jeremy parsons, md

Apheresis Basic Science

Jeremy Parsons MD

Presbyterian Healthcare, Albuquerque, NM

Confession

I am a museum tour guide for this topic. The curators in the back who know everything will be giving talks later throughout the meeting.

I may not know the answer to your questions off the top of my head. I do know where to find out.

I have no financial disclosures today.

Barely scratching the surface today

Outline

1)Hematology and Coagulation 2)The Immune System and Blood Antigens 3)Blood Component Therapy 4)Electrolyte Physiology 5)Common Clinical Laboratory Testing 6)Introduction to Fluid Replacement

Hematology and Coagulation

Historically blood was considered the essence of life.

Hippocrates described 4 humors: blood, phlegm, black bile and yellow bile 400 BCE.

Bloodletting was common to remove contamination and evil humors.

Breathing a Vein. James Gillray, published by H. Humphrey, St James’s Street, London, January 28, 1804.

Hematology and Coagulation

Blood consists of multiple parts

Plasma

Erythrocytes (RBC)

Leukocytes (WBC)

Platelets

Image courtesy of Fairview.org

Plasma

Liquid portion of blood

Many substance dissolved or carried

Proteins (albumin, globulins, etc.)

Nutrients (glucose, lipids, ammino acids)

Gases (CO2, N)

Metabolic waste (urea, lactic acid)

Electrolytes (Na, K, Cl)

Plasma Proteins

3 groups

1) fibrinogen / coagulation factors

2) albumin and small transport proteins

3) globulins

Erythrocytes (RBCs)

Small biconcave discs

Hemoglobin for O2 transport

Carbonic anhydrase to convert CO2 to carbonic

acid and bicarbonate

Erythrocytes produced in bone marrow

2-3 million per second.

Red blood cells. Credit: Photos.com/Rice University

Erythrocytes

Average concentration of RBCs in blood is

4.5 - 6 x 106 cells/μl.

Average life span of 120 days

Leukocytes (WBCs)

Average total WBC count in 3.2-10 X 103 cells/μl

Made up of 5 main cell types

Neutrophils

Eosinophils

Basophils

Lymphocytes

Monocytes

Photo from Wikipedia Commons

Granulocytes

Neutrophils 50-80% of WBCs

Use phagocytosis to clear blood of bacteria and foreign particles

Eosinophils 1-4% of WBCs

Main defense against parasites

Basophils <1% of WBCs

Release inflammatory chemicals such as histamine when activated by IgE.

Mononuclear Cells

Lymphocytes 20-40% of WBCs

B-cells involved with antibody production

T-cells involved with cellular immunity

Monocytes 2-8% of WBCs

Also phagocytic, circulate before migrating to tissues to become macrophages.

Stem Cells

Normally localized to the bone marrow.

Circulate in minute numbers in peripheral blood.

Can be mobilized from marrow space into the peripheral blood (steroids, GCSF, plerixafor).

CD34+

Platelets

Cellular fragments from megakaryocytes

Normal platelet count 150-400 x 109/L.

Average circulating time of 7-10 days

Primary Hemostasis

Bind to damaged endothelium of vessels via vWF

Recruit other platelets to create a platelet plug


Coagulation

Platelets are involved in primary hemostasis to create a temporary plug to seal vascular injuries

Secondary hemostasis involves plasma proteins in what is known as the coagulation cascade.

Coagulation Cascade

3 pathways in the testing model

Intrinsic (contact activation) aPTT

Extrinsic (tissue factor pathway) PT

Common

Factors I through 13 typically depicted by Roman numerals (factor IV is Calcium) (factor VI is actually activated V)

Immunity

1) Innate immunity: non-specific first response

Physical and chemical barriers

Phagocytic cells

Compliment and cytokines

2) Acquired or adaptive: specific secondary response

Lymphocytes

Immunoglobulins (Antibodies)

Acquired Immunity

T cells activated when presented with antigen in association with MHC

Important in self detection and cellular immunity

HLA recognition

B cells form plasma cellss when stimulated by antigens

Plasma cells secrete immunoglobuins

Immunoglobulins

Antibodies to specific antigens

5 types

IgA

IgD

IgE

IgG

IgM

Blood Groups

RBC membranes express membrane structures called antigens

More than 20 can be clinically significant

Play important role in transfusion, organ transplant, hemolytic disease of the newborn

RBC antigens

ABO

Antigens expressed and plasma antibodies produced by each blood type.

Photo courtesy of the University of Utah Genetics and Science Learning Center

ABO Group Prevalence

ABO GROUP European Ethnicity African Ethnicity

O 45% 49%

A 40% 27%

B 11% 20%

AB 4% 4%

Adapted from Cooling L. ABO, H, and Lewis blood groups and structurally related antigens. Technical Manual 17th ed p. 364

RBC antigen antibodies

Antibodies to A and B RBC antigens are said to be naturally occurring as they are present soon after birth and do not require exposure to the RBC antigens.

Other RBC antigens such as RH, Kell, Duffy and Kidd require exposure of the immune system

Blood Component Therapy

Can be collected via whole blood donations or aphereis procedures.

Most common types

(1)Red blood cells

(2)Plasma

(3)Platelets

(4)Cryoprecipitate

(5)Granulocytes

Red Blood Cell Units

Stored at 1-6º C with shelf life up to 42 days in additive solution

Will raise HCT 3-4% and Hgb approximately 1 g/dl

Requires either serological or electronic crossmatch


Plasma

FFP frozen to -18º C within 8 hours of collection

FP24 frozen within 24 hours

Usually used within 24 hours of thawing

Cryoprecipitate-poor plasma is the byproduct of cryoprecipitate production.

Used to help with bleeding by correcting coagulation factor deficiencies


Platelets

Stored at room temperature (20-24º C) for up to 5 days

Can be collected via whole blood donation or apheresis (5-6 whole blood platelets are approximately equal to 1 apheresis platelet unit)

Expected to raise platelet count of 70-kg adult by 20-40k/μL

Cryoprecipitate

Prepared by slowly thawing FFP at 1-6ºC

The precipitate is separated and then refrozen for a 1 year shelf life

Commonly pooled in groups of 5+.

Contains fibrinogen, FVIII, FXIII, and vWF

Granulocytes

Collected by apheresis typically

Stored at room temp for 24 hours

Very rare therapy for special situations regarding bacterial or fungal infections in patient with low absolute neutrophil counts.

Require irradiation to prevent TAGVFD


Blood Product Modifications

Irradiation: Use of radiation to inactivate donor lymphocytes to prevent transfusion associated graft vs host disease.

Leukoreduction: Use of filters to remove donor leukocytes from blood products. Leukoreduced blood units are deemed CMV safe.

Washing: Removes plasma from units of RBCs. Used Primarily in IgA deficiency to prevent anaphylactic reactions.

Electrolyte Physiology

Sodium Na primarily extracellular

Normal serum range 135-147 mEq/L

Plasma change does not change levels typically

Potassium K primarily intracellular

Normal serum range 3.5-5.2 mEq/L

0.25 mEq/L decrease with albumin replacement

0.7 mEq/L decrease with plasma exchange.

Electrolyte Physiology

Chloride Cl primarily extracellular

Normal serum range 95-107 mEq/L

4 mEq/L increase with albumin replacement

6 mEq/L increase with plasma exchange.

Bicarbonate HCO3 pH buffer of blood

Normal Serum range 22-29 mEq/L

6 mEq/L drop with albumin replacement

3 mEq/L increase with plasma exchange.

Citrate

Used as anticoagulant in apheresis procedures

Binds calcium to inhibit the coagulation cascade

Acid Citrate Dextrose solution (ACD)

ACD A 20.6-22.8 g citrate/ml

ACD B 12.4-13.7 g citrate/ml

Calcium

Electrolyte most affected by apheresis.

Most circulating calcium is bound to albumin

Physiologically active ionized Ca is small fraction


Calcium

During apheresis ionized calcium decreased by three mechanisms.

1)removal of ionized Ca from plasma itself

2)binding of ionized calcium by citrate in the ACD solution

3)replacement fluid

Albumin replacement binds calcium

Plasma replacement introduces more citrate

Hypocalcemia

Single plasma exchange using ACD infused at rates of 1.0-1.8 mg/kg/min will drop ionized Ca 25-35%

Some replace with oral calcium carbonate

IV calcium gluconate or calcium chloride

Common Lab Test for Apheresis

• CBC (complete blood count)

• aPTT (activated partial thromboplastin time)

• PT (prothrombin time)

• Fibrinogen

• LDH (lactate dehydrogenase)

• Ionized calcium


Common Lab Test Cont.

• Hemoglobin electrophoresis

• Serum Viscosity

• ADAMTS-13

(a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13)

• Specific analytes that are your target (antibody titers, IgM, etc.)

Introduction to Apheresis Replacement Fluid

Albumin

• Typical 5% solution

• Purified from pooled plasma

• Maintains oncotic pressure

• Lacks coagulation factors

• Most commonly used


Replacement Fluids

Plasma

• Has coagulation factors

• Used when patient has underlying coagulopathy

• Special case with TTP the plasma contains the ADAMTS-13 enzyme

• Risk of infection (blood product)


Replacement Fluid

Saline

• 0.9% NaCl

• 2 major uses

• To reduce viscosity

• Replace volume during cytoreductions

• Lacks oncotic pressure


Replacement Fluid

Red Blood Cells

• Red cell exchanges

• Babesiosis

• Malaria

• Sickle cell disease

• Blood prime

Review

Very basic overview barely scratched the surface.

Apheresis science is highly technical and well studied.

My most active referring neurologist doesn’t ask me to remove antibodies from his patients. He asks for me to remove the bad humors from his patient.

References

This presentation is an overview of the book chapter

Parsons, Jeremy. (2014). Basic Science. In Walter Linz (Ed), Principles of Apheresis Technology 5th ed. (pp.1-22). Vancouver, BC: ASFA.

All references for the chapter are in page 22 of text.

Apheresis Principles and Practice AABB Press

Chopek M, McCullough J. Protein and biochemical changes during plasma exchange. In: Berkman EM, Umlas J, eds. Therapeutic hemapheresis. Washington, DC: AABB, 1980:13-52.

Thanks

Walter Linz and Kendall Crookston for giving me the opportunity to work on this chapter.

My apheresis mentors

Kendall Crookston @UNM

Sara Koenig @UNM

Leonor Fernando @UC Davis

Questions?

jeremy parsons, md

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