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None • Marleen None Vrielink & Neyrinck; ASFA May 2016 1 This presentation is intended for teaching purposes solely and not for publication.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 Vrielink & Neyrinck; ASFA May 2016 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/ Vrielink & Neyrinck; ASFA May 2016 3 http://nl.depositphotos.com/1701375/stock-photo-varicoloured-marbles.html 8 4 Separation of blood components Centrifugal separation: • Separation based on the presence of cells • Separation based on specific gradients Vrielink & Neyrinck; ASFA May 2016 Hematology, Immunology and Blood transfusion Basics for Apheresis Copyright © Marleen Neyrinck Hans Vrielink 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 Vrielink & Neyrinck; ASFA May 2016 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) Vrielink & Neyrinck; ASFA May 2016 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 Vrielink & Neyrinck; ASFA May 2016 8 Mononuclear cells www.pathologystudent.com www.profelis.org Vrielink & Neyrinck; ASFA May 2016 Polymorphonuclear cells Eo Baso www.enc.edu Neutro www.vetmed.ed.edu www.classconnection.s3.amazonaws.com Vrielink & Neyrinck; ASFA May 2016 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 Vrielink & Neyrinck; ASFA May 2016 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 Vrielink & Neyrinck; ASFA May 2016 10 White blood cells: Function • Monocytes • Phagocytosis cellular debris and pathogens • Activation T-cells Vrielink & Neyrinck; ASFA May 2016 White blood cells: Function • Basophils • Allergic and antigen response • Eosinophils • Parasitic infections & allergic reactions • Neutrophils • Bacterial and fungal infections Vrielink & Neyrinck; ASFA May 2016 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% Vrielink & Neyrinck; ASFA May 2016 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) Vrielink & Neyrinck; ASFA May 2016 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 Vrielink & Neyrinck; ASFA May 2016 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 Vrielink & Neyrinck; ASFA May 2016 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 Vrielink & Neyrinck; ASFA May 2016 15 Blood types • AB0 • Rhesus (CcDEe) •Kell •Duffy •Kidd •MNSs • Lewis Karl Landsteiner (1868 – 1943) • Lutheran •P • Others Vrielink & Neyrinck; ASFA May 2016 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 Vrielink & Neyrinck; ASFA May 2016 Blood types • AB0 Natural/regular antibodies • Rhesus (CcDEe) •Kell •Duffy •Kidd •MNSs Irregular antibodies • Lewis • Lutheran •P • Others Vrielink & Neyrinck; ASFA May 2016 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 Vrielink & Neyrinck; ASFA May 2016 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 Vrielink & Neyrinck; ASFA May 2016 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) Vrielink & Neyrinck; ASFA May 2016 19 40 Vrielink & Neyrinck; ASFA May 2016 20 Vrielink & Neyrinck; ASFA May 2016 42 Vrielink & Neyrinck; ASFA May 2016 21 Platelet activation http://courses.washington.edu/conj/bloodcells/platelets.htm Vrielink & Neyrinck; ASFA May 2016 Von Willebrand Factor •Produced in • Endotheleal cells (Weibel-Palade bodies) • Megakaryocytes (α-granulae platelets) Vrielink & Neyrinck; ASFA May 2016 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) Vrielink & Neyrinck; ASFA May 2016 48 Vrielink & Neyrinck; ASFA May 2016 24 Platelet aggregation platelet platelet platelet platelet von Willebrand factor fibrinogen Endothelial cells 49 Vrielink & Neyrinck; ASFA May 2016 Fibrinogen binding receptor HPA-1/24? Vrielink & Neyrinck; ASFA May 2016 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 53 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