Laboratory Tests of Platelet Function

H. JAMES DAY, M.D. and HOLM HOLMSEN, Ph .D. Hematology Laboratories, Temple University School of Medicine Philadelphia, PA 19140

Introduction portant adenine nucleotide released is ADP, There has been growing interest in the a powerful platelet aggregating substance. role of platelets in the hemostatic process. Platelets also contribute a major source of Following injury to the blood vessel wall, phospholipids required for platelet coagula the platelets are exposed to some substance tion and this has been identified as platelet in the basement membrane, which may or factor 3 or platelet thromboplastic factor. may not be collagen, which leads to the This phospholipid is not normally available rapid formation of a platelet plug. The on the platelet surface and only becomes steps involved here are those of adhesion so when the platelets are altered or acti to the site of injury and exposed basement vated. Hardisty and Hutton1 have shown membrane, then aggregation of the plate that Kaolin makes this thromboplastic activ lets during which time the platelets un ity available and Spaet and Cintron24 have dergo a release reaction. Formation of a shown that connective tissue suspensions permeable platelet plug then occurs and are equally active. Platelets are also re finally, the subsequent development into a quired for clot retraction, the energy for hemostatic plug with the incorporation of which is derived from the breakdown of fibrin strands. It is important to note that platelet ATP to ADP. During this time, the time needed for the formation of a plate fibrin strands form which tend to radiate let plug correlates well with the bleeding between the clumps of platelets which act time measured from the beginning of the as the nidus of clot retraction. To date, clot infliction of the wound to the time the retraction has only been shown to be abnor bleeding ceases, i.e., approximately 6 min mal in those patients who have a defect utes. Thus, in performing a bleeding time, known as thrombasthenia and in those who the steps are measured which are necessary lack a normal number of platelets, i.e., for platelet plug formation. In vitro the thrombocytopenia. It is uncertain which function of platelets is tested by measuring role clot retraction plays in the mainte platelet adhesion to glass, aggregation with nance of normal hemostasis. various agents, the release reaction, clot re 5-Hydroxytryptamine (5-HT), a power traction, platelet-phagocytosis and platelet ful vasoconstrictor, is also carried by the uptake of numerous substances. platelets and released during the platelet Platelets contain large amounts of ade release reaction. The role of 5-HT in nor nine nucleotides which are released during mal hemostasis is not known, but recent the platelet release reaction, an important evidence has shown that a low platelet step in hemostasis. Possibly the most im serotonin level is often associated with low 63 64 DAY AND HOLMSEN releasable nucleotide levels. Thus, plate disease, etc. It is not in the scope of this lets are unable to release aggregating mate paper to discuss these in detail. More rials and hemorrhagic manifestations result. detailed general review papers on this Such a defective release reaction is typical subject should be consulted.12- ls- 16- 20 of thrombopathy or thrombocytopathy, a clinical condition characterized by hemor Methods rhagic manifestations. This paper will describe those tests of By definition, patients having defects in platelet function which are simple to per platelet function (also called qualitative form and yield the desired information. All platelet disorders) are those who have a are now available for use in clinical normal platelet count, but a prolonged laboratories. bleeding time owing to abnormal function ing platelets. It is generally accepted that Collection of Blood Sample bleeding phenomena do not occur unless the platelet count is below 2 0 ,0 0 0 per mm3. It is important to collect a sample of However, some patients, specifically those blood from a normal subject at the same with thrombopathy, may have platelet time as that of the patient if detailed tests counts that are in the range of 75 to 150,000 of platelet function are to be performed. per mm3 and have bleeding phenomena The volume of blood to be drawn for study even with this relatively minor thrombocy of platelet function may vary anywhere topenia. Under these circumstances, the from 9 to 38 ml. As performed in our platelets can often be shown to have a laboratory, 18 or 38 ml of blood are drawn functional defect. by gravity into plastic conical tubes by free The major diseases of platelet function flow into 2 ml or 4 ml of 0.11 M sodium that have been identified to date are classi citrate (31.3 g per 1 trisodium citrate 'H^O) fied in the following manner: with a teflon coated needle attached to a 1. Thrombasthenia — a disease which plastic tubing. The tube is then capped seems to be characterized by a defec with parafilm and inverted several times tive platelet membrane resulting in a for adequate mixing. Two blood smears failure of platelet aggregation. are also made at this time for staining with 2. Thrombopathy — in which there is a Wright’s stain and examined with particular failure of the release mechanism in reference to platelet number, platelet mor platelets and a failure to make plate phology and the presence or absence of let factor 3 (PF3) available. platelet aggregates. A sample of the whole 3. Von Willebrands disease or pseudo blood is then submitted for platelet count hemophilia—which is characterized by ing by standard electronic counting tech niques. Plastic equipment or siliconized a prolonged bleeding time and a low glassware is used for all platelet transfers. factor VIII. Strictly, this is not a platelet functional abnormality in as Preparation of Platelet-Rich and much as the platelets function per fectly well, but is included mainly Platelet-Poor Plasma because of a prolonged bleeding time. Citrated whole blood is centrifuged for These patients lack a plasma factor ten minutes at 2 0 0 x g at room temperature. called von Willebrand’s factor. It is important that the platelets not be Many platelet functional abnormalities subjected to extreme cold. The platelet-rich are seen as secondary to other disease plasma (PRP) is then transferred to a sec states. These include renal diseases, para- ond plastic tube, care being taken to avoid proteinemias, thrombocythemia, iatrogenic transferring any red cells. A platelet count LABORATORY TESTS OF PLATELET FUNCTION 65 is performed on this PRP which is kept Bleeding Time at room temperature until tested. Approxi Introduction mately 5 ml of PRP is re-centrifuged at 4°C for 30 minutes at 2,000 xg to remove The bleeding time is perhaps the best platelets and obtain platelet poor plasma screening test for defects of platelet func (PPP), which is collected and kept at 22 tion. As mentioned previously it measures to 25 °C until tested. the entire sequence of events in the forma tion of the hemostatic platelet plug. When Capillary Resistance the bleeding time is abnormal, other tests of platelet function are performed which Principle yield specific information as to the specific The test for capillary resistance, called type of hemostatic defect. the tourniquet test, is performed every time a venous blood specimen is obtained with Principle venostasis. It is best quantitated by putting A standard incision is made through the a blood pressure cuff on the arm and inflat skin and the time necessary for stopping of ing it to a point mid-way between systole bleeding is carefully measured. As useful and diastole (about 100 mm Hg). The as is this test, it must be rigidly controlled. cuff is left in place for 3 to 4 minutes with The method of Ivy et al. 13 is preferred in an area approximately the size of a half- as much as it is better controlled than any dollar, circumscribed on the volar aspect others. Recent modifications1’ 4- 17 have in of the forearm. The number of petechiae troduced even further methods for quanti in this area are then counted following tating the bleeding time. Strict attention deflation of the cuff. Some petechiae also must be paid to detail and each laboratory occur on the dorsum of the hand, this area must establish its normal range. should be observed closely. At the same time petechiae may develop in the area to Procedure1, 4 which the cuff is applied. A sphygnomanometer is placed around Discussion the patient’s arm and inflated to 40 mm of mercury. Following adequate cleansing of The test is not very useful in assessing the volar surface of the forearm, three platelet function because the results may parallel cuts are made on the forearm with be abnormal in many disease states, only a #11 Bard-Parker blade. These cuts some of which are related to abnormal should be 5 to 6 mm long, 1 mm deep and hemostatic processes . It is generally abnor 1.5 cm apart. Blood is carefully absorbed mal in thrombasthenia and von Wille- with the edge of a filter paper every 30 s brand’s disease, but more commonly it is until bleeding ceases, the wound itself not pathological in patients with severe throm being touched. Wounds which bleed pro bocytopenia or increased vascular fragility. fusely from the start probably represent Patients who develop petechiae under the perforated veins. Those which do not cuff or on the dorsum of the hand usually bleed for at least 2 minutes are assumed to have increased vascular fragility. be incorrectly performed and therefore dis regarded. Two separate end points may Normal Values be considered: a) the longest time taken to Less than 5 petechiae on the area under stop bleeding and b ) the mean time for all observation on the forearm is within the three wounds. The former is to be pre normal range. ferred. The modification of Mielke et al.17 66 DAY AND HOLMSEN utilizes a template which controls the limit of 8 minutes might be set for normal length and depth of the incisions made with values. the surgical blade. With this method, Clot-retraction bleeding time is longer than might nor Introduction mally be seen using a puncture method. Probably one of the oldest tests of platetet Discussion function, clot retraction, has been surpassed by more specific and more useful tests. Sev The bleeding time is the premier test for eral methods for clot-retraction are em determining whether or not a patient with ployed using whole blood, plasma clot, a normal platelet count has a platelet func dilute clot, etc. The test is affected by red tion defect. All diseases of platelet function cell volume and plasma fibrinogen concen have abnormal bleeding times at some pe tration. Therefore, it is not solely a test of riod in their disease. If the clinical circum platelet function. Generally, the more sim stances suggest a defect in platelet func ple the test, the easer it is to interpret. The tion, several bleeding times may have to method listed is a modification of the be done if the original one is normal. This original method of McFarlane14 as pro is particularly true for von Willebrand’s posed by Hardisty et al.4- 12 disease where daily variations in the bleed ing time have been recorded. Thrombas- Method thenias and thrombocytopathias, unless of Exactly 2 ml of whole blood is allowed an extremely mild nature, always have pro to flow into each of two plastic conical longed bleeding times. tubes which are then placed in a water bath at 37°. Blood may be allowed to flow Sources of Error directly from the needle-plastic tube com In addition to those mentioned pre bination suggested in the general procedure viously, the status of the patient’s subcu section. In another method, 5 ml may be taneous tissues must be considered. Patients drawn into a plastic syringe, 1 ml discarded receiving intravenous fluids, patients with and exactly 2 ml added to two plastic or generalized edema, etc. may have bleeding siliconized tubes. A stainless steel metal times which are not truly reflective of the wire (made from a paper clip) is immedi hemostatic status. Non-steroidal, anti-in- ately placed in each tube. Exactly 60 min flammatory drugs, such as acetylsalicylic utes after the blood has clotted, the clot is acid have been shown to prolong bleeding removed with the wire spiral and is al times by as much as 2 to 3 minutes. Pa lowed to drain for 15 seconds without tients must be carefully questioned as to the pressing it against the side of the tube. recent ingestion of these drugs. It has been The volume of expressed serum is meas demonstrated that the effect of aspirin will ured with a graduated pipet and recorded as a percentage of the original 2.0 ml. The last for as long as 7 to 10 days. test is always performed in duplicate. Normal Values Discussion As performed in these laboratories, the Clot retraction is a function of the retrac longest time recorded in all three wounds tile protein, thrombosthenin, in blood plate is 5.5* 2 minutes and the mean of all three lets. Clot retraction is generally abnormal wounds is 4.8* 2 minutes. As the single in states where the platelet count is de reading is usually the longest, this seems creased. The only specific disorder of to be the optimal value for assessing the platelet function in which clot retraction patient’s bleeding time. An arbitrary upper is abnormal is thrombasthenia. It is gen- LABORATORY TESTS OF PLATELET FUNCTION 67

erally not held to be useful in assessing one of the commercially available aggrega platelet function defects, either primary or tion meters may be utilized. Platelet ag secondary, and has been replaced by many gregation is a function of the final concen more specific tests. tration of the aggregating agent used. The method described is based on several Sources of Error methods.2' 19-21 •25

If the equipment used is not clean, the Reagents clot will adhere to the sides of the tube and not retract. It is probably best to rim Platelet-rich and platelet-poor normal and the clot in the tube after clotting has test plasma. Citrated platelet rich plasma occurred to allow for maximum retraction. is prepared as stated. The platelet count should be adjusted to a set number in the Normal Ranges range of 150 to 300,000/mm3 by dilution of the plasma under test with the appropriate After 60 minutes the clot should have platelet poor plasma (PPP). retracted 48 to 64 percent of its original volume. Adenosine phosphate (ADP) (Sigma Chem ical Co., St. Louis, MO). Platelet Aggregation Stock solution: 400 ¡¿g per ml in saline. This is stored in 0.5 ml volumes at Introduction —20°C. The development of aggregates upon ex Working solution: posure to various agents is considered to be one of the major functions of platelets a) 8 fjig per ml. The entire content of and of cardinal importance in hemostasis. one of the 0.5 ml tubes of the 400 ¡a,g per ml stock is now diluted to In vivo, the source of aggregating agents 25 ml total volume with saline, are probably collagen from the vessel wall and adenosine diphosphate (ADP) from b ) 4 ¡jig per ml. 10 ml of the 8 ¡¿g per other platelets. The platelet’s ability to ml working solution is diluted with respond to various aggregating agents may an equal volume of saline. be measured in vitro with specialized in Epinephrine. Epinephrine bi-tartrate, (Cal- strumentation as originally described.2 biochem ). Stock solution: 0.5 mg per ml of saline. Principle This is stored in volume of 0.5 ml at Platelet rich plasma (PRP) is slightly —20°C. turbid due to the presence of platelets in Working solution: One tube of the stock suspension. When the platelets aggregate, solution is diluted to 50 ml total volume the turbidity decreases. A light beam is with saline. Final concentration: 5 fig directed through PRP in a cuvette and the per ml. percent transmission or optical density Collagen. Crude equine tendon extract recorded on a linear recorder. The contents (Sigma Chem. Corp., St. Louis, MO). of the cuvette have to be stirred constantly May be prepared by any one of two and kept at 37°. When ADP, epinephrine methods. or collagen is added to PRP, the formation of platelet clumps produces an increase in Method (1)1S percentage transmission or a decrease in Four g of crude collagen (Sigma) is optical density which is then recorded. A placed in 50 ml of 0.9 percent NaCl in photoelectric colorimeter, absorptimeter or a Waring blender at 0°C. The blender 68 DAY AND HOLMSEN

is run at top speed for 3 separate one cuvette and the initial optical density or minute periods making sure that not percentage transmission recorded. The re too much heat is generated. The mix corder should initially be calibrated rela ture is well shaken for 5 minutes in the tive to the platelet poor plasma blank. A cold and then centrifuged at 1800 X g normal control should always be run at the for 15 minutes at 5°C. The material is same time that a patient’s plasma is being to be made up each day the test is to tested. The plasma is continuously stirred be run. for 2 minutes while being heated to the desired 37 °C. A small volume (1/10 of the Method(2)16 PRP volume) of aggregating solution is This method avoids the necessity of added to the platelet-rich plasma and having to make up new collagen each changes in the percentage transmission or day. It employs a relatively stable optical density continuously recorded until collagen suspension prepared by solu the maximal changes have occurred. The bilizing collagen in dilute acid. One test is performed using epinephrine, col gram of collagen (Sigma) is homoge lagen and both concentrations of ADP. nized in a Waring blender with 100 ml Considerable experimentation with final of 8.35 mM acidic acid for 30 s at concentrations may have to be done in 20,000 revolutions per minute at 0°C. order to achieve maximal aggregation. If Following this, 100 ml of cold distilled no recorder is available, the opical density water is added and the mixture re readings may be made every 10 s for 4 homogenized for another 30 s followed minutes and then plotted on graph paper. by dilution with 4 volumes of 1.67 mM acidic acid. The resulting gel is stored Calculation at —20°C. Immediately before use, Several formulae have been proposed for the collagen solution is thawed and re measuring the degree of aggregation. homogenized with a teflon pestle-glass Weiss25 measures the initial (I) and maxi homogenizer for 5 to 10 s and diluted mal (M) transmittance which is then con with 1 volume of 1.67 mM acidic acid. verted to optical density (O.D.) and the This mixture is referred to as soluble values collagen and is kept at 4° (ice-bath) during experiments. Instantaneous transformation to insoluble collagen fibrils occurs upon addition of this taken as an estimate of the platelet aggre material to saline or plasma. gation. Rozenberg and Holmsen17 measure Special Apparatus the fall in light absorption occurring in the first 50 s and record this value as the delta Aggregometer* 50. To observe the pattern of the aggrega Linear Response Recorderf tion curves and compare these to the nor mal PRP run-in parallel is probably the Procedure easiest method of calculation. Depending upon the aggregometer used, one volume (0.4 ml or 1 ml) of citrated Discussion platelet rich plasma is pipetted into the Platelet aggregation is probably the most useful test of platelet function currently * Platelet aggregometer, Chronolog Corporation or EEL platelet aggregometer. performed. The curves recorded consist of f Bausch and Lomb, Vom-6 . a rapid increase in light transmission due LABORATORY TESTS OF PLATELET FUNCTION 69 to initial aggregation which is a function AGGREGATION WITH ADP of the platelets’ primary aggregating ability (figure 1). If the final concentration of aggregating agents is low, rapid disaggre gation occurs (figure 1). This is referred to as a first phase of aggregation. Using ADP, the final concentration may be so adjusted that following first phase aggrega tion, a plateau and then a second phase of aggregation occurs. This second phase rep resents the release reaction of platelets where more ADP accumulates in the plasma as released from the platelets them selves.18 Epinephrine similarly has a bi- phasic pattern but not as pronounced as that seen with ADP (figure 2). Epineph rine induced aggregation is sub-optimal in those conditions characterized by the re lease failures such as thrombopathia. Col F ig u r e 1. Platelet aggregation curves ob tained from platelets of a normal control and a lagen-induced aggregation may be quanti patient with thrombopathia. ADP induced aggre tated by varying the final concentration of gation. Final concentrations of ADP are given collagen suspension. After a lag of a few for each type of curve. Note that the greatest aggregation (expressed as highest percent trans seconds, aggregation occurs in only one mission) is obtained with highest concentration. phase and the aggregates do not dis-aggre- The normal control shows a first phase aggregation gate. Collagen induces aggregation by with 0.8 /¿M and 1.2 /iM ADP (final concentra tion) but a second phase with 1.6 /¿M. The being phagocytosed by the platelet by thrombocytopathic platelets fail to show a second which the cell releases ADP which is then phase aggregation even with 3.2 /iM ADP. primarily responsible for the aggregation.10 Platelets from patients with thrombasthenia Sources of Error do not aggregate with any modality. In thrombopathia, characterized by failure of Aggregation tests are best performed in release process, a failure of second phase the time period of 30 to 180 minutes after aggregation is seen. As may be seen in fig drawing the blood and preparation of plate ure 1, first phase ADP-induced aggregation let rich plasma. All patients must be ques does occur with a patient with thrombo tioned about the recent ingestion of non pathia, but there is no evidence of second steroidal anti-inflammatory agents since phase aggregation. Epinephrine and col these will block release and interfere with lagen induced aggregation are sub-optimal the aggregation patterns. Thrombocyto in this condition (figures 2 and 3). Patients penia will also yield poor aggregation since who have been on non-steroidal anti-inflam the test is dependent on the platelet con matory drugs such as phenylbutazone or centration in the PRP. aspirin may have inadequate or sub-optimal aggregation with epinephrine and collagen, Normal Ranges and lack of second phase ADP-induced The type of changes in aggregation are aggregation. Conditions such as uremia, best illustrated in the changes in aggrega macroglobulinemia, thrombocytosis, and tion patterns. Each laboratory must estab scurvy are also characterized by abnormal lish its own normal patterns with each ag aggregation patterns. gregating agent used. 70 DAY AND HOLMSEN

AGGREGATION WITH EPINEPHRINE the addition of calcium. During the incu bation with kaolin, PF 3 is released from the platelets and the plasma contact factors are activated. At a specific time period, calcium is added to the mixture of platelet rich and platelet poor plasma and a recalcification time done immediately.

Reagents Platelet rich normal and test plasma. Pre pared as described previously. Platelet count is performed on this plasma and should be in the range of 150,000 to 400,000. Platelet poor and test plasma. Prepared as described previously. Kaolin. (Fischer Scientific Co.) 5 percent (w/v weight/volume) 5 mg per ml made up in 0.05 M imidazol buffered normal saline, pH 7.3. Calcium chloride. 0.025 M.

Procedure6, 12, 25 Two separate 75 X 12 mm glass test F ig u r e 2 . Epinephrine induced aggregation of tubes are set up in a water bath at 37°. platelets from a normal control and a patient with thrombopathia. The control platelets show a typi Tube #1 contains 0.1 ml of test platelet cal two-phase aggregation curve while the pa rich plasma and 0 .1 ml of normal platelet tient’s platelets yield only a first phase. poor plasma. Tube #2 contains 0.1 ml of test platelet poor plasma and 0 .1 ml of Platelet Factor 3 (PFS) Availability normal platelet rich plasma. These tubes differ only in the source of the platelets. Introduction Precisely 0.2 ml of the 5 percent kaolin Platelets function in the coagulation se suspension is added to each tube and the quence by supplying phospholipid neces incubation carried out for 2 0 minute periods sary for the generation of thrombin. This of time with occasional shaking. At the lipid material is called platelet factor 3 end of this incubation period, 0 .2 ml of the (PF3). The platelets make the PF3 avail calcium chloride solution is added to each able for the coagulation process by the tube and the clotting times of the recalci same release mechanism that yields ADP. fied plasma recorded with a second stop Therefore, the measurement of PF3 avail watch. ability from the platelets is a measure of the platelet release reaction.5 Discussion As the plasma factors are maximally acti Principle vated, the recalcification time performed The re-calcification time of platelet rich in these tests reflects the amount of PF3 plasma is considerably shortened since the released from the platelets. Conditions in plasma is incubated with kaolin prior to which the platelets are unable to release LABORATORY TESTS OF PLATELET FUNCTION 71 their phospholipid are thrombocytopathy, COLLAGEN INDUCED AGGREGATION thrombasthenia and various clinical condi tions, such as uremia, protein abnormali ties, scurvy, etc. Blockers of the release reaction, such as aspirin or phenylbutazone, may inhibit the platelet factor 3 availabil ity. An absolute deficiency of platelet fac tor 3 probably does not occur.

Sources of Error As with all tests of platelet function, prior ingestion of drugs can markedly affect any laboratory result. Since this test indirectly measures the same platelet function (the release reaction) as a second phase of aggre gation, the sources of error in platelet ag gregation also apply. It is important to note that thrombocytopenia will cause an abnormality of the recalcification time which would be recorded as a failure of platelet factor 3 release.

Normal Ranges The recalcification time after 20 minutes of incubation should be between 45 and 50 s. A failure of platelet factor 3 release F ig u r e 3. Collagen induced aggregation of platelets from a normal control and a patient with is characterized by prolonged recalcifica thrombopathia. Note the suboptimal aggregation tion times of 80 to 100 s. response to soluble collagen manifested by this patient. Platelet Glass Retention (Platelet Adhesiveness) between the glass beads and the number retained is a measure of the platelet adhe Introduction siveness. The procedure is better called the The initial step in hemostasis involves platelet glass retention test. platelets adhering to the site of a damaged vessel. Platelets have the function of ad Principle21' 22 hering to many seemingly diverse sub With the method of Hellem,7' 8 citrated stances such as glass, collagen, latex parti whole blood is passed at a constant speed cles, glass wool fibers, etc. Wright26 was through a glass bead column. The per the first to quantitate the platelet glass centage of platelets retained in this column adhesion by rotating heparinized whole is estimated from the differences between blood in a glass bulb and observing a de platelet counts carried out before and after crease in the platelet count. Recent modi the passage of blood through the column. fications have allowed the passage of native whole blood, anti-coagulated whole blood Procedure or anti-coagulated PRP through columns The procedure used is a modification of containing small glass beads. In this proc the method of Hellem.7’8 A motor driven ess, the platelets are retained on or trapped device is used such as a Harvard Constant n DAY AND HOLMSEN

Infusion Pump* which delivers 1 ml of (X = ml of citrate solution — H = hemat blood in 23.5 s from a 5 ml syringe. This ocrit). This formula is expressed in such pumps the blood through a glass bead col a way that the amount of citrate concentra umn containing 5 g of beadsf with a diam tion in the plasma fraction of the blood eter of 0.5 ml, in a plastic tubing with an is always constant since the results are internal diameter of 5 mm. Both ends of dependent upon the amount of ionized the column are provided with filters of calcium. Exactly 12 minutes after the with nylon cloth to prevent loss of beads from drawal of blood, citrated blood is trans the column. The number of beads in the ferred to a 5 ml plastic syringe to which a column is about 15,000 with a total glass short plastic tubing is attached. The syr surface area of 12,000/mm2. It is impor inge is then placed in a machine; the tant that the columns be well packed with motor is started and for 23.5 s the blood a good fit; there should be no more than is drained directly into a glass tube contain 1.2 ml of free space available. The time ing 19 ml of isotonic citrate solution. The required for a platelet to pass through this tube is covered with parafilm and inverted column is about 30 s and this contact time 10 times before it is placed on the bench must be strictly controlled. The rate of for sedimentation of the red cells. The syr blood flow through the column is 28 cm per inge is now connected to the glass bead minute and is constant regardless of blood column and 1 ml of blood is collected after viscosity. For whole blood estimations, the its passage through the column in another blood is collected into plastic or siliconized glass tube containing 19 ml of isotonic ci glass tubes with a 10 ml mark. Platelet trate. After 2 to 3 hours, the platelet count counting is done as described previously. is performed on the supernatant plasma. The hematocrit of the patient is very im Duplicate tests are always performed, the portant and must be estimated by a micro results being rejected if they differ more method before blood collection. At a nor than 10 percent. The difference of the mean mal hematocrit (45 percent), 1 ml of 0.11 value of the platelet counts before and after M sodium citrate solution is pipetted into passage through the column gives the num the collecting tube with a mark at 10 ml. ber of adhesive platelets. This is usually A venipuncture is performed. After the expressed as percentage of the initial count. first 2 to 3 ml of blood has been discarded, the tube is filled to the 10 ml mark directly Other Methods from the end of the plastic tubing attached to the needle. The tube is then covered Several methods are presently available with parafilm and inverted 10 times to mix for estimating platelet glass retention. The method of Salzman23 is most commonly the contents. The ratio of citrate solution to plasma in the blood sample is now 1 to used in this country, but in our laboratory 4.95 and must be constant. In patients with it has been the most difficult to quantitate. an abnormal hematocrit, the amount of This method, using a vacuum tube system, citrate which is pipetted into the collecting draws the blood directly from a vein tube is adjusted according to the formula through a pre-packed glass bead column. The method of Bowie3 et al. combines the infusion pump principle and heparinized whole blood and seems to be reliable. The type of anti-coagulants used, flow rate of * Model 975, Harvard Apparatus Company, the blood through the glass bead column, Dover, MA. size of the glass beads, etc. have been shown I Type 070, Superbrite, Beads, Minnesota Min ing and Manufacturing Corporation, St. Paul, MN. to influence results obtained. LABORATORY, TESTS OF PLATELET FUNCTION 73

Discussion tion is included in table I. All of these disorders, except for the ingestion of non Platelet adhesiveness is abnormal in all steroidal and anti-inflammatory drugs, are disorders of platelet function. This test has characterized by abnormal bleeding times. a diagnostic spectrum as broad as the bleed ing time. It serves best as a screening test The latter has a prolongation of a bleeding for detection of platelet function abnormali time over the control, non-drug state, but ties. It, combined with the bleeding time, this may or may not be abnormal. The probably serves as the best tool to substan degree of abnormality with non-steroidal, tiate a diagnosis of von Willebrand’s dis anti-inflammatory drugs varies in different ease, a condition characterized by pro patients. However, the effects of a single longed bleeding time, low factor VIII drug dose may last up to as long as a levels and a decreased platelet glass bead week.27 retention. Clot retraction is only abnormal in throm basthenia. Platelet factor 3 availability is Sources of Error abnormal both in thrombasthenia and The test is difficult to standardize. Any thrombopathia. Epinephrine-induced ag one attempting to perform this procedure gregation (2 phase) and collagen-induced should consult detailed descriptions of aggregation are both abnormal in thrombo methodology.24 pathia and in thrombasthenia. Platelet re Normal Range tention to glass beads has the same degree of specificity as the bleeding time and Retention of platelets by the column therefore is abnormal in the same condi is 25 to 40 percent. tions. Von Willebrand’s disease has an Summary of Laboratory Tests of abnormality only in platelet retention to Platelet Function glass beads and in the bleeding time. In A summary of the laboratory abnormali addition, low factor VIII values are often ties found in the disorders of platelet func found in these patients.

TABLE I L a b o r a t o r y S t u d ie s o n P l a t e l e t F u n c t io n a l D e f e c t s

Platelet Platelet Platelet Platelet Aggrega- Clot Bleeding Retention Factor 3 Aggregation tion by Epinephrine Retraction Time on Glass Availability by ADP and.Collagen

Thrombasthenia Abnormal Abnormal Abnormal Abnormal Abnormal Abnormal

Thrombopathia Abnormal Normal or Normal Abnormal to Abnormal (First Abnormal Thrombocytopathy Normal Phase )

Von Willebrand’s Disease Normal Abnormal Abnormal Normal Normal Normal

Non-Steroidal, Normal Normal Anti-inflamma Normal to Normal Normal ( First Abnormal tory Drugs Abnormal Phase) 74 DAY AND HOLMSEN

References 15. M a r c u s , A. J.: Platelet function. New Eng. J. M e d . 280: 1213-1220, 1278-1284, 1330-1335, 1. B orchgrevink , C., E g e b e r g , O., G o d a l , H. 1969. C., a n d H jo r t , P. F.: The effect of plasma and Cohn’s fraction I on the Duke and Ivy 16. M ic h a l , F . a n d F i r k in , B. G.: Physiological bleeding times in von Willebrand’s disease. and pharmacological aspects of the platelet. Acta Med. Scand. 173: 235-242, 1963. Ann. Rev. Pharm. 95: 118, 1969. M i e l k e , H ., K a n e s h ir o , M . M ., M a h e r , 2. B o r n , G. V. R.: Aggregation of blood plate 17. C. lets by adenosine diphosphate and its reversal. I. A., W e in e r , J. M ., a n d R a p a p o r t , S. I.: Nature 194: 927-929, 1962. The standardized normal Ivy bleeding time and its prolongation by aspirin. Blood 34: 3. B o w ie , E. J. W., O w e n , C. A., T h o m p s o n , J. 204-215, 1969. H., a n d D i d is h e im , P.: Platelet adhesiveness in von Willebrand’s disease. Amer. J. Clin. 18. M i l l s , D. C. B., R o b b , C. A., a n d R o b e r t s , Path. 52: 69-77, 1969. G. C. K.: The release of nucleotides, 5-hydroxytryptamine and enzymes from human 4. H a r d is t y , R. M., D o r m a n d y , K., a n d H u t blood platelets during aggregation. J. Physiol. t o n , R. A.: Thrombasthenia. Brit. J. Haemat. (London) 195:715-729, 1968. 10: 371-387, 1964. 19. M u s t a r d , J. F., N e g a r d t , B., R o s w e l l , H . 5. H a r d is t y , R. M. a n d H u t t o n , R. A.: Platelet aggregation and the availability of platelet C., a n d M a c M i l l a n , R . K.: Effect of adeno factor 3. Brit. J. Haemat. 12: 764-776, 1966. sine nucleotides on platelet aggregation and clotting time. J. Lab. Clin. Med. 64: 548-555, 6 . H a r d is t y , R. M. a n d H u t t o n , R. A.: The 1964. kaolin clotting time of platelet-rich plasma. A test of platelet factor 3 availability. Brit. J. 20. M u s t a r d , J. F. a n d P a c k h a m , M . A.: Factors Haemat. 11: 258-269, 1965. influencing platelet function: Adhesion, re lease and aggregation. Pharmacology Rev. 7. H e l l e m , A. J.: Platelet adhesiveness. Ser. 22: 97-187, 1970. Haemat. 1: 99-145, 1968. 21. O ’B r ie n , J. R.: Platelet aggregation. II. Some 8 . H e l l e m , A. J.: The adhesiveness of human results from a new method of study. J. Clin. blood platelets in vitro. Scand. J. Clin. Lab. Path. 15: 452-455, 1962. Invest. 12(Suppl. 51): 1-117, 1960. 22. R o z e n b e r g , M. a n d H o l m s e n , H .: Adenine 9. H o l m s e n , H., D a y , H. J., a n d S t o r m , E.: Adenine nucleotide metabolism of blood plate nucleotide metabolism of blood platelets. II. lets. VI. Subcellular localization of nucleotide Uptake of adenosine and inhibition of ADP- pools with different functions in the platelet induced platelet aggregation. Biochim. Bio release reaction. Biochim. Biophys. Acta 186: phys. Acta 155: 342-352, 1968. 254-266, 1969. 23. S a l z m a n , E. W.: Measurement of platelet 10. H o l m s e n , H ., D a y , H . J., a n d S t o r m o r k e n , adhesiveness. A simple in-vitro technique H .: The blood platelet release reaction. Scand. demonstrating an abnormality in von Wille J. Haemat. (Suppl. # 8 ), 3-26, 1969. brand’s disease. J. Lab. Clin. Med. 62: 724- 11. H o v ig , T.: Aggregation of rabbit blood plate 735, 1963. lets produced in vitro by saline “extract” of 24. S p a e t , T. H. a n d C in t r o n , J.: Studies on tendons. Thromb. Diath. Haemorrh. 9: 248- platelet factor 3 availability. Brit. J. Haemat. 263, 1963. 11: 269-275, 1965. 12. H u t t o n , R. A.: Tests of platelet function 25. W e is s , H . J.: Platelet aggregation, adhesion and their use in the investigation of bleeding and adenosine diphosphate release in throm- disorders. J. Med. Lab. Tech. 23: 161-185, bopathia (Platelet factor 3 deficiency). Amer. 1966. J. Med. 43: 570-578, 1967. 13. I v y , A. C., N e l s o n , D., a n d B u c h e r , G.: Their standardization of certain factors in the 26. W r ig h t , H. P.: The adhesiveness of blood cutaneous “venostasis” bleeding time tech platelets in normal subjects with varying con nique. J. Lab. Clin. Med. 26: 1812-1816, centrations of anti-coagulants. J. Path. Bact. 1940. 53: 255-259, 1941. 14. M a c F a r l a n e , R. G.: A simple method of 27. Z u c k e r , M. B.: Tests of platelet adhesion, measuring clot retraction. Lancet i: 1199- aggregation and release. Thromb. Diath. 1202, 1939. Haemorrh. In press.