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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