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The Science Behind the Success Development of a Continuous Flow Blood Cell Separator
Jeane P Hester, MD
Cohn de Laval Award Lectureship
Accepted on behalf of Dr Hester and presented by April G Durett, MSc
1st International Congress World Apheresis Association
May 20-23, 1986 Tokyo Japan
Crown Prince and Princess Takamata 3
Chronology Development of Blood Components
1914 – 1940 Need for red blood cells for anemic patients Search for anticoagulants – sodium citrate and mechanism of calcium
binding
1940 – 1950 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
1950 – 1960 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
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Chronology Development of Blood Components
1960 – 1980 1965 NCI and IBM (Freireich
and Judson) development of first
continuous flow blood cell separator - IBM 2990 for collection of leukocytes from normal donors
1977-79 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 2990 / George Judson IBM Archives www.03.ibm.com
COBE Spectra www.TerumoBCT.com
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IBM 2997 at MDACC
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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
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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
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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
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IBM 2990 Used at MDACC
Bowl is mounted in a well
Operator controlled pumps
Plasma
RBC
Inlet
ACD-A
Saline
Relationship between RPM and Leukocyte Separation
RPM WBC
(E3 per µL)
3,200 1,700
1,500 17,000
600 150,000
400 258,000
Relationship established with 2990
Data from CML ‘donors’
Best separation at low RPM
EJ Freireich, G Judson, and RH Levin Separation and Collection of Leukocytes Cancer Res 25(9) : 1516, 1965
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Effect of Hematocrit (HCT) on Leukocyte Differential
Relationship established with 2990
Data from Normal donors
EJ Freireich, G Judson, and RH Levin Separation and Collection of Leukocytes Cancer Res 25(9) : 1516, 1965
Sample WBC
(E3 per µL)
MNC (%)
PMN (%)
HCT (%)
1 7,700 92 7 0%
2 16,200 91 9 0%
3 12,500 45 44 2.4%
4 13,400 17 76 4.5%
5 3,100 32 65 9.3%
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IBM 2997
First unit of the 2997 manufactured was donated to MDACC Apheresis Clinic by IBM
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Single Stage Collection / Depletion Leukocytes Plasma Exchange RBC / Buffy Coat interface stabilized
Dual Stage Collection single donor platelets
Transfusible quantity Ready for infusion
Disposable channels with Circumferential blood flow
Jeane P Hester et al, Blood 54(1) : 254, 1979
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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
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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
ACD-A Dose Administration to Donors & Patients
ACD-A mg / kg per min
Total Citrate mg
per min
ACD-A mL / L BV per min
Reference
1.0 70 0.73 CD Bolan et al, Transfusion 42 : 935, 2002
1.36 95.2 0.99 S Haddad et al, Transfusion 45 : 934, 2005
1.5 105 1.09 CD Bolan et al, Transfusion 43 : 1403, 2003
1.6 112 1.16 CD Bolan et al, Transfusion 41 ; 1165, 2001
2.2 155 1.61 CD Bolan et al, Transfusion 42 : 935, 2002
1.36 96 1.0† JP Hester WBC / PLT
1.64 115 1.2‡ JP Hester PLT Deplt
1.91 134 1.4‡ JP Hester TPE
† Standard Dose for 2997 & Spectra ‡ Calcium replacement Blood Volume estimates – H Chaplin & PL Mollison, Blood 7 : 1227, 1952
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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 1.2 – 1.4 mL per L Blood Volume
per min
Hyopcalcemic symptoms – early and more severe
Calcium replacement – intravenous Calcium Chloride
Calcium Distribution
Ion mM per L % Total
Ionized Ca++ 1.18 47.5% ‡Protein Bound 1.14 46.0%
Ca-Phosphate 0.04 1.6%
Ca-Citrate 0.04 1.6%
Unidentified Prothrombin Factor X Factor IX Factor VII Factor XI Factor XII Platelets
0.08 3.2%
‡ Albumin / Globulins
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Calcium Distribution
Unstimulated Platelets 8.60 x104 high affinity Ca++ binding sites per platelet 2.89 x105 low affinity Ca++ binding sites per platelet
Activated Platelets 1.83 x105 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++
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Citrate Concentration in Whole Blood (WB)
Citrate Solution Ratio
Citrate : WB mg Citrate per mL WB
Apheresis Procedures 6 3.55
ACD-A 8 10 13 15 20
2.66 2.13 1.64 1.42 1.06
Sodium Citrate (1940s) 5.2 3.7
ACD-A (1950s) 7.4 2.9
Sodium Citrate (PT†) 9 2.4
ACD-A + Citric Acid‡ 4.2 5.0 †Prothrombin Time ‡ Aster et al, J Clin Invest 43 : 843, 1964
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Citrate
mg per mL
Ratio
ACD-A : WB
% Ca++
Bound pH
0.5 43 59 - 71% 7.340
1.0 21 80 - 86% 7.231
1.5 14 87 – 91% 7.121
2.0 11 92 - 94% 7.038
2.5 8.5 94 - 96% 6.911
3.0 7 95 - 97% 6.794
5.0 4 96 - 98% 6.398
Citrate Concentration in Whole Blood (WB)
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
0%
20%
40%
60%
80%
100%
0 1 2 3 4 5
Ionized Calcium Bound
Ionized Citrate (mg per mL)
Ratios above 1:15 may not bind sufficient calcium to inhibit platelet aggregation and coagulation protein function
Binding of Ionized Calcium in Peripheral Blood Related to Citrate Concentration
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Therapeutic Plasma Exchange
Routinely exchanged 1.5x Patient’s Plasma Volume Biological marker – bilirubin in plasma Patient had undergone partial hepatectomy for a malignancy
Robert Kellogg’s mathematical model vs Plasma Bilirubin Levels
Kellogg & Hester, Plasma Therapy & Transfusion Technology 8 : 283,1987
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Mobilization from the Extravascular Space
Increase in concentration – result of movement from extravascular space to intravascular space following exchange
NOT rebound synthesis of pathological molecule
Kellogg & Hester, Plasma Therapy & Transfusion Technology 8 : 283,1987
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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
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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
COBE Spectra
www.TerumoBCT.com
Therapeutic Cytoreduction First Procedure approved for Spectra
Nikolai Kalinin, MD – collaborator in NIH-Russia Scientific Exchange Program
Jeane Hester, MD
Patient undergoing platelet reduction
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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
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Collaboration with Professor Peter Jacobs University of Cape Town, South Africa
Circa 1990 : (back row) Lucille Wood, Professor Jacobs, Dr Hester
Assisted in establishing Apheresis Unit 1979 obtained COBE Spectra Jeane Hester and Lucille Wood set collection standards
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MDACC circa 1990 Apheresis Clinic & Research Laboratory Staff
“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 1561-1626 31