QIA REVIEW SESSION: BASIC SCIENCE IN APHERESIS Nicole Aqui, M.D. Chief, Transfusion and Apheresis Services Division of Transfusion Medicine Department of Pathology and Laboratory Medicine University of Pennsylvania

Disclosure • No relevant conflicts.

Outline • Immunohematology/Genetics

• ABO • Blood component therapy

• Immunology • HLA • Antibodies • Antibody-mediated diseases

• Apheresis

Immunohematology/Genetics: ABO

Adapted from U.S. National Library of Medicine

A and B Antigens • Carbohydrates antigens • On RBC membrane and secreted GPs • Copies per RBC

• A1 800,000 – 1,170,00 • A2 240,000 – 290,00 • B 610,00 – 830,00

ABO Antigen Structure

Gal GlcNAc

Gal

Fuc

Gal

Gal-NAc

Gal GlcNAc

Gal

Fuc

Gal GlcNAc

Gal

Fuc

transferase

A

B

H Antigen (O) A Antigen

B Antigen

A and B Antibodies • IgM • Naturally occurring

• Exposure to bacteria • Detected in the first months of life

• 95% have isohemagglutinin by 1 yr age • Anti-A,B is IgG found in type O

ABO Summary of Antigen/Antibody

Why do we bother with ABO typing?

End Organ Damage

Immunohematology/Genetics: ABO

Hemolysis

ABO Frequency and Compatibility

+ - - + 41 A,O A, AB

- + + - 10 B,O B, AB

+ + - - 4 All AB

- - + + 45 O All AB

A B

O

α-A

A1

Freq (%) Comp RBCs

Comp Plasma

Reagent anti-sera

Reagent red cells

Measurement of Agglutination

Distribution in different populations

Immunohematology/Genetics: Blood Component Therapy

Blood Component Therapy Component Storage

Temp/Shelf Life Vol Indication

Whole blood 2-6oC / 35 days

500 ml Acute hypovolemic anemia, exchange transfusions

Packed red cells 2-6oC / 35 - 42 days

250 - 300 ml

Symptomatic anemia

Single donor platelets

20-24oC / 5 days

150 - 300 ml

Bleeding due to thrombocytopenia or platelet defects

Fresh frozen plasma

-18oC / 12 months

250 ml Replacement of multiple coagulation factors, DIC, TTP, warfarin reversal

Cryoprecipitate -18oC / 12 months

5 - 10 ml Replacement of fibrinogen, FVIII, vWF

Immunology - The HLA System • Human leukocyte antigen

(HLA), also referred to as MHC

• Highly polymorphic, polygenic, clustered on chromosome 6

• 3 sets each of class I genes (A, B, C) and class II genes (DR, DQ, DP)

• Thus, most individuals express 6 different MHC class I molecules and 6 different MHC class II molecules (co-dominant expression from maternal and paternal chromosomes)

Expression of MHC Class I vs. Class II

MHC-T cell interactions

T cell activation requires 2 signals

TCR

CD40L CD40

Antigen

MHC Class II CD4+

T cell

Dendritic cell

T cell responses are restricted

T cell responses are restricted, however...

Mechanisms of Allorecognition

Immunology - Antibodies

Antibody Functions

Mechanisms Antibody-Mediated Diseases

Examples of Antibody-Mediated Diseases • Myasthenia gravis • Guillain-Barre syndrome • Chronic inflammatory demyelinating polyneuropathy

(CIDP) • Goodpasture’s syndrome • Transplant rejection

FINALLY - Apheresis • Apheresis is Greek for “to take away” or “subtract” • In hemapheresis we take away part of the blood

• Plasmapheresis – remove plasma • Leukopheresis – remove white blood cells • Erythropheresis – remove red blood cells • Plateletpheresis – remove platelets

• Originally performed discontinuously • Now performed with continuous removal and

separation of blood components

Modern Apheresis

Alan (Jack) Latham, Jr.

Edwin Cohn

Developed cold ethanol fractionation to produce albumin

Methods of Separation • Centrifugation • Membrane Filtration

Methods of Separation: Centrifugation

Methods of Separation: Membrane Filtration

Outline of an Apheresis Procedure • IV access obtained (single vs. double needle) • Drawn blood is anticoagulated (citrate or heparin)

prior to entry into separator • Anticoagulated blood is pumped into bowl/belt

chamber • Blood components are separated by density and

desired component is removed • Remaining blood is returned, mixed with

replacement fluid if required.

Plasma Exchange Circuit

Centrifugation vs. Filtration

Centrifugation

• Low blood flow rate • Citrate • Special equipment, but

more versatile

Filtration

• High blood flow rate • Heparin • Existing dialysis

machines • Simpler • Limited to plasma

exchange

Replacement Fluid in Plasma Exchange • Cystalloid (normal saline)

• Cheap • Hypo-oncotic • No coagulation factors or immunoglobulins

• Colloid (albumin) • EXPENSIVE • Slightly hyper-oncotic, can result in volume expansion • Very low risk of infectious disease transmission • No coagulation factors or immunoglobulins

• Plasma • Cheaper than albumin • Iso-oncotic • Associated risks of all blood product transfusions (infectious

disease, allergic reactions, TRALI)

Selective removal/adsorption column • Plasma separated by centrifugation or filtration • Passed over a selective column/filter

• LDL Apheresis: dextran sulfate column

Plasma Line

LIPOSORBER® Column

Plasma Pump

LIPOSORBER® SYSTEM

Blood Pump

Heparin Pump

Blood Return

Plasma Separator

Regeneration Pump

Re-Priming Solution

Regeneration Solution

Waste Line

Kanaka Pharma America Corporation

Effects of Plasma Exchange

From McLeod; Apheresis: Principals and Practice (2003)

• Dose = Plasma volume (PV) • PV = BV x (1-Hct) = (70ml/kg x Wt) x (1-Hct) • 1 PV = ~60% removal • No benefit in exchanging > 2

PV

Alteration in Blood Constituents

From McLeod; Apheresis: Principals and Practice (2003)

Constituent Percent Decrease from Baseline

Percent Recovered at 48 Hours

Clotting factors 25 – 50 80 – 100

Fibrinogen 63 65

Immunoglobulins 63 ~45

Paraproteins 33 – 60 Variable

Liver enzymes 55 – 60 100

Bilirubin 45 100

C3 63 60 – 100

Platelets 25 – 30 75 – 100

Removal of Drugs • In general, drugs with a low volume of distribution

(Vd) and/or a high rate of protein binding are most likely to be removed during plasma exchange • Cisplatin • Vincristine • Propanolol • Gentamycin • Vancomycin • Rituximab • IVIG

Pharmacotherapy 2007;27(11):1529–1549

Adverse Reactions • Vascular Access

• Hematoma • Clotting of line • Insufficient for pressures required

• Central line • Fistula • Graft

• Infection • More problematic with central lines than with

anticubital access

Adverse Reactions • Vaso-vagal reactions

• Pallor • Hypotension • Diaphoresis • Bradycardia • Nausea/vomiting

• More common with plasmapheresis than with cytapheresis

• ACE inhibitors exacerbate

Adverse Reactions • Citrate toxicity

• Temporary decrease in Cai2+

• Tingling, numbness, nausea • Calcium supplementation: calcium carbonate, calcium

gluconate, calcium chloride • Coagulation alterations

• Daily TPE without plasma replacement can deplete coagulation factors and increase bleeding

• Consider FFP replacement with daily exchange and in patients with coagulation abnormalities

• Monitor fibrinogen

Summary • ABO is the most important blood antigen group. • The HLA system is complex, highly polymorphic and

polygenic. • Antibodies have several protective functions, but

can also cause disease. • It is important for the apheresis operator to have an

understanding of basic transfusion medicine, as well as immunology and the mechanisms underlying broad categories of disease.

Acknowledgments UPENN Transfusion Medicine Faculty • Don Siegel, MD PhD • Una O’Doherty, MD PhD • Taku Kambayashi, MD PhD • Vijay Bhoj, MD PhD • Andrew Fesnak, MD PhD • Carl June, MD • Bruce Levine, PhD

UPENN Apheresis Staff • Leah Irwin, Nurse Manager • Lita Jamensky • Caitlin Cahill • Melissa Murter • Jane Mason • Jennifer Green • Marcia Hole • Kevin Schell • Jennifer Schwartz • Julie Colanero • Kelli Hines • Colleen Henry