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
Photo from Wikipedia Commons
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
Photo from Wikipedia Commons
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
Photo from Wikipedia Commons
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
Photo from Wikipedia Commons
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
Photo from Wikipedia Commons
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
Photo from Wikipedia Commons
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
Photo from Wikipedia Commons
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)
Photo from Wikipedia Commons
Replacement Fluid
Saline
• 0.9% NaCl
• 2 major uses
• To reduce viscosity
• Replace volume during cytoreductions
• Lacks oncotic pressure
Photo from Wikipedia Commons
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?