Download Immune

Antibody Formation in
Transfusion Therapy
Michael Passwater
SC: 812
Cincinnati, Ohio
November 5, 2016
Vidant Medical Center
Greenville, North Carolina
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Partnership with Brody School of Medicine
at East Carolina University
909 licensed beds
Centerpiece of 8 hospital system
29 County Service Area (population ~1.3 million)
Outline
• Pathophysiology of antibody formation
(Immunology Review/Update)
• Significance to Transfusion Therapy
• Effects on Pretransfusion Testing
• Infusion Nurse Assessment
– At-risk patients
– Recognition of rejection
• s/s acute or delayed reactions
• Management Strategies
Basic Immunology Review
• Innate Immunity
– Alternative Complement System
– Lectin System
• Adaptive Immunity
– Antibodies
– Cells
– Environmental influences (“soup”)
• Passive Immunity
– IVIG, RHIG
– Immunotherapies
Innate Immunity
(fixed set of bodyguards)
• Barriers
– Skin, GI Tract
• Lectin System
– Bind to common antigens on many bacteria and plant
proteins
– Abundant in intestines and respiratory tract to keep out
particles
• Complement System
– attach to foreign or sick cells to destroy or to mark for
destruction
– may attach to immune cells to activate adaptive immunity
Innate Immunity
(fixed set of bodyguards)
• Granulocytes / Macrophages / Dendritic Cells
– Phagocytize particles marked for destruction if activated by
inflammatory signals
– Secrete cathelicidin and other potent antimicrobial peptides
• Effective, but lacks Memory and Flexibility
– Great at clearing what they can clear
– Unable to adjust/adapt to new/different threats
Adaptive Immunity
• Complex – many classes of proteins, receptors,
cells, cofactors
• Flexible – remarkable interaction between local
environment and immune cells to recognize the
situation and select the response
• Interactive – Distress signals from invaded or
damaged tissue, neuroendocrine influences, multiple
cells and proteins, each with multiple purposes,
converge to clear the problem and rebuild
Adaptive Immunity - Antibodies
• IgA
– IgA1, IgA2
– Main antibody in exocrine secretions (milk, respiratory and
intestinal mucin, genitourinary tract, tears, saliva)
• IgE
– Associated with allergic reactions and anaphylaxis
• IgD
– Least abundant isotype; may be present, along with IgM, as
co-receptor on B Cell membranes
Adaptive Immunity - Antibodies
• IgM
– Initial immune response
– Intravascular - Stays within the circulatory system
– Great at agglutinating and binding complement
• IgG
– Secondary immune response
– IgG1, IgG2, IgG3, IgG4
– Evenly distributed throughout tissues
• intravascular and extravascular
– May bind complement
• influenced by Ag-Ab density and IgG isotype
Adaptive Immunity - Antibodies
• Glycoprotein
– 2 heavy chains
– 2 light chains
– Disulfide bonds join chains together
• Y-shaped
– 2 “claws” on variable end to bind with specific antigen
(epitopes)
– Other end (constant region) serves as biologic effector –
may bind complement or activate other immune cells
Antibody Structures
(images from www.abcam.com )
Adaptive Immunity - Antibodies
• B Cells mature into Plasma Cells and produce
antibody
• Each B Cell is “programmed” to produce
antibodies with one single specificity.
– May switch isotypes (e.g. from IgM to IgG), but specificity
stays the same
– May be activated to divide into numerous “offspring”, but
the “cell line” will all produce the same antibody specificity
– Mechanisms for immune system to destroy B cells with selfspecificity
Adaptive Immunity - Cells
• B Cells – Plasma Cells – Memory Cells
• T Cells – T4/CD4 (Helpers), T8/CD8 (Suppressors),
Natural Killers
• Antigen Presenting Cells (APCs)
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Monocytes – Macrophages
Dedritic Cells – Plasmacytoid dendritic cells
T Cells
Endothelial Cells
• Gatekeeper of lymphocyte movement out of intravascular
space
• Key player in NO, procoagulant and anticoagulant regulation,
and immune responses
Adaptive Immunity - Cells
• Immune cells are influenced by
environmental signals
– Receptors expressed on membranes varies
– Output signals vary
• secretion of Cytokines, Chemokines, Antibody
– Cell activation may trigger:
• rapid division
• expression of “suicide receptors”
• nothing
Adaptive Immunity – Putting it together
• APC, T Helper Cell, B Cell, antigen-antibody
complexes form
• “Soup” of Cytokines, Chemokines,
neuroendocrine influences, and other
cofactors determines receptor expression on
cells, which then influences the outcome of
Cell-Cell-Ag-Ab complex interactions
Under the right conditions, APC activates T cell
T cell then activates B cell to produce antibody
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By Altaileopard - Own work, Public Domain,
https://commons.wikimedia.org/w/index.php?curid=12193556
Immune System Concepts
• “Danger Hypothesis” – immune
system responds to clues that an
infection has taken place before
responding strongly to antigens; foreign
substances may be invisible to the
immune system unless accompanied by
danger signals (infection/inflammation)
Immune System Concepts
• “Cross-reactivity” – antibodies
selected for one antigen (epitope) may
also bind to similar antigens
– Ex: Sugars on some bacteria membranes
are similar to sugars on some Red Blood
Cell membranes
– Antibody production carries risk of damage
to self-cells (auto-immunity)
Immune System Concepts
“Healthy Immune Cells”
• Your Grandmother may have been an immunologist
– Iron is essential for white blood cells – not just RBCs
– Sunlight (vitamin/hormone D) is important - Immune cells
have 1,25(OH)2D receptors; shifts response towards innate
immunity rather than adaptive immunity (inhibits Th1 and
Th17, enhances Th2 and Treg)
– Ascorbic acid is 80x more concentrated in granulocytes than
in plasma; is essential for T Cell maturation and division, and
for phagocytosis; also important for Endothelial Cell function
– Mg2+ and Ca2+ ions are essential for complement activation
and many enzymatic reactions of the immune system
Immune System Concepts
• Antigen factors influencing Antibody production
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“Foreignness”
High Molecular Weight (>6,000 Da), and Complexity
Dose and Route of Entry
Genetic background of the individual
• Large (or small and bound to larger protein),
different substances, inside an “inflamed” area of the
body, may draw the attention of an Adaptive
Immune Response.
Immune System
Self-Defense vs. Self-Destruction
• Many control mechanisms are triggered to restore
the immune system to a resting state when the
response to an antigen is no longer required
• Brain, anterior chamber of the eye, testes lack
adaptive immunity
– Risk of “collateral damage” and auto-immunity is less with
innate immune responses than adaptive immune response
• Macrophages can initiate, promote, prevent,
suppress, and terminate immune responses
Flexible and Interactive Membranes
Electron Photomicrographs courtesy of Debra Laisch, Vidant Medical Center
Blood Transfusion… or Transplant?
• Growing popularity for the concept of
Blood as an Organ
• More comprehensive: Vasculature
(Endothelium + Blood) is an Organ
– RBC, WBC, Platelets, Plasma, and
Endothelial cells are critical to Hemostasis
and overall health (no flow = no go)
– Regulate the 3 gases of life (O2, CO2, NO)
Big Numbers:
Stars, Grains of Sand, and Blood Cells
• Typical adult has a blood volume of 5L
– 10 “pints” of whole blood
• 1 billion WBC in typical unit of whole blood
– 1,000,000,000 or 1 x 109
• 1 million WBC in typical leukoreduced RBC unit.
– 1,000,000 or 1 x 106
• Healthy adults have 20-30 trillion RBC
– 30,000,000,000,000 or 2-3 x 1013
• 300 billion Platelets in one unit of apheresis platelets
– 300,000,000,000 or 3 x 1011
Blood Transplants – “Foreignness”
• 8 ABO/Rh(D) Types
– O Pos, O Neg, A Pos, A Neg, B Pos, B Neg, AB Pos, AB Neg
• Tens of thousands of different
lymphocyte (HLA) antigens
– e.g. A, B, C, DR, DQA, DQB, DP alleles
• Hundreds of “minor” RBC antigens
– e.g. Rh, Kell, Kidd, Duffy, MNSs, Lewis systems
• Dozens of Platelet antigens
– HPA system
Transfusion and Antibody Formation
• Inflammation
– Sickle Cell crisis
– Sepsis
– Major Surgery
• Chronic Exposure
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Sickle Cell / Hemoglobinopathies
Warm Autoimmune Hemolytic Anemia
Chemotherapy
Pregnancy
Erythrophagocytosis and RBC Agglutination
Group O Recipient vs. Group A Donor
Transfusions and Antibodies - Prevention
• Avoid transfusions when possible
– IV iron, B12, Folic acid, ESA
– Conserve patient’s blood
– Manage anticoagulants & anti-platelet medications
• Prestorage Leukoreduction
– Minimizes exposure to WBC / HLA antigens
– Minimizes buildup of inflammatory proteins in donor bag
during storage
• Extended phenotype matching for chronically
transfused populations (minimizes “foreignness”)
• Minimize passive antibody transfer
– Male donors for plasma and platelet products
– Platelet Additive Solution (PAS) products
Transfusions and Antibodies - Detection
• Blood Type
• Antibody Screen
• Direct Antiglobulin Test (DAT)
– IgG antibodies attached to patient RBCs
– Complement attached to patient RBCs
• Antibody Identification
• Crossmatch
• Good history of treatments and blood bank workups
Transfusions and Antibodies - Detection
Transfusion Reactions
Most Common Types of Reactions
• Often cannot tell cause of reaction from clinical
symptoms alone; further investigation required
• Allergic
– Mild (hives) to Severe (anaphylaxis)
• Respiratory Distress: TACO, TRALI, Sepsis
• Hemolysis: Immune or non-Immune
• Tingling, Numbness: electrolyte disturbance
– Ionized calcium deficiency risk with large volume
transfusions (citrate toxicity)
Immune Hemolytic Reactions
• Intravascular Hemolysis (Acute)
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IgM (or high titer IgG)
Pain (flank and/or infusion site)
Fever and chills may be present
Hemoglobinuria (red, brown, black urine)
• Risk of renal failure
– New or increased bleeding (DIC)
– Free hemoglobin in plasma binds NO irreversibly
• Risk of vasoconstriction & shock
– Haptoglobin is decreased (typically undetectable)
– Risk of “bystander hemolysis”
• Strong complement activation causes complement mediated
destruction and phagocytosis of autologous RBCs in addition to
transfused cells
• Post-transfusion Hematocrit lower than pre-transfusion Hematocrit
Immune Hemolytic Reactions
• Extravascular Hemolysis
– Delayed (typically 3 days to 3 weeks after transfusion)
– IgG antibodies
– Jaundice, Anemia
• Increased Total & Indirect Bilirubin
• Decreased Hematocrit & Hemoglobin
– May have Positive DAT and/or Positive RBC
Antibody Screen
Transfusions and Antibodies – Reaction Response
• STOP the transfusion
• Maintain IV access
• Recheck Patient and Blood Product ID
– Clerical errors are leading cause of ABO incompatible
transfusions
• specimen collection & blood administration
• Notify Patient’s Provider and Blood Bank Lab
• Support patient vitals
• Collect blood specimen(s) and observe or collect
urine as directed
• Send blood product(s), IV tubing, & specimens to
Blood Bank Lab
Transfusions and Antibodies - Challenges
• Delays – hours to days
– More extensive testing
– More extensive search to find compatible donors
• Larger sample requirement for pretransfusion testing
• More expensive workups and transfusions
• Increased risk of transfusion reaction / shortened
RBC survival
– Undetected antibody(ies)
• Antibodies to low frequency antigens not present on
screening cells
• Antibody titer(s) may decrease over time
Transfusions and Antibodies – Passive Abs
• Rh Immune Globulin
– Anti-D; may contain detectable levels of additional Rh
antibodies
• IVIG
– Contains anti-A, anti-B
– Often contains detectable levels of other RBC and WBC/HLA
antibodies
• Daratumumab (anti-CD38)
– CD38 is heavily expressed on myeloma cells; some
expression on RBCs
– May cause positive DAT and RBC antibody screen; interfere
with Blood Type testing
Transfusions and Antibodies – Passive Abs
• Rituximab (anti-CD20)
– Inhibits antibody producing B cells
– Autoimmune, Transplant, and acute Transfusion
Reaction applications
• Erlizumab (anti-CD18)
– Targets lymphocytes and LFA-1 integrin
– Immunosuppresive, designed to reduce
inflammation
Other Antibodies:
Drug-induced immune thrombocytopenia and/or anemia
• Heparin Induced Thrombocytopenia (HIT)
– Antibodies form to Heparin-Platelet membrane complexes
– Both patient & donor platelets cleared by antibodies in presence of heparin
– Less common with LMWH
• Post Transfusion Purpura (PTP)
– Exposure to platelets in transfused blood products leads to antibodies that
destroy both transfused and native platelets
• Cross-reactivity and/or adsorption of donor antigens by recipient cells
– Rare; typically associated with HPA-1a or HPA-5a antigens
• Other Medications
– Antibiotics (esp. cephalosporins, penicillen), Platinum containing
chemotherapies, Methyldopa, and numerous other drugs may induce
antibodies that react with RBC or PLT
– Antibodies may require drug/metabolite to react, or may react with
membrane antigens independent of the drug’s presence
Illustration generously provided by Patricia Arndt of
the American Red Cross
Summary
• Intravenous transplantation of foreign RBC, WBC,
and Platelet antigens into the presence of an
activated adaptive immune response increases the
likelihood of RBC, WBC, and PLT antibody formation
– May complicate current and future transfusions
– May induce transient or chronic autoimmunity
• Transfusion associated immune destruction may
manifest minutes to weeks after transfusion
– Attention to Patient ID during specimen collection and blood
administration, and prompt recognition of reactions, is
critical to optimize transfusion safety
Summary
• Future research will likely discover
better ways to:
– optimize wanted immune responses
– minimize unwanted immune responses
– provide more specific therapies to minimize
the need to transplant foreign blood
elements
Thank You! / Questions?
Michael Passwater
[email protected]
(252)847-4229
References / Further Reading
•
Blood Transfusion Therapy: A Guide to Blood Component Administration, 3rd
edition. AABB Press. 2014.
• Antibody Production. ThermoFisher Scientific. 2016.
www.thermofisher,com/us/en/home/life-science/protein-biology
•
“Blood Bank Guy” (Dr. Chaffin) www.bbguy.org
• “Red Cell Antigens”, accessed 5/29/2016
www.ualberta.ca/~pletendr/tm-modules/immunology/70imm-ag.html
•
David Male, Jonathan Brostoff, David B Roth, Ivan Roitt. Immunology, 7th
edition. Mosby Elsevier. 2006.
•
Richards AL, Hendrickson JE, Zimring JC, Hudson KE. “Erythrophagocytosis by
plasmacytoid dendritic cells during inflammation” Transfusion 2016;56; 905-916.
•
Daniel Bikle. “Nonclassic Actions of Vitamin D”. Journal of Clinical Endocrinology
Metabolism. 2009 Jan; 94(1):26-34.
References / Further Reading
•
Dzik WH. “The air we breathe: three vital respiratory gases and the red blood
cell: oxygen, nitric oxide, and carbon dioxide.” Transfusion 2011; 51: 676-685.
•
Cobb BA and Kasper DL. “Characteristics of carbohydrate antigen binding to the
presentation protein HLA-DR”. Glycobiology vol 18 no 9 pp 707-718, 2008.
•
Luisa Mohle et al. “Ly6Chi Monocytes Provide a Link between Antibiotic-induced
Changes in Gut Microbiota and Adult Hippocampal Neurogenesis”. Cell Reports
15, 1945-1956. May 31, 2016.
•
Maroin E. Reid, Christine Lomas-Francis, Martin L. Olsson. The Blood Group
Antigen Facts Book, 3rd edition. Academic Press. 2012.
•
Buck MD, O’Sullivan D, Pearce EL. “T cell metabolism drives immunity”. The
Journal of Experimental Medicine 2015 vol 212 No 9 1345-1360.