Apheresis Basic Science

Hans Vrielink, MD, PhD Sanquin Blood Supply, Amsterdam, The Netherlands

Marleen Neyrinck, RN AZ Delta, Roeselare, Belgium

Disclosure of Relevant Financial Relationships

• Hans None • Marleen None

Vrielink & Neyrinck; ASFA May 2016

1 This presentation is intended for teaching purposes solely and not for publication.

Vrielink & Neyrinck; ASFA May 2016

Basic science

• Hematology • Physiology

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2 Apheresis

Method of obtaining one or more blood components by machine processing of whole blood in which the residual components of the blood are returned to the donor / patient during or at the end of the process.

Guide to the preparation, use and quality assurance of blood components European committee on Blood Transfusion 2015-18th Ed.

Vrielink & Neyrinck; ASFA May 2016 Transfusion August 2000

Separation

https://www.pinterest.com/jufjanneke/knikkerbanen/

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3 http://nl.depositphotos.com/1701375/stock-photo-varicoloured-marbles.html

4 Separation of blood components

Centrifugal separation: • Separation based on the presence of cells • Separation based on specific gradients

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Hematology, Immunology and Blood transfusion

Basics for Apheresis

5 Blood

• White blood cells / Leukocytes • Lymphocytes (T / B-cells) • Monocytes • Granulocytes (Baso, Eo, Neutro) • Stem cells (CD34 positive cells) • Red blood cells / Erythrocytes • Platelets / Thrombocytes •Plasma

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STEM CELLS DEVELOPMENT STADIA MATURE BLOOD CELLS

IL-1 IL-2 IL-6 B-LYMPHOCYTE

Lymphoid Precurser- Stem cell lymphocyte IL-2 IL-6 Hematopoiesis T-LYMPHOCYTE

Pluripotent Stem cell IL-3 IL-3 IL-6 IL-6 GM-CSF Monocyt MACROPHAGE GM-CSF M-CSF

G-CSF GM-CSF Myeloid NEUTROPHIL GRANULOCYT Stem cell

IL-1 GM-CSF IL-3 Promyelocyte EOSINOPHIL GRANULOCYTE IL-6 GM-CSF G-CSF

IL-3 BASOPHIL GRANULOCYTE

IL-1 EPO IL-3 GM-CSF Erythroblast ERYTRHOCYTES EPO Red blood cells

IL-3 IL-6 IL-1 IL-3 IL-1 IL-2 THROMBOCYTES12 GM-CSF IL-6 IL-3 IL-6 Platelets EPO EPO Megakaryocyte EPO GM-CSF GM-CSF

6 Bone marrow

http://neurobio.drexelmed.edu Vrielink & Neyrinck; ASFA May 2016

Mobilization & Homing

http://circres.ahajournals.org/content/106/5/854/F2.large.jpg Vrielink & Neyrinck; ASFA May 2016

7 Leukemia

• Lymphoid leukemia • Acute lymphoid leukemia (ALL) • Chronic lymphoid leukemia (CLL) • Myeloid leukemia • Acute myeloid leukemia (AML) • Chronic myeloid leukemia (CML)

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White blood cells / Leukocytes

• Lymphocytes • B-cells Mononuclear cells • T-cells (MNCs) • CD34 +ve cells / Progenitor cells • Monocytes • Granulocytes Polymorphonuclear cells • Basophils (PMNCs) • Eosinophils • Neutrophils

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8 Mononuclear cells

www.pathologystudent.com

www.profelis.org

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Polymorphonuclear cells Eo

Baso

www.enc.edu Neutro

www.vetmed.ed.edu

www.classconnection.s3.amazonaws.com

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9 Leukocytes

• Many types www.en.wikipedia.org

• 3-10 x 109 cells / liter (3,000 – 10,000 cells / mm3 or / µL)

• Size 6 – 30 µm

• Life span of 2 days – many years

• Function: defense against both infectious disease and foreign materials

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White blood cells: Function

• B-lymphocytes • Antibody production & assist activation T-cells • T-Lymphocytes • T-helper (CD4+ve)  activate and regulate B-cells • CD8+ve  viruses and tumor cells • ɣσT-cell  first line of defense • Regulatory T-cells  prevents autoimmunity and back regulation to normal • Natural killer cells  viruses and tumor cells

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10 White blood cells: Function

• Monocytes • Phagocytosis cellular debris and pathogens • Activation T-cells

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White blood cells: Function

• Basophils • Allergic and antigen response • Eosinophils • Parasitic infections & allergic reactions • Neutrophils • Bacterial and fungal infections

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11 Vrielink & Neyrinck; ASFA May 2016

Red blood cells (erythrocytes)

www.withfriendship.com • RBCs released from the bone marrow as reticulocytes

• Reticulocytes: normally 0.5 – 1.5%

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12 Red blood cells

• Oval and flexible biconcave disks

• ± 5 x 1012 cells / liter (5,000,000 cells / mm3 or / µL)

• Size 6-8 µm

• Life span of ± 120 days

• Function: Oxygen transport (hemoglobin protein)

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Hemoglobin

CH322CH CH COOH

N CH22CH CH2 CH COOH NNFe

CH3 CH3 N

CH2 CH CH3 Vrielink & Neyrinck; ASFA May 2016

13 Hemoglobin

• 90-95% of the dry content of RBCs

• Females 12.0 – 16.0 g/dL www.en.wikipedia.org 7.4 – 9.9 mmol/L

• Males 13.5 – 17.5 g/dL 8.4 – 10.9 mmol/L

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Hematocrit

• The volume percentage of red blood cells in blood

• Females 0.36 – 0.46 L/L (36 – 46%)

• Males 0.41 – 0.53 L/L (41 – 53%)

www.docstock.com

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14 http://www.interactive-biology.com/wp-content/uploads/2012/07/RedBloodCells3.jpg

RBC membrane

OUTER SURFACE CELL Oligosacharid- chain

Glycolipid Hydrophil protein segment

Protein phospholipid

Hydrophobic Protein segment Cholesterol

INNER SURFACE CELL

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15 Blood types

• AB0 • Rhesus (CcDEe) •Kell •Duffy •Kidd •MNSs • Lewis Karl Landsteiner (1868 – 1943) • Lutheran •P • Others

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AB0 antigen

A A antigen

Gal NAGA Red Blood Cell Fuc H H antigen

Gal Gal

Precurser Fuc substance

B antigen

Gal Gal

Fuc B Vrielink & Neyrinck; ASFA May 2016

16 Rhesus D antigen

http://www.ncbi.nlm.nih.gov/projects/gv/rbc/images/systemimages/rhmodel.gif

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Blood types

• AB0 Natural/regular antibodies • Rhesus (CcDEe) •Kell •Duffy •Kidd •MNSs Irregular antibodies • Lewis • Lutheran •P • Others

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17 AB0 blood type

BLOOD TYPE ABAB0

GENOTYPE AA and A0 BB and B0 AB 00

ANTIGENS ON A antigen B antigen A and B antigens No A, No B (H) RED BLOOD CELL

ANTIBODIES No anti-B IN BLOOD Anti-B Anti-A No anti-A Anti-A and Anti-B

CAN RECEIVE BLOOD FROM A and 0 B and 0 A, B, AB and 0 0

CAN DONATE A and AB B and AB AB A, B, AB and 0 BLOOD TO

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Blood transfusion: RBCs

0 pos 0 neg A pos A neg B pos B neg AB pos AB neg 0 pos 0 neg A pos A neg B pos B neg AB pos AB neg

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18 Blood transfusion: Plasma

0 pos* 0 neg A pos* A neg B pos* B neg AB pos* AB neg 0 pos 0 neg A pos A neg B pos B neg AB pos AB neg *Provided that RBCs < 1x108/unit Vrielink & Neyrinck; ASFA May 2016

Platelets

• Irregularly shaped clear cell fragments

• 150-400 x 109 cells / liter (150.000 – 400.000 cells / µL or / mm3)

• Size: 1-3 µm

• Lifespan of 7 - 9 days

• Function: primary hemostasis

• Increased risk for bleeding if < 10 x 109 cells / liter (10.000 cells / µL)

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21 Platelet activation

http://courses.washington.edu/conj/bloodcells/platelets.htm

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Von Willebrand Factor

•Produced in • Endotheleal cells (Weibel-Palade bodies) • Megakaryocytes (α-granulae platelets)

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22 http://stroke.ahajournals.org/content/43/ 2/599/F1.expansion.html Von Willebrand Factor

• Glycoprotein • Circulates in plasma • Size 850 20,000 Dalton 80 subunits of each 250,000 Dalton

• Size ±100 μm https://upload.wikimedia.org/wikipedia/com mons/thumb/3/3c/PBB_Protein_VWF_imag • Only “stretched” large multimers are functional e.jpg/250px-PBB_Protein_VWF_image.jpg

https://www.tcd.ie/IMM/haemostasis/projects/ von-willebrand-factor.php Vrielink & Neyrinck; ASFA May 2016

Platelet adhesion (adhesion to vascular endothelial cells)

platelet von Willebrand factor

Endothelial cells

https://classconnection.s3.amazonaws.com/445/flashcar ds/491445/jpg/platelet_adhesion1314219847599.jpg Vrielink & Neyrinck; ASFA May 2016

23 Von Willebrand Factor

• “Regulation” by cleaving protein (ADAMTS 13)

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48 Vrielink & Neyrinck; ASFA May 2016

24 Platelet aggregation

platelet

platelet von Willebrand factor

fibrinogen

Endothelial cells 49 Vrielink & Neyrinck; ASFA May 2016

Fibrinogen binding receptor

HPA-1/24?

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25 51 Vrielink & Neyrinck; ASFA May 2016

QUANTITATIVE IMPORTANT ELEMENTS OF PLASMA

Water 91% Albumin 3.2 – 5.0 g per 100 ml Plasma Fibrinogen 0.20 – 0.45 g per 100 ml Globulins • ±5% body weight Alfa 0.40 – 0.98 g per 100 ml • Composition Bèta 0.56 – 1.06 g per 100 ml • ±91% water Gamma 0.44 – 1.04 g per 100 ml •salts Glucose 61 - 130 mg per 100 ml • minerals • carbohydrates Cholesterol 128 - 347 mg per 100 ml •fats Bilirubin 0 – 1.1 mg per 100 ml • proteins (>100 different) Urea 13.8 – 39.8 mg per 100 ml • Function: various Sodium 310 - 356 mg per 100 ml Potassium 12 - 21 mg per 100 ml Calcium 8.2 – 11.6 mg per 100 ml Iron 0.04 – 0.21 mg per 100 ml Viscosity of plasma 52 Vrielink & Neyrinck;Chloride ASFA May 2016355 - 381 mg per 100 ml

26 Clotting

Clotting factors

FXII FXIIa

FXI FXIa Tissue factor Ca2+ FIX FIXa http://www.vivostat.com/images/vivostat-fibrin-sealant/fibrin-thread.JPG?sfvrsn=2 FVIIa FVII

Ca2+ FIXa-FVIIIa Tissue factor‐FVIIa Ca2+ FX FVIII FVIIIa Ca2+ Tissue factor FXa Ca2+ FV FVa FXa-FVa Ca2+ prothrombin thrombin

Vrielink & Neyrinck; ASFA May 2016 fibrinogen fibrin

27 Vessel damage

Other clotting factors Platelets become activated Fibrinogen

Prothrombin Thrombin

Calcium Fibrin

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Interference in the hemostasis

To avoid hemostasis we interfere in • The function of the platelets • The clotting cascade

http://www.discoverymedicine.com

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28 Applied Physiology in Apheresis

Prevention of blood clotting by citrate

1914

Luis Agote Albert Hustin

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29 FXII FXIIa

FXI FXIa Tissue factor Ca2+ FIX FIXa FVIIa FVII

Ca2+ FIXa-FVIIIa Tissue factor‐FVIIa Ca2+ FX FVIII FVIIIa Ca2+ Tissue factor FXa Ca2+ FV FVa FXa-FVa Ca2+ prothrombin thrombin

Vrielink & Neyrinck; ASFA May 2016 fibrinogen fibrin

Citrate

Trisodiumcitrate • Flavoring and buffering agent in drinks / food (E330) • Prevention of blood clotting in disposable / machine • Laxans • WHO “oral rehydration solution”

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30 FXII FXIIa

FXI FXIa Tissue factor

Ca2+ FIX FIXa FVIIa FVII

Tissue factor‐FVIIa Citrate Ca2+ FIXa‐FVIIIa Ca2+ FX FVIII FVIIIa Ca2+ • Completely dissolved in plasma Tissue factor FXa • No binding to cells Ca2+ FV FVa FXa‐FVa • Chelates calcium and magnesium Ca2+ • Prevents coagulation protrombin trombin fibrinogen fibrin “Neutralized” by: • Distribution throughout extra cellular fluid • Excretion by the kidneys (± 20%) • Rapid metabolism in mitochondria kidneys, liver and skeletal muscle

T1/2 = 36 ± 18 minutes Jeff Winters, AABB Oct. 2007

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Calcium

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31 Calcium

• 99% in the bones  calcium phosphate (± 24,500 mmol)

Extra cellular fluid: 22.5 mmol  9 mmol in plasma (2.2 – 2.6 mmol/L) • Ionized (free) calcium 1.1 – 1.4 mmol/L (4.5 – 5.6 mg/dL) • Remainder bound mainly to albumen (± 50%)

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Calcium metabolism

• Active intake in intestines • Excretion via Kidney: • 250 mmol/day in pre-urine Parathyroid hormone (PTH) • Reabsorption of 245 mmol/day • Exchange blood – bone

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32 Calcium metabolism

http://www.en.wikipedia.org Vrielink & Neyrinck; ASFA May 2016

Function of Calcium

• Structural function  bones • Signaling function  messenger for some hormones • Enzymatic function  co-enzyme for clotting factors • Function in transmission of nerve impulse • Function in the contraction of muscles Using citrate to inhibit • Stabilization of cellular membranes hemostasis during apheresis

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33 Serum calcium & citrate infusion

Bolan, Transfusion 2002 Vrielink & Neyrinck; ASFA May 2016

Serum PTH and Calcium

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34 Serum PTH, iCa and citrate during plt apheresis

McLeod BC, Szczepiorkowski ZM, Weinstein R, Winters JL, eds; Apheresis: Principles and Practice, 3rd edition; Bethesda, MD: AABB Press, 2010 Vrielink & Neyrinck; ASFA May 2016

Function of calcium

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35 Hypocalcaemia by citrate

•Decrease in ionized calcium results in increased excitability of neurons to the point of spontaneous depolarization.

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Citrate → Metabolic Alkalosis & Hypokalemia

Additional factors to consider • Presence of citrate in replacement fluids (e.g. FFP for TTP) • Large volume PBSC collections (average drop Ca2+ of 11.3±7%) •Citrate  Bi-carbonate  Alkalosis

Ca2+

Mg2+

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36 Citrate metabolism

• Metabolism in mitochondria of kidney, liver and skeletal muscles • Excretion by kidneys (± 20%) • Half-life of citrate: 36 ± 18 minutes

Calcium in urine ↑

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FXII FXIIa

FXI FXIa Tissue factor

Ca2+ FIX FIXa FVIIa FVII

T1/2 = 36 ± 18 minutes Jeff Winters, AABB Oct. 2007

Vrielink & Neyrinck; ASFA May 2016

37 Serum magnesium during plateletapheresis

Bolan, et al. Transfusion 2001;41:1165-1171 Vrielink & Neyrinck; ASFA May 2016

Magnesium

Involved in: • Synthesis of nucleic acids • Synthesis of proteins • Intermediary metabolism • Specific actions in • Neuromuscular systems • Cardiovascular systems

Mg2+ competes with Ca2+ for binding sides on proteins and membranes

Competitive inhibition of calcium

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38 Magnesium

Affects: • Muscular contraction and relaxation including the heart and vascular muscles. • Electrical activity of myocardial cells • Stabilization of the axon • The release of neurotransmitters

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Therapeutic apheresis procedures

• Collection volume 1000 – 5000 mL • considerable volume of this is anticoagulant

COMPENSATION / REPLACEMENT IS NEEDED

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39 McLeod BC (editor). Apheresis: Principles and Practice, 2nd edition. AABB Press, Bethesda, Maryland, 2003.

Intra- and extra vascular compartments Capillary wall

extra Intra cellular compartment

Colloid osmotic pressure

intra 3 ltr 9 ltr 27 ltr

Hydrostatic pressure Vrielink & Neyrinck; ASFA May 2016

40 Intra- and extra vascular compartments Capillary wall

extra

Colloid osmotic pressure

intra 3 ltr 9 ltr

Hydrostatic pressure Vrielink & Neyrinck; ASFA May 2016

McLeod BC (editor). Apheresis: Principles and Practice, 2nd edition. AABB Press, Bethesda, Maryland, 2003.

41 Replacement fluids

• Crystalloids • Solutions of mineral salts or other water-soluble molecules, e.g. NaCl 0.9% • Colloids • Fluids with larger insoluble molecules, e.g. starches, gelatins, albumin, plasma

Vrielink & Neyrinck; ASFA May 2016

Replacement fluids

Van Zundert, et al. CPD Anaesthesia, 2006;8:131-149.

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42 Saline 0.9% vs Lactated Ringer’s in Volunteers SodiumN=18 Chloride (cross over) Poisoning 90 80 70 60 Confusion % 50 Headaches 40 30 Nausea and Vomiting 20 10 Abdominal pains 0 CNS changes Abdominal discomfort

Saline Ringer's

Williams et al, Anesthesia and Analgesia. 1999 Vrielink & Neyrinck; ASFA May 2016

Exchange fluids and volume effects Initial volume effects (%) Duration (hrs) Crystalloids NaCl 0.9% 20% 0-1

Colloids HES 130 6% 100% 3-6 HES 200 6% 100% 3-4 HES 200 10% 145% 3-4

Gelofusin® 70-80% 2-3 Geloplasma® 60-80% 2-3 Haemacel® 70% 2-3

Dextran 5% 100% 2-4 Dextran 10% 200% 2-4 Dextran 6% 120% 6-8

Albumen 20% 300% 2-8

Adapted from: van Zundert, et al. CPD Anaesthesia, 2006;8:131-149. Vrielink & Neyrinck; ASFA May 2016

43 Finalizing

• Hematology • Blood cells • Blood types • Hemostasis

• Physiology •Citrate • Calcium • Magnesium • Plasma replacement fluids • Fluid compartments