Cohn de Laval Award Lectureship
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2 Cohn de Laval Award Lectureship The Science Behind the Success Development of a Continuous Flow Blood Cell Separator Jeane P Hester, MD Accepted on behalf of Dr Hester and presented by April G Durett, MSc
3 1 st International Congress World Apheresis Association May 20-23, 1986 Tokyo Japan Crown Prince and Princess Takamata 3
4 Chronology Development of Blood Components Need for red blood cells for anemic patients Search for anticoagulants sodium citrate and mechanism of calcium binding Military needs in 2 wars emphasized need for expanding RBC collection and storage Glucose / dextrose added to sodium citrate for RBC metabolism Citric Acid added to reduce K + loss Tullis describes development of Cohn Fractionator resin column removed Ca ++ and platelets allowing passage of RBC and plasma Bierman uses fractionator to deplete leukocytes from a patient Freireich uses single unit leukocytes from CML patients to treat neutropenic infections 4
5 Chronology Development of Blood Components NCI and IBM (Freireich and Judson) development of first continuous flow blood cell separator - IBM 2990 for collection of leukocytes from normal donors Hester and IBM (Kellogg & Mulzet) development next generation continuous flow blood cell separator - IBM 2997 Hester and COBE/BCT development of COBE Spectra continuous flow blood cell separator IBM 2997 at MDACC IBM 2990 / George Judson IBM Archives COBE Spectra 5
6 Design Goals of the First Continuous Flow Cell Separator IBM 2990 Sedimentation in a centrifuge - Leukocytes should be separated from WB at a reasonable efficiency Processing large quantities of blood operation performed on a continuous flow basis Avoid arterial puncture vein-to-vein procedure Anticoagulant that does not require systemic anticoagulation Minimize Platelet, Red Cell and Plasma loss allow processing large volumes of WB from a single donor Entire system should contain 500mL of blood System should allow control by a single operator 6
7 IBM 2990 Bowl Citrated blood enters the bowl, passes vertically to bottom Separated at 850rpm into Packed RBC Leukocyte-platelet rich buffy coat Plasma 3 peristaltic pumps draw separated components to surface for collection or returned to the donor Separate pump infused ACD-A 7
8 Initial Clinical Trials at MDACC Collection of leukocytes from CML patients Provide information on safety and operating effectiveness Potential citrate toxicity Flow rates <50mL per min no adverse events Higher flow rates Parenthesia (face and fingers) Normal ECG QT intervals plasma citrate levels <25-35mg% Decreasing flow rate prompt reversal 8
9 IBM 2990 Used at MDACC Operator controlled pumps Plasma RBC Inlet ACD-A Saline Bowl is mounted in a well 9
10 Relationship between RPM and Leukocyte Separation RPM WBC (E3 per µl) 3,200 1,700 1,500 17, ,000 Relationship established with 2990 Data from CML donors Best separation at low RPM ,000 EJ Freireich, G Judson, and RH Levin Separation and Collection of Leukocytes Cancer Res 25(9) : 1516,
11 Effect of Hematocrit (HCT) on Leukocyte Differential Relationship established with 2990 Data from Normal donors Sample WBC (E3 per µl) MNC (%) PMN (%) HCT (%) 1 7, % 2 16, % 3 12, % 4 13, % 5 3, % EJ Freireich, G Judson, and RH Levin Separation and Collection of Leukocytes Cancer Res 25(9) : 1516,
12 IBM 2997 First unit of the 2997 manufactured was donated to MDACC Apheresis Clinic by IBM 12
13 Disposable channels with Circumferential blood flow Single Stage Dual Stage Collection / Depletion Leukocytes Collection single donor platelets Plasma Exchange Transfusible quantity RBC / Buffy Coat interface stabilized Ready for infusion Jeane P Hester et al, Blood 54(1) : 254,
14 Developments to Improving Granulocyte Yields Hydroxyethyl Starch (HES) Rouleaux formation improved separation of RBC and Granulocytes High molecular weight derivative starch amylopectin Deposition in skin and spleen that possibly led to toxicity Dexamethasone steroid granulocyte mobilizing agent Donors selected from primary family members HLA shared antigens Benefits observed of Granulocyte replacement to neutropenic patients Inability to establish benefits with certainty Limitations in available donors Restrictions in frequency of donation Patient variability uncertainty of response to antibiotics Diversity of pathogenic organisms Duration of neutropenia Currently GCSF increase granulocyte yields Low molecular weight HES shortened excretion time 14
15 Collection of Platelets using Dual Stage Channel Mid 1960s and Early 1970s Pooled units of platelet concentrates from single units whole blood Manual technique involved 2 centrifugation steps Units of packed red blood cells Platelet-poor plasma Platelet concentrates Platelet clumps units left standing until platelets re-suspended Major goal of IBM 2997 perform these steps in continuous process Question : ACD-A flow rate to assure donor safety Question : Citrate concentration in blood being processed to keep platelets in suspension 15
16 ACD-A mg / kg per min Total Citrate mg per min ACD-A Dose Administration to Donors & Patients ACD-A ml / L BV per min Reference CD Bolan et al, Transfusion 42 : 935, S Haddad et al, Transfusion 45 : 934, CD Bolan et al, Transfusion 43 : 1403, CD Bolan et al, Transfusion 41 ; 1165, CD Bolan et al, Transfusion 42 : 935, JP Hester WBC / PLT JP Hester PLT Deplt JP Hester TPE Standard Dose for 2997 & Spectra Calcium replacement Blood Volume estimates H Chaplin & PL Mollison, Blood 7 : 1227,
17 ACD-A Dose Administration to Donors & Patients Standard 1 ml ACD-A per L Blood Volume per min over 90 min procedure Modest increase in citrate 15%-20% reduction Ionized Calcium Symptoms (if present) mild and transient in donors 4.0L TBV Increase ACD-A flow rates ml per L Blood Volume per min Hyopcalcemic symptoms early and more severe Calcium replacement intravenous Calcium Chloride 17
18 Albumin / Globulins 18 Calcium Distribution Ion mm per L % Total Ionized Ca % Protein Bound % Ca-Phosphate % Ca-Citrate % Unidentified Prothrombin Factor X Factor IX Factor VII Factor XI Factor XII Platelets %
19 Calcium Distribution Unstimulated Platelets 8.60 x10 4 high affinity Ca ++ binding sites per platelet 2.89 x10 5 low affinity Ca ++ binding sites per platelet Activated Platelets 1.83 x10 5 new Ca ++ binding sites per platelet Membrane proteins IIb and IIIa for Ca++ dependent complex Ca ++ required for binding of fibrinogen to its receptor Platelet aggregation Presence of Ca ++ and fibrinogen Removal of Ca ++ from surface during mixing with ACD-A inhibits platelet function Calculation of intravasclar platelet pool in a helathy adult 250,000 platelets per µl contains ~10mg Ca ++ 19
20 Citrate Concentration in Whole Blood (WB) Citrate Solution Ratio Citrate : WB mg Citrate per ml WB Apheresis Procedures ACD-A Sodium Citrate (1940s) ACD-A (1950s) Sodium Citrate (PT ) ACD-A + Citric Acid Prothrombin Time Aster et al, J Clin Invest 43 : 843,
21 Citrate Concentration in Whole Blood (WB) Citrate mg per ml Ratio ACD-A : WB % Ca++ Bound ph % % % % % % % Keep Platelets from clumping 1964 Richard Aster lower WB to ph6.5 Jeane Hester proposed ACD-A : WB ratio 6-8 with >2.66mg Citrate per ml 21
22 Binding of Ionized Calcium in Peripheral Blood Related to Citrate Concentration 100% 80% Ionized Calcium Bound 60% 40% 20% 0% Ratios above 1:15 may not bind sufficient calcium to inhibit platelet aggregation and coagulation protein function Ionized Citrate (mg per ml) 22
23 Therapeutic Plasma Exchange Robert Kellogg s mathematical model vs Plasma Bilirubin Levels Kellogg & Hester, Plasma Therapy & Transfusion Technology 8 : 283,1987 Routinely exchanged 1.5x Patient s Plasma Volume Biological marker bilirubin in plasma Patient had undergone partial hepatectomy for a malignancy 23
24 Mobilization from the Extravascular Space Kellogg & Hester, Plasma Therapy & Transfusion Technology 8 : 283,1987 Increase in concentration result of movement from extravascular space to intravascular space following exchange NOT rebound synthesis of pathological molecule 24
25 Therapeutic Plasma Exchange (TPE) Acute Promyelocytic Leukemia Associated with disseminated intravascular coagulopathy and severe hemorrhage TPE to remove multiple procoagulants Fresh Frozen Plasma and Cryoprecipitate replacement fluids Diffuse hemorrhage ceased Rapid rise in plasma fibrinogen concentration Onset of chemotherapy-induced leucopenia Tumor Lysis Syndrome Renal failure avoided with TPE and leukocyte depletion Acute Rhabdomyolisis Renal failure avoided with TPE Acute Respiratory Distress (ARD) Patient with essential thrombocytothemia Onset of pulmonary failure due ARD following surgery for gastric perforation Sudden platelet drop 700,000 per µl to <100,000 in absence of active hemorrhage Consumption process which responded to TPE 25
26 COBE Spectra Principle objective automation of all procedures Creation of algorithms for apheresis procedures Blood is heterogeneous mixture Cell and plasma molecules of various size, density, & function that could influence device performance 26
27 Therapeutic Cytoreduction First Procedure approved for Spectra Nikolai Kalinin, MD collaborator in NIH-Russia Scientific Exchange Program Jeane Hester, MD Patient undergoing platelet reduction 27
28 COBE Spectra Peripheral Blood Stem Cell (PBPC) 1986 PBPC from patients with Multiple Myeloma Bone marrows not used due to infiltration of plasma cells or presence of hypoplasia Chemotherapy mobilization before GCSF and flow cytometric identification of CD34 + cells All patients engrafted earlier than marrow transplants Auto and Allo transplant strategies developed Early 1990s processing bone marrow harvests Objective retain maximum stem cells while reducing volume, erythocte and myeloid fractions Hurdle convince transplant group to use ACD-A instead of heparin >90% mononuclear fraction freshly harvested marrow was retained MNC fraction efficiently collected in aged marrow collected in heparin platelet function lost during transportation 28
29 Collaboration with Professor Peter Jacobs University of Cape Town, South Africa Assisted in establishing Apheresis Unit 1979 obtained COBE Spectra Jeane Hester and Lucille Wood set collection standards Circa 1990 : (back row) Lucille Wood, Professor Jacobs, Dr Hester 29
30 MDACC circa 1990 Apheresis Clinic & Research Laboratory Staff 30
31 What is required in order to advance our knowledge of the natural world is following controlled and systematic procedures. First, we must observe the facts, record our observations and amass a body of reliable data. This is more effectively done by many people working in communication with one another than by individuals working alone hence the need for scientific societies and colleges. At this stage we must be careful not to impose our ideas on the facts, but to let them speak for themselves. When we have amassed enough of them they will begin to do so : regularities and patterns will begin to emerge, casual connections will reveal themselves and we shall start to perceive the laws of nature at work in the particular instances..sir Francis Bacon
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