.4 ANALYSIS OF 91 3.4.1 GENERAL FEATURES OF ridau AND AN APPROACH FOR ANALYSIS 93 3.4.2 THE T| PARAMETERS USED 93 • T|r AT TWO EXTREME y's (0277 r|r AND 128 \) 93 • GRADIENTS (GA AND GD) 94 • INTERCEPT POINT (IY) 94 3.4.3 PROBLEMS ASSOCIATED WITH 94 • PLATEAU EFFECT 96 • TRANSITION EFFECT 96 3.5 EXPERIMENTAL ASSESSMENT OF THE ANALYSIS AND PROPOSED PARAMETERS 98 • A BRIEF LOOK AT THE IY PARAMETER 98 • TESTING PARAMETERS AND LOOKING AT EFFECTS OF INCREASING FIBRINOGEN CONCENTRATION 99 • TESTING PARAMETERS AND LOOKING AT EFFECTS OF INCREASING DEXTRAN CONCENTRATION 104 •WHOLE BLOOD 104

CONCLUSIONS AND DISCUSSION . 107 85 CHAPTER 3 : A STUDY OF THE CONTRAVES LS30 VISCOMETER

AIM

The general workings and standard operation of the Contraves LS30 Viscometer were given in §2.7.1, and here a study is presented that extends this in two ways. Firstly, some methodology is given that briefly explains how measurements were made on Newtonian liquids, and on various RBCSuip's ranging from non-aggregating up to hyperaggregating tendencies. The second aspect of this study is analysing viscosity data for these RBCSusp's using the log-log relationship between r|r and y (§1.5.1). Potential parameters from the regressed data are described and assessed with preliminary testing to show some advantages/disadvantages and successes/failures of the different parameters. The discussion and conclusion brings together all of the findings and looks at analysis developed by other researchers.

FOREWORD

What should not be lost here is that the aim of this thesis is to investigate mechanisms involved in RBC aggregation. The necessity of this chapter was to establish an analysis system for the capable of giving parameters that were adequate to- cover the vast range of aggregation investigated in this thesis. Using q ^ for monitoring aggregation is well established, but many problems were encountered in measuring and analysing various RBCsugp's, especially those where aggregation was grossly elevated. These problems meant reassessing how t| measurements were made and what analysis could be carried out on the qdJll. Such an in depth study revealed many new aspects of the q ^ , and the misuse of parameters representing q ^ at times by other researchers.

Some rjdlta used in this chapter will be presented more completely in chapter 6. Briefly, enzymes trypsin (T), chymotrypsin (CT), bromelain (B) and neuraminidase (NA) were used to degrade the RBC surface, leading to large increases in aggregation compared to normal washed RBC's (W) under the same conditions. Three such treated systems were NA, B or T treated RBC's with 15g/l dextran 70 present to induce aggregation (ie NA, B or T+15g/l dextran 70); the NA RBCSujp showed the largest levels of aggregation, followed by B and 86 then T, and all showed far more aggregation than untreated RBC's (ie W+l 5g/l dextran 70). Viscometric measurements of these hyperaggregating systems was not simple, and methodology and analysis had to be developed as will be discussed below.

STUDY:

3.1 VISCOSITY ( ti) MEASUREMENTS

After initially setting the Viscometer up (§2.7.1), the sample was pipetted into the cup which the bob, suspended on a wire, was then lowered in to. The cup was then made to rotate, which applied a shear on the sample that in turn acted on the bob, twisting it by an amount dependent on the sample r|. A control device allowed the speed of cup rotation, and thus y on the sample, to be selected, and whilst being applied the instrument continually displayed a reading related to the torque on the wire. From this the t| of the sample could be obtained via a conversion table.

The torque readings are not given instantaneously of course, nor are they constant for various reasons (eg thixotropy). When a y is first applied to a solution the torque reading can be seen to rise continuously until a maximum is reached which is where readings are normally taken. However, if the solution is an aggregating RBCSuip, at LSR's the maximum may take 30s, or more, to reach and it normally lasts a short time (~ls) before dropping (figure 3.1b). The delay at the peak was a useful indication that the maximum readings were being obtained. The subsequent drop is due to the combined effect of settling and the build up of a cell free zone between the cup and bob surfaces and the sample. The usual approach to making r| measurements on RBCSusp's is to use the maximum reading. In all suspensions looked at by the author, at HSR's there was no problem since the maximum was quickly reached and maintained.

3.1.1 DEVELOPED TECHNIQUES FOR MAKING ti MEASUREMENTS

LOW n NEWTONIAN LIQUIDS

For Newtonian liquids, like PBS, solutions of dextran etc, after applying a y (between 5.96- 87 69.5s*1) the torque reading quickly reached a maximum where values were taken (figure 3.1a). The average reading of all calculated t|’s was used to give the measure of the solution T|. Going below 5.96s*1 generally produced irregular low readings, and going above 69.5s*1 normally produced artificially high readings, thought to be due to vortexing that occurs at HSR's in such low viscous solutions.

NON AQQREQA UNORBCs^S.

To measure the r\ of washed RBCSusp's, with no aggregating agent present, readings were used between y's 0.945-128.5s*1. Here as a LSR was applied, it caused an artificial overshooting of the shear stress on the sample, and thus the bob. This reading was very short lived, dropped rapidly and finally increased again to a maximum which was the reading taken (figure 3.1c). Readings at y's below 0.945s*1 were certainly possible, but were generally less consistent and so were not used by the author in any analysis.

Figure 3.1: Demonstrating response of Viscometer with time after applying a LSR to (a) a Newtonian liquid, (b) a normally aggregating RBCSu,P, (c) a non-aggregating RBCs^p or (d) a hyperaggregating RBCsu.p-

88 NORMAL AGGREGATING R B C ^

Measurements of various normal aggregating RBCSusp's were carried out at several y's, as described above, and were generally straight forward because the maximum reading was always easily obtained and used (figure 3.1b).

HYPERAGGREGA TING SYSTEMS

Generally the Viscometer produces excellent, highly reproducible, t\ measurements between 0.277-128.5 s *1 on blood samples and RBC^p's. However, at LSR's with hyperaggregating RBCSusp's (eg T, B or NA+15g/l dextran 70) obtaining readings was much more difficult as the maximum reading was highly variable. After applying the y the reading rises to a maximum, taking about 15s, and drops without the usual delay, giving the impression of being artificially low (figure 3.Id). Hyperaggregating RBCSlup's required more vigorous mixing, for longer times, to achieve the momentary delay associated with the maximal point, as described in §2.7.1.

3 . 2 T) D A T A (T|dlU)

The non-Newtonian behaviour of blood, ie decreasing rj with increasing y, was briefly described in §1.4. Here a more complete assessment of the behaviour of r j^ is given and various parameters considered. As mentioned in §1.5, relative viscosity (r|r) is used because it is believed that it allows the r| characteristics of RBC's suspended in different supernatant q's to be compared.

A plot of T|r against y is shown in figure 3.2a for three RBCSusp's with very different levels of aggregation (washed RBC's in PBS, W and CT+15g/l dextran 70). It is not so easy to see what information ri^ may provide when presented in this form; eg to quantify shear thinning (decreasing r\ with increasing y - §1.5.1). Features, such as shear thinning are made much clearer when plotted on a log-log scale, as shown in figure 3.2b. It can be seen that, to a good approximation, there are two "apparent" linear regions; ie data behaves biexponentially. This biexponential behaviour has previously been acknowledged by a

89 Figure 3.2: (a) Linear-linear plot of Hr against y for non-aggregating washed RBC's (W - PBS), normal aggregating RBC's (W+15g/l dextran 70) and hyperaggregating RBC's (CT+15g/l dextran 70). (b) Data from (a) plotted on log-log axes.

90 number of researchers131,45,47, 123J, but appears not to have been more rigorously investigated. The majority of published work has looked at only limited aspects of and particularly there has been little work aimed at quantitating more completely the shear thinning properties. Here shear thinning has been looked at more thoroughly, with the thought that it may be a better representation of the aggregation process occurring in the various JH3CSutp s.

3.3 CALCULATING q

In §2.7.1 it was mentioned that readings from the Viscometer were converted to r| by a conversion table supplied by Contraves. The author looked at ways of improving the conversion process and was able to derive the following equation from the conversion table:

q - £XP[-4.5627*(/2*1.6095)-Zm(y )] • Torque Reading (3.1) whereR and y are the range and shear rate at which the torque reading was taken.

Also the author wrote a computer program (BASIC), called 'AnalEta', that allowed experimental data (ie range, y's, readings and supernatant q) to be input, and then using equation 3.1 it calculated r) and qr. Above it was mentioned that the q ^ appears to decrease biexponentially with increasing y (figure 3.2b), and hence two power law equations of the following form:

n - c. y ° (3.2) were used to represent q ^ through the two "apparent" linear regions. The two linear regions have been termed "A-Phase" and "D-Phase" because the former is considered to be dominated by aggregation and latter by deformation. AnalEta used a method of least squares algorithm to fit a power law equation through data points in the A-Phase and D- Phase regions; ie:

cA-Phase - LSR '*) Ln(r\r) - GA*Ln{ y) ♦ CA (3.3)

(D-Phase - HSR 's) - GD»Ln (y ) ♦ CD (3.4)

91 Figure 3.3: Some potential parameters associated with rid.u presented in figure 3.2b : intercepts 0 )» gradients (GA & GD) and extreme n's at 0.277 and 128.5 s'1 (°277r)r & 128 5r|r )•

Figure 3.4: Demonstrates how computer program (AnalEta) allows to be investigated, by providing user selected limits where points can be ignored from the linear regressions.

92 AnalEta then displayed plots of T) or r)r against y on a loge-loge scale along with the regressed lines. Also displayed were the calculated parameters for these equations (GA, CA, Go, Co), the regressed T|r values at y's 0.277 and 128.5s*1 (ie 0277qr and ,28-5qr respectively), and the y at which the two regressed lines intercept (ie Iy). These parameters are shown in figure 3.3.

3 .4 A N A L Y S IS O F TidlU

3.4.1 GENERAL FEATURES OF qdllU AND AN APPROACH FOR ANALYSIS

In developing AnalEta, the obvious question that occurred was how good the biexponential behaviour for blood was, and which points should be used in each regression. By looking at T| measurements at more y 's, it became apparent that not all data points fall into either linear regression; assessing the extent of the deviation and deciding how to take these points into consideration for a better representation of shear thinning was a problem. As an aid to investigating such points, AnalEta was modified to allow points at the lowest y's and points at the intercept region of the two linear regressions, to be removed from the regression calculation. The facility to remove such points came in the form of user-adjustable boundaries as shown in figure 3.4, where it can be seen that three initial points, and six points at the region of overlap, were removed from regressions; clearly these points fail to fit into either regression. For the purpose of discussion, points not falling into either regression have been described as being due to the "plateau effect" and the "transition effect" respectively and will be further discussed later.

3.4.2 THE T| PARAMETERS USED n. AT TWO EXTREME y's (°:77n and1:85nJ

The most widely used parameters in the literature to represent q ^ is a single q value at a LSR and/or HSR; here the y's 0.277 and 128.5s*1 were chosen (ie °'277q r and 128 5q r)11241. Hct has a large influence on these parameters (§1.5.1), but fixing hct allows them to represent measures of aggregation and deformation and can be defined as follows:

93 BBCSuIp Viscosity at 0.277 x*1 Index of Aggregation 0.277 ^r Suspending Phase Viscosity (3.5)

BBC ^ Viscosity at 12E.5/*1 Index of Deformation 1M.5„ Suspending Phase Viscosity (3.6)

il values for the above two parameters were always obtained from the regression analysis to improve accuracy as mentioned in §3.3. A further index of aggregation used in the literature is to divide these two quantities (ie o-277^ /12*-5^)^24]. However, there is nothing in the literature to decide if °-277r)r or a 277 T|/128 -5T| is the better index of aggregation to use.

GRADIENTS (Ga ANDGqL

Clearly GA and GD are potential parameters for quantitating the shear thinning properties of blood. Here it will be shown that these parameters provide further interesting information, that may give better indication of the aggregating and deforming tendencies of the RBCSujp’s compared to other parameters (ie °‘277r|p 128 5t|r etc).

INTERCEPT POINT a y)

The y-component of the intercept point of the two regressed lines (7y) gives more shear thinning information, albeit crude, about the transition from A-Phase to D-Phase.

3.4.3 PROBLEMS ASSOCIATED WITH iidlU

It has already been implied that is not OfortJ fa. biexponential, and two defined deviations come in the form of Q plateau andAtransition pciAh . The only way of dealing with points of the plateau or transition in the above analysis, is to remove them from the regressipn. It should be made clear here that derived \ equations only truly represent data in the y boundaries of the regression. Any extrapolation beyond these y's was done purely to provide data for the parameters. It is not, and should not be, assumed that extrapolated points represent the behaviour of the RBCSusp beyond the limits of the regression.

94 Figure 3.5: Loge-log* plots of Hr against y for various h\peraggregating RBC&ap's (a) Shows the plateau effect found with T, B & NA+15g/l dcxtran 70 at LSR's. (b) Shows how regression through the four points of A-Phase, which is all that is available with B+15g/l dcxtran 70, represents all A-Phase points of 10g/l dextran 40. Hence, even four points can give an accurate measure of shear thinning during the A-phasc.

95 PLATEAU EFFECT

One instance where the plateau effect was found to occur, was with hyperaggregating RBC^'s. Examples of three such suspensions are NA, B and T+15g/l dextran70 as shown in figure 3.5a. It is probable that the initial plateau, at least in part, was artifactual due to enhanced settling and cell free layer next to the Viscometer bob and cup surfaces (§2.7.1). How much of the plateau represents the true r\ behaviour at these y's was not investigated.

Looking at the data shown in figured.5a it is difficult to see how well the limited.number of points, between the plateau and transition effects, would represent the A-Phase shear thinning properties of the RBCSusp or act as a measure of aggregation. As a way of assessing the use of only the limited number of points available with hyperaggregating RBCSujp's, B+15g/l dextran 70 ri^ is shown in figure 3.5b along with B+10g/I dextran 40 t^ , where there was no plateau effect. In both cases regressions are carried out through only four point in the A-Phase, but the regressed line through B+10g/l dextran 40 can be seen to closely represent all of the points of lower y's. Hence it is possible to obtain the parameters even if only four points were available.

fointsAwere part of the plateau effect, simply chosen by progressively ignoring initial points from the calculation of the- A-Phase Uhl if i regression line,* points were m h o j u e r e then . ignored tn A the final analysis.

TRANSITION EFFECT

The full extent of the transition effect is shown in figure 3.6 for three RBCSusp's: W+15g/l dextran 70 (Aj), T+15g/l dextran 70 (A2) and B+15g/l dextran 70 (A3). More Aah* f>Qi*h cfpuiak. f&L hieppcn&jhai relcdio^inip huh hxuulFon appeal? occur oh Similes

96 Care was taken in choosing which points in this region to ignore, and with the majority of data where a large number of points were available the answer was to ignore points between y's 11.02-5 1.2 s' 1, as was seen in figures 3.3, 3.5b and 3.6. Even if transition was much reduced, these boundaries were always used for consistency.

It is probable that the increased transition with increased aggregation was a reflection of the increased difficulty in separating the aggregates. Hence, transition may offer some useful information about this aspect of the shear thinning properties of the RBCSusp. However, information^ relating to the transition is lost with the above approach and no further quantitation was attempted.

97 3.5 EXPERIMENTAL ASSESSMENT OF THE ANALYSIS AND PROPOSED . PARAMETERS

Now some t)^ is used to test the parameters, but it should be understood that the data was not obtained for such a task, and some of the results shown here are of some importance in themselves. Hence, there is a dual purpose here: to test the parameters and to present some interesting results on aggregation.

A BRIEF LOOK A T THE Iy PARAMETER

1^ will not be discussed in great detail for a number of reasons including the uncertainty as to usefulness or meaning of this parameter, its lack of sensitivity. No rigorous study was carried out on Iy, but values are given here from a few sets of data. To save confusion later, all of the observations concerning the IY parameter are given here. Using values of normal human RBC's as a reference point, at 45% hct unless otherwise stated, findings for IY are as follows:

• normal human RBC's with 15g/l dextran 70 gives 18.53il.14s*1 SD (n=5), with 6g/l fibrinogen gives a higher value at 27.80il.21s*1 SD (n=4) and in only PBS (ie non- aggregating) gives a much lower value at 7.67 ± 0.63s*1 SD (n=7). • the response of IY to increasing dextran 70 concentration on normal human RBC's is shown in figure 3.7. There appears to be little change until above 75g/l where IY increases (n=5). • hyperaggregating normal human RBCSusp's only showed small differences; eg T+15g/l j$-s3 v-fo dextran 70 increased^to 21.20il.89s*1 SD (n= 6) and T+ 6g/l fibrinogen decreased^to 24.3 lil.97s *1 (n=4). • I for hcts of 30 and 50% are 12.42il.33 and 20.99i0.70s*1 SD respectively for normal human RBC's and 22.71i0.90 and 23.65i0.21s*1 SD respectively for hyperaggregating (T+15g/l dextran 70) RBCSusp's (n=2). For normal RBC's IY is seen to increase with hct, but little difference is found with hyperaggregating systems. • increasing supernatant t) on non-aggregating washed RBC's causes IY to decrease, ie for RBC's with 15g/l albumin, IY was 7.88s *1 that progressive decreases to 3.74s *1 at 75g/l albumin (n=l). 98 IY probably acts as a measure of the change from aggregation to deformation in the various RBC^'s. A few instances were seen above where IT showed large changes, and explaining these changes was not always easy. For example, the most reasonable explanation as to why was much higher for fibrinogen than for dextran 70, appears to be different mechanisms involved in aggregation induced by these to aggregating agents; this is further supported by data presented in chapter 6 where other differences are found between the actions of dextran and fibrinogen. Another interesting observation here was with non-aggregating RBCSufp's where much lower values in IY were found. A possible explanation here is the small forces that deform the monodispersed RBC's may not be enough to break up small aggregates, which would act against deformation thus causing a later transition point. Also in aggregating RBCSujp's it is uncertain where deformation effects start to occur.

There is potential to make a number of observations from the small amount of data presented here, but this is a complicated parameter and more studies are needed to see exactly what IY represents, how sensitive and reproducible it is, etc. However, it appears that IY has the potential to provide more insight into the shear thinning phenomenon.

TESTING PARAMETERS AND LOOKING AT EFFECTS OF INCREASING DEXTRAN CONCENTRATION

A number of experiments were carried by the author, to look at the effects of increasing dextran concentration on normal and CT and T modified RBC's. Figure 3.8 shows how normal RBC's behaved with increasing dextran concentration for the two parameters 0 277 rjr and °-277t)/128 5t|. It can be seen that both parameters give the same form of curve which agrees with what Chien et al found as was described in §1.9.3. However, differences in characteristics of the two parameters are evident, with a slightly different shape and a different peak position, which raises the question of which is the more correct representation of the aggregation measurement of the RBCSusp. Taking a step backwards and looking at the ri^ on a log-log plot of r|r against y (figure 3.9), it immediately becomes apparent that there is a problem that would affect both parameters, making their use in this instance misleading. The is progressively shifted down as dextran concentration, and hence supernatant r|, increases due to increased RBC deformation, as caused by the greater forces acting on the RBC's1311. The effect of increasing dextran 70 concentration was also looked 99 Figure 3.7: Shows how the intercept point (I?) and HSR gradient (Go) vary with increasing dextran 70 concentration. (n=5 : ±SD) (A/or*^ Me f/)

Figure 3.8: Shows how o:77r)r and 0277n/,:!8 5r) vary with increasing dextran 70 concentration. (n=5 : ±SD) (AAW tftfC'J

100 Figure 3.9: Logt-log* plots of r|r against y for RBC's suspended in various concentrations of dextran 70 (n=4). The cur\'es are shifted down as concentration increases, indicating 0277Tir is not a tenable parameter for representing aggregation in such instances.

Figure 3.10: Showing effects of increasing dextran 70 concentration on T or CT modified RBC's (all average of n=4 : ♦ SD except at concentration 130 which was n=l). Shows how 0277nr and a277n/1285 again differ, and both show a drop at high concentrations of dextran 70 which is believed to be an artifact associated with the breakdow-n of these parameters at high supernatant n's. 101 at with CT and T modified RBC's and figure 3.10 shows the results using 0277 r|r and 0277 T)/128 5r|. Here differences between the two parameters are exaggerated, but both show a drop at higher concentrations of dextran 70. The drop here is certainly due to the break down in the parameters because of their inability to compensate for increased deformability. In §1.9.3 it was seen that NA treated RBC's behaved differently to untreated RBC's in that there was no disaggregation of RBC's above a concentration of ~35g/l dextran. A microscopic index of aggregation was used here, but the author would have expected that had t)r been used it would show the same drop as found above with CT and T modified RBC's. However, in one early paper by Jan and Chien 11251 they presented the NA treated RBC's data using T|r and claimed it showed the same pattern as they found with their microscopic index etc.

In looking at the data on AnalEta, GA appeared to be uninfluenced by supernatant t| . Figure 3.11a shows normal and CT and T modified RBC's with increasing concentrations of dextran 70. It can be seen that for untreated RBC's the form of curve for GA is the same as in figure 3.8, agreeing more closely with 0-277 Ti/,28 -5r| (je the same peak position). However, figure 3.11a shows that GA gives a completely different response to the other parameters when CT and T modified RBC's are looked at. Here, GA reaches and maintains a plateau for both T and CT, which interestingly is approximately the same (ie -0.7). The picture becomes even more interesting when using dextran 110 instead of dextran 70 as shown in figure 3.1 lb. Here the peak position of normal RBC's is approximately the same as for dextran 70, and CT and T treated RBC's peak at the same position of -0.7. This apparent maximal value for GA may indicate a maximum level of shear thinning or even a maximum level of aggregation.

Finally, the response of GD with increasing dextran 70 concentration is shown in figure 3.7. Gd is .190±.015 SD at 15g/l which remains unchanged until ~50g/l dextran 70, after which it is seen to decrease linearly with increasing dextran 70 concentration to 0.092±0.008 at 100g/l (n=5). Such a decrease is believed to be possibly due to the increased deformation of the RBC's due to the increased forces of the higher supernatant r|'s, and would indicate that Gd was to some extent acting as a measure of deformation.

102 Figure 3.11: Plots of Ga against (a) dextran 70, or (b) dextran 110 concentrations for W, T or CT treated RBC's. n=4 : ±SD for all dextran 70 concentrations except at 130g/l which is n=l. n=l for all dextran 110 concentrations.

103 TESTING PARAMETERS AND LOOKING AT INCREASING FIBRINOGEN CONCENTRATION fiOft ^ Previously Rampling has shown that increasing fibrinogen concentration^caused a linear GofJ increase in RBC aggregation Here, the author has been able to reach much higher concentrations of ~40g/l.

The parametersa 277 r)/128-5T| and GA are shown in figure 3.12a and b respectively. One point that is apparent is that by ~20g/l, aggregation has reached a maximum level that appears to be maintained until at least 40g/l. also confirms these findings as shown in figure 3.13. Mmto)AS)1 gives strange results because of the poor response of this parameter as will be discussed in chapter 4. 0 277r|r showed a drop at higher concentrations of fibrinogen (not shown), again supporting the belief that this parameter is not suitable for describing aggregation in RBCSusp's where supernatant t) increases substantially.

Finally, no change was found in GD for increasing fibrinogen concentration. At 6g/l fibrinogen, GD was .200±.018 SD and at 30g/l it was .196±.011 SD (n=2).

WHOLE BLOOD

All of the above work has looked at artificial systems involving RBC's suspended in different media. However, an obvious important point is whether the plateau and transition effects are found with whole blood ri^. No plateau was found with normal whole blood at the y's looked at by the author, but it is likely to be found with hyperaggregating whole blood samples. Also, the transition effect has been observed to some degree in every set of whole blood T^ia that the author has seen. For instance, figure 3.14 shows the average of eight normal whole blood samples, corrected to 45% hct, that clearly shows the transition effect (Ramplings' data - personal communication). Other data found in the literature (see conclusions) also shows this effect.

104 0.7 X

o 5 10 15 20 25 30 35 40 Fibrinogen Concentration (g/1)

Figure 3.12: Plots of 0277r|/,‘85n (a) of GA (b) against fibrinogen concentration for normal human RBC's (n=l at 35 and 40g/l, n=2 at 15, 20, 30g/l, and various n for all other concentrations). Both parameters appear to describe the aggregating tendencies of the RBCSuiP's showing a plateau is reached at approximately 20g/l which appears to be maintained.

105 Figure 3.13: Plot of Mln<0 *nd 3)ASyi against fibrinogen concentration for normal human RBC's (n as for figure 3.12).

M y)

Figure 3.14: Loge - log* plot of Hr against y for data from whole blood, demonstrating the occurrence of the transition effect. Data is the average of 8 subjects each corrected to 45% haematocrit. Data obtained by Rampling and used here with permission (personal communication).

106 CONCLUSIONS AND DISCUSSION

METHODOLOGY

The methodology was included here because there appears to be nothing in the literature to guide researchers in making r| measurements o f widely different aggregating R B C Sufp's, and clearly slightly different techniques are required for different suspensions (especially hyperaggregating RBCSusp's). A number o f studies o f r) measurements on hyperaggregating systems have appeared in the literature, where results may be artificially lower because o f the difficulty in making q measurements. Such methods as included here should be brought to the attention of Haemorheologists to ensure correct use o f the Viscometer.

ANALYSIS SYSTEM

The main aspect o f the above work was a reassessment o f q ^ analysis. A computer program, 'AnalEta', was written to automate calculation and enhance investigation o f q ^ .

Certain characteristics o f q ^ were shown by AnalEta (ie transition and plateau effects) that posed problems to analysis o f RBCSusp's and whole blood samples. AnalEta was modified to allow points in the plateau and transition regions to be removed from analysis, such that their extent could be investigated. The analysis system was based around linear regressions through two regions that appear to behave linearly, especially at normal levels o f aggregation. However, there are no a priori reasons to assume such linearity, and clearly in hyperaggregating systems there are few points that behave linearly because o f the plateau effect and the increased degree o f transition effect. In spite o f these problems, and with the lack o f a simple alternative analysis system, quantitation o f data was kept as simple as possible by using the analysis system described above. Having made the decision to ignore points in the plateau and transition regions a number o f parameters (a277q n 128'5qn a277q /128-5q, G a, G d and I Y) were derived from data and assessed.

PLATEAU AND TRANSITION EFFECTS

Above it was described that the plateau effect was probably at least partly due to settling o f aggregates and increased cell free layers next to the bob. However, an obvious point is that

107 r) would certainly not continue to increase indefinitely as y decreases and Quemada predicted that at some point it would reach a plateau11261. Hence, there is probably an element o f truth in the plateau effect seen with the hyperaggregating systems. An interesting point that contributes here can be seen in figure 3.9, where is shown for various concentrations of dextran 70. At 100g/l the plateau effect can clearly be seen, yet it is known from the work here, and by other researchers, that aggregation is much reduced here, and so such a plateau could not be caused by settling. Hence, what is probably seen here is an approach towards Quemada's theoretical limit.

The transition effect is certainly not a surprise, and ignoring its presence would make little difference to the outcome o f studies. However, it is an effect that appears to always be present to some degree in q ^ , and is something that Haemorheologists should be aware of. It would be interesting to be able to quantitate this effect as it may provide useful information about the shear thinning region o f the transition from aggregation to deformation. For instance it would show a further interesting fact seen with W +100g/l dextran 70 in figure 3.9 where there appears to be very little transition effect.

TESTING OF PARAMETERS 0277 n. 0277 n',28-5m GA andGD

Above, changes in 0'277T|r and 0-277 t|/128-5t| were looked at for various RBCSlBp's with increasing dextran concentrations. On untreated RBC's the same pattern was obtained as

Chien found using a number o f techniques as was described in §1.9.3. However, one difference with the untreated RBC's was the dextran concentration at which maximum aggregation occurred. Above, this was seen to be at ~50g/I for GA with both dextran 70 and dextran 110. However, this peak position is higher than was found above with a277T|p and what Chien and et al[55J and Whittingstall et al found1921 using different techniques; these findings showed aggregation peaks at a dextran concentration o f ~35-40g/l. W ith enzyme modified RBC's, it has already been discussed in some detail how a277r|p and to a lesser extent a277r)/128-5r), differed from G A in how they described the aggregating tendencies o f enzyme modified R B C Susp's.

The second set o f data, looked at RBC's suspended in increasing fibrinogen concentration.

Both 0 277r|/128 5Ti and G A increased with increasing fibrinogen concentrations and reached

108 bs. a maximum at ~20g/l which appeared to maintain up to 40g/l. Here 0277r|/12®'5r| coped

with the levels o f supernatant q, although the response again takes on a different shape to

g a.

The different responses o f the 0 277 t|, a277q/1285q and GA complicates the decision as to which

parameters or techniques best represent the aggregation behaviour o f the RBCSufp's.

However, because o f the dependancy o f 0,277qr and 0,277q /12,5q on supernatant viscosity etc,

the author feels that G A is the best o f the parameters looked at for representing the

aggregating tendencies o f RBCSutp's where supernatant q's change.

ANALYSIS QE.noouJLQ.THER RESEARCHERS

There has previously been much interest in whole blood T| quantitation, but the authors

approach differs from these in both how q ^ quantitation was carried out and in

interpretation o f derived parameters. W ork by others o f particular concern to the author

is based on their fitting a monoexponential curve, o f the form o f equation 3.2, through

, points over a wide range o f y's (ie 0.0404-94.5/s) as have Bemasconi et al1127,1281 and other

researchers1129,1301. This is surprising because as far as the author can tell, it is fairly well

accepted that q ^ behaves biexponentially! There are numerous reports in the literature to

such biexponential behaviour and the behaviour relates directly to shear thinning.

I A ll o f the data presented above, is clearly behaving biexponentially. In order to assess

whether different Viscometers or different researchers techniques may affect the behaviour,

two sets o f Bemasconis data, taken from one o f his papers11271, are shown in figure 3.15a

and b. Also shown is one monoexponential regression through all points (fit 1), as

Bernasconi used, and one through points approximately in the limits the author normally

used in A-Phase (fit 2). Clearly in both sets o f data the last two points are higher than fit

2; ie ther? is a transition effect. Using these points in the analysis, as Bemasconi and other

workers have, 4xr ^ the viscosity behaviour o f the blood sample. There is no

doubt here that within the limits, fit 2 describes the data much better than fit 1.

109 Figure 3.15: Log-log plots of r| against y for two sets of data, (a) and (b). taken from one of Bemasconi et al papers11275. Two regressions are shown: One through all the points as with Bemasconis' analysis ( ------). The second regression through only points that are approximate to the limits of A-phase used by the author ( ...... ). Clearly data exhibit transition effects.

110 There is a further lesson that can be learnt from Bemasconi's paper. Above, the author

made it clear that extrapolating values beyond the limits o f y's used in regressing the lines

is very dubious. Bemasconi claimed that, to comply with IC S H Guidelines for making r|

measurement11241, that suggest making r| measurements up to 200s*1, his monoexponential

equations could predict what the q would be at this y. Clearly because o f not considering

the transition effect his estimation would be far different to reality as shown in figure 3 .15a

and b; ie for the averaged data given in figures a and b, his equation predicts q

=~2.827m Pa.s at 200s*1, whereas using the two points o f transition available as a rough

estimate, the value obtained was ~3.972mPa.s that would certainly be closer to, but

probably not actually, the truth!

CQ&CIMDMGJmtAMS

When using the artificial RBCSusp's, it is easy to avoid hyperaggregating systems by using less

effective aggregating agents; ie dextran 40 instead o f dextran 70. The main reason for

persevering with these higher aggregating suspensions, other than for investigating the shear

thinning properties, was to give an analysis system that would work in clinical studies

involving hyperaggregating blood samples.

The breakdown in parameters 0277T)r and 0277t|/ 12* 5t) at high supernatant q's may affect much

published work, including studies o f increasing dextran and fibrinogen concentrations and

clinical studies o f disorders where aggregation and/or plasma r\ is abnormally raised.

However, in the work presented above °*277*r|/128-5r| only broke down at excessively high

supernatant T) levels, as with 100g/l dextran 70, and would thus be the better of the two

parameters for studies comparing RBC's in different supernatant t|'s .

Having seen and assessed a number o f parameters for representing different aspects o f r ) ^ ,

the important question is which of the parameters will be used in the remainder o f the thesis.

The two parameters 0 277r|r and 128 sT|r are used because none o f the work that follows is

concerned with drastically different supernatant q's, and also values for these parameters can

easily be converted back to 0 277r) which helps relate the results to other researchers work, if wJ tecavft it to the effects o f hct (chapter 5), etc. GA/has been seen above to be very useful in that it apfip/ed l&T affected by supernatant rfs, it was reproducible, etc. GD will also be looked at, although

111 because it is derived from only three points, the sensitivity o f this parameter is questionable.

Analysis for 0 277rj/128 5r| is only shown once (§6.6), as it offers no further information elsewhere. For the reasons given above, I y is not used in the remainder o f the thesis.

112 CONTENTS OF CHAPTER 4: A STUDY OF THE MYRENNE ERYTHROCYTE AGGREGOMETER (MEA), THE SYLLECTOGRAM AND METHODS OF ITS ANALYSIS

A IM 114

F O R E W O R D 114

S T U D Y : 116

4.1 T H E S Y L L E C T O G R A M 117

• CHANGES IN SYLLECTOGRAM CURVES 117

• CURVE FITTING TO THE SYLLECTOGRAM 117

• POTENTIALLY USEFUL PARAMETERS 117

4.2 ANALYSIS SOFTWARE FOR THE SYLLECTOGRAM 119

4.3 PROBLEMS ASSOCIATED WITH ANALYSING SYLLECTOGRAMS 121

• VARIABILITIES IN A SYLLECTOGRAM 121

• FITTING BIEXPONENTIAL CURVES TO MONOEXPONENTIAL

D A T A 125

• CONTINUALLY DECAYING OR FLAT SYLLECTOGRAM 125

• SYLLECTOGRAMS OBTAINED AT 6s*1 - THE ODD ONE OUT! 127

• INCREASING y ON AGGREGATING RBCSusp'S 127

4.4 SOME PROBLEMS ASSOCIATED WITH SYLLECTOGRAMS

OBTAINED FROM THE MEA 129

4.5 REPRODUCIBILITY OF THE SYLLECTOGRAM FROM THE MEA 131

4.6 MEA RESPONSE TO HCT VARIATION FOR WHOLE BLOOD 136

4.7 COMPARING THE MEA AND THE CONTRAVES LS30 VISCOMETER

REVISITED! 138

• SUBJECT VARIABILITY 13 8

• ■ EftWO/NOr T o Hiw CWElf OF AGGREGATION 141

CONCLUSIONS AND DISCUSSION 144

113 CHAPTER 4: A STUDY OF THE MYRENNE ERYTHROCYTE

AGGREGOMETER (MEA), THE SYLLECTOGRAM AND METHODS OF ITS

ANALYSIS

AIM

This chapter describes and assesses a system developed on an IB M PC for analysing data, the syllectogram, captured from the M E A as described in §2.7.2. The analysis uses monoexponential and biexponential curve fits to provide parameters for representing various aspects o f the whole blood samples, or R B C Suip's, aggregation process including measures o f the level and dynamics o f aggregation. Such parameters are described and tested.

The biggest limitation o f the M E A is the small range over which it will measure aggregation1104,1311, and it was the main aim o f this study to look at the possibility o f removing this limitation. Further aims include better understanding o f artifacts and problems found with the M E A , assessing variabilities found in the syllectogram and looking again at one o f the most fundamental o f questions: "how reproducible is the syllectogram from the

M E A under different circumstances?".

FOREWORD

Some justification is needed to explain why such an extensive study was carried out on the

M E A when the point o f interest in this thesis was investigating mechanisms o f RF. There are a number o f reasons, but the most important, is that aggregation in some o f the

R B C Susp's could not be measured properly because it was at a level either below or above the limits of the manual MEA. The limited range of the M EA was shown in a paper by

Rampling and Whittingstall, where they compared five methods for measuring aggregation, two being the Viscometer and the M E A (figure 4.1)11041. As can be seen, the M EA was found to respond very differently from the Viscometer; ie only responding between 2.5 -

10g/l o f fibrinogen and then sigmoidally rather than linearly like the other methods. In fact out o f all the methods assessed in this paper, the M E A was the odd one out. In this thesis, the M E A was needed to act as a second device for measuring aggregation in order to compare with the r| measurements o f aggregation, and so possibilities o f extending the range

114 40 Shear Rale 0.27s Hcl 45 %

FIBRINOGEN CONCENTRATION (mg/dt)

Figure 4.1: A comparison of viscosity and manual ME A measurements of aggregation of normal human RBC's in various concentrations of fibrinogen. Notice the lack of response of the MEA at low and high levels of aggregation. Taken from a paper by Rampling and WhittingstallII04,.

had to be investigated. In attempting to do this, everything about the M E A became more complicated with numerous problems including artifacts, reproducibility o f the syllectogram etc. Hence, a small task turned into the big study presented here.

The M E A , and its data the "syllectogram" were shown in figure 2.9 and 2.10 respectively, and were described in §2.7.2. Briefly, the M E A optically measures RBC aggregation by monitoring the change in light transmission (L T ) through a sample from the start o f aggregation over 5s. The M E A can be operated in either "manual mode" or "computer mode". In "manual mode", following a 600s*1 H SR phase, the sample can be subjected to a y o f 0 (MO mode) or 3s’1 (M l mode), and it displays a value relating to the area under the syllectogram during the aggregation part (C -D in figure 2.10) o f this phase (ie Man(0)A s>1 or

Min<3)Asyi). In "computer mode" software on an Apple IIGS computer controls the MEA, and after the 600s*1 H S R phase, applies a user specified y (0-600S*1) on the sample and captures the L T data. The L T data is then passed to an IB M PC for analysis with a

115 specially devised analysis system as will be described a little later (§4.2). For the majority

o f the data obtained from the M E A , two computer runs were carried out each on a different

aliquot o f sample. Each run consisted o f 10 cycles; one cycle represents a 10s H S R phase

followed by a 10s LSR phase. The LSR's used for run 1 were 0, 3, 6, 9, 12, 12, 9, 6, 3, 0

s'1 and for run 2 were 0, 6 ,1 2 ,1 8 ,2 4 ,2 4 , 18, 12, 6, 0 s*1; each run wa.s followed by manual

readings. The previous work o f Schmid-Schonbein et alI,32‘1361 and Bauersachs et al[1371 has been based on making a number o f measurements on the same aliquot o f blood, which

initially suggested that this protocol used by the author was acceptable. However, some o f the findings presented in this chapter suggest measurements may change with time, and thus recommendations for a better protocol for measuring aggregation will be given at the end

o f this chapter.

As in the last chapter, some data needs to be used here that will be more fully presented in chapter 6. Briefly, RBC's have been modified with the enzymes trypsin (T ), bromelin (B) or neuraminidase (N A ) and it will be seen that the modification leads to an increase in the

RBC's aggregating tendencies. Extreme increases in aggregation were found with N A , B or T treated RBC's compared to normal washed RBC's (W), with dextran present

(N A >B >T>W ). Also B or T treated RBC's with fibrinogen present, were found to increase in aggregation from W (B>T>W ). However, increases in aggregation with fibrinogen were far less than with dextran.

STUDY:

As with the last chapter, because o f how complex this study became, and to attempt to avoid confusing the reader, some findings and discussion are presented together, but further discussion and conclusions are given at the end o f the chapter, to bring everything together.

116 4.1 THE SYLLECTOGRAM

CHANGES IN SYLLECTOGRAM CURVES

The shape normally associated with the syllectogram, obtained from an aggregating R B C Siap, was shown in §2.7.2, but in fact the syllectogram varies considerably depending on the level o f aggregation, y being applied and the suspending phase T|. Figure 4.2 shows some syllectograms for various levels o f aggregation S5>S4>S 3>S2>S 1, where S, represents non­ aggregating samples. It is such diversity that makes devising an analysis system particularly difficult.

CURVE FITTING TO THE SYLLECTOGRAM

In w ork carried out by the author, and other researchers11371, it appeared that monoexponential and/or biexponential curves acted as a good fit to syllectogram data, and so were used here in the analysis system. Hence, equations were o f the following form:

LT = K „ - 4.1

LT = Kb- ab*Exp[-bb-{t-tmr)] - cb*Exp[-db*(t-t^] 4.2

Where L T is light transmission,^/-/*,,* is the time after the start o f the fit (units ds) and K , a , b , c and d are the calculated coefficients o f the monoexponential and biexponential equations (subscript m and b respectively). Despite the diversity of the syllectograms, one or both o f these equations succeeds in closely fitting, and thus describing, most o f the syllectogram. However, equation 4.2 proved to be a better fit in most cases as demonstrated in figure 4.3.

POTENTIALLY USEFUL PARAMETERS

The M E A has many problems, as already mentioned, and so before becoming too adventurous the author felt that one should return to the basics and reassess the M E A .

Hence, to keep things as simple as possible only a few parameters associated with individual syllectog'rams are looked at here as shown in figure 4.4.

117 Figure 4.2: Various forms of syllcctogram which have to be analysed. Syllectograms taken from RBCsu*p's with aggregation levels as follows: S1

Figure 4.3: A syllcctogram with an associated monoexponential and biexponential curve fits. Clearly the latter is a better representation of the syllcctogram curve.

118 Figure 4.4: The syllectogram and some potentially useful parameters calculated by the computer analysis system.

Tw o parameters, based on measuring the area under the syllectogram, were given by the

M E A in "manual mode", Man(0)A S)1 and Man(3)A Syl, as previously mentioned. The area gives a gross measure of aggregation, and is clearly useful, so an equivalent parameter was calculated from data captured in "computer mode" (Comp(Y)ASyl). Also briefly looked at were the curve fitted decay constants, that give information on the dynamics o f the aggregating process. This information may help understand the mechanisms o f RF such as the initial rate o f aggregation, whether secondary, end on side, aggregation occurs, how long RF continues to increase, whether clumps act differently etc. Finally, two further parameters were briefly looked at, corresponding to the minimum position of the syllectogram (t^ , L T J , that gives an idea o f how fast the sample recovers from the H S R dispersion phase.

4.2 ANALYSIS SOFTWARE FOR THE SYLLECTOGRAM

As already mentioned, a specialised analysis system was devised on an IB M PC for analysing

M E A data based upon monoexponential and biexponential curve fitting. A shareware

119 package N L R E G 1' (version 3.3) was used for curve fitting and, according to the developers, uses the "state o f the art" non-linear curve fitting algorithm based upon the Dennis-Gay-

Welsch method11381; this method is a combination of the Gauss-Newton and

Levenberg-Marquardt non-linear curve fitting methods, but is very often an improvement on both of these methods. As is normally the case with non-linear curve fitting algorithms, starting values were required for each parameter which was a problem because o f the diversity o f the curves. To keep things simple the author used initial values o f K m equal to

L T during the 600s'1 disaggregation phase (always -3000), am=500 and b = 0.1; these proved to be a good starting point for all syllectograms o f types S3 - S5 o f figure 4.1. For syllectograms o f the type S, or S2 these starting values are obviously not so good, and

N L R E G normally made the decay constants very small (ie produced straight line fits).

However, this proved adequate for extending the lower limits of aggregation of the M E A as will be described later. The monoexponential coefficients calculated by N L R E G were then used as the starting point for the biexponential curve fit for parameters K b, c h, d b together with coefficients ab= 50 and bb= 0.0001, whose value were aimed at forcing N L R E G to make them the secondary slower phase o f the syllectogram curve; ie bb is referred to as the "slow decay constant" and db as the "fast decay constant" and here only db will be assessed because o f simplicity, and also because bb only normally changes a little between syllectograms. When N LR E G performs the curve fitting procedure, it finds the best possible fit by carrying out up to 50 iterations, slightly adjusting the parameters each time to improve the fit.

A computer program, 'ViewSyl', was written by the author (PASCAL) to read and display the syllectogram data. Automatically calculated boundaries, over which curve fitting was to take place, were calculated which the user could change if necessary. Curve fitting information and data points, within the limits selected by the user, were written to a file in a format N L R E G recognised, and then monoexponential followed by biexponential curve fits were performed. A further program was written to collect all o f the results and calculate areas over 5s from t ^ , and these were stored in a file that could be imported to other packages (eg Microsoft Excel).

’Available from many anonymous FTP sites (eg garbo.uwasa.fi) or the registered version from P.Sherrod, 4410 Gerald Place, Nashville, TN 37265-3806, USA.

120 4.3 PROBLEMS ASSOCIATED WITH ANALYSING SYLLECTOGRAMS

Before assessing the parameters produced by the above analysis, a number o f problems associated with the syllectogram needed to first be looked at.

VARIABILITIES IN A SYLLECTOGRAM

N ot all syllectograms were as "ideal" as those seen up to this point. One variation came in the form of continuous irregularities (noise) as shown in figure 4.5a; also shown is the biexponential regression that still closely represents the data. Noise is present in most syllectograms, but interestingly it is nearly totally removed for syllectograms obtained between y's 0 and 12s’1 when aggregation is enhanced; noise is still present at higher y's (ie

18 and 24s*1). This implies that it may be associated with the continuous motion, interactions etc o f RBC's.

Another variation came in the form o f apparent regular "dips" in the syllectogram as shown in figure 4.5b. Again the curve fitting copes fairly well with these dips as shown. However, problems arise when a dip appears close/at the point where the minimum point for curve fitting is chosen (figure 4.6). Choosing the minimum point in these cases affects the decay constants and artificially rises areas. The only solution was to avoid these initial dips and choose to start just after the dip finishes.

Finally, occasionally blips would occur in the syllectogram; figure 4.7 shows 9 such instances in a set o f experiments where 700 syllectograms were obtained. Initially this was thought to be caused by the mains supply, but the problem still occurred after the M E A power supply was passed through a filter. ViewSyl allowed these blips to be removed so they were no problem as regards to analysis.

121 Figure 4.5: Some potential problems found with the syllectogram obtained from the MEA. (a) An example of noise in a syllectogram. (b) An example of regular dips in a syllectogram. In both cases the analysis system copes reasonably well, cUps u-uq. t/arrow h\cjovtr/6s & 's os

122 Figure 4.6: Shows two syllectograms with dips at the beginning of the aggregation phase, making choosing the minimum and regression start position difficult. Attempts are shown of the curve fits calculated by the MEA analysis system over a range specified by the author.

123 Figure 4.7: 9 examples of syllectograms showing the *blip' artifact, out of a total of 700 syllectograms from a days experiment. The cause of this artifact remains unknown. 124 FITTING BIEXPONENTIAL CURVES TO MONOEXPONENTIAL DA TA

One o f the downfalls o f the devised analysis system was if a monoexponential curve fit closely described the data, then attempting to fit a biexponential curve to the same data sometimes caused problems. The majority o f the time N L R E G just made the decay constants, bb and dh the same as bm with the amplitudes, ab and ch adding up to that ofa m.

How ever, occasionally one o f the two exponential curves would fit through a 3 or 4 syllectogram data points which normally gave an elevated amplitude and decay constant.

Hence, it was always obvious to identify when this happened, and b m was then used instead o f d b. Comp(lf)A SyJ was unaffected by this problem, and was still calculated from the biexponential coefficients.

CONTINUALLYDECA YING OR FLAT SYLLECTOGRAMS

In figure 4.2 two syllectograms, S, and S2, were shown that were continually decaying and flat respectively. S, was taken from a non-aggregating R B C Susp and S2 from a R B C Susp having a little aggregation, and in both cases ^ ^ A ^ and ^ ^ A ^ gave readings of zero.

To measure these small degrees o f aggregation, ie extend this lower range o f the M E A, some means o f quantifying the transition from S, to S2 was needed. The approach finally used was very simple but from the analysis, was apparently effective. Basically, if a curve continuously decayed, was flat or was slightly rising but the minimum position was not obvious, then the point 35ds was chosen to be the cut-off, and all points after this used in the regression. There was no "scientific" reason for choosing point 35, but it was generally found that for RBCSusp's where only a little aggregation occurred, the beginning o f the syllectograrn (recovery phase) was more erratic, especially at higher y's, and by 35ds the

/torec & < u i j Figure 4.8 demonstrates how, for Comp(Y)A Syl, this approach succeeds in showing a difference between S, and S2; ie has succeeded in extending the lower range of the MEA. The curve fitting hof pmhk/hs , because it resolves the exponential equations to a linear equation, but it was adequate enough to show a larger negative area for non-aggregating RBCSusp's, than for RBCSusp's with a small amount o f aggregation. It is anticipated that the sensitivity o f this approach would be enough to show the progression from continually dropping to flat syllectograms. Furthertesting is not given here, but it will be seen to work well at numerous times throughout the remainder o f the thesis where negative or very small Comp

125 Figure 4.8: Shows syllectograms Si and S2 from figure 4.2. These syllectograms are below' the lower range of sensitivity of the manual MEA, but shown is a potential method for extending this; ie for continually dropping, or flat syllcctograms, 35ds is chosen as the point to start the curve fitting. For no aggregation (SO, the calculated area is veiy negative, but for slight aggregation (S2) the negative value is less.

Figure 4.9: Three syllectograms at y's 0. 6 and 12s'1, taken from one human whole blood sample. Demonstrates aggregation phase for 6s'1 occurs later than for higher (eg 12s'1) and lower y's (eg Os'1).

126 SYLLECTOGRAMS OBTAINED A T 6s' - THE ODD ONE m i l l

Out o f all the syllectograms the author has encountered at the y's examined, those obtained

at 6s*1 have always appeared different, as shown in figure 4.9, and also have been the hardest

to analyse. It can be seen that it takes longer for the aggregation phase to start at 6s*1,

compared to other y's; ie t ^ occurs much later. Hence, 6s*1 is the optimal y for acting

against the initial coming together o f normal aggregating RBC's. Another interesting point

surrounding syllectograms obtained at 6s*1 was that biexponential curve fits nearly always

"insisted" on having one positive and one negative decay constant, even if all initial points,

and more, about t,^ were removed. Finally, when aggregation is substantially increased, as

with hyperaggregating systems (T+15g/l dextran 70 etc), the delayed onset o f aggregation

with 6s*1 disappears; it is not known if the late onset o f aggregation moves to another y in

such systems. These observations are important when considering analysing the

syllectograms from the M E A , and because o f this unique behaviour o f the syllectogram

decay constants, these were not used in any analysis. However, good curve fits were still produced and so it was acceptable to consider Comp(6)A Sy,.

INCREASING y ON AGGREGATING RBC,IIJr'S

Analysis by others has shown potentially useful information can be obtained from changes in the syllectogram with increasing y (see discussion). The effects o f increasing y on the

R B C Susp T+15g/l dextran 70 was looked at here and figure 4.10 shows a typical response to increasing y from 20 to 200s*1. Clearly there are some technical difficulties in the form o f oscillations; these appear to increase in amplitude and frequency with increasing y, becoming most obvious at -80s*1 onwards. Other researchers have made use o f these higher y's on the M E A 1132*1371, and have not reported such problems, which may indicate a local problem unique to the M E A being used by the author. Any analysis o f such syllectograms would be highly dubious, but as far the rest o f this thesis is concerned such HSR's are never used (ie no y's above 24s*1) and so the problem was o f no relevance.

127 Figure 4.10: 10 syllectograms from a single computer run on a hyperaggregating RBCSuJp- Each carried out at the given y, and followed a HSR (600s*1) disaggregation phase. Shows the occurence of oscillations, that increase in frequency and amplitude as y increases. 128 4.4 SOME PROBLEMS ASSOCIATED WITH SYLLECTOGRAMS OBTAINED FROM THE MEA

The manual M E A and the analysis developed by Bauersachs et al were both designed to work on a "black box" approach. The author feels this has a certain danger associated with it, concerning whether the syllectograms are behaving as a user might expect they would.

For instance, the occurrence o f the oscillations, dips etc seen above could very easily be lost in a black box system. There are a number o f further instances when data produced from the M E A does not behave as one might expect, and by not being restricted with a black box system these instances can be observed and investigated.

One such instance that caused some concern was when very different results were produced from different aliquots o f the same RBCSusp; ie one aliquot o f a sample may give a value for

M>n(3)As>1 of 3.6, whereas another aliquot would give the value 16.4. Figure 4.11 shows the syllectograms from these two aliquots, and it can clearly be seen that there are big differences between the syllectograms from the different aliquots. The syllectograms whose recovery phase goes down to an L T o f -1 2 0 0 in 6ds is much lower than normal for aggregating RBCSusp's, and furthermore this was shown to be the abnormal value when the

M E A data for this set o f experiments was correlated with r ^ . Hence, these were believed to be the "abnormal" syllectograms, and the unusual L T drop to 1200 supplied a method for recognising such "abnormal" syllectograms. Such unacceptable inconsistencies only occurred occasionally, but syllectogram data was always recognised from the large L T drop, and based on this it seemed reasonable to recognise and discard such data. Also this problem was normally recognised at the time o f the experiment from the manual readings, and further runs would clarify the correct value. The author is unsure o f the origin o f such inconsistencies, but it may be related to trapped air or a badly distributed blood sample.

Another inconsistency was found with non-aggregating RBC's suspended in either PBS or a low rj suspension such as 10g/l dextran 40. Here, instead o f producing a value o f zero as would be expected, the manual M E A (Mm,(3)a s>.1) displayed high readings; ^ ^ A ^ gave the expected zero value. Whittingstall showed with increasing small concentrations o f fibrinogen, Min(3)A Sy) dropped in value, and then above a certain concentration the value increased again due to aggregation (figure 4.12)1'391; Anwar also had similar findings11401.

129 Figure 4.11: Compares the syllcctograms (6s’1 and 12s*1) from two different aliquots of one blood sample (W+15g/l dcxtran 70), where M,n(3)Asyi gives large differences in results for each aliquot as shown. It is not normal for the recovery phase to drop to a light transmission as little as 1100, and also the low reading did not fit when MEA data was correlated with Hd.u

Figure 4.12: The response of M,n<0)Asyi and “"^Asyi for RBCsu»P's of increasing fibrinogen concentrations (0-12g/l). Of interest here is at zero, and very low, concentrations of fibrinogen, Min(3)Asyi gives high readings, but adding a little fibrinogen (protein) removes these high values. ^^A syi shows no response. It is thought that these high values are caused by crenation. Data taken from thesis by Whiltingstall (1 typical subject)11041. 130 It is not certain what causes the initial high results, but it seems probable to be caused by crenation o f the RBC's on the glass slide. This problem produced particular difficulties with

10g/l dextran 40, as will be seen in chapter 6, and for the purpose o f discussion it will be referred to as the "crenation problem". It was hoped that the syllectograms may offer further insight into the cause o f these results, but when high Ml^3)ASy, values were produced, the syllectograms looked normal (figure 4.13). One further observation made by the author was that this problem appeared to be subject dependant; ie high readings were rarely found with the authors washed RBC's (1 in 6 runs), but were nearly always found with another subject (5 in 6 runs).

Finally, it was likely that a problem o f settling would occur in hyperaggregating R B C Suip's, particularly in view o f the thin film o f blood in the system. I f settling did occur then the syllectogram would show the initial re-orientation phase, an initial aggregation phase, but then a continuously decaying L T signal due to the settling aggregates reducing LT. Such a pattern was found with untreated RBC's in the presence o f dextran 500 (15-25g/l), but only at y's o f 9,1 0 and 12s'1 for the two subjects looked at (figure 4.14). Interestingly such dextran concentrations are known to cause clumping type aggregation which would show greater settling, compared to linear aggregation, because o f the higher concentration o f

RBC's over a globular area*751. Settling was also found, to a lesser extent, with B treated

RBC's, with 15g/l Dextran 70 present, at the same y's and microscopic observations revealed clumping type aggregation. These syllectograms were analysed by using only points before the start o f the drop, and will be seen later to appear to work very well.

4.5 REPRODUCIBILITY OF THE SYLLECTOGRAM FROM THE MEA

The syllectogram has previously been described as being a highly, or extremely, reproducible curve1141,1421. However, reproducibility o f the syllectogram from the M E A does not appear to have been investigated in much detail, and the author found a number o f instances when clearly reproducibility was not good. Initially, to investigate reproducibility two aspects o f the syllectogram were considered: the expected natural variation between replicates and the variations with time. To investigate these, whole blood was obtained from the author via a fingerprick, immediately heparinised and used. One computer run was performed per aliquot o f whole blood sample, which consisted o f 10 different cycles; one cycle consisted

131 Figure 4.13: Shows two syllectograms, obtained at 0 and 6s'1, from RBC's suspended in only PBS. Demonstrates how for y's greater than Os*1, the ME A produced syllectograms that suggested aggregation. The syllectograms generally look no different from those obtained with aggregating RBCSu,p's, but are possibly caused by crenation of the RBC's to the glass slide.

Figure 4.14: Syllectograms obtained at 12s*’ from washed normal human RBC's suspended in 15 or 25g/l dextran 500. This response is believed to be caused by the settling the RBC clumps known to be formed at these concentrations of dextran 500. Shown is the user selected point which determines which syllectogram data points are used in the regression. All points after the indicated position are ignored. 132 o f a HSR (600s*1) disaggregation phase followed by a user selected LS R phase. A computer

run used the same LSR's for all ten cycles (either 0, 3, 6, 8, 10 or 12s*1).

Tte C V between five syllectograms o f the same y, and o f the same cycle number (eg cycle

1 from five aliquots o f a blood sample), were calculated for the various parameters. For

y=10s*1 CV's calculated at cycle 1 and cycle 10 were as follows: Comp

9.0% respectively, LT,^ =6.94% and 6.32% respectively and for db (fast decay constant)

= 42.2% and 29.4% . The C V for Comp(Y)ASy! and db significantly decreased with cycle

number, thus demonstrating variations with time, and L T ^ showed little change. The reason for the high C V for db can be seen in figure 4.15 which shows db for each cycle of

a computer run o f 10 cycles; syllectograms o f very different values for db, can be seen to have very different initial rates o f aggregation. The CV's also appeared to be dependent on the y being used; eg 0s*1 had much higher CV's than found at 10s’1. These results suggest that the syllectogram obtained from the M E A is not as reproducible as previously thought.

Also the change in natural variation between replicates with cycle number indicates changes with time, which suggests computer runs o f a number o f cycles on one aliquot o f blood may be prone to artifactual results.

The above findings were with whole blood, but it was also important to assess whether there were any time variations for hyperaggregating RBCSuip's. Again the extreme case of B treated RBC's with 15g/l dextran 70 present was looked at, so that any differences would be more obvious; two computer runs, from one subject, are shown in figure 4.16a-e (y= 0-

12s*1) and figure 4 .16f-j (y=0-24s*1). Clearly time has had major affects on syllectograms at 0, 3 s’1 and 6 s*1 but the other y's appear to be less affected, possibly because the time differences are less. Hence, this shows further problems o f changes in the syllectogram with time.

The above work shows that syllectogram data does change with the time the sample is in the M E A , and hence the protocol o f computer runs o f a number o f cycles on one aliquot o f a blood sample may not be tenable. The most striking consequence o f this concerns the syllectogram analysis developed by Schmid-Schonbein et al[132*1361 and used by other researchers such as Bauersachs et al[,37]. As will be discussed at the end o f this chapter,

Schmid-Schonbein developed parameters that use information obtained from the

133 LT.Bin-2229 LTm ” 2197

L T ^ -2 1 2 3 LT.rt-2175 3000 2800 2600 2400 2200

2000

3000 2800 2600 2400

2200 2000

Figure 4.15: Syllcctograms of a single computer run of 10 cycles, all of which were at a y of 10s*1 , from a sample of whole blood. It can be seen that the fast decay constant (db) varies considerably, but differences appears to be related to the syllcctograms. db always showed a large variation. 134 with 15g/l dextran 70. It can be seen that for y's 0, 3 and 6 there are large differences between syllectograms of different cycles; ie they show severe time variation. The other y's appear to show no such change. 135 syllectograms obtained from runs o f a number o f cycles. Time changes in the syllectogram raises the question o f the validity o f such parameters. A possible explanation for these changes could be a badly distributed sample, the H S R 600s*1 monodispersion phase failing to monodisperse the RBC's, etc. However, as far as work in this thesis is concerned, these problems are mostly avoided by using Comp(,2)A Sy,.

4.6 MEA RESPONSE TO HCT VARIATION FOR WHOLE BLOOD

To save confusion later, and to present some more uncertainties o f the M E A , the response o f the M E A to hct variation for whole blood is looked at here. Chapter 5 will look in more detail at hct variation o f the M E A , and the Viscometer, for R B C Susp's.

Figure 4.17 shows a plot o f Klan(0)ASy) and Man(3)A Sy1 against hct, where the readings were collected in two ways. Method 1 used the original protocol o f performing a computer run o f 10 cycles followed by manual readings. Method 2 involved making manual readings as soon as the sample was put into the M E A . Clearly figure 4.17 shows there are large differences between results from the two methods. One difference was with method 1, where manual readings between 35 and 60% hct decreased by only 23.6% for ^ ^ A ^ and

10.7% for ^ ^ A g y,, yet for method 2 the decrease was much higher at 49.5% and 35.8% respectively. The only consistency between the two methods was with mode ^ ^ A ^ at hcts o f 45% and greater. Also at low hcts, method 1 gave readings close to zero at 20% for both ^ ^ A ^ and Mjn(3)AS)t, yet with method 2 readings were far from zero. Figure 4.18 shows that Comp(,2)ASy, between 35 and 50% hct decreased by 25.6% , which was similar to the response o f M,n(3)A S)1 immediately after being put into the M E A . The same reasons as mentioned above could have caused these findings, which again questions the goodness o f performing a computer run o f a number o f cycles on an aliquot o f blood. However, the similar response o f Comp(,2)A S)1 and Man(3)A s>1 measured immediately, again suggested

Comp(12)A Sy, to be a good parameter to use.

136 Figure 4.17: Plot 0f Mtn(0)Asyi and Man(3)Asyi against haematocrit for whole blood (n=2 : ±SD). One set of results were taken "immediately” after putting an aliquot of the sample in the M E A , and the second set of results after 10 computer cylcs. Large differences can be seen between the two methods, due to time variations.

20000

18000 -

16000 -

* 14000 - |< 3 12000 -

10000 -

8000 -

6000 -- 15 25 35 45 55 65 Hatmatocrit (*/■ )

Figure 4.18: Plot of Comp(,2)Asyi against haematocrit for whole blood (n=2 : ±SD). Shows similar pattern to manual data obtained immediately after putting the sample into the M E A as shown in figure 4.17. 4.7 COMPARING THE MEA AND THE CONTRAVES VISCOMETER

REVISITED!

It was hoped that the computer analysis system described above (§4.2), would provide the means of removing the differences between measurements from the MEA and other methods, described in the foreword, such that the MEA would at last "fall in line" with the other methods. Some data is used here to test the MEA analysis system, and furthermore is compared with analysis of results from the Contraves LS30 Viscometer which was the topic of the previous chapter.

SUBJECT VARIABILITY

It is now well known that some intrinsic factors of the RBC change from one individual to another, and leads to the RBC's aggregating to different levels, as was mentioned in §1.9.3. However, previous work only appears to show subject variability for measurments made on one instrument, and so it is feasible that such differences are in some way associated with experimental error and/or the instrument used for the measurement. To gain some evidence against such a possibility, aggregation of RBC's from various subjects were measured on both the Viscometer and the MEA.

Washed RBC's were prepared from five individuals and each suspended in 25g/l dextran 70 at 45±2% hct. Measurements were made on the Viscometer and the MEA in both computer and manual modes. Figure 4.19a and b shows a direct linear relationship between ^ ^ A ^ and °’277 r|r and Nlan( 3)AS)1 and GA respectively; note all parameters have been corrected to a standard hct of 45%, which greatly improves the relationship, as will be described in chapter 5. Figure 4.20 shows how Comp(Y)A S)1 varies with y for the same five subjects. It can be seen that at 0 and 3s*1 the response is variable and at 6, 9 and 12s *1 there is some consistent differentiation between subjects, and good correlation with 0 277 r|r for 6 and 12s*1 can be seen in figure 4.21. At 18 and 24s*1 the correlation is once again seen not to be so good.

138 Figure 4.19: Plots showing good corelation between M,n(3)A syi and 0277 r|r (a), and MaT,(3)A s>i and GA (b) for washed RBC's with 25g/l dextran 70 present (n=5). Data was adjusted to a standard hct of 45% for small variations (±2%) caused by experimental error (chapter 5). The correlation between results from the two instruments supports the existence of subject variability.

139 Figure 4.20: Plot of Comp0f)Asyi against y for W+25g/l dextran 70 (n=5). Shows how low’ y's (ie 0, 3 and 6s*1) are insensitive to the subject variability, but after about 6s*1 variability is clearer.

Figure 4.21: As for figure 4.19 but for Comp(6 ■nd ,:)ASyi against 0277nr. Shows the same good correlation between the two parameters and again values are hacmatocrit corrected (chapter 5).

140 These results show good correlation between the MEA and the Viscometer, and also give added support to the existence of RBC variability between subjects. It should be noted that here aggregation levels are measured adequately by the MEA in manual mode, but it was seen that at 6, 9 and 12s '1 the computer analysis works as well as the manual MEA. It is at higher and lower levels of aggregation that problems occurred, and the former of these will now be looked at.

Exretf0/M6- /y&ifuffMf/tfT 7b m /w w m r

As already mentioned, extending the upper limit of the MEA is the main reason for carrying out this study, and here it will be seen if the analysis gives any insight into how the upper limits of the MEA can be extended. Results of B treated RBC's from one subject were looked at with 6g/l fibrinogen, 10g/l dextran 40 or 15g/l dextran 70 present, where in chapter 6 it will be seen that aggregation levels follow the pattern fibrinogen

141 Figure 4.22: Plot of Comp( ^syi against y for bromelain treated human RBC's with either dextran 40, dextran 70 or fibrinogen present to induce aggregation. Demonstrates how- M*n<0 Bnd 3)ASyi and Comp(0-3-6 ind 9)ASyi fails to confirm Hd.u because the upper limit of these parameters has been reached, and how higher y's (12, 18 and 24s'1) demonstrate a difference similar to Data from one subject, but a second subject showed the same pattern.

142 Figure 4.23: Plots of Comp(9,12,18 *nd :4 )AS)i against 0 277rjr (a) and against GA (b). For both parameters, Comp(9)Asyi fails to show correlation, probably because the upper limit for this parameter has been reached. However, the higher y's show good correlation, and hence successfully extends the upper limit of the M E A . Data from one subject, but a second subject showed the same pattern. 143 CONCLUSIONS AND DISCUSSION

7 should bww the Myrenne , I designed itV H. Schmid-Schonbein (9th European Conference on Clinical Haemorheology, Siena. 1995)

Without doubt the MEA has the potential to become a much better instrument for measuring RBC aggregation and furthermore to provide more information about the process of aggregation. It is an instrument worth developing because of its many advantages over other techniques including a compact design, working on minute quantities of blood and being quick, simple and reproducible. However, there have been many problems found with the MEA including the numerous artifacts and limitations as mentioned above. A number of points presented above were explained and discussed at the time, and so will not be mentioned again here.

THE ANALYSIS SYSTEM

The computer analysis system described and tested above, was designed around keeping one aspect of the manual MEA that was felt to be particularly important; ie being quick. Hence, syllectogram data was only collected over 10s, and it was hoped that this would be adequate to provide enough information to perform the necessary analysis; as was seen above, this generally appeared to be true. When the author first started working on the computer analysis system of the syllectogram, there was much excitement over what information might be obtained about the aggregation process of RBC's in the various RBCSujp's. It was particularly exciting when considering the parameters developed by Schmid-Schonbein et al, that provided various information about the aggregating process. This has already been (pa$Z Up) mentioned and will be discussed further later. However, it soon became apparent that there were too many problems and inconsistencies that confused the issue, including problems and artifacts associated with the MEA, the variability of the syllectogram, etc. Hence, it was necessary to keep the analysis as simple as possible by keeping the number of parameters small etc, and it was also necessary to return to basics by performing simple testing of the MEA to see exactly what it was capable of.

144 PROBLEMS ASSOCIA TED WITH SYLLECTOGRAMS OBTAINED FROM THE MEA

Two instances were discussed above where there were problems with data obtained from the manual MEA. The first was high readings being produced in the absence of an aggregating agent, and was thought to be caused by crenation of the RBC's on the glass, and hence will be referred to as the "crenation" problem. The second was from an occasional large difference with supposedly identical runs; it was less certain what caused these differences, but was possibly caused by a badly distributed sample. In computer mode the syllectograms in these cases could be directly observed and were seen to be very different; one set of syllectograms showed a lower LT,,^ than normally obtained, and furthermore results from these syllectograms were found to be the abnormal data by correlating all the MEA data for the experiment with r^ . Hence, a very low LT,^ may be a way of identifying abnormal MEA data, so that it can be removed from the analysis, and this approach was used in a number of cases.

REPRODUCIBILITY

At first sight, the syllectogram from the MEA appears to be highly reproducible. However, the above study showed Clearly that there were considerable natural syllectogram variation between replicates, and more importantly variations timeA. Above it was seen that the CV for Comp1, L T ^ and db were all found to increase in value with time (not shown). The more worrying problem was found with hyperaggregating systems where severe time variations were found for syllectograms obtained at 0, 3 and 6s*1. To discover what caused such large variations with time would require more investigation, and remains a concern for the analysis developed by others as will be discussed a little later.

THE MEA vs THE CONTRA VES LS30 VISCOMETER

Two sets of data were presented above to assess the new analysis for the MEA, comparing it to analysis of the rj^. The first set of data looked at subject variability of RBC's that had previously only been observed on individual instruments. Here the same samples were 145 measured on both instruments and, after slightly correcting both and MEA data for variations in hct (±2%), caused by experimental error (chapter 5), a very good linear relationship was found between the analysis from the two instruments. This gave supporting evidence for the existence of RBC variability between subjects and also tested the new analysis system. However, the MEA was measuring aggregation within its manual limits of sensitivity, and so here the analysis was carried out only to show agreement between parameters in computer and manual modes, and to see how well the new analysis worked within this range.

The second set of data really aimed to show the limited range of lower y's (ie 0, 3 and 6s*1) and to assess if higher y's would extend the upper limit of the MEA such that analysis agreed with that found with r)^. The above analysis achieved this remarkably well, where y's of 12, 18 and 24s*1 showed direct agreement with the 0 277 r|r and GA. Further testing is carried out in chapters 5 and 6, but at this stage everything looks hopeful.

It is interesting to look at the history of the MEA to see the problems, the confusions etc and to try to see 'what went wrong' in the design and development of the MEA.

THE MEA: THE PAST THE PROBLEMS AND THE CONFUSIONS

The MEA has been around for a number of years now, and the problem of the limited range known for almost the same amount of time; so "why has Myrenne GmbH not investigated the limitation and overcome it?". It is amusing when one realises that all Schmid- Schonbeins original work in the 1970's1132*1361 was carried out at 0 and 7s*1, and yet in the final design of the MEA 0 and 3s *1 were chosen. Had the original 7s *1 been chosen, the manual MEA would have worked much better over longer ranges.

It is interesting to look at the many different configurations of the MEA that have been developed by Myrenne GmbH; the continuous change in design of the MEA would suggest that Myrenne GmbH are aware of some of the problems described above. Initially in 1982 the model was the HAEMA 60, designed to shear the sample at 500s*1 for 30s and measure area at stasis over the next 10s, and said to give readings up to 10011421. Meiselmans' laboratory has the MEA Model MA-1 that initially had a HSR phase of 460s*111101, which 146 changed to 500s'1 at some stage11371, and aggregation was measured over 1 Os; the papers do not say what y was being applied during this time. The MEA used by the author was different again, and applied a HSR of600s'1 and then measured aggregation at 0 or 3 s '1 over 5s. The above differences helped piece together one of the mysteries concerning the variation between different MEA's. Differences in the MEA's could be due to different HSR's, different y's being applied during the aggregation process and different times periods over which areas are calculated.

OTHER ANALYSIS SYSTEMS

As part of Schmid-Schonbein^ studies in the 1970'sf132'1371 he proposed a number of different parameters that could be obtained from the syllectogram data. Many of these are not looked at here because of wanting to keep this study simple. However, one immediate concern comes to mind with some of his parameters, because they rely on there being no &€h* ie

Bauersachs et al is the only other researcher, to the authors knowledge, to have developed software for the MEA,137l This was an extensive analysis system implementing many of the parameters that Schmid-Schonbein introduced in his earlier papers. One parameters was the flow to stasis ratio (FSAR), calculated by dividing LT at 10s '1 by LT at 0s'1, where in both cases the average LT was taken between 57 and 60s after the HSR phase; this parameter acted as an index of aggregation under low shear conditions. Another parameter was yTmin, calculated by collecting syllectogram data at a number of y's, and estimating the y that induces the minimum LT after 30s; this parameter was a measure of the mechanical strength of the RBC aggregates. These both rely on there being no change with time. Bauersachs analysis system meant a complete computer run on one aliquot of a sample takes ~6 min to complete, and so blood is in the MEA for far longer than looked at above, and also individual computer cycles are much longer; these would both presumably enhance changes due to time, settling, etc.

147 If only one cycle is carried out on the MEA, ie a HSR followed by a LSR phase, time variations, and the failure of the HSR monodispersion, is not so important. However, if one becomes more adventurous and carries out a number of cycles, as with the authors, Bauersachs and Schmid-Schonbeins systems, then it could be of much importance to the results obtained.

RECOMMENDATIONS

The course of action from this study is obvious. Myrenne GmbH need to change the manual mode of the MEA it two ways: (1) to work at y's around 12s '1 to extend the upper range of sensitivity. (2) to extend sensitivity at the lower levels of aggregation by working at Os '1 and implementing a similar method described above; ie for continuously decaying or flat syllectograms analysis should be carried out on all points after 35ds, such that decreasing negative area indicates increasing aggregation. There appears to be no other way to extend the lower level of aggregation over such short time periods, but this will be seen to work very well in chapter 6. The second action will never happen, but the MEA needs modifying to all have the same specifications and produce similar results on the same samples. Another possibility here would be to devise a standard suspension that could be run on the different MEA's and act as a reference point or calibration factor. It is vital for MEA's to be standardised in some form, so that results from different studies can be compared, and without such a change the use of the MEA as a research tool is limited.

From all of the investigations of the MEA data presented above, it is hard to know the best protocol to use. Certainly more work is needed, but if only measures of aggregation are required, then from the work carried out so far it is apparent that it depends on the level of aggregation. For systems of very low aggregation, because of the "crenation" problem, it would be best to perform a computer run of one cycle at Os'1. For systems with normal, or even very high levels of aggregation it may be best to perform a computer run of a few cycles at 12s'1. In each case, computer runs should be carried out on at least two aliquots of a blood sample, and the averages used.

148 FINAL COMMENTS

The contents of this chapter forms a long overdue study of the MEA, and appears to provide answers to some of the problems that have been encountered by various researchers, as seen in publications and have arisen in a number of personal communications (M.Rampling [London], T.Fisher [Los Angeles], R.Guillet [Paris], etc).

Here a new analysis system has been developed for analysing the syllectogram obtained from the MEA, aimed at being quick (20s) at each y, and appears to work in what it was set out to do; ie extend the limits of the MEA to "fall-in-line'' with measurements of aggregation made with other techniques. The author realises that the study is very detailed and complex, but this was necessary to the thesis and to the further development of the MEA.

Extending the limitations of the MEA is by no means something only of use in artificial studies (ie using enzymes to induce hyperaggregation). If measurements were made on blood samples where aggregation was much reduced, the lower limits of the manual MEA would certainly not cope with these samples. One such example would be found with neonatal blood and the small volume requirements of the MEA make it particularly appealing here. Furthermore, if measurements were made on blood samples with hyperaggregating tendencies, the upper limits of the manual MEA would not cope with these samples, and the result would be artifactually low readings.

Another point not yet mentioned, concerns a very important limitation of the authors current analysis system. Since it is based on obtaining syllectogram data over a very short time (10s), then measures of aggregation are highly dependent on the velocity with which the RBC's aggregate. For RBC's with slow aggregating tendencies, the analysis system would produce artificially low measures of aggregation. This will be seen to be what occurs with NA treated RBC's in chapter 6, because it is believed that during NA treatment the RBC membrane is stiffened, which in turn acts to slow the aggregation process down. This needs further investigation, and acts as a problem for the MEA providing measures of the level of aggregation,* syllectogram data collected over longer periods of time may provide a solution to this problem. However, it also shows the importance of not only considering measures of gross levels of aggregation, as important about the aggregation A 149 process of the sample is lost. Clearly it is also important to consider the velocity of aggregation. This will be further discussed in chapter 6 when work with NA treated RBC's is more fully presented.

Finally, it was decided for simplicity, etc not to show results for LT,^ or t^ in the remainder of this thesis. Also the decay constants were not used because of their need for further investigations. In chapter 5 Min<0 and 3)A Syl and Comp(0, * 12 *Bd 24)A Syl w ere used m ainly to give a complete picture of the response of the MEA. In chapter 6, because all that is required is measures of aggregation, Comp(12)ASyl and sometimes Comp<24)ASy, are used, except for systems of low aggregation (ie 1 Og/1 dextran 40), when Comp(0)ASyl is used to avoid the "crenation" problem mentioned above. The changes in time were not considered for simplicity, and because with the protocol used Comp<12)ASyi appeared to be unaffected.

150 CONTENTS OF CHAPTER 5:

HAEMATOCRIT EFFECTS ON VARIOUS BLOOD SUSPENSIONS

A IM 152

F O R E W O R D 152

RESULTS: 5.1 THE INFLUENCE OF HCT ON THE MEA FOR NORMAL AND HYPERAGGREGATING RBCSusp's 153 5.2 THE INFLUENCE OF HCT ON ti MEASUREMENTS OF WHOLE BLOOD AND VARIOUS RBCSuip's 157 5.2.1 HCT CORRECTION FOR r)r MEASUREMENTS OF WHOLE B L O O D 157 5.2.2 HCT CORRECTION FOR rir MEASUREMENTS OF RBCSusp'x \6o 5.2.3 HCT CORRECTION FOR Tir MEASUREMENTS OF NON-AGGREGATING RBCSu,p’s 163 5.3 Tjr VARIATIONS OVER A WIDE RANGE OF HCTS (30-50%) FOR NORMAL AND HYPERAGGREGATING RBC SUSPENSIONS 163 5.4 TESTING THE HCT CORRECTION WORK 166

CONCLUSIONS AND DISCUSSION

151 CHAPTER 5: HAEMATOCRIT EFFECTS ON VARIOUS BLOOD

SUSPENSIONS

AIM

The main aim of this chapter is to present work carried out looking at the effect of hct on whole blood and artificial RBC suspensions (RBCSujp's), as measured on the MEA and the Contraves LS30 Viscometer at various y's. Previously, Chien et al has carried out much work looking at how hct affects whole blood129, ^ but a major concern here was how these effects might change for the various RBCSuip's, particularly as the level of aggregation increases beyond that found in normal blood; does hct affect hyperaggregating RBCSuip's in the same way as normally aggregating RBCSusp's?

It was of much importance in the analysis of the data presented in chapter 6, to assess the necessity of adjusting r\ and MEA measurements on the various RBCSujp's, for small differences in hct (±2%) from the standard hct of 45% used here; such hct differences were caused by experimental error in preparing the suspensions. Correcting r\ measurements of whole blood for these small changes in hct is already accepted to be important, but there appears to be no work that has made such an assessment for normal or hyperaggregating RBCSujp's on the Viscometer or MEA. Hence, the aim here was to make such an a assessment.

Also of interest here was the possibility of predicting rj's for whole blood, and some RBCSuip's, at a LSR and HSR of 0.277 and 128.5s*1 respectively, and furthermore to derive a predictive equation that works over a range of y's. Much research has previously been carried out by other researchers to derive various equations for predicting whole blood r) from various parameters1126,143*1481. However, the important difference here is to look at hct effects on the various RBCSusp's, and predictive equations were derived that allowed the small corrections for hct differences to be calculated more easily.

FOREWORD

Again this chapter uses some facts that will be more fully presented in chapter 6. Briefly,

152 RBC's have been modified with the enzymes chymotrypsin (CT) and trypsin (T), and it will be seen that the modification generally leads to an increase in the RBC's ability to aggregate, compared to untreated washed RBC's (W), when dextran 40 or dextran 70 are present (Wr\cX [A e rtji'ikT CoMp/uvi. For the MEA work presented here, the response of the normally aggregating untreated washed RBC's (W) are compared with hyperaggregating T treated RBC’s, where aggregation is induced by 15g/l dextran 70; ie W +15g/l dextran 70 and T+15g/l dextran 70 respectively. The same protocol was used for obtaining data from the MEA as was described in the last chapter. Two computer runs were carried out, each on a different aliquot of a sample and consisting of 10 cycles; one cycle represents a 10s HSR phase followed by a 10s LSR phase. The LSR's used for run 1 were 0, 3, 6, 9, 12, 12, 9, 6, 3, 0 s"1 and for run 2 were 0, 6, 12,18, 24, 24, 18, 12, 6, 0 s’1; each computer run was followed by six manual readings.

RESULTS;

5.1 THE INFLUENCE OF HCT ON THE MEA FOR NORMAL AND

HYPERAGGREGATING RBCSusp's

The response of the MEA to changes in hct for whole blood has previously been looked at by many researchers, and findings were generally the same; Man(0)ASyi and KUn(3)ASyl increased with increasing hct, peaking at -35% and then falling away again. The same response was shown in §4.6, if manual MEA readings were taken immediately, and was also found with Comp(0, 6, 12 & 24) a

The majority of the work carried out in this thesis was concerned with RBCSu3p's, and it was of some interest and importance to assess if effects of hct variation on the MEA was different for these systems. Particularly, it was necessary to know if small variations in hct (±2%) from the standard hct of 45%, due to experimental errors, had significant affect on the MEA data and if so what could be done to correct for these variations. Another question was whether hyperaggregating RBCSusp's responded differently to normal systems.

153 Figure 5.1a and b respectively shows KUn(0 *nd 3,Agy| and Comp(0, * 12 and ^ A ^ for W +15g/l dextran 70 (n=l). It can be seen that generally the same pattern is found here as for whole blood, as described above, except for Comp(24)ASy, (and Comp(1,)ASyi - not shown), that gave a linear response for reasons that are not clear. For hyperaggregating RBCs^'s, T+15g/l dextran 70 (n=l), figure 5.2a and b interestingly show exactly the same pattern for all parameters (ie peaking at 35s*1 etc), except for Comp(24)ASyl (and Comp(18)ASy, - not shown) which changes from their linear behaviour to give the same pattern as the other y's. \]&

What is useful from this data, as regards this thesis, is to know the extent to which Man

Nlan(0 and 3) a Comp

0s*1 3s*1 0s*1 6s*1 12s'1 24s-'

AASyl (43-47% ) 0.75 1.55 750 1400 1800 30 00 WB (n= 2) P eak(% ) 31.3 33.3 32.3 30.5 3 5 .0 29.5 w AASyl (43-47% ) 0.81 1.41 1000 30 00 2000 - (n=l) P eak(% ) 35.7 35.3 34.1 35.2 34.1 -

AASy1 (43-47% ) 1.1. 4 .5 0 23 00 5100 5 7 00 4 3 00 T (n= 2 ) P eak(% ) 37 35.2 35.8 34 .0 36 .6 37.4

Table 5.1: Shows changes in NMOand3)As>1 and Comp(0,6-12 “ d 24)ASyl for whole blood (W B), or washed (W) or trypsin (T) treated RBC's with 15g/l dextran 70 present, between 43 and 47% hct (AASy1). Also shown is the approximate hct at which the peak value occurred.

154 washed RBC's with 15g/l dextran 70 (n=l). All, except Comp( 24)Asyi, give a similar response to whole blood as shown in figure 4.17. Lin zi u v ? /*»♦ «3rd cstkr polynomial curut/.-)b,

155 Figure 5.2: As for figure 5.1a and b but for the hyperaggregating RBCSlap T+15g/l dextran 70 (n=l). It can be seen that hct affects the parameters here far more than with the normal aggregating RBCsu»P W+15g/l dextran 70. Again the pattern is similar to whole blood, (including Comp< 24)Asyi). Uftrt

156 5.2 THE INFLUENCE OF HCT ON qr MEASUREMENTS OF WHOLE BLOOD AND VARIOUS R BC ^s

It was shown in §1.5.2 that LSR q's vary considerably with hct, and due to this the ICSH Guidelines for making measurements11241 stated the importance that each laboratory should set up tables of blood q ^ over a range of hcts (30-60%). These tables could then be used to calculate small corrections to q ^ for any hct differences (±2%). Primarily it was the authors aim to automate the calculation of corrections to q ^ from whole blood by fitting equations to the data for the various y's. A necessary modification to this, that will become apparent, is the need to collect similar data for the various RBCSujp's that aggregate to differing degrees; ie normal aggregating RBCSusp’s require far less correction than hyperaggregating RBCSusp's. Only a few RBCSuap's with very different levels of aggregation were looked at, because it is infeasible and unnecessary to investigate all RBCSusp's.

5.2.1 HCT CORRECTION FOR qr MEASUREMENTS OF WHOLE BLOOD

Previously Rampling collected whole blood q ^ , at various y's, from a group of normal subjects at their native hcts (38.5-47.5%) and also at a corrected hct of 45% (personal communication). Also calculated were their respective plasma q's, thus allowing qr to be calculated. Rampling then used the semi-logarithmic relationship between qr and hct (equation 1.12 - ie Ln(qr)=k' + k"*H) to compile reference 'calibration' graphs, allowing adjustments in qr to be estimated for small deviations in hct (±2%) (qr correction values - A t|) from the standard 45% used in this work.

Primarily the aim, with permission from Rampling, was to automate, and thus simplify, the calculation of A q by regressing a line of the form of equation 1.12 through the data (figure 5.3) and thus obtain the coefficients (table 5.2). However, for reasons that will become obvious later, it was of much benefit and interest to use a modification of this equation as follow s:

Z^q,) - w1 .H (5.1) where nq is the gradient (figure 5.3 and table 5.2). The assumption here was k' of equation 1.12 equals ln(supematant q) at all y's. Other researchers have used the same 157 simplification1146,1471, some of whose work will be discussed at the end of this chapter.

Number of subjects Y k \ k" mi 29 0.2 7 7 -0.157, 0.0796 0 .0 7 6 0

17 0.695 -0.590, 0.0659 0 .0 6 5 9

19 1.747 -0.283, 0.0630 0 .056 6

17 4 .3 9 0 -0.121,0.0504 0 .047 6

19 11.02 0.209, 0.0361 0.040 8

17 27 .70 0.182, 0.0307 0.034 8

17 69.50 -0.013,0.0302 0 .0 2 9 9

29 128.5 0.155, 0.0247. 0.0283

Table 5.2: Lists the coefficients of equation 1.12 (ie Ln(qr)=k’ + k"*H) and the coefficient of equation 5.1 (ie Ln(qr)=m,*H), as derived from whole blood. These values allowed changes in q (Aq) to be calculated for small hct (±2%) variations.

However, the main reason for using equation 5.1 was because it led to a further convenient linear relationship between Ln(m,) and Ln(y) as shown in figure 5.4. A single linear equation appeared to describe all points, but closer inspection showed the point at 128.5s'1 to be slightly higher. Including or not including this point made very little difference to the goodness of the linear regression, however for consistency with the next section points were split into two distinct ranges, corresponding to the aggregation phase (0.277-27.7s*1 - PHASE A) and deformation phase (69.5-128.5s*1 - PHASE B) of shear thinning. Figure 5.4 shows a plot of the data and linear regressions that demonstrates the excellent correlation (Pearson correlation, for phase A, gives -.99969). The regression equations from Ramplings data were as follows:

PHASE A: In(m,) - -0.171 .Ln(y) - 2.79 (5.2)

PHASE B: Ln(m{) - -0.0895 .L n ( y) - 3.13 (5.3)

These equations can each be substituted into equation 5.1 to give a convenient relationship betw een q n hct and y. By simple rearrangement the following equation was obtained:

- h ./'. k x (5.4) 158 two linear regressions through data points: one freely regressed (equation 1.12) and the other regressed with a fixed constant equal to Ln(plasma n) (equation 5.1). (Data used with permission from Rampling) Ln(y)

Figure 5.4: Log* - Log* plot of mj against y- Demonstrates the excellent linear relationship between Ln(mO and Ln(y) in two phases labelled A and B. One line regressed through all points would have been almost as good (dashed line),'but for consistency with work carried out later the regression was split into the two phases A and B. 159 where the coefficient A, is the gradient and K, the exponential of the constant of equations 5.2 and 5.3. Table 5.3 shows the values of these coefficients for whole blood.

PHASE A PHASE B -0.1710 -0.0895 K, 0.06142 0.04370 Table 5.3: Showing coefficients Aj and K! of equation 5.4 for whole blood. 5.2.2 HCT CORRECTION FOR qr MEASUREMENTS OF RBCSusp’S

Previously when researchers have corrected for small hct variations, normal whole blood Tjdaia appears to have been used regardless of the level of aggregation. However, in looking at how well Ramplings correction data worked for the various RBCSuip's the author generally found this to be inadequate; ie for RBCSusp's with much higher or lower levels of aggregation, using correcting equations derived from whole blood data would either under or over correct for hct differences respectively. Hence to extend calculation of Aq correction the author applied the same principle as above to some of the different RBCSusp's. It was difficult to know how far to take this study, ie how many different RBCSlup's to calculate Aj and K, for. It was unfeasible, and unnecessary, to perform this for all RBCSujp's, since levels of aggregation in some RBCSusp's were similar. Hence, it was decided to only look at the RBCSiap's shown in figure 5.5, because they covered a wide range of aggregation. To correct a RBCSujp other than those shown in the table 5.4, it seemed reasonable to use values of A! and K, of the RBCSusp's of table 5.4 which showed similar levels of aggregation., etc.

Unfortunately the relationship between Ln(m,) and Ln(y) was found not to work over all y's for trypsin treated RBC's with 15 dextran 70 (T+l 5g/l dextran 70). Basically, nq values for y's of 1.737s'1, or less, failed to fit in with the loge-Iog* relationship between nq and y; ie the values produced were lower than the regressed line as can be seen in figure 5.6. The source of this breakdown can be seen in figure 5.7, when at LSR's free regression lines are very different to those forced through zero. Hence, the necessary modification here was to

160 limit the range over which the derived equation was used for correction (2.37-128.5s'1). However, because the y of 0.277s'1.was chosen to represent the LSR r\ behaviour of the blood sample, it was important to be able to correct this y for small hct changes. Here, it was decided to use equation 5.1 where n^ was calculated to be 0.0980.

Monodispersion Phase Deformation Phase A, K, Ai K, W+15g/l dextran 70 -0.129 0.0638- -0.106 0.0583 (n-13) (0.277-37.61') (51.2-128.5*') CT+15g/l dextran 70 -0.160 0.0710 -0.114 0.0600 (n-12) (0.512-27.7*1) (37.6-128.5s1) T+15g/l dextran 70 -0.206 0.0841 -0.104 0.0578 (n-ll) (2.37-37.6s') (51.2-128.5*') Table 5.4: Coefficients of equation 5.4 obtained from untreated (W), and enzyme (CT or T) treated RBC's with 15g/l dextran 70 present. Measurements made on n subjects at various hcts.

161 Ln(T)

Figure 5.6: Log, - Log,, plot of mj against y for the hyperaggregating RBCsu,p, trypsin treated RBC's with 15g/l dextran 70. A-B and C-D indicate the y's used for the linear regressions for phase A and B respectively, y's below A are not described by the equations and for y's between B-C can be obtained by either phase A or B equations.

Figure 5.7: Log, - linear plot of Hr against hacmatocrit for n measurements of the hyperaggregating RBCsuip, trypsin treated RBC's with 15g/l dextran 70, at various y's. One subject (one subject run twice).

162 5.2.3 HCT CORRECTION FOR r\r MEASUREMENTS OF NON­ AGGREGATING RBCsusp’S

If the logg-loge relationship between n^ and y is looked at in RBCSutp's with no aggregant present, it was found to fall into two phases, corresponding here to the flat or monodispersion phase, and deformation phase (figure 5.8). Table 5.5 summarises these results and figure 5.5 demonstrates how W relates to other RBCs^'s and whole blood. This pattern was also found with W and CT+10g/l dextran 40, as no aggregation normally occurs in these RBCSusp's.

Monodispersion Phase Deformation Phase (1.747-5.961') (11.02-128.5i') A, K, A, K, w .00131 .0434 -.0773 .0511 CT -.00036 .0446 -.0839 .0529 T -.00835 .0454 -.0870 .0536 T+CT -.0112 .0474 -.0947 .0560 CT+T -.0138 .0475 -.0944 .0559 Table 5.5: Coefficients of equation 5.4 from various RBCSu,p's with no aggregating agent present; ie No aggregation. (n=5)

5.3 T)r VARIATIONS OVER A WIDE RANGE OF HCTS (30-50%) FOR NORMAL AND HYPERAGGREGATING RBCSusp’S

A comparison between normal and hyperaggregating RBCSusp's has already been looked at to some extent, but with an emphasis in terms of generating hct correction equations. However, it was of interest to briefly look more directly at the affects of hct on °' 277 rjn and also the gradient parameter, GA, proposed in chapter 3. In §1.5.2 it was described how qp at 0.3s*1, changed by -500% between hcts 30 and 50%. °- 277 qr for washed RBCs with 15g/l dextran 70 (W+15g/l dextran 70) was found by the author (n=l) to show a similar change, between 30 and 50% hcts, of-528% and furthermore GA changed by -137%. However, for trypsin treated RBC's with 15g/l dextran 70 (T+15g/l dextran 70), changes (n=l) were

163 Ln(Y> 0 0.5 1 1.3 2 2.5 3 3.5 4 4.5 5

Figure 5.8: Plot of Ln(mi) against Ln(y) for various enzyme treated RBC's without any aggregating agent present.

Figure 5.9: Log* - log, plot of 0277 r|r against y for the normally aggregating RBCSlBp's W+15g/l dextran 70, and the hyperaggregating RBCSu*p T+15g/l dextran 70. It is shown (n=l) that between 30% and 50% haematocrits, 0277 r|r for W+15g/l dextran 70 changed by 528% whereas it changes by only 335% for T+15g/l dextran 70 (Gi changed by 135% and 29% respectively). 164 Figure 5.10: Demonstrates the improvement of making het corrections for both 0 277rjr and M*n(3)A sy Plots show correlation between 0277 r|r and Man 0 )ASyi for data without het corrections (a), with only 0277r)r corrected (b), and with 0277nr and Mina)Asyi corrected (c). Data obtained from washed RBC's from five individuals and suspended in PBS with 25g/l dextran 70. 165 much less at -335% and -29% respectively (figure 5.9). Hence, hct appears to have less affect on hyperaggregating systems. However, even though the % change of 0277 rjr between 30 and 50% hcts was less forT+15g/l dextran 70 than for W+15g/l dextran 70, the actual correction values per % change in hct was greater for T+15g/l dextran 70.

5.4 TESTING HCT CORRECTION WORK

It was seen in §4.7 how a good linear correlation was found between Man( 3)ASy, and 0 277 r)r for the RBCSuip W+25g/l dextran 70 (n=5). The very close correlation between the two instruments was in fact the outcome of correcting for the small changes in hct (± 2%), caused by experimental error, for both parameters. Figure 5.10a shows the data before correction, figure 5.10b after correcting 0277 r|r and figure 5.10c after correcting both °‘277T)r and M,n( 3)ASyl. It can clearly be seen from the Pearson correlation values given, that both corrections act to improve the correlation. A similar response was found for GA (not shown).

CONCLUSIONS AND DISCUSSION

The above work was far more complicated than anticipated and more detail is given than originally intended; this was certainly not meant as a chapter in its own right! However, in order to make the points clear, and not detract from the main issue of other chapters, this was the obvious solution. There are a number of important points presented here concerning the preparation of the data in the next chapter.

EFFECTS OFCHANGES JNHAFMA TOCRITON MEA DA TA

There are a number of studies that have used the MEA to measure aggregation of whole blood, yet ignore the effects of the wide physiological hct variation (eg 37-54% for normal subjects) on the MEA. For example, one study looked at the influence of ethnicity on haemorheolgical factors of diabetics11491. In this study, because of the large number of subjects involved (>300), it was considered infeasible to make physiological hct corrections, and hence hct differences were ignored. Significant differences were found, but one can_not be sure as to whether this was caused by possible hct differences between the two ethnic 166 groups. However, for the small study presented above (§5.4), correcting for hct differences appears to offer much improvement (figures 5.10a-c). Also, when aggregation was significantly raised (eg T+15g/l dextran 70), hct affects were found to increase to a level that is less feasible to ignore (table 5.1). Hence, the author feels that hct correction of MEA data should be considered in the analysis as is the case for the analysis of ri^ . The inventor of the MEA, H. Schmid-Schonbein, appears to share this opinion, because at a recent conference (9th European Conference on Clinical Haemorheology, Siena. 1995) he criticized work carried out on the MEA that ignores changes in hct. It would not be difficult to devise a system for correcting for hct differences, ie using a correction table devised from a polynomial fit for a large number of subjects, and it would then be more acceptable to carry out MEA work at the natural hct.

From the brief investigation presented above, due to time and restricted data, obtaining standardized correction equations was not attempted. Instead a very simple system was devised for correcting for changes in hct, involving considering differences in MEA responses between 43 and 47% hct to be linear, and calculate values for hct correction from table 5.1. Another complication was that data was only obtained for two RBCSutp's; the normal aggregating W+15g/l dextran 70 and the hyperaggregating T+15g/l dextran 70. Modifications to the remainder of RBCSuip's was made by using values based upon the level of aggregation; ie for aggregation that falls in between W and T+15g/l dextran 70 (eg CT+15g/l dextran 70), a correction value half way between that obtained for W and T was used, and for aggregation «greater than T+15g/l dextran 70 there was no choice but to use that obtained for T+15g/l dextran 70. This is certainly not ideal for a number of reasons, but was considered better than doing nothing.

EFFECTS OF CHANGES INHAEMA TOCRJT ON ^

The need for adjusting r|r for small changes in hct has been accepted for some time, and as already mentioned, is advised in the ICSH Guidelines for making measurements11241. However, as the levels of aggregation rises the corrections of qr for % change in hct rises, such that the degree of correction for normal aggregating samples is far less than for hyperaggregating samples.

167 Graphs were compiled, and equations derived, for a few RBCSusp's at different y's. The need for this preparation work is made clear above, which shows the large variations in blood rj for W, CT and T treated RBCs with 15g/I dextran 70 at different hcts.

Another complication was whether GA needed to be changed for hct variations, and if so how. This was not really investigated in great detail, mainly because GA appeared to be less sensitive to hct changes than 0,277qn and for simplicity it was an early decision to ignore hct effects on GA. Hence, all GA data presented in chapter 6 was not corrected for hct changes. However, benefits of correcting GA for small hct differences were found from preliminary tests (not shown) and so this needs to be reconsidered and further investigated. However, this is of little importance in the work presented here, since differences are much large than any hct effects on GA.

EQUATION5,1: LtrfnJ^mfH

Equation 5.1 appeared to describe the hct dependence of q ^ over a limited range of hcts, and had a number of advantages associated with it. The most striking of these advantages, was the unexpected progression to equation 5.4 that contained the parameters r|p H and y, thus allowing one equation to correct q ^ over a wide range of y's. This proved useful in the analysis and presentation of q ^ .

However, a further advantage here was m, could be estimated from RBCSusp's where q ^ was only available over a small hct range (43-47%). It can be seen in figure 5.5 that this appeared to offer a good approximation. T+15g/l dextran 70 was looked at over a wide range of hcts, as an example of a hyperaggregating RBCSusp, and was seen to obey the same sort of relationship, although only for y's above 1.747s*1. The breakdown of equation 5.1 here was considered to be related to the different behaviour of q ^ below ~ 1.747s*1 for T+15g/l dextran 70 etc (§3.1.1), but this was not further investigated here.

168 OTHER ffr PREDICTIVE EQUATIONS FOR WHOLE BLOOD. AND HOW WELL THESE WORK ON VARIOUS RBC^/S

This section falls into two parts. The first will look at other work using the simplified equation 5.1, and the second part briefly mentions other predictive equations.

There has been a fair amount of work along the same lines of that presented here. One of the earliest researchers was Weaver et al, who in 1969 used a Capillary Viscometer and appeared to obtain a good linear-log relationship between mt and y i,50]. Whittington et al extended this to obtain a relationship linking r|, y, H for capillary viscosity measurements as follows:

~ t,r • ( • /- ) " (5.5) A y) A y) v 9 where f(y) is a function of y and A is a constant11451. However, Aarts dismissed this work: "...these calculations were extrapolated from capillary viscosity measurements and based on normal values of older studies"11291. The authors' data shows a different relationship from Weavers; ie a log-log relationship between m, and y. When regressing a log-log line through the whole blood data obtained by Rampling (figure 5.11), clearly it does not describe the data very well. However, the coefficients were almost identical to those obtained by Weaver et al (ie m1=0.030-0.0076*log(y))11491. Undoubtably figure 5.4 shows that a better linear relationship is found between log(m,) and log(y). Hence Whittingtons equation is also wrong and should be in the form of equation 5.3 above, which means that a reassessment of his theoretic work is needed.

Matrai et al also carried out studies assessing how well the equation of the following form predicted rjr at 45% hct:

45 (5.6) ^ r 45 ‘ r not) where qr 4J and t|r are the relative viscosities at 45% and native hct (H^,). This is just a simple manipulation of equation 5.1, that cancels out m/1471. The goodness of Matrais equation was compared to the authors' equation 5.4, to indicate if the latter was an 169 Figure 5.11: Linear-log plot of mi against y for Rampling's whole blood data, with a linear regressed line, that can be seen to poorly describe the data points. Weaver et al concluded from a paper that such a relationship worked well; the linear regression shown produces coefficients almost identical to Weaver et al's[150].

170 improvement on the former. Again Rampling's data was used and the results are shown in table 5.6 along with the predictions of equations 5.4 and 5.6. It can be seen that the authors equation 5.4 is an improvement of Matrai's equation 5.6. They are both derived from the same source, but equation 5.4 has the advantage of being shear dependent and has been gained by averaging several y's. It is not a surprise that equation 5.4 is an improvement as it is derived from the data being tested, but being based on the average of several y's, for a number of subjects etc, has to offer improvement.

n . °-277 Tirnat °'27,ti, 45% Eq. 5.4 pred Eq. 5.6 pred 41 23.92 34.27 32.60 32.16 46 32.17 30.00 29.83 29.68 46.5 35.3 30.98 31.50 31.54 47 35.88 30.00 30.71 30.81 44 27.42 29.84 29.72 29.56 42 31.35 36.51 37.76 40.10 41.5 19.10 26.32 26.44 24.49 38.5 18.90 36.76 31.14 31.04 40 18.15 28.52 28.08 26.07 Mean ± SD 31.47 ± 3.60 30.80 ± 3.16 30.67± 4.40 Table 5.6: Comparison of the Authors and Matrais predictive equations on Ramplings data.

As already mentioned, the problem with predictive equations is that they represent the group of subjects from which they are derived, yet they do not necessarily work for other groups of subjects. To demonstrate this, consider a study Matrai carried out comparing the hct dependance of normal subjects to patients with peripheral vascular disease (PVD). For the patients with PVD (n=31), Matrai found m, at 0.7s ’1 was 0.067±0.012 and at 94.5s '1 was 0.032±0.007. However, Rampling obtained similar values from a normal group of subjects; ie 0.066 and 0.029 respectively. Hence Rampling's normal subject group was the same as Matrais' PVD patients, thus demonstrating the difficulty of developing predictive equations.

171 Many other workers have tried to develop different mathematical equations to predict whole blood r| from similar parameters and also from MCV and protein concentrations ( and IgG). Easthope et al,144) reviewed eleven consfitutive equations for blood and found the Walbum and Schneck equation 11431 to be the best, followed closely by an equation by Quemada11261. Quemadas equation appears to be the most widely used equation today, mainly because the parameters have the advantage of having physical meaning. These equations were not compared to the authors' equation, because of their greater complexity and also it was moving too far away from the aims of this thesis.

FINAL COMMENTS

One important difference here, from work carried out by other researchers, was that most of the variables found with whole blood were removed by investigating RBCSlttp's; ie washed or enzyme treated RBCs with an aggregating agent. This appears to be a better approach, from many previous researchers, to developing predictive equations rather than immediately attempting to consider all of the variabilities of plasma, etc. By looking at RBCSusp's, only changes in the intrinsic aggregating properties of the RBC need to be considered, and it was found that as the aggregating tendencies of RBC increases, the influence of hct changes as mentioned above. Hence, this suggests that a better predictive equation may have a parameter related to the intrinsic aggregating tendencies of the RBC. However, this needs further investigation.

For all laboratories to go to the extremes of this chapter is obviously unfeasible, which raises the question as to what should be done? Clearly the MEA is not affected as greatly as the Viscometer, so it depends on the instrument being used. It also depends on the project; ie the range of aggregation in samples being looked at. If the differences between systems are large, as with the different enzymes and aggregating agents, then little concern is necessary because big differences would show up regardless. However, as already mentioned, when wanting to assess smaller differences (eg subject variability), then this is far more important. There is currently no real answer, as there are a number of unanswered questions and further investigations needed.

172 CONTENTS OF CHAPTER 6 : USING ENZYMES TO DEGRADE THE RBC SURFACE TO INVESTIGATE CELLULAR FACTORS OF IMPORTANCE TO ROULEAUX FORMATION / AGGREGATION.

A IM 174

FOREWORD 174

RESULTS: 6.1 INVESTIGATING THE MECHANISMS OF RF USING T AND CT TO DEGRADE THE RBC SURFACE 176 6.1.1 ENZYME INCUBATION TIME COURSE STUDIES 6.1.2 DEXTRAN INDUCED MEASUREMENTS OF AGGREGATION 176 6.1.3 INVESTIGATING CHANGES TO THE RBC's 181 6.1.3.1 CELLULAR FACTOR MEASUREMENTS 181 6.1.3.2 r| CHANGES IN NON-AGGREGATING RBC's 184 6.2 USING ENZYMES AS A TOOL FOR INVESTIGATING THE ACTIONS OF AGGREGATING AGENTS ON RBC's - A COMPARISON OF DEXTRAN AND FIBRINOGEN 186 6.3 BRIEF ASSESSMENT OF THE EFFECTS OF OTHER ENZYMES (Th, B AND NA) ON THE AGGREGATING TENDENCIES OF RBC'S 188 6.4 CORRELATION OF UE AND nr's FOR THE VARIOUS RBCSllsp'S 192 6.5 DOES ALBUMIN INDUCE AGGREGATION OF T, B OR NA TREATED RBC's 195 6.6 CT AND T INCUBATION TIME COURSE STUDIES REVISITED! 197

CONCLUSIONS AND DISCUSSION 204

173 C H A P T E R 6 : USING ENZYMES TO DEGRADE THE RBC SURFACE TO INVESTIGATE CELLULAR FACTORS OF IMPORTANCE TO ROULEUAX FORMATION / AGGREGATION.

AIM

The aim of this chapter is to present work that has attempted to use enzymes as a tool for investigating possible mechanisms involved in rouleaux formation (RF) or more generally aggregation. Cellular factors (size, deformability and surface charge) were measured on normal washed RBC's (W), and on RBC's degraded with enzymes trypsin (T), chymotrypsin (CT) or cocktails of these; ie T then CT (T+CT) and CT then T (CT+T). The aggregating tendencies of these RBC's were measured with either dextran 70, dextran 40 or fibrinogen present, thus allowing an assessment of the cellular factors of influence to aggregation to be made. Also, the aggregating tendencres of RBC's treated with thrombin (Th), bromelain (B) or neuraminidase (NA) were briefly investigated. Looking at the actions of both fibrinogen and dextran on these RBC's allowed these two aggregating agents to be compared. Finally, albumin, which is known not to induce aggregation on untreated RBC's, was investigated in various concentrations with either T, B or NA treated RBC's, to see if it would induced aggregation of these RBC's.

FOREWORD

One purpose of the previous three chapters was to establish methods for analysis of the aggregation data for the various RBCSusp's. The main reason for the necessity of these chapters in this capacity, was due to various problems associated with the hyperaggregating enzyme modified RBC's. A summary of these chapters will now be covered briefly.

In chapter 3, a detailed study was presented of viscosity data (ti^ ) from a number of aggregating and non-aggregating RBC suspensions (RBCSusp's). A new PC based analysis system was developed, involving a computer program (AnalEta), to investigate the log.-log,. relationship between r|r and y, and produced a number of parameters. These parameters were derived from regressing .through data points at different y's, avoiding points that fail to fit into the log-log relationship; ie the points of the plateau and transition regions. The 174 parameters that will be used here from this analysis are a277 r)r, 128 5r|r, GA and GD.

In chapter 4, a study was presented on the MEA, which is an optical device for measuring aggregation. It works by detecting the transmission of light through a newly dispersed blood sample, which has a user specified y placed across it. The curve produced from the detected transmission is known as the syllectogram and is made up of a reorientation and aggregation phase; for simplification, here the term syllectogram will only refer to the aggregation phase. The MEA has two modes of operation: the manual and computer modes. In manual mode it displays a value corresponding to the area measured under the syllectogram, obtained from a sample sheared at either 0 or 3s'1, and these parameters were referred to as Man( 0)ASv) and Man( 3)ASyl respectively. However, these parameters are known, and were shown again in chapter 4, to only describe a limited range of aggregation of a sample, and will only be used here when computer data is not available. In computer mode, the MEA is controlled by a computer which also captures the syllectogram data. The general protocol was to set the computer to collect data from 10 cycles at various y's between 0 and 24s'1. A new PC based analysis system was developed for investigating the captured syllectogram data. The analysis system was based on viewing the data (ViewSyl), and fitting monoexponential and biexponential.curves (NLREG33) to the user specified region of the syllectogram. A number of parameters were derived from the analysis and investigated, but for simplicity only a few of these were used here. The study showed that calculated areas under syllectograms obtained at 0, 12, 18 or 24s*1 (Comp{0, 1118 or 24)ASyI), appeared to extend, maybe even remove, the limits with which the MEA would accurately measure aggregation. Comp( 12)ASvl was used for the majority of the work of this chapter, but comP(0)ASyl and Conip( 2',)ASyl were used if Comp( 12)ASyl produced uncertain results, as was found with very low or extremely high levels of aggregation.

In chapter 5, investigations were carried out on the effects of haematocrit (hct) variation on the aggregating tendencies of whole blood samples and the various RBCSusp's, as measured on the Viscometer and MEA. It was established that r)^ and MEA data benefitted from correcting for small changes in hct (± 2.0%) due to experimental error in sample preparation. Hence, values were obtained for correcting and MEA data over these small hct changes and were used in all of the data presented here.

175 6.1 INVESTIGATING THE MECHANISMS OF RF USING T AND CT TO DEGRADE THE RBC SURFACE

6.1.1 ENZYME INCUBATION TIME COURSE STUDIES

Previously Ertan et al carried out a time course study (up to 2 hours) of T treated RBCs, and found from rj measurements, with 15g/l dextran 70, and electrophoretic mobility (UE) measurements, that trypsin action was completed within 30 minutes (figure 6.1)I151]. The author used this same protocol to look at time course studies of T and CT and obtained similar findings. As a result of this, RBC's were subjected to enzyme treatments for two hours at 37°C, and all enzyme treatments in the following work used this protocol.

6.1.2 DEXTRAN INDUCED MEASUREMENTS OF AGGREGATION

Here dextran-induced-aggregation is investigated before and after treatment with the enzymes CT,T, T+CT or CT+T, and changes are related to cellular factors that were also measured on these RBC's.

J rISCO SIT)' MRA SIJREMENTS

Initially 15g/l dextran 70 was used as the aggregating agent and °' 277 r)r is shown in figure 6.2 for the various enzyme treated RBC's (n= 8 ). Significant increases were found with CT treated RBC's, and even higher increases with T, compared to untreated RBC's. However, T treated RBC's with 15g/l dextran 70 aggregate to very high levels and so detecting further, possibly smaller, increases in aggregation with the cocktails (T+CT and CT+T) was felt to be very difficult. So it was advantageous to lower the level of aggregation to increase sensitivity for highly aggregating RBC's and for this 10g/l dextran 40 was used.

For RBCSusp's with 10g/l dextran 40 (n=7), no aggregation occurred for untreated and little (or nothing) for CT treated RBC's. T modified RBC's always showed significant increases in °'277 r|r and cocktails showed a further significant increase from T, although no significant difference was found between the two cocktails (figure 6.2 and table 6.1). Also no significant changes were found in 128 5r|r. 176 Figure 6.1: Viscosity (o:"r|r) and electrophoretic mobility (UE) data showing time course study of trypsin. Both show trypsin action on RBC are finished after 30 minutes. Data taken from a paper by Ertan et al[151J.

Figure 6.2: Plot of o:77nr against various enzyme treatments ofRBCs with either 15g/l dextran 70 (n=8) or 10g/l dextran 40 (n=7) present to induce aggregation ( * s o )

177 W CT T T+CT CT+T ’ 15g/l 30.39*3.02 46.86*3.75* 77.59*15.1” Dextran 70 - - (n = 8) 4.95*0.36 4.80*0.22 4.91±0.23 10g/l 7.31*0.62 9.66*0.87’ 27.53*4.71*’ 35.82±5.75”x 36.19*7.8 l*’x Dextran 40 (n=7) 4.83*0.25 4.92*0.25 4.96±0.32 4.84*0.18 4.86*0.24

Table 6.1: o: 77 r)r and 128'5T}r data for the various RBCSusp's (n subjects : *SD). significant from W ,’: significant from CT, x: significant from T. No difference was found for 128-5T )r between W and the other suspensions. No significant difference was found between any of the cocktails, (paired t-test)

Ga gave the same pattern as shown in table 6.2. Hence aggregation measurements of the different RBC's followed the pattern: W < CT < T < (T+CT = CT+T) Gd could only be obtained by regressing through two or three points, which made this parameter variable (table 6.2). However, in non-aggregating RBCSusp's more points are available, because of the shifted intercept (transition) point as mentioned in §3.5. The changes in GD for the different enzyme treated RBC's will be looked at later (§ 6.1.3.2), when non-aggregating RBCSusp's are specifically looked at.

\v CT T T+CT CT+T lSg/l -0.343*0.023 -0.441*0.048* -0.578*0.032*’ Dextran 70 - - (n = 8) -0.187*0.024 -0.192*0.029 -0.180*0.023 10g/l -0.009*0.020 -0.056*0.040* -0.324*0.034*’ -0.395*0.045”x-0.396*0.052**x Dextran 40 r- ii c -0.132*0.027 -0.177*0.018* -0.187*0.019’ -0.205*0.015** -0.185*0.027’ Table 6.2: GA and GD for the various RBCSusp's (n subjects : *SD). *: significant from \V, ’: significant from CT, x: significant from T. (paired t-test)

MEA MEASUREMENTS

ancj Nlan(3>As>1 data showed the same pattern for W, CT and T but could not provide agreement for the cocktails as the limit of sensitivity was reached with both dextrans used (data not included). However, Comp(i:)ASyl generally gave supporting results for both 178 dextrans. as shown in table 6.3, apart from T+CT that was not significantly greater than T. Due to the problems found with making MEA measurements of RBCSut's with 1 Og/1 dextran 40 (ie the crenation problem), CoTTip( 0)ASvl was looked at, but here gave the same pattern as for Compn 2)ASyl (table 6.4).

YV CT T T+CT CT+T 10898 24086 15g/l Dextran 70 43961 - - (n=3) ±8305 ±8865* ±9346" 10g/l Dextran 40 5792 7090 14657 16336 18625 (n=4) ±7686 ±7042 ±6996" ±6026" ±3042"x Table 6.3: Comp(,2)ASvI for the different enzyme treatments with either 10g/l dextran 40 or 15gl dextran 70 present to induce aggregation (n subjects : ±SD). *: significant from W, significant from CT, x: significant from T. (paired t-test)

YV CT T T+CT CT+T | 10g/l Dextran 40 -2554 -1014 6926 8504 8905 I (n=4) ±1070 ±402* ±5719" ±5393" ±4524"x | Table 6.4: Comp(0)ASvl for the different enzyme treatments with 1 Og/1 dextran 40 present to induce aggregation, (n subjects : ±SD) *: significant from W, significant from CT, x: significant from T. (paired t-test)

ESR MEASUREMENTS

ESR measurements were carried out mainly to confirm, with yet another method, the large changes in aggregation caused by the enzymes T and CT. Figure 6.3a shows that ESR measurements agree with the above r) and MEA work as shown for W, CT and cocktails. 7g/l dextran 70 or 5g/l dextran 110 were also used, and the results for the various RBCSusp's are shown in figures 6.3b and c respectively, and show the differences between W, CT and T as before. Due to the rate of settling of the hyperaggregating 'cocktail' suspensions, any small differences between the cocktails and T RBCSusp's would be very hard to show with this method.

179 0 100 200 300 400 500 600 700 | Time (minutes) j

Figure 6.3: ESR data for normal RBC's (W) and enzyme modified RBC's (CT. T. T+CT and CT+T) with either (a) 15g/l dextran 70. (b) 5g/l dextran 110. or (c) 7g/l dextran 70 present. All show large differences between W. CT and T (and/or cocktails), but it is uncertain about differences between T and the cocktails. n=l for each set of data.

180 6.1.3 INVESTIGATING CHANGES TO THE RBC’s

6.1.3.1 CELLULAR FACTOR MEASUREMENTS

With the differences in aggregation between the enzyme modified RBC's known, the next stage was to investigate the cellular factors of the RBC. As already mentioned (§ 1.9.3), the potential cellular factors of influence to RBC aggregation are the RBC size, deformability, surface charge and the number of adsorption sites. The first three of these were measured, using standard methods, before and after enzyme treatments (CT, T, T+CT or CT+T) and any changes related to the enzyme induced increases in aggregation. Assessing changes in the number of adsorption sites was more difficult, and the work of the next chapter was cl&icjneJ to achieve this. However, it was hoped that some insight into changes in the adsorption sites, could be gained from the other three cellular factors failing to explain all findings.

MEAN CELL VOLUME (MCV)

The RBC size, MCV, was calculated from equation 2.7; ie by dividing hct of the sample by the number of cells per liter of sample as obtained from the Coulter Counter Zbi (§ 2.6.1). No significant changes were found after enzyme treatment in the 8 subjects looked at (table 6.5). When these measurements were made, RBC's were taken from their PBS environment, in which all other measurements were made, and suspended in Isoton n, the standard solution used by the Coulter Counter. A concern here was whether RBC's changed, due to changes in osmolarity etc, when placed in Isoton II and so a further method of assessing changes in MCV was used. This involved measuring hct of the RBCSusp before any enzyme was added, and then again after 2 hours incubation. This showed no change in hct, which therefore implies no large change in MCV. An alternative way of assessing changes in the RBC size and also obtain information about the RBC size distributions, was to look at data captured from the Coulter Channelyzer. Two parameters were used to represent the distribution data, which were measures of the distribution width (o) and the mean position (M) which was equivalent to MCV. These were calculated in Microsoft Excel, and results (n=3) are shown in table 6.5. Again there is no evidence of the enzymes changing the RBC size, and furthermore the enzymes do not appear to change the size 181 distributions. All these findings suggest size played little, if any, part in the increased aggregation caused by the enzymes.

W CT T C T + T T + C T

M C V (fl) 97.3+ 7.6 94.5+1 1.8 96.3+9.1 95.7+ 9.1 (n=8) 95 .6+ 9.0

Size 0 13.98+ 2.57 13.S 0i2.91 14.24+3.58 13.48+ 1.88 13.20= 1.73 Distribution (n=3) M 27.58+ 0.71 2 7 .44 + 0 .40 27.68+ 0.10 26 .31 = 2 .43 26 .03 + 2 .54

rFR(O) O O cn .580+ .038 .586+ .072 n .5 99+ .04 9 .599+ .035 (n=4) Table 6.5: The cellular factors RBC size (MCV and RBC size distribution width (o) and mean (M)) and deformability (rFR(O)) data (n subjects : +SD). Paired t-test showed no significant difference between any enzyme treated RBC's and control.

DEFORMABILITY

Deformability was measured on the St Georges' Filtrometer and represented by the index of deformability 'rFR(O)' (§2.6.2). For the four subjects looked at, no significant changes in the deformability were found after enzyme treatment (table 6.5). Hence, deformability plays no, or little, part in the increased aggregation.

SURFACE CHARGE

The RBC surface charge was assessed by measuring the electrophoretic mobility (UE), on the Malvern Zetasizer En (§2.6.3). Initially UE was measured for normal RBC's suspended in a number of different ionic strength (7) buffers, and UE was found to exponentially decrease with increasing 1 buffer which was in agreement with Seaman and co-workersI23‘153] (figure 6.4). The different enzyme treatments generally caused UE to decrease in accordance with increasing aggregation; ie surface charge for the different RBC’s was as follows: W > CT > T = (T+CT = CT+T) No significant difference was found between the cocktails and T treated RBC's. The decrease for CT and T, at 7=0.172, was again in agreement with Seaman123,153], but he never looked at cocktails of the two. These results are shown in table 6.6 and figure 6.5.

182 | 3.5 -

j1 0.5 - ; I 0 ------1------. I 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 Ionic Strength (M) i______Figure 6.4: Electrophoretic mobility (UE) against ionic strength of the suspending phase for normal human RBC's taken from one subject. Shows exponential decay in electrophoretic mobility with increasing ionic strength. (n=l)

4 -

~ 1.5 -

0.172 M F------1 ------±------•

W CT T T+CT CT-T RIJC Treatment Figure 6.5: Electrophoretic mobility (UE) for various enzyme modified RBC's measured at two different ionic strengths 0.172 (n=3 : = SD) and 0.0043 M (n=5 : = SD). Error bars represent SD's in results. For simplicity CV at 0.172 M averages to 9.3= 1.2% and at 0.0043 M to 15.6=3.1% except for CT+T at 0.172 M that was low at 2.6%. 183 1 W CT T T+CT CT+T 0.172 M 0.78 ±0.08’ 0.70 ± 0.07’* 0.68 ± 0.06*" 0.64 ± 0.02’* (n=3) 0.93 ±0.07 (16.2%) (25.3%) (27.6%) (31.0%) 0.0043 M 3.01 ±0.35* 2.60 ± 0.35*" 2.47 ±0.46’* 2.45 ±0.53’* (n=5) 3.32 ±0.42 (9.3%) (21.8%) (25.6%) (26.1%) Table 6.6: UE data of RBC's before and after enzyme treatments at two ionic strengths (7) 0.172 and 0.0043 M. Values correspond to average of 3 or 5 subjects (±SD). Also shown is the % decrease in UE for the various RBC treatments compared to untreated RBC's. *: significant difference from control,* : significant difference from CT. (paired t-test)

6.1.3.2 T) CHANGES IN NON-AGGREGATING RBC’s

It seemed probable that the reduction in surface charge would modify the interactions between the different RBC's, even in non-aggregating RBCSusp's, and this should be seen in the shear thinning properties of the RBCSujp's. Hence, GA, GD, °‘277 T]r and 128,5r)r of non­ aggregating RBC's were investigated before and after T, CT and cocktail treatment and averaged t) ^ (n=4) is shown in figure 6.6. Significant differences between the cocktails and the control (W) RBCSusp's were found with 0-277 T)r. GD was found to increase from control (W), but significant differences were only found with the cocktails.. 128 5,nP again showed no significant change. Results are summarised in table 6.7. The main point of interest here was with GD that gave some indication of the RBC-RBC interactions; ie GD increased as surface charge decreased, and thus RBC interactions increased. Hence, the initial thought that GD may act as an index of deformation is no longer tenable as it is complicated by these cell interactions.

W CT T T+CT CT+T “" t). 7.092±0.455 7.523±1.113 8.149±2.383 8.763±1.559* 8.977±0.654* 128.5—. U r 4.833±0.130 4.858±0.242 4.838±0.329 4.883±0.183 4.992±0.212 g a -0.0053 -0.0007 -0.0163± -0.0190 -0.0280 (3 data points) ±0.0192 ±0.0185 ±0.0360* ±0.0300* ±0.0191* g d -0.134 -0.147 -0.153 -0.169 -0.170 (5 data points) ±0.010 ±0.021 ±0.043 ±0.022* ±0.015* Table 6.7: Enzyme treated RBC's with no aggregating agent present (n=4: ±SD). Results demonstrate how enzyme treatment alters the shear thinning properties of the RBC. significant from W (paired t-test) 184 Figure 6 . 6 : Loge-log* plot of 0 2 7 7 r|r against y for nonnal and enzyme modified RBC’s without any aggregating agent present.

i

Figure 6.7: Plot of o : 7 7 r)r against various enzyme treatments of RBC's with either 6g/l fibrinogen (n=4 : zSD) or 10g/l fibrinogen (n=2 : *SD) present to induce aggregation.- Also shown is the dextran data from figure 6.2 to demonstrate different responses between the two aggregating agents.

185 6.2 USING ENZYMES AS A TOOL FOR INVESTIGATING THE ACTIONS OF AGGREGATING AGENTS ON RBC's - A COMPARISON OF DEXTRAN AND FIBRINOGEN

Previously dextran has been used as a model for fibrinogen as an aggregating agent, because it is cheaper and easier to work with, yet this seems dubious as the two aggregating agents are structurally very different (eg fibrinogen in negatively charged and dextran is neutral). This was investigated here by comparing the aggregating tendencies of fibrinogen and dextran on various enzyme treated RBC's.

W CT T T+CT CT+T 15g/l 30.39+3.02 46.86+3.75* 77.59±15.f* Dextran 70 (n=8) 4.95+0.36 4.80+0.22 4.91±0.23 10g/l 7.31+0.62 9.66+0.87* 27.53±4.71’+ 35.82+5.75**x 36.19+7.81*^ Dextran 40 (n=7) 4.83+0.25 4.92+0.25 4.96±0.32 4.84+0.18 4.86+0.24 6g/l 26.21+6.57 26.41+4.65 32.35±7.27** 36.30+5.91*^ 38.12+8.98*"x Fibrinogen (n=4) 4.95+0.36 4.94+0.31 5.04±0.28 4.96+0.24 5.09+0.35

10g/l tfi-?3: <&.n SoJoj $f’£o b l'llf' S6-SO (,l-7f)X-93 Cl io Fibrinogen (n=2) S-/6 j f - ( S ; f-3Sj b - if M l ) Q T L Table 6.8 : a277 r)r and 128 5T|r data for the various RBCSusp's (n subjects : ± SD). *: significant from W ,+: significant from CT, x: significant from T. No difference was found for 128'5r)r between W and the other suspension. No significant difference was found between the cocktails, (paired t-test)

Initially 6g/l of fibrinogen was used because it produced the same level of aggregation (ie °-277 r|r) as that of 15g/l dextran' 70 on untreated RBC's. Results (table 6.8 and figure 6.7) show the actions of fibrinogen to be different from dextran in a number of ways: with fibrinogen no significant difference was found between CT and W RBCSuap's, the increases in T and cocktail treated RBC's was much less than found with dextran 70 and there were no significant differences between T and the cocktails, ie: (W = CT) < T = (T+CT = CT+T) 10g/l of fibrinogen was also used (n= 2) to attempt to exaggerate any differences, but this 186 showed exactly the same pattern as for 6g/l. Again no significant change was found in 1285r)r between the various treated RBCSusp's. These results are shown in table 6.8 .

TAe ejh * lata {J&iv b A Awith the above fmdincs, *" but showed sicnificant *" increases in both cocktails from T treated RBC's with 6g/l or 1 Og/1 fibrinogen. GD shows the same pattern as with the various non-aggregating RBC's. Both GA and GD are shown in table 6.9.

W CT T T + C T C T + T 15g/l -0.343*0.023 -0.441*0.048* -0.578*0.032** D extran 70 - - (n = 8) -0.187*0.024 -0.192*0.029 -0.180*0.023 10g/I -0.009*0.020 -0.056*0.040* -0.324*0.034** -0.395*0.045**x -0.396*0.052’** D extran 40 (n=7) -0.132*0.027 -0.177*0.018* -0.1S7±0.019* -0.205*0.015** -0.185*0.027*

6g/l -0.332*0.036 -0.321*0.043 -0.353*0.060** -0.387*0.048**x -0.387*0.051*** F ib rin o gen (n=4) -0.176*0.017 -0.181db0.018 -0.190*0.016 -0.203*0.029 -0.172*0.035 10g/l -OAU;-0’t7L -0 -0 A & ;-° ‘t e7 -0.4$7; -o-yor -0.49 2 ;-o -tfl F ib rin o gen (n=2) -0.137/-*77?. -0.1 (f3j~0'!73 -0.1 68/-0-/S7 -0.17/ ;-o-226 Table 6.9: GA and GD for the various blood suspensions for n subjects. *: significant from W ,significant from CT, x: significant from T. (paired t-test : *SD)

Comp(12)As>1 agreed with most of the ti^ as shown in table 6.10. However, there were some disagreements: Compa 2)As>1 was found to be higher for W+15g/l dextran 70 than for W+ 6g/l fibrinogen, whereas a 277 r|rand GA were approximately the same; also CoTnp( 12)ASvl for W+ 6g/l fibrinogen was lower than for CT+15g/l dextran 70 whereas 0277 r|rand GA, showed no significant difference. Comp( 12)As>.l for lOg/l fibrinogen did not show such a difference here. The reasons for these differences remains unclear.

187 W CT T T+CT CT+T 15g/l Dextran 70 10898 24086 43961 (n = 3) ±8305 ±8865* ±9346“ 10g/l Dextran 40 5792 7090 14657 16336 18625 (n=4) ±7681 ±7042 ±6996** ±6026“ ±3042“ x 6g/l Fibrinogen -1003$ £21 (»j t loO »; 79; (7602; (n=2) u+is £772.' l l t f l 1330 10g/l Fibrinogen 30866 27605 40240 43929 46760 (n=l) Table 6.10: Comp(12)ASyl data for the various RBCSusp's (n subjects : ±SD). Shows general agreement with ri^ shown in table 6.S and 6.9, except for W+ 6g/l fibrinogen that was not equivalent to 15g/l dextran 70. *: significant from W, *: significant from CT, x: significant from T. (paired t-test)

6.3 BRIEF ASSESSMENT OF THE EFFECTS OF OTHER ENZYMES (Th, B AND NA) ON THE AGGREGATING TENDENCIES OF NORMAL RBCS

Throwhiti (Th)

Th was briefly investigated because it is another serine protease with some functional similarities to trypsin, and also any effect on the RBC's may have important physiological implications as will be discussed later. Th action on RBC's was again assessed by measuring r)'s before and after incubating RBC's with Th (~90 U/ml Th /ml cells for 2 hours at 37°C), with 15g/l dextran 70 present. Density (age) fractionated RBCs were used, for reasons given in the conclusions. Th treatment appeared to cause no changes in aggregation for the different fractions of RBCs, for the two subjects looked at (table 6.11). Unfortunately, this work was not carried out on the MEA as this was still being assessed at the time.

188 . . . W+15g/I Dextran 70 Th+15g/l Dextran 70 Young Middle Old Young Middle Old

ZS-Hf J iv e s v

g a • o - J W fc - t 9 4 -O -} 7 0 / 0 -j 90 -O 'btfjO 'Xt “ O -v o Z j ~ o -3 *l

“ ’Hr I f O - j W L f > l f j (f U S i/; C-7? ir - W jW

GD •O -K'Jj-O lti - O 't o g j- o - io C -0-ns;~ o)% Table 6.11: Age separated RBC's before and after Th treatment with 15g/l dextran 70 present to induce aggregation in order to assess effects of Th on the different RBC's (n =2 :±SD).

Bromelain and Neuraminidase

Changes in aggregation were also briefly looked at for B and NA treated RBC's because the specificity of the former is similar to a combination of T and CT, and the latter removes most of the RBC sialic acid and therefore surface charge. As already mentioned in chapter 4, B and NA treated RBC's showed enormous tendencies towards aggregation and a 277 T|r, and Ga results (n=2) are shown in table 6.12. With either dextran 40 or dextran 70 present, Ga and °-277tir suggest that B treatment causes RBC's to aggregate more than T, and NA treatment causes RBC's to aggregate more than B; ie: T

189 T B NA “•27X GA GA °'277 Tlr g a 15g/l 77.59 -0.578 /(f-Z-SZj 173' xOj - 0 - 7 Olj Dextran 70 ±15.1 ±0.032 IU-77 - O d 6 / lo l-U

10g/l 27.53 -0.324 - o -SjJ/ iih j} - < 0 6 2 ^ Dextran 40 ±4.71 ±0.034 - o r e 6 - 0 6 7 2 6g/l 32.35 -0.353 -T O -7 7 ; “ O J C O . (kl'tSj -o -rry Fibrinogen ±7.27 ±0.060 cr-if - O -J 2 7 is-n ~o-j*og Table 6.12 : 0277 r)r and GA results for bromelain (B) or neuraminidase (NA) modified RBC's with either 15g/l dextran 70, 10g/l dextran 40 or 6g/l fibrinogen present to induce aggregation. (n=2 : ±SD). Also shown, for comparison, is T results from table 6.8 .

1285r|r and Gd results for T, B or NA treated RBC's are shown in table 6.13. Earlier it was found that no significance change was found in 128'5T)r between W, CT, T, T+CT or CT+T treated RBC's. The response of 128"V)r for NA treated RBC's, was variable and the numbers small (n=2), so it is uncertain if changes occur for this parameter. GD for B treated RBC's appears little different than for CT, T etc (table 6.2), but GD for NA treated RBC's appeared to be much greater than all other treated RBC's; possible reasons for this increase are the reduced surface charge allowing more cell-cell interactions and/or stiffening of the RBC's.

T B NA. 1385n r GD 12^ r GD ,28 5n r g d 15g/l 4.91 -0.180 «1/ M 7; -O -27 7y Dextran 70 ±0.23 ±0.023 ~Ol6ct - 0 - 2 7 4

10g/l 4.96 -0.187 “C ’IfOj r-% - O . U 4 ; Dextran 40 ±0.32 ±0.019 ic~n -O-HS -0270

6g/l 5.04 -0.190 1(4.0• 4*74; -Ollfj Fibrinogen ±0.28 ±0.016 4 <1 'O'M 4-*?o - 0 2 / 6 Table 6.13: 128V|r and GA results for bromelain (B) or neuraminidase (NA) modified RBC's with either 15g/l dextran 70, 10g/l dextran 40 or 6g/l fibrinogen present to induce aggregation. (n=2 : ±SD). Also shown, for comparison, are results from T treated RBC's taken from tables 6.8 and 6.9.

The MEA data for T, B or NA treated RBC's is shown in table 6.14. In agreement with ri^ it shows the increasing aggregation tendencies between B and T for both dextran and fibrinogen (dextran > fibrinogen) Also confirmed, is the small change in aggregation

190 Figure 6.8: Summary plots of (a) o:"nr and (1>) GA for all of the enzyme treatments investigated.

191 between NA and B treated RBC's induced by 6g/l fibrinogen. However, Comp(l 2)As>1 differs by not showing the increase in NA compared to B treated RBC's with either dextran 40 or dextran 70; Comp{ 12)As>1 forNA is slightly less than for B treated RBC's with dextran 70, and much less with dextran 40. To investigate if this was a problem of being the upper limit of Compfl2)As>1, as was found with Ktar 3)As>1 (§4.7), Conipf24)ASvl was also looked at, but showed the same pattern (table 6.14). The reason for the difference here is believed to be due to the stiffening of NA treated RBC's which would lead to slower aggregation. This stiffening is suggested above with GD, and also has been found by other researchers11541. This was an important finding regarding interpreting results from the MEA for a number of reasons, and will be discussed in more detail at the end of this chapter.

T B NA

Comp(12) a Comp(24) * Comp(12) a CompQ4) a Comp(12) a Comp(24) a A Syl ^ S y l A SyI A Syl A Syl A Syl /»*} *1-3 15g/l Dextran 70 43961 4493-3 syou) m i ; ±9346 ±11134 W o 6 tfSW Vtott rev /i-v 14657 13102 M l; 2S6J1; 3 W ; 10g/I Dextran 40 ±6996 ±11249 W 7 (063g/ larU ; 6g/l Fibrinogen 1/9?/ l(f]/3 3n& i m i o 3 W UK Table 6.14: Comp(12)ASv, and Comp( 24)ASvl results of NA or B modified RBC's with either 15g/l dextran 70, 10g/l dextran 40 or 6g/l fibrinogen present to induce aggregation. (n=2 : ±SD). Also shown is^omp( 12)ASy, for T treated RBC's taken from table 6.10, and Conip( 24)As>1 shown here for completeness.

6.4 CORRELATION OF UE AND tid ,u FOR THE VARIOUS RBCSusp'S

The main reason for briefly looking at B and NA, was to increase the range of RBC surface charge over which aggregation was measured, thus obtaining greater insight into the role of surface charge in aggregation. UE was not measured for B or NA modified RBC's, partly because of time, and partly because values obtained for T and CT treated RBC's by the author differed little to those of Seaman121153]. Therefore, Seamans' estimates that UE is reduced by -51% for B,153) treatment and -80% for NA 11531 treatment were used. Figure 6.9a and b shows a plot of °' 277 rir for the different RBCSusp's against UE at the ionic strengths 0.172 and 0.0043 M respectively. Figure 6.10a and b shows the same but for GA. For the dextran 40 and 70 RBCSusp's, as UE decreases aggregation continuously increases; for °': 77 r|r

192 Figure 6.9: Plot of o:7'nr against electrophoretic mobility (U e ) at ionic strengths (I) 0.172 M (a) and 0.0043 M (b). 027'Hr values used from table 6.8. and Ue values from authors measurements for W, CT, T, T+CT and CT+T treated RBC's (0.172 M - n=3:*SD; 0.0043 M - n=5:iSD) and from Seamans' measurements for B and NA treated RBC's. CV for Ue measurements are shown in figure 6.5. 193 IO 15g/l Dcxtran 70 0.9 T-KTI NA j A J Og/1 Dextran 40 CT+T CT w | I □ 6gfl Fibrinogen I I I 0.8 \ X I0g1 Fibrinogen

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 (a) Ut (pm.s'.V'.cm) 1=0.172 M

! O 15g1 Dextran 70 0.9 i A lOg^l Dexiran 40 T+CT CT+T CT W |D 6 g 1 Fibrinogen 0.8 !X lO gl Fibrinogen S i

Figure 6.10: Plot of GA against electrophoretic mobility (U e ) at ionic strengths (I) 0.172 M (a) and 0.0043 M (b). Ga values used from table 6.9. and UE values from authors measurements for W. CT. T, T+CT and CT+T treated RBC's (0.172 M - n=3:rSD: 0.0043 M - n=5:rSD) and from Seamans' measurements for B and NA treated RBC's. CV for UE measurements arc shown in figure 6.5.

194 the increase is linear, but for GA the increase plateaus for T, B or NA treated RBC's. For fibrinogen RBCSusp's, both 0 277 r|r and GA show the story is more interesting because the changes are smaller, and there are two regions where there appears to be no change at all; ie from W to CT treated RBC's, where surface charge changed by -16%, and from B to NA treated RBC's where surface charge changed by -30%. The lack of change in aggregation in these two regions could be explained in a number of ways, such as there being no change in adsorption sites, by fibrinogen charge interactions between the RBC surface and fibrinogen, etc.

6.5 DOES ALBUMIN INDUCE AGGREGATION OF T, B OR NA TREATED RBC's?

It is known that albumin does not induce aggregation of normal RBC's151,52], and this is thought to be due to its small size and globular structure. However, the author was interested to see if removing large amounts of the RBC surface charge would cause albumin to induce aggregation; ie an obvious extension of Chiens' CBH.

For T modified RBC's there was no indication that albumin induced any aggregation at all concentrations looked at (15-75g/l) (figure 6.11). The only change was the decrease in the transition intercept point as shown (§3.5). The MEA,Man(3)ASyl, produced confusing results here as shown in figure 6.12; these results suggest aggregation does occur. Klan( 0)As>1 remained zero during this time. To remove any doubt, samples were examined under the microscope and no aggregation was observed. Hence results from the MEA were, once again, in some way artifactual.

B and NA treated RBC's, with various concentrations of albumin present, were also looked at microscopically, but again there was no evidence of any aggregation.

195 Figure 6.12: ^'^Asyi for trypsin treated RBC's with 15. 25. 50 or 75g/l of albumin present (n=l : i SD of measurements).

196 6.6 CT AND T INCUBATION TIME COURSE STUDIES REVISITED!

With the development of improved methods of analysis for MEA and Viscometer data, from some of the results and from various aspects of the literature, the author felt it important to re-evaluate time incubation effects of the enzymes CT and T on RBC's. For this re- evaluation the protocol was modified in two ways: ( 1) incubation times were increased to 5Vz hours to see more clearly if the enzyme digestion was complete. ( 2) the level of aggregation^lowered by using 10g/l dextran 40, which made rj measurements much easier at the high levels of aggregation found with T treated RBC's with 15g/l dextran 70 present; ie T+15g/l dextran 70.

For the two subjects looked at with the modified protocol, suggested that T W perfbrwzch Much of-ffo vjqc {< wilhi/) bo uc) huh con.hnuec1 MCrOMt U/\hl }xr (figure 67 ^ £°r ^ S0(n1 should be used, and was seen to give the same results is shown in figure 6.14b.

(Xfpeirt j /WAflr 3Ofii/nak( ufu'k The above results showed that T effects complete a fte r 1 hour ^ CT effects (to-Jo*i2 vdux. vi wHi T M Ih/i krPi ■ o\ atfKcjf cplx>SA h o u r s Hence, CT was further investigated (n=2) over the same time period, but because it apparently caused RBC's to aggregate much less than for T, 15g/l dextran 70 was used once again. Also, two CT concentrations were used to help establish the maximum effect of CT; ie 5mg/ml as above, and 15mg/ml. Figure 6.15a shows a plot of °' 277 r)r against CT incubation time, and in the absence of CT there is no large change, and also suggests 5mg/ml and 15mg/ml CT appeared to still be having effects after SVz hours. Yet figure 6.15b shows that GA suggests that effects of 15mg/ml CT had finished after 3]/z hours and 5mg/ml was still rising. °’ 277 r|r /128 5r|ris shown in figure 6.15c to see how this described the data, and was similar to a 277 r)r.

Figure 6.16 shows that Comp(12'ASy, generally agrees with GA, but after SVz hours shows a drop; exactly the same pattern was found with Nbn( 0)As>1 and M3n(3)ASV| (data not shown). The drop is believed to be caused by the stiffening of the RBC's, which would act to slow the 197 aggregation process down, as seen with the MEA data, but not necessarily change the final level of aggregation, as seen with the T)^. Gd and , 28'5Tir both increase, which appear to support this finding as shown in figure 6.17a and b respectively.

Finally, a control (Omg/ml CT) was carried out to assess how much RBC's changed due to the incubations at 37°C. The results are shown in figures 6.15 and 6.16 for the various parameters, and again suggest a stiffening of the RBC's. An interesting observation with this data is that untreated RBC's appears to stiffen more than for CT treated RBC's.

The overall impression was that 15mg/ml CT had reaches optimal effect after 314 hours and 5mg/ml CT does not reach optimal effect in the 514 hours looked at. It should be noted that because of the late reassessment of CT effects, CT results shown above were obtained with 5mg/ml CT for ~2 hours incubation times; ie CT action was not exhausted.

198 Figure 6.13: Effects of different incubation times of chymotrypsin (CT) or trypsin (T) on the RBC's from two subjects. Changes assessed by viscosity measurements, (a) 0 277 r|r and (b) GA, of RBC's with 10g/l dextran 40 present.

199 Figure 6.14: MEA data to compare with Hdau presented in figure 6.2. (a) Comp<12)Asyi results are odd probably because of the "crenation problem" (b) Comp{0)As)i shows general aggreement with ridau as show'n in figure 6.13.

2 0 0 0 -Omg/ml CT-M ■ * O- - • Omg/ml CT-F . X ------A- — 5mg/ml C T-M • * O- - • 5mg/ml CT-F ---- K—— 15mg/ml CT-M •• X : - • 15mg/ml CT-F

100 150 200 250 350 Chymotrypsin Incubation Time (minutes)

0.6 — e— “ Omg/ml CT-M - - Q - Omg/ml CT-F -----A- - 5mg/ml CT-M - - O- - • 5mg/ml CT-F -----H— 15mg/ml CT-M - - *- - • 15mg/ml CT-F

- - Q 0.35 50 100 150 200 250 300 350 (b) Chymotrypsin Incubation Time (minutes)

20 ■■ -O— Omg/ml CT-M - • O- - • Omg'ml CT-F -----A — 5mg/ml CT-M - - O- - * 5mg'ml CT-F -----K— 15mg/ml CT-M 15mg/ml CT-F

100 150 200 250 300 350 Chymotrypsin Incubation Time (minutes) Figure 6.15: Chymotrypsin lime (0-330 min) and concentration (0? 5 and 15mg/ml) studies on RBC's from two subjects (M and F) with 15g/l dextran 70 present to induce aggreation. Plots of (a) 0 27 ; r|r (b)G- nnd(c) 027V‘85n. 201 Figure 6.16: ^"^'Asyi for clivmotrypsin incubation time and concentration studies for RBC's from two subjects (M and F). Gives similar response to GA in figure 6.15b.

202 Figure 6.17: Plots of (a) GD and (b) i:s 5r|r against time and concentration studies of chymotiypsin modification of RBC's from one subject (M). Another subject (F) showed the same pattern.

203 CONCLUSIONS AND DISCUSSION

TIM E INCUBATIONS

The pilot study carried out by Ertan, mentioned above, showed changes with time in °-277

These lower values are probably a consequence o f inadequate mixing o f the sample before a measurement at each y, and by using points in the plateau and transition regions in the analysis (§3.4.3). This is an example o f why more rigorous guidelines are needed for using the Viscometer and analyzing data, especially when aggregation is particularly high.

It was found that for enzyme concentrations o f 5mg/ml RBC's, T action was complete after

1 hour but CT action was incomplete after 5Vi hours. C T was further investigated by looking at effects o f higher concentrations o f CT; ie 15mg/ml. Here °'277T|r implied enzyme action was not complete after 5 V 2 hours, but GA and Cornp02)ASyl implied that enzyme action was complete after 314. The discrepancy here is believed to come from the stiffening o f the

RBC's at these higher incubation times as was shown with the control (Omg/ml). Further investigations are needed for time course studies o f the action o f C T on the RBC's.

Hence, the CT work presented here is from RBC's that have only partly been degraded with

CT, but this has some advantages; ie by only removing -1 6 % o f the surface charge o f the

RBC, fibrinogen showed no significant increases in aggregation. However, a study o f the aggregating tendencies o f completely CT modified RBC's, induced by dextran or fibrinogen, could be very useful.

Before moving on, it is worth highlighting a failing here, that appears common in the literature, which was to consider the concentration o f the enzyme used as mg/ml. This bears little relation to enzyme activity, and it is more useful to consider the enzyme activity U/mg used, ie activity from 5mg/ml T was much less than activity from 5mg/ml CT. I f time course studies are carried out, such that the point in time when optimal effect o f the enzyme is reached, then this gives a good estimate o f the concentration o f substrate removed.

However, this is complicated by different enzymes having different efficiencies under

204 different conditions (eg different temperatures, pH's, etc).

imTSTJGA TING THE MECHANISMS OF AGGREGA TJON

The initial study was concerned with dextran and showed that aggregation increased with

the different enzyme treatments as follows: W < C T < T < (T + C T = C T + T ). It was found that

the cellular factors size and deformability did not change significantly for these treated

RBC's and so played little part in the large increases in aggregation found. It seems likely

that increases in GD were caused by increased cell-cell interactions due to the decreased

surface charge. Hence, even though GD may have a component o f deformability, it is

complicated by these interactions.

Changes in U E, surface charge, for C T and T modified RBC's are well known in work by

Seaman, as already mentioned, and were also found by the author. N o significant changes

between RBC's modified with the two cocktails, and between the cocktails and T. A However, the Malvern Zetasizer was not consistent enough with measurements o f RBC's,

as seen above with the large error bars (figure 6.5), and in work by other researchers[154].

0,"77T|r showed dextran-induced-aggregation linearly increased with decreasing surface

charge. However, GA again gave a different response by showing plateauing at the higher

levels o f aggregation (T onwards). A possible reason for this is that the RBC's are

approaching a point o f maximum aggregation, and there are two supporting reasons for this

thought. Firstly, the difference between the aggregating tendencies of dextran 40 and

dextran 70 on N A treated RBC's is much less than for B, T, C T etc treiated RBC's. The

other reason is that GA for the RBCSuspNA + l 5g/l dextran 70 was the same as the maximum

found with high concentrations o f dextran 70 and dextran 110 on C T and T treated RBC's

(§3-5).

Understanding 'dextran-induced aggregation' itself is important, to some degree, because

it offers insight into the basic physiochemistry o f cell-cell interactions, and to a lesser extent

because o f its clinical role as a volume expander. However, these are not as important as

further understanding the action o f fibrinogen, and many researchers have made the mistake

o f using dextran, an uncharged polyglucose, as a model for fibrinogen, a negatively charged

protein; the above findings clearly show this to be unacceptable. The influence o f surface

charge towards aggregation is very complicated, and the fibrinogen-RBC charge interactions

205 certainly complicate the issue as already discussed. Another difference between dextran and fibrinogen, was that the upper limit o f aggregation for fibrinogen was much less than for dextran 70 or dextran 40. It is possible that this represents maximum adsorption, and thus aggregation, o f fibrinogen; ie below 50% of normal surface charge, any dominating exclusion of fibrinogen from the RBC surface is removed. There are numerous possibilities at this stage, and as always further investigations are needed.

Having said that aggregation o f NA treated RBC's is much greater than for B or any o f the other R B C Susp's, the M E A data gave a response that suggested the N A modified RBC's aggregate less than for B modified RBC's. The different response o f the M E A data could be caused by N A stiffening the RBC membrane, that would act to slow down the aggregation process, giving a slower syllectogram aggregation phase and thus a lower comP(i2and24)As^; even though the aggregation was slower the equilibrium measurement o f aggregation, ie °‘277r)r or GA, is not necessarily affected by cells being stiffer. The belief that

N A has caused the RBC's to stiffen is supported by work by other researchers11541, and the belief that stiffer RBC's have a slower aggregation process is supported again by work o f other researchers, involving heat treated RBC's which are stiffer and aggregate slower156*581.

O f particular relevance here is some work carried out by Bohler and Linderkamp11311, where they found the manual M E A gave lower values for N A than T treated RBC's washed and resuspended in autologous plasma; observing the syllectograms also implied this. They used a Rheoscope to show the reverse o f this was true. They explained their findings by the

600s*1 HSR dispersion phase not being adequate. Schmid-Schonbein11551 commented on this work and acknowledge the HSR phase may not totally disperse RBC's in hyperaggregating systems, but stated how complicated the whole process was (ie involving thixotropy, yield stress etc), and that a number o f things may lead to these differences. However, he did not mention the complications involved in the stiffening o f RBC's, and this would explain the findings probably better than the failure of the HSR dispersion phase. This highlights another aspect o f the M E A system developed in chapter 4; ie the M E A gives measures o f aggregation, but because it was developed to obtain results over short times, this is dependent on the velocity o f aggregation. Hence, anything that affects the change in aggregation speed (eg stiffening) causes the M E A to give low readings. I f what is required is a measure o f gross aggregation, this would be a false low reading. The fast decay- constant (db) was not included here for simplicity, but it is certainly an important parameter

206 that should not be ignored especially in view o f the M E A response to N A treated RBC's.

An ideal analysis system for the M E A , which with further research is possible, would be a system that provides both gross measures o f aggregation and measures o f velocity of aggregation. This may possibly be achieved by collecting M E A data over longer periods of time; ie 60s as opposed to 10s. However, there are problems that need to be considered, associated with sampling over longer periods o f time, such as settling etc as described in

§4.3.

GGREGA TION OR ROIJLEA lIX FORMA TJOW

In the past, generally the aggregate morphology has been largely ignored. This may be an important issue to consider for a number of reasons; eg the process involved in different forms o f aggregate may not be the same, different aggregating agents may act to form different types o f aggregate, etc. Above, RF was observed in all cases except for B and N A with 15g/l dextran 70 where clumping was observed. This may be relevant and could even explain why fibrinogen plateaus at a relatively low level o f aggregation; ie no clumping was observed with fibrinogen-induced-aggregation o f NA or B treated RBC's, which may mean that fibrinogen is unable, at 6g/l at least, to induce clumping. Somewhere after T, and the cocktail, treatment yet before B treatment, clumping started to occur and could the reason why aggregation continued to rise with dextran.

ACTION OF THE VARIOUS ENZYMES ON THE RRC SURFACE

The specificities o f the enzymes used above are well known:

• T cleaves the hydrophilic side chains lysine and arginine.

• C T cleaves the hydrophobic, aromatic bulky side chains phenylalanine, tyrosine etc

• B cleaves lysine, arginine, phenylalanine, tyrosine etc (ie combination o f T and CT).

(the actions o f this enzyme appears to be less well defined).

• N A cleaves the sialic acids.

• Th cleaves only arginine-glycine combinations.

Furthermore, what some o f these enzymes (T, CT and N A ) remove from the RBC internal and external surface has been extensively investigated115'211. However, only what is removed

207 from the external surface o f the RBC is o f importance here

CT has been found to act on glycophorin A dimer (88kD) and glycophorin A B heterodimer

(65kD), reducing them to -6 8 and ~47kD respectively. CT also acts on band 3 (88-98kD ) reducing it to ~62kD. T has been found to act on glycophorin A dimer (88kD ) reducing it to~40kD. It also removes both glycophorin C monomer and dimer1201. Some workers have found that T has no affect externally on band 3 on intact RBC's116’20], whilst others have found small changes118,19). What is further interesting here is cocktail studies o f the two.

This showed that T and CT both act on the same glycophorin substrate but at different loci

(T lower than CT). Hence if T was used after CT (CT+T) a further reduction in the remaining glycophorin substrate occurred, and conversely (T + C T ) induced no further change in the remaining glycophorin substrate, but band 3 would be reduced.

N A is known to remove most of the sialic acids from the RBC surface123,153]; what T and CT remove o f the sialic acids is only part o f what N A removes. Finally, what B removes from the RBC surface has never, to the authors' knowledge, been investigated, but is likely to be more vigorous that either C T or T.

The investigation o f Th was another enzyme of interest because it is serine protease, it cleaves arginine as T does and there are conceivably physiological benefits to its modifying

RBC's; ie Th is formed when blood loss occurs (§1.2.2), and it would be advantageous for

RBC's to aggregate more at such times to help plug the damaged vessel faster. Density

(age) fractionated RBC's were used here because o f the possibility that thrombin may affect certain populations o f RBC's, particularly young RBC's; ie it was thought that Th treatment may act to cause RBC's to aggregate more; ie age the RBC. However, there was little evidence above for such changes.

208 FINAL COMMENTS

There has been an enormous amount o f work investigating different aspects o f enzyme , heist earlier H\if tUykr modified RBC's. Some of this work, o f most relevance here, has recentlyA been reviewed in eu'e'/|** a paper by Rampling and Pearson11561.

The above information is useful and helps understand more about what might lead to the

increase in aggregation observed with the modified RBC's. The above work has not really

succeeded supporting one hypothesis above the other. Certainly the dextran data, increasing

aggregation with decreasing surface charge, can not differentiate the mechanism of

aggregation. However, there appears more possibilities with the fibrinogen data. For

instance, by the non-adsorbing D L H why was no change in aggregation found between W

and CT and B and N A treated RBC's.

209 CONTENTS OF CHAPTER 7:

A METHOD FOR DIRECTLY INVESTIGATING THE MECHANISMS

OF ROULEAUX FORMATIONS.

A IM 211

METHODS:

7.1 PR EPA R IN G SO LU TIO N S 211

7.1.1 FLUORESCENT FIBRINOGEN - (4)^) 211

7.1.2 RADIOACTIVE FIBRINOGEN - (I12S) 213

7.1.3 FIXATIVES 213

7.2 TECHNIQUES AND EQUIPMENT USED 214

7.2.1 THE TEST TUBE METHOD OF FIXATION 214

7.2.2 THE MICROSCOPE SLIDE METHOD OF FIXATION 214

7.3 ASSESSING ABSORPTION TO THE RBC SURFACE 215

7.3.1 MICROSCOPIC OBSERVATIONS 215

7.3.2 BETA COUNTER 217

RESULTS:

7.4 THE TEST TUBE METHOD AND (f)n:5 217

7.5 FLUORESCENT STUDIES 220

7.5.1 LABELLING EFFECTS ON FIBRINOGEN 220

7.5.2 FIXATIVES AND AUTOFLUORESCENCE 222

7.5.3 THE TEST TUBE METHOD AND 222

7.5.4 THE MICROSCOPE SLIDE METHOD AND 222

7.6 DOES THE DISTANCE BETWEEN RBC's IN ROULEAUX INCREASE

WITH DEXTRAN SIZE ? 225

CONCLUSIONS & DISCUSSION 226

210 FOREWORD

Previously there has been much work carried out to investigate adsorption of proteins to the RBC surface. These studies are either based on how much protein remains in supernatant or is associated with RBC’s after centrifugation. Janzen et all82J has reviewed much of the work associated with these methods, and concluded both methods had problems associated with them as was highlighted by the variable results.

One of the biggest problems with previous work is that because methods of measurement were not direct it was never certain what was being measured. It was hoped that by using fluorescent fibrinogen, direct observations would give clearer insight as to whether fibrinogen adsorbs to the RBC surface. The radioactive fibrinogen work presented here was mainly to assess the best method of fixation, not to act as a method of quantifying adsorbed protein. CHAPTER 7: A METHOD FOR DIRECTLY INVESTIGATING THE

MECHANISMS OF ROULEAUX FORMATION.

AIM

The aim o f the work carried out in this chapter was again to investigate the mechanisms behind rouleaux formation (RF), or more generally aggregation, but by using specially developed methods that allows more direct investigations o f the process o f aggregation.

The main approach was to investigate the interactions o f radioactively or fluorescently labelled fibrinogen with the RBC surface. Radioactive measurements were restricted by not being able to directly observe the fibrinogen etc, but was a very sensitive and quantitative approach. The fluorescent fibrinogen studies had the potential of directly showing how fibrinogen induces RF, but was restricted by optics, etc. Furthermore, one o f the developed methods was manipulated to attempt to assess whether the distance between RBC's o f a rouleaux, increased with increasing dextran size (and thus length) as the C B H would suggest. All o f this work was to offer fresh insight into the mechanisms behind RF and to help sort out the current confusion with the hypotheses of this mechanism; ie the C B H and

D L H as explained in §1.9.1. Note: printed images for this chapter are in the sleeve o f the back cover.

METHODS:

7.1 PREPARING SOLUTIONS

7.1.1 FLUORESCENT FIBRINOGEN - (

This study used fluorescently labelled fibrinogen.

Human fibrinogen (Chromogenix), had to be prepared slightly differently from that described in §2.1.4: 0.35g o f fibrinogen was dissolved in 11.25ml o f carbonate- bicarbonate buffer (CBB) - 56.6 mM Na2C03 (anhydrous) and 345.8 mM Na:H C 03

(pH 9.0). The labelling process (conjugation) is most efficient at this pH. This was left undisturbed to dissolve over a heat bath o f 37°C and then dialysed against CBB overnight.

211 The main fluorescent dye used was sulphorhodamine B derived from the commercial lissamine rhodamine B (Sigma). This was the cheap option, but first had to be purified from its major contaminant, dextran, because it interferes with the labelling (conjugation) process as described by N aim ,157J. Purification involved dissolving the lissamine rhodamine B in

70% ethanol, and then filtering. The impurities are insoluble, and so are kept on the filter, while the pure rhodamine (inactive form) passes through. The pure rhodaminesolution was dried on a glass sheet overnight, collected and ground to a fine powder with a mortar and pestle. Then, in a fume cupboard, because of unpleasant fumes, lg PC15 was added per 2.5g ground rhodamine (5 times more than Naim used to minimise diluting fibrinogen) and was ground with a mortar and pestle, gently at first and then more vigorously, until a thoroughly mixed paste w-as formed. 5ml acetone was added and stirred for two minutes, and the solution then filtered. This final solution was the active form o f rhodamine, sulphonyl chloride, and w-as stored in a stoppered, dark, container at -20°C and used within six months. The optical absorption o f a 1:100 dilution o f rhodamine in acetone, followed by a further 1:100 dilution in PBS was measured at 565nm on a spectrophotometer, and using, the molar extinction factor 73*1 0 3 the concentration o f dye wras obtained (~120mg/ml).

Sulphorhodamine B was used for the majority of the work because o f its cheapness, its good photostability and efficient quantum yield.

This active form o f rhodamine was added slowly to the stirring fibrinogen CBB solution

(~30g/l) at ~0°C in such a concentration as to obtain a fluorochrome to protein ratio o f 3:1

(ie a conjugation o f ~3) and left stirring for one hour. A variable amount o f precipitation was produced during conjugation, but was always negligible compared to the total fibrinogen concentration. The labelling fibrinogen solution was passed through a G25 column (Sephadex, Pharmacia) with PBS to obtain labelled fibrinogen free o f unbound dye

(^Rh-Lab) in a PBS solution (pH 7.4). Sometimes, depending on availability, this column was attached to standard Chromotography equipment LKB213S Uvicord S/LKB2210

Potentiometric Recorder and LKB2112 RediRac (LKB, Produkter AB, Box 305, A-161 26

Bromma, Sweden).

The [R],.Lab] could not be measured using the thrombin clotting technique described above for normal fibrinogen (§2.1.4), because the dye would change the absorption at 2S0nm and thus drastically affect the result giving a'false high reading. Estimating [4 )^ .^ ] from

212 changes in volume after passing it through the column proved to be very inaccurate. The most satisfactory method used to measure [4>Rh.l-ab], was to use the M E A in manual mode

(§2.7.2). The basic principle here was to adjust the concentration o f unlabelled fibrinogen

(4>Unlab) to produce the same degree o f RBC aggregation as that o f 4>Rh.L»b- This had the added advantage of showing the fibrinogen was still functional, as regards to forming rouleaux, and also o f a sufficient concentration.

The problem of passing (tw^b through the column, was that it became considerably diluted and a small amount was lost by sticking to the gel and the column wall. These losses, with the additional loss due to precipitation during the labelling procedure, caused [4>Rh-ub] to sometimes be less than required. For convenience, fibrinogen levels o f the order o f 6 g/1 are desirable, and so the majority o f the time 4>Rh.L*b was concentrated up using polyethylene glycol (PEG) 6000 obtained from B D H (B D H Chemicals,Ltd, Poole, U K ), 4>Rh-ub solution was put in dialysis tubing and covered with PEG. The PEG had a tendency to draw the PBS from inside the tubing leaving the 4>Rh-Lab inside, which thus becames more concentrated.

7.1.2 RADIOACTIVE FIBRINOGEN (125I) - 4>I125

Fibrinogen 125I (4>n2sX recently time expired for in vivo use, was obtained from the

Thrombosis Research Unit, King's College Hospital, London. UK. It was used in various volumes (concentration unknown1) and it was assumed that free iodine levels would be very low because o f its clinical role. Dialysis was not carried out since free iodine was considered at the time to be negligible.

7.1.3 FIXATIVES

Three fixatives were used in the work contained in this chapter as follows:

1. EM Grade 25%-glutaraldehyde (CHO.(CH2)3CHO) was obtained from Agar

Scientific Ltd (66a Cambridge Road, Stanstead, Essex. U K ) and stored at 4°C. This

was diluted in PBS to various concentrations (2.5% , ]% , 0.5% , 0.1%).

’The concentration o f 4>n25 really was not important, as this was only used to assess the method o f fixation. 2. Paraformaldehyde ( (H C H O )2 ), obtained from Agar Scientific Ltd, was prepared

by adding 20g to 200ml PBS (10%), and heating to 60°C stirring continuously until

dissolved. Drops of 1M NaOH were added until the milky solution cleared. It was

left to cool in the fridge and used within three days11581. This was diluted to 1% with

PBS and 0.5% for use in the experiments.

3. Formaldehyde (H C H O ) was prepared by diluting 40% formalin, obtained from

BDH, down to 10% with a 0.1M NaH2P04 + 0.1M Na2HP 04 buffer solution11581.

This was further diluted to 1% and 0.1% with PBS.

7.2 TECHNIQUES AND EQUIPMENT USED

7.2.1 THE TEST TUBE METHOD OF FIXATION

In a test tube, various concentrations of (J)1125 or 4 )^ .^ were added to RBC's and then either formaldehyde (F), paraformaldehyde (P) or glutaraldehyde (G) fixatives were mixed in.

After leaving the RBC's to fix for various amount of time, they were washed three times

(figure 7.1a). The control for this was the same protocol, but without any fixative being added. This method was referred to as the Test Tube M ethod (T T M ) and measurements using this method are described in §7.3.

7.2.2 THE MICROSCOPE SLIDE METHOD OF FIXATION

A big deciding factor that the T T M really was not adequate was that fixed rouleaux were never found. This observation, and the belief that the interaction between RBC's is extremely weak and certainly disrupted when the fixatives are added and mixed in, led the author to devise a method more suitable for capturing the weak interactions between RBC's.

Such a method had to be one in which the RBC's were disrupted as little as possible during the fixation process and the following was devised.

214 10|il packed washed RBC's (either normal or typsinised RBC's) were mixed on a microscope slide2 with either lOpl o f or c))Un]ab or one o f the dextran stock solutions, and spread out to approximately form a 2cm square. RBC's were left to aggregate for 2 minutes, and then one drop of 1% glutaraldehyde was carefully added to each comer and allowed to diffuse through the sample for 5 minutes, fixing the RBC's in whatever state they were in (ie. rouleaux, RBC's in point contact, free RBC's etc). The fixed sample was then washed o ff the slide into 10ml o f PBS, and used to make slides for microscopic observations; ie 10pl sample was pipetted onto a slide, covered with a cover slip, sealed with clear nail varnish and left to dry for a few minutes. This is demonstrated in figure 7. lb and is referred to as the Microscope Slide Method (MSM).

7.3 ASSESSING ABSORPTION TO THE RBC SURFACE

7.3.1 MICROSCOPIC OBSERVATIONS

RBC-dextran fixed suspensions were observed under a normal light microscope (xlO) simply to assess the degree o f aggregation fixed by the M S M . For the fluorescent microscopy studies, a Leitz Aristoplan fluorescent microscope was used with a xlOO fluohar

(N A 1.3) immersion oil based objective for its improved definition. Also it allowed shear forces to be applied on the RBC's by slight continuous adjustment o f the focus mechanism.

Fluorescence was stimulated with a 50W mercury lamp and the appropriate excitatory filter for the fluorescent label being used. Attached to the microscope was an image-intensified

CCD camera (Photonic Science Ltd, Roberstbridge, East Sussex, T V 32 5LO. U K ), with a further xlO magnification, which in turn was connected to an IB M compatible computer, where 24bit frame grabbing software (Neotech Image Grabber, Neotech Ltd, Eastleigh,

Hants. S053 2UG. U K ) captured and stored images in TA R G A (.T G A ) graphics format for later analysis (figure 7.2). All o f the fluorescent work presented here is qualitative, no quantitation was possible partly because o f time, and partly because o f the difficulty in quantitating the features found.

in itially silicon coated microscope slides were used to avoid any artifacts caused by glass interactions, but later normal microscope slides were used for convenience as no difference was observed in the results.

215 \ -A ; ;! ▼ 777 i y RBC'S + F1XATOE ADDED WASHED C U ~ 3 » u . (PBS) (a ) THE TEST TUBE METHOD (TTM)

(10ml PBS)

(b ) THE MICROSCOPE SLIDE METHOD (MSM) Figure 7.1: Schematic representations of two methods for preparing RBC's for investigating RBC-fibrinogen interactions: (a) the test tube method (TTM) and (b) the microscope slide method (MSM).

\

Figure 7.2: Schematic representations of the imaging and data capture equipment used for investigating the fluorescent fibrinogen-RBC interactions.

216 7.3.2 BETA COUNTER

For samples prepared by the T T M , radioactive counts o f RBC's, with any adsorbed fibrinogen, and their associated control were made on a beta counter (Count Ratemeter

M S 3 10, J & P Engineering) before and after several washes. The measure o f how much c})n:s was adsorbed to the RBC surface was assessed by the difference in counts between the test tube+RBC's+4)n25 after 3 washes and the empty tube (ie after RBC's+chj^s removed).

This is also presented as a percentage o f initial count.

RESULTS:

7.4 THE TEST TUBE METHOD AND 4>,,25

The T T M method was chosen because it originally looked to be the best method available.

Although a small amount o f free iodine was probably present, this was believed not to be responsible for the findings, and nonetheless some insight can still be gained, such as the effectiveness o f the method for different types o f fixative at different concentrations and incubation times, etc.

Paraformaldehyde (0.5% and 1%) and (0.1% and 1%) formaldehyde (fixation times 1 min -

IV2 hr) failed to significantly fix any adsorbed 4)1125 to the surface o f the RBC (figure 7.3 and table 7.1). 4% formaldehyde appeared to fix a little fibrinogen to the surface o f the RBC

(figure 7.4 and 7.5b and table 7.1). However, both paraformaldehyde and formaldehyde caused the RBC's to severely deform as observed under the microscope.

Glutaraldehyde (0.1%, 0.5%, 1% and 2.5% for between 1 min - 1% hr) turned the RBC's brown, but otherwise left the RBC's looking normal (healthy). Glutaraldehyde appeared to succeed in fixing <})I125 to the surface of the RBC (figure 7.4 and table 7.1). Also glutaraldehyde concentration and fixation time variation were carried out as shown in figure

7.5a and 7.5b respectively. Lower concentrations of glutaraldehyde (0.1% and 0.5% ) failed to produce much

217 Calculated by subtracting the radioactive count (after three washes) of RBC's+u 25+fixative from RBC's+fes (ie with no fixative added). (n=l)

blood. Fixation for 1 hour. The measure of adsorption was calculated as for figure 7.3. (n=l)

2 1 8 Figure 7.5: (a) Time course study of u 25 fixation to the RBC surface with 1% glutaraldehyde (G) when 10p.l 4)n25 was added to whole blood (n=l). (b) Concentration course study of fixation to the RBC surface with various concentrations of formaldehyde (F) or G when 20pi (foizs is added to washed RBC's; F fixed for 60 minutes and G for 30 minutes (n=l). fJdforbtJ 4^lU- osf*<

2 1 9 FIXED TO RBC CONTROL % of initial count % o f in itial count

F(.l% )-20nl <(>„« 1.22 0.84 (Washed RBC's) (n-D (n-l) F (l% )-20|il m5 3.58 2.81 (Washed RBC’s) (n=l) (n=l)

F (4 % )-5 MI 4>m.« 0.74±0.18 0.97±0.91 (Whole Blood) (n=6) (n-3) F (4 % )-1 0 fil (f.1155 0.48±0.11 1.21±1.02 (Whole Blood) (n-6) (n-4) P (.5 % )-1 0 MI 4>ms 1.61 1.91 (Whole Blood) (n-1) (n-l) P (l% )-10,tl „25 1.28 1.18 (Whole Blood) (n=l) (n=l) G (.l% )-2 0 fil 4>m5 1.10 0.53 (Washed RBC's) (n=l) (n=l) G (.5% )-20fil I12J 2.39 0.76 (Washed RBC’s) (n=l) (n-l) G(1.0% )-20nl 4>nJ5 7.09 1.31 (Washed RBC’s) * (n=l) (n-l) G(2.5% )-5jtl (}>„,< 6.44±1.60 0.63±0.32 (Whole Blood) (n=4) (n=3) G(2.5%)-10nl 6.32±1.52 0.85±0.62 OMiole Blood) (n=4) (n=4)

Table 7.1: Showing concentration of 4)1125 fixed to RBC's, calculated by dividing the difference between the test tube+RBCs+cJ),,^ after three washes and the test tubes after the RBC's+ 4)1125 are washed away, by the initial radioactive count. (mean±SD if a number o f suspensions were made up). This is shown for suspensions with and without (control) adding fixatives.

7.5 FLUORESCENT STUDIES

7.5.1 LABELLING EFFECTS OF FIBRINOGEN

It was important to make sure that there was no change in the structure o f fibrinogen during the labelling process. One fact supporting no structural change was that was always found to be thrombin clottable, and furthermore induced aggregation, indicating it was still functional. Another supporting fact came from passing and 4>Un]ab through chromatography equipment, in approximately the same concentration; the initial rise of absorption detected was approximately the same, indicating no large change in fibrinogen molecular weight (M W ) (figure 7.6).

220 Figure 7.6: The absolution curve produced by passing (a) adfocpht* (Yny/s) M&e&rts cd Me /a***t? />aC ^ b. 0 // puf’ Ctifu#jo. '7X*AXoid js> (h) if Jut /q, /5^

221 7.5.2 FIXATIVES AND AUTOFLUORESCENCE

Glutaraldehyde caused the RBC's to autofluoresce to varying degrees at the wavelength being used for observing the fluorescent fibrinogen (575nm) (picture set 7.1). To try to avoid this autofluorescence, two other wavelengths were looked at 595nm and 495nm, as used by the fluorescent dyes Texas Red and Fluorescein IsoThiCyanate (F IT C ) respectively.

However, this failed to improve the situation as RBC's also autofluoresced at these wavelengths.

Another approach was to look at various concentrations o f glutaraldehyde to try to reduce some o f the autofluorescence. However, little change was observed until below 0.5% glutaraldehyde concentration which did not induce much fixation as seen above with <})I125

(figure 7.5b); this was also observed with the M S M in that no rouleaux or fluorescent features were found.

A final approach was to look again at formaldehyde and paraformaldehyde even though they structurally changed the RBC's. However, these fixatives failed to produce any fluorescent features on the RBC's indicating again they were not suitable as suggested above

(figure 7.4).

7.5.3 THE TEST TUBE M ETHOD AND cf>Rh Llb

The T T M failed to produce any fluorescent observations of 4 ) ^ ^ fixed to the RBC surface, but there were problems caused by the autofluorescence that would hide any small

amount o f bound 4>Rh.ub-

7.5.4 THE MICROSCOPE SLIDE METHOD AND 4)Rh L>b

The M S M immediately provided what was required; with glutaraldehyde, RBC's were fixed at various stages o f the rouleaux forming process. Various combinations o f formaldehyde and paraformaldehyde were again tried with this method, but these failed to fix any rouleaux or give any fluorescent observations that glutaraldehyde gave.

222 In obtaining a protocol for this, the radioactive work presented above suggested 1% glutaraldehyde would be adequate. Initially here 0.1%, 0.5%, 1% and 2.5% glutaraldehyde were looked at for fixing times up to ten minutes. It was found that 0.1% and 0.5% failed to produce fixed rouleaux, and 2.5% appeared to produce more autofluorescence than 1%.

So the final protocol used was 1% glutaraldehyde for 5 minutes fixation time, as this appeared to work well and give the best balance between autofluorescence effects and time available for experiments. In all o f the observations that follow, the control (^u^b) showed the general autofluorescence but none o f fluorescent features. Also the fluorescent features observed on RBC's where Rh.L.b was present was much brighter than any autofluorescent features observed. The following observations were made:

■ Whenever RBC's were found in point contact with each other, there was strong

fluorescent emission at the point of contact as shown in picture set 7.2. These point

contacts were found to be very strong, and impossible to break when strong

mechanical forces were applied, through vigorously adjusting the microscope focus

mechanism.

■ Fluorescence was found between a distinct region of overlap between two RBC's

in the process o f forming or breaking a rouleau, as was apparent from their

deformed shapes. An example is shown in picture set 7.3.

■ Points of fluorescence were found on single RBC's (picture set 7.4a), on the sides

and ends o f rouleaux (picture set 7.4b) and in different planes o f view o f a

dissociated rouleau (picture set 7.4c).

■ Picture set 7.4b also shows that for complete rouleaux no fluorescence was

observed between the RBC's above the autofluorescence. Another interesting

observation made, worth mentioning here, is shown in picture set 7.5. Here a

normal looking rouleau is shown that has two regions o f higher fluorescent

emission. It so happens that at these fluorescent regions, the rouleau had some

freedom to move as again was shown by adjusting the focus. ■ Asa quick comparison, trypsin treated RBC's were compared to untreated RBC's.

The reason for this was to compare two systems o f very different levels o f

aggregation, since it was seen in chapter 6 that trypsinised RBC's form much more

aggregation than untreated RBC's. I f the two systems were compared, then it was

apparent that more points o f fluorescence were observed with trypsinised RBC's

compared to untreated RBC's. A system for quantitating this would be very

difficult, and as such this was only an observation o f the author. All o f the picture

sets included here were o f trypsinised RBC's, mainly because the increased number

of fluorescent features optimized the potential for different observations.

As soon as the samples were exposed to the fluorescent exciting light (575nm) the autofluorescence and 4>Rh-ub started to fade, ie were being bleached. This bleaching was a slow process taking upto about twenty minutes, in some cases, before the fluorescence disappeared. It was thought that this could be a way o f assessing if there was a diffuse layer o f fibrinogen adsorbed across the surface o f the RBC; if there was any adsorbed 4>Rh-ub then this should cause a different rate o f bleaching to that o f the control (autofluorescence).

Generally RBC's with 4>Rh-L»b bleached slower than with 4>Unlab, such that after ten minutes

RBC's with (jjRh.ub still fluoresced, and RBC's with c|)Unlab had disappeared (not shown).

However, as already stated, autofluorescence varied considerably between RBC's and this affected bleaching times considerably making comparisons very difficult. Also RBC's would probably have a varying amount o f ^Rh-ut, adsorbed to the surface, and hence RBC's would show different levels o f bleaching.

One immediate use o f bleaching, with RBC's + 4)Rh-Lab> was t0 remove some o f the gross fluorescence using a little bleaching, and sometimes this revealed more fluorescent spots.

These were generally fainter and lasted a shorter time than immediately obvious spots and were difficult to capture.

To try to improve the contrast between the different fluorescent features and the general fluorescence across the RBC, different labels were looked at. F IT C and Texas Red (Sigma) were used, having maximum excitatory wavelengths of 495nm and 595nm respectively.

These could be added directly to fibrinogen (50pg/mg fibrinogen), as opposed to /Ae labelled profon Rhodamine B that had to be purified first, and^was then prepared as for rhodamine. These were again found to be thrombin clottable. N o picture sets are available but a few observations showed that when FITC was used, the fluorescent features were harder to see

(lacked contrast) and for texas red there was no obvious difference from Rhodamine.

7.6 DOES THE DISTANCE BETWEEN RBC's IN ROULEAUX INCREASE

W ITH DEXTRAN SIZE ?

It was described in §1.9.1 how Chien et al showed, from electron microscopy studies, that the distance between RBC's of a rouleau increased with increasing dextran fraction M W (ie size). There are a number o f concerns about this study, such as sample preparation, measurements etc, which have left researchers questioning these results.

Here, a manipulation o f the M S M provided a completely .different approach to testing if the distance between RBC's o f rouleaux does in fact increase. This manipulation involved using dextran instead o f fibrinogen and worked on the principle that dextran would not be fixed to the RBC surface by glutaraldehyde. It was important that this was the case, and so to confirm this a control was carried out using F ITC labelled dextran 40 and trypsin modified

RBC's3. Results from this experiment showed the RBC's fixed as rouleaux, but no fluorescence was observed between RBC's of overlap, at point contacts etc as was found with c^Rh.Lab- It was believed that with dextran 40 the glycocalyx o f adjacent RBC's were overlapping sufficiently for glutaraldehyde to fix them together, and thus fix the RBC's in rouleaux.

By the CBH, it is feasible that a dextran of sufficient size would leave the glycocalyx too far apart to be fixed together by glutaraldehyde, and thus larger dextrans were used to see if this was true. Dextran 70, 110 and 500 were used on trypsinised RBC's4 and again the method fixed the RBC's as rouleaux. However, when dextran 2000 was used, any attempt to fix the

RBC's in their formed aggregates failed, which suggested that here the glycocalyx o f adjacent RBC's were too far apart to be fixed together. As a final test to see if RBC's o f

3Trypsinised RBC's were used because dextran 40 induces a large degree o f aggregation on these RBC's, but only very' little on untreated RBC'sI3,J.

4Trypsinised RBC's were used this time to avoid being concerned with the disaggregation effect found with untreated RBC's as was seen in §3.5.

225 larger surface charge made any difference, 35g/l o f dextran 500 was used on normal RBC's, but .RBC's were fixed in their rouleaux state. Hence there is some evidence here that supports the CBH.

4///A A> Lj/ihcj ^ Another interesting fact^came from trying to fix T treated RBC's, in the presence o f dextran 40, with formaldehyde. Formaldehyde failed to fix the aggregating RBC's together, which means that the fixative really is o f no use with RBC's.

CONCLUSIONS & PISCUSSTON

RADIOACTIVE

The radioactive work was mainly carried out using the T T M and the results suggest that above -0.5% glutaraldehyde, cj)1125 was fixed to the RBC's. It was also seen how formaldehyde and paraformaldehyde failed to fix any significant amount o f fibrinogen to the

RBC surface. Little more could be interpreted from these results, but there is potential for using (f>n25 with the M S M in the future in order to quantitate the level o f adsorbed fibrinogen.

FLUORESCENCE

The glutaraldehyde induced autofluorescence, assumed to be caused by the fixed haemoglobin, really was a problem in these experiments, which is why so much time was spent attempting to find an alternative fixative, that was equally effective and did not cause autofluorescence. The different fixatives formaldehyde and paraformaldehyde were tried at various concentrations and although these caused no, or little, autofluorescence, they left the RBC's looking very unhealthy. Most biconcave features were lost, and they took on the appearance o f crenated RBC's. The real 'nail in the coffin'' so to speak for these fixatives, came from the fact that they failed to fix the RBC's as rouleaux in instances where glutaraldehyde succeeded; ie with fibrinogen or dextran and the M S M . The fluorescence work appears to give evidence in support of the CBH in a number o f ways, such as the fluorescence at points o f contact and between RBC's o f overlap. It may be argued that this could be trapped c^rm ^ but the author feels the fluorescence is very bright and localised to the points o f contact for this to be the case. In the case o f overlapping

RBC's (ie forming or breaking a rouleau) to see such a concentration o f in the region o f overlap appears to contradict the D L H .

The points found on free RBC's or RBC's in complete or dissociating rouleaux seemed to suggest that another phenomenon is involved. It is possible that this was some kind o f artifact, although there seems to be no real explanation for such an artifact. Also it was thought that this was some kind o f lateral diffusion o f fibrinogen, caused by cell movement etc112,13]. Another possibility, was that the observed spots were a collection o f clustered fibrinogen from RBC separation, ie as RBC's move apart the fibrinogen is dragged with them, clustering to a point at which the RBC's are last in contact (figure 7.7); RBC's are seen in the pictures before the point is broken (ie when they are at point contacts), and the spots on free RBC's when the contact is broken.

The fact that there appeared to be more spots on trypsinised RBC's was not surprising because there was more aggregation, that would mean more instances where RBC's parted leaving these historical points o f concentrated fibrinogen. It may also mean increased adsorption, but this requires further studies to establish. The two fluorescent regions on the rouleau in picture set 5 could again be explained by clustering, in that the rouleau was breaking in these two regions, which would again bring the fibrinogen together at the places the RBC's are still bridged.

The bleaching effect could be o f much value in contributing to observations, and making some kind o f estimate o f the concentration of fibrinogen distributed across the cell. The fact that RBC's with bleached at different rates would be an indication o f how much fibrinogen was adsorbed to the surface o f the RBC. The rate o f bleaching for the control

(autofluorescence) also varied, but in preliminary studies this never appeared to be as great as with RBC's with 4 )^ ^ . More investigations and a method o f quantitation is needed, but there is much potential here.

227 OTHER RESEARCHERS

Recently Bronkhurst et al,159J carried out some work that supports and contributes to these findings. They used the 'Optical Trapping Method'11601, that allowed RBC's to be manipulated (moved around) using lasers. The basic principle is that a laser (1064 nm) is finely focused to a point (tweezer) and when objects (RBC's), o f refractive index greater than the suspending phase, are in this beam, they experience a gradient force towards the focal point. Using this technique Bronkhurst controlled two RBC's in various suspensions

(several concentrations o f plasma, serum, 4 mg/ml fibrinogen, 40 mg/ml albumin etc), brought them together, and left them to aggregate under their own devices. One interesting new phenomenon they found was that when two RBC's were brought together and formed a rouleau, then the longer the RBC's were together the stronger the rouleau became as assessed by the sliding velocity with which they could be moved apart. This may suggest conformational changes in fibrinogen adsorbed to the RBC surface. They extended this by moving the RBC's apart, which was only possible by slowly sliding the RBC's across each other in opposite directions, until they were only in point contact with each other, and any attempts to separate the RBC's further resulted in a tether forming between the RBC's

(figure 7.7). The tether was very strong and could only be broken in systems where weak aggregation occurred (ie 25% plasma). They deduced from their work that the bridging fibrinogen is dragged with the RBC's, clustering at the point of contact which would strongly hold them together. This fits very well with the fluorescent work described above where very strong fluorescence was seen between RBC's in point contact. The most likely explanation to these findings was that o f support for the CBH.

Another method previously used to manipulate RBC's to investigate cell-cell adhesion interactions is micropipetting. In a paper by Buxbaum et al|161], the process o f forming and breaking aggregates o f two RBC's were investigated and they found that more energy was needed to separate the RBC's than was needed in the formation process. This fitted well with the belief that conformational changes o f adsorbed fibrinogen takes place when RBC's form rouleaux. Also for neuraminidase treated RBC's in the presence o f dextran, a very strong point (or tether) like bond was found between RBC's during separation which also fits with Bronkhurst et al's work.

2 2 8 A B

binding between membranes clustering of binding sites during pulling

C D

further clustering very strong binding and point attachment

Figure 7.7: Demonstrates how fibrinogen is dragged with RBC's as they move apart from a rouleaux. The arrows indicate how optical traps were used by Bronkhurst et al to drag RBC's apart from a formed rouleaux. Taken from Bronkhursts thesis11”1.

ASSESSING DISTANCE BETWEEN RBC'S /A7 A G G REG A TES

The fact that typsinised RBC's with dextran 40 fixed as rouleaux can only be explained by the RBC's being close enough to link the extending glycocalyx o f the R B C surfaces 'with glutaraldehvde. The method also succeeded in fixing rouleaux with dextran 70, 110 and

500, but not with dextran 2000. It is interesting that even dextran 500 (length=150nm) still

left the glycocalyx close enough to be fixed together, but dextran 2000 (length=290nm) left the RBC's too far away to be fixed as rouleau. Chien et al's electron microscopy measurements of the intracellular distance between RBC's o f rouleau showed that there was only -2nm difference between the distance for RBC's suspended in dextran 500 and dextran

2000. Hence if Chiens' work is correct, it is interesting how this small difference was

enough to keep the glycocalyx far enough apart to prevent glutaraldehvde fixing them together.

2 2 9 FINAL COMMENTS

It is worth mentioning here that the above observations are only the start. The M S M has much potential and really requires many different sets of experimental observations to be carried out to give more insight into what is involved in the RBC's forming rouleaux. Also using 4>n:s with the M S M may provide the most accurate means o f quantitating adsorbed fibrinogen to the RBC surface. Further possibilities are to carry out further investigations into the bleaching o f the RBC's, looking at different proteins, applying shear forces to the

RBC's as they are being fixed, etc. There is much potential here!

2 3 0 CHAPTER 8: DISCUSSION

THE DEVELOPMENT OF IMPROVED TECHNIQUES FOR

QUANTITATING RBC AGGREGATION 232

AN INVESTIGATION INTO THE MECHANISMS OF RF -

REVISITED! 234

WHAT IS THE MECHANISMS INVOLVED IN RF? 235

FUTURE WORK.... 236

231 CHAPTER 8: DISCUSSION

As is apparent from the thesis title, there were two broad aims of this thesis. One aim concerned the new improved techniques for quantitating and investigating rouleaux formation, or more generally aggregation. Chapters 3 and 4 presented new ways of analyzing data from two well known instruments for measuring aggregation, the Contraves LS30 Viscometer and the MEA respectively; as far as this thesis is concerned the most important aspects of these chapters concerned parameters representing levels of aggregation. In chapter 5 methods were presented for making hct corrections to measures of aggregation, necessary to correct for small deviations in hct (= 2%) due to sample preparation error. All of this work went towards preparing the results of chapter 6, which was concerned with the second aim of this thesis; ie investigating the potential mechanisms involved in aggregation. Chapter 7 was a self contained study, with the same aims of chapter 6, but using a very different, more direct, approach.

A detailed discussion was included in each chapter, and will not be repeated here. Instead what is to follow is intended to provide a brief overview of the work.

THE DEVELOPMENT OF IMPROVED TECHNIQUES FOR QUANTITATING

RBC AGGREGATION

There is one main reason for the investigations of chapters 3, 4 and 5, that appears to have previously been neglected or ignored. The reason being that of dealing with "hyperaggregating" RBC's, that require a different approach to "normal aggregating" RBC's.

MSCOSITY ANALYSIS (CHAPTER 3)

The analysis system, AnalEta, was developed to investigate r i^ and provide various parameters representing different aspects of the ri^, two of which were 0-277T|r and GA. The former of these is an index of aggregation, and is simply the r\ at one low y, in this case 0.277s'1, and is a much used index in the literature. However, there are limitations and problems associated with this parameter, because it represents, only one point of the tj behaviour of the blood sample. It was felt that GA may give a better indication of the aggregation behaviour of a blood sample, since it was derived from a number of r\ measurements at different y's, and it was much less affected by increases in supernatant r|, hct and stiffening of the RBC membrane. It was thought to be more than just an index of aggregation, as it represented the shear thinning properties of the sample during the aggregation process. In the majority of cases GA and 0,277 ,nr gave similar trends, but there were occasions when they differed. It was generally thought that GA was the more reliable of the two parameters for the reasons given above, and because it agreed more closely with the MEA parameter Comp02)ASyl.

MEA ANALYSIS (CHAPTER 4)

For chapter 4, the difficulties and demands were far greater. Here the analysis system was based around data from the MEA: the syllectogram. Analyzing the syllectogram data in itself proved much harder than ever anticipated. Although many parameters could be derived from the syllectogram and showed potential for the quantitation of aggregation, for simplicity only parameters derived from measuring areas under the syllectogram were made use of here (ie ^ " d 3)ASyi and 1210,124)ASyl). It was seen that the MEA works well within the normal range of aggregation, but when aggregation was much lower or higher than normal, then the MEA failed to give a correct measure of aggregation. With the aid of the analysis system (ViewSyl) continuously decaying or flat syllectograms were recognized, and it was found that by using ^^Agy,, the lower range of aggregation was extended. Also the upper limit of aggregation was extended by simply using Comp( 12)ASyl. Even though the analysis used here is based on the same principle of the original MEA parameters, ie using areas (Klan(()and 3)As>1), it is the different approach, and the use of different y's, that have succeeded in extending the limits of the MEA.

Purely from the manner in which the MEA works, and how the analysis system is configured, the response to increasing y is very different to that of the Viscometer. With the Viscometer at LSR's, aggregates are given time to form to the maximal possible level at a given y and then the measured r\ used. With the MEA, aggregation is only given 1 Os to occur and this is monitored through the entire time; ie at stasis, RBC's have 10s to find each other and aggregate under natural forces. Hence, y will have various influences on the process of aggregating RBC's. Aggregation would be facilitated by an applied low y, because the number of cell-cell interactions is increased and thus measures of aggregation in the given 1 Os increases. However, over a certain y forces will act against aggregation.

233 Hence, by measuring different aspects of aggregation, as the MEA and Viscometer clearly do. the results may not necessarily follow the same trend.

HAEM A TOCR1T CORRECTIONS (CHAPTER 5)

There were a number of different aspects to work in this chapter based around investigating hct effects on r i^ and MEA data. The necessity of this came from having to correct T)^ for small changes in hct (±2%), from the standard hct of 45% used here, due to sample preparation error. It was of some interest and importance to see whether MEA data needed, or would benefit, from a similar correction, and it was shown here that significant improvements were obtained if hct correction was made.

The necessity of such a detailed study again came from analyzing hyperaggregating RBCSusp's. It was found that these small hct variations had a much greater affect on both r)^ and MEA data, which highlighted the fact that the level of aggregation had to be taken into consideration when making such hct corrections, or in the case of the MEA whether hct variations could be ignored.

AN INVESTIGATION INTO THE MECHANISMS OF RF - REVISITED!

ENZYME DEGRADATION OF THE RBC SURFACE (CHAPTER 6)

As already mentioned, chapter 6 is the core of this thesis and was where the original emphasis lay, before problems with hyperaggregation, the MEA etc occurred. The study involved making RBC aggregation measurements before and after enzyme treatment, with either dextran 40, dextran 70 or fibrinogen present to induce aggregation. The cellular factors RBC size and deformability were investigated for a number of the enzyme treatments, and were found not to change and thus played no, or little, part in the increased aggregation. Surface charge was also investigated for a number of the enzyme treatments, and was found to continually decrease in accordance with an increase in dextran-induced- aggregation. Fibrinogen-induced-aggregation was found to behave differently to dextran in a number of ways. Most striking of these was the lower increase in aggregation with the decrease in surface charge, and the fact that this change only occurred between -18-50% decrease in surface charge. FLUORESCENT FIBRINOGEN STUDIES (CHAPTER 7)

When work was originally started on this problem, it was believed that by simply showing the existence of adsorption or non-adsorption of fibrinogen to the RBC surface, would be enough to prove either the DLH or the CBH correct. However, as already mentioned it is not necessary for there to be non-adsorption for the DLH to be true.

It was seen here that there is a new technique for directly investigating the actions of proteins, eg fibrinogen, on the RBC surface. So far it has provided some fascinating, and new observations concerning the aggregation process, which is supported by the work of Bronkhurst et al,159). The fact that the author found that fluorescent regions occurred between RBC's in point contact with each other, and Bronkhurst found tethering between RBC's being pulled apart from a rouleau, could apparently only be explained by fibrinogen bridging.

WHAT IS THE MECHANISMS INVOLVED IN RF?

Previously there have been two proposed hypotheses for describing the mechanisms behind RBC aggregation: one involved the bridging together of the RBC's by macromolecules adsorbed to the RBC surfaces (ie CBH), and the second involved overlapping regions of macromolecular depletion surrounding RBC's that induces an osmotic gradient which acts to push the RBC's together (DLH) -§1.9.1. The principle of the former is relatively simple, but the latter remains unclear. There appears to be two potential ways in which the regions of depletion surrounding RBC's can form. The first stems from non- (or little) adsorption of the macromolecule to the RBC surface and the second from an adsorption equilibrium such that no further adsorption can occur (depletion stabilization). From the work presented here, and by others, it appears that the former of these is no longer possible. The author, and Bronkhurst et al, have shown that fibrinogen is adsorbed to the RBC surface and is actively involved in the process of aggregation as described above. Hence, the fibrinogen would have to already have adsorbed to the RBC surface before the process of aggregation occurs, because there would be no apparent way in which the fibrinogen could get between the RBC's once rouleaux had formed. From the enzyme work presented here using dextran as an aggregating agent, there is little to support one hypothesis above the other; the results can be explained by both hypotheses as being caused by a reduction in surface charge. However, should an instance be found when aggregation increased significantly with little change in surface charge, then this would clearly support the CBH.

Therefore this leaves the two remaining hypotheses: the CBH and depletion stabilization. The former of these is clearly involved, but there are good reasons to believe that RBC's initially may be brought together under the influence of osmotic forces. For instance the work of Baumler et al suggests RBC's do have regions of depletion surrounding them. Hence, the author proposes that it is osmotic forces that brings the RBC's together initially, and once together the bound fibrinogen conformationally changes to cross-bridge the RBC's together. These later conformational changes would explain the time changes in the strength of the aggregate that Bronkhurst et al and Buxbaum et al found.

FUTURE WORK....

To get closer to a hypothesis that accommodates all of the latest research, there are a number of further experiments that are needed. For Bronkhurst et al's work11591, an obvious advancement is to use fluorescence work to establish if the clustering of fibrinogen occurs, and if it becomes concentrated at the tethered point; Baumler et al's workf98] needs to be extended to use proteins as opposed to the artificial dextrans that have been used to date. Finally the work carried out by the author could be extended in a wide variety of ways. The enzyme work could be extended to look at other enzymes, aggregating agents etc. Here an aim is to find a situation where changes in aggregation can not be explained by changes in surface charge. Further fluorescent work could be carried out with fibrinogen, and other proteins, on normal and enzyme treated RBC's, particularly bromelain treated RBC's as fibrinogen-induced-aggregation was found to be maximal here.

Finally, it would be useful to develop some analysis for quantitating the fluorescent features of a RBC. To this end, it would be useful to have the ability to create time integrated pictures; ie summing images captured at different time intervals. This would hopefully remove some of the restrictions of the autofluorescence, and reveal further features. Also further radioactive work could be performed with the MSM, which could act as an accurate way of measuring adsorbed fibrinogen to the RBC surface.

2 3 6 REFERENCES:

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248 ACKNOWLEDGEMENTS

I would like to start by thanking the Medical Research Council (MRC) for funding this research and Charles Michel for giving me the chance of undertaking this PhD.

A special thanks to my supervisor Mike Rampling for his help, support and guidance whilst undertaking this PhD, and also for his considerable help towards the writing of this thesis.

Thanks to the technicians Enid, Gill, Chris and Matt and to all of the victims who kindly "donated" their blood, some of whom gave willingly.... Also to Phil Gribbon for his help with using the instruments at the PFSU, and to Peter Winlove for his encouragement, help and enthusiasm.

I would like to thank my parents for their love, support and understanding for all times, but particularly for the last few years, and for helping me obtain the computer on which this thesis was bom. Also to the "outlaws" Archie and Rosemary for their love and support.

Finally I would like to thank my wife Fi for everything....

To my love of fantasy and the unknown

249 Picture Set 7.1: Three instances (a-c) of white light (left) and fluorescent light (right) images of RBC's fixed, by the MSM and with glutaraldehvdc. in the presence of unlabelled fibrinogen: ic demonstrates RBC autofluorescence induced by glutaraldehyde.

250 Picture Set 7.1: Compares white light (left) and fluorescent light (right) images of RBC's in point contact with a rouleau, as fixed in the presence of (a) unlabelled fibrinogen or (b) rhodamine labelled fibrinogen. In (a) no fluoresence was seen at the point of contact, yet in (b) fluorescence was seen suggesting fibrinogen was holding the RBC's together.

251 Picture Set 7.3: Overlapping RBC's arc seen under white light (top left), that are in the process of forming or breaking away from a rouleau. On the same focal plane under fluorescent light (top right) no features are seen, but upon refocussing (bottom left) a strong fluorescent region is found between the overlapping RBCs. Refocussing again (bottom right) reveals a fluorescent spot.

252 Picture Set 7.4: Shows a number of instances w here rhodamine labelled fibrinogen is concentrated at a single point on the surface of a RBC or rouleau, (a) show two planes of view of RBC's under white light (left), and the fluorescent image (right) shows fluorescence at the point of contact between RBC's. Also seen is a fluorescent spot on a single RBC. (b) show s a single RBC and a rouleau both of which have a fluorescent spot, (c) show s tw o and (d) three fluorescent spots at different planes of view.

2 53 Picture Set 7.5: Shows a large rouleau under white light (left) and fluorescent light (right). Only two regions of fluorescence were found, which corresponded to the two positions at w hich the rouleau moved when forces w ere applied

254