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Immunohematology Chapter 5 Notes

I. OVERVIEW

  • Immunohematology: study of blood group antigens/antibodies, HLAs and antibodies, pretransfusion testing, identification of unexpected alloantibodies, immune hemolysis, autoantibodies, drugs, blood collection, blood components, cryopreservation, transfusion-transmitted viruses, tissue/organ transplantation, blood transfusion practice, safety, quality assessment, records, blood inventory management, and blood usage review.
  • Immune system concepts:
    • Acquired immunity: specific antibody response to an immunogen; plasma cells produce antibodies.
    • Innate immunity: nonspecific defense; barriers (skin, mucous membranes) and cells (neutrophils, monocytes/macrophages, NK cells).

II. GENETICS

  • Key definitions:
    • Chromosomes, mitosis, meiosis, blood group systems, phenotype, genotype, pedigree, gene, genetic locus, alleles, antithetical alleles, polymorphic, codominant, recessive, dominant, autosomal, sex-linked.
  • Mendelian inheritance:
    • Law of independent segregation; law of independent assortment.
    • Punnett squares predict blood group inheritance; homozygous vs heterozygous.
    • Dosage effect: stronger agglutination in homozygous vs heterozygous cells.
    • Linkage and haplotypes: linked genes inherited as units; haplotype defined; amorphs and related concepts.
  • Population genetics and paternity:
    • Phenotype frequency calculations; combining probabilities for multiple antibodies to predict compatible units (product rule).
    • Parentage testing uses HLA polymorphism and exclusion statistics.

III. ABO AND H BLOOD GROUP SYSTEMS AND SECRETOR STATUS

  • Landsteiner’s Rule: If an antigen is present, the corresponding antibody is absent (universally true with few exceptions).
  • ABO antigens:
    • Located on RBCs, lymphocytes, platelets, tissues; can be secreted in secretors.
    • Glycolipids/glycoproteins; develop in utero ~5–6 weeks; full expression by age 2–4 years.
    • ABO antigen expression frequencies by population (Table 5-2): O ~45–49%, A ~40–27%, B ~11–20%, AB ~4% in Caucasian/African American groups.
  • Inheritance and development:
    • H antigen is the building block for A and B; H gene with alleles H and h; H more common (~99.99%); A and B require the H acceptor with added sugars.
    • Subgroups: A1 (A and A1) and A2 (A only); A1 ~80%, A2 ~20%; anti-A1 can be present; subgroups detectable with Anti-A,B and Anti-A1 lectin; A3 can show mixed-field reactions.
    • A and B are codominant; O is amorphic (no transferase).
    • Anti-A and Anti-B are IgM; typically formed in response to absence of the antigen; can activate complement; may cause room-temperature hemagglutination.
  • Routine ABO typing:
    • Forward typing (RBCs with Anti-A/Anti-B)
    • Reverse typing (serum with A1/B RBCs)
  • Subgroups and clinical implications:
    • Wrong transfusion consequences if subgroups are not detected; weak A subgroups can be problematic in O recipients.
  • Secretor status:
    • Se and se alleles determine whether A, B, H antigens appear in secretions (saliva, urine, tears, etc.); nonsecretors lack secretor antigens in fluids.

IV. RH BLOOD GROUPS

  • System overview:
    • Controlled by RHD and RHCE genes; D antigen is most immunogenic; other Rh antigens include C/c, E/e, and others (e.g., Cw, G, V, RH variants).
    • Antigens are proteins; haplotype terminology includes Fisher-Race and Wiener systems.
  • Antigen/phenotype vs genotype:
    • Phenotype: antigens detected on RBCs with antisera; Genotype: genes on chromosomes.
  • D antigen and variants:
    • Weak D and mosaic D: weaker D expression; detected by IAT; position effect, partial D, and weakly reactive D concepts.
    • Partial D: only part of D antigen present; risk of making anti-D after exposure to D-positive blood; often detected with monoclonal reagents.
    • Weak D recipients usually receive D-negative blood; donors testing weak D are labeled as D-positive per AABB standards.
  • Other Rh system antigens:
    • f, Ce, rhi, Cw, V, G, Rh29, Rhnull, Rh variants, D deletion, Rhnull phenotype and its implications (RBC membrane abnormalities, reduced survival).
  • Rh antibodies:
    • IgG; usually do not activate complement; optimally react at 37°C; AHG phase; enhanced by LISS, PEG, enzymes; dosage effects common; can cause HTRs and HDN; often clinically significant.
    • Rh antibodies can cause HDN; RhIG (Rh immune globulin) given postpartum and after potential feto-maternal bleed to prevent sensitization.

V. OTHER BLOOD GROUP SYSTEMS

  • Kell (K/k, Kpa/Kpb, Jsa/Jsb, Kx):
    • Antigens: K, k, Kpa, Kpb, Jsa, Jsb, Ku; K is highly immunogenic; anti-K common and can cause HTR/HDN. Kellnull (K0) lacks Kell antigens but has Kx; McLeod phenotype (X-linked Kx alteration) with low Kell expression and abnormal RBCs.
  • Duffy (Fy):
    • Antibody FyA and FyB; clinically significant; Fy(a−b−) confers some malaria resistance; Fy phenotypes and dosage effects.
  • Kidd (Jk):
    • antibodies Jka/Jkb; AHG phase; can cause HTR and mild HDN; exhibit dosage; antibodies may deteriorate in storage and cause delayed HTR.
  • Lutheran (Lu):
    • Lua and Lub antibodies; Lua IgM, Lub IgG; variable enzyme effects; generally not clinically significant but Lub can be.
  • Lewis (Le):
    • Le system; Lea and Leb antigens; secretor status influences expression; Lea often secreted by Lea+, nonsecretors can show Le(a+b−) phenotype; associations with Bombay phenotype and anti-Le antibodies.
  • I, P, MNS, Diego, Cartwright, XG, Scianna, Dombrock, Colton, Chido/Rodgers, Gerbich, Cromer, Knops, Vel, JMH, Sid, and other high-incidence or low-incidence antigens summarized with their clinical significance, antigens, and common antibodies.
  • I Blood Group System (I):
    • Anti-I is IgM, usually nonclinical; binds to I antigen; associated with some infections (e.g., Mycoplasma pneumoniae). IS and occasionally 37°C reactivity.
  • P system (P1):
    • Anti-P1 typically not clinically significant; certain combinations (P1 with P and Pk) can be clinically significant; autoanti-P (Donath-Landsteiner) linked to paroxysmal cold hemoglobinuria; some individuals with p phenotype have anti-P1 or anti-P1Pk; management may require blood warmer in cases of autoanti-P.
  • MNS system (M/N, S/s, U):
    • M/N usually IgM and clinically insignificant; S/s and U mostly IgG and clinically significant (HDN/HTR); dosage effects observed for anti-M (IgG is clinically significant, IgM not).
  • Miscellaneous: Diego, Cartwright, Xg, Scianna, Dombrock, Colton, Chido/Rodgers, Gerbich, Cromer, Knops, Vel, JMH, Sid, and other high-incidence antigens with clinical implications.

VI. BLOOD BANK REAGENTS AND METHODS

  • Principle: Ag + Ab forms an immune complex.
  • Routine testing procedures:
    • ABO/Rh typing: forward (cell) typing with anti-A/anti-B; reverse typing with A1 and B cells.
    • Antibody screen: screens for clinically significant antibodies using reagent screening cells.
    • Antibody identification: panel (10–20 cells) to identify specific alloantibodies.
    • Crossmatch: donor cells with recipient serum to assess compatibility.
  • Reagents:
    • Reagent RBCs with known antigens; antisera with antibodies; antiglobulin reagents; potentiators to enhance reactions.
  • Reagent production regulation:
    • FDA licenses reagents; potency and specificity regulated.
  • Antisera types and uses:
    • Polyclonal vs monoclonal vs blended monoclonal; ABO antisera; D typing reagents; AHG reagents; Check cells (IgG-coated controls) per standards.
  • ABO antisera table (example):
    • Anti-A and Anti-B performance with RBCs typed Type A, B, AB, O.
  • Additional testing modalities:
    • Gel technology, microplate methods, solid-phase adherence methods, IAT (indirect antiglobulin test).
    • Potentiating media: LISS, albumin, PEG, proteolytic enzymes (papain, ficin, bromelin).
  • DAT (Direct Antiglobulin Test): detects antibodies/complement on patient RBCs; EDTA preferred for sample collection.

VII. DIRECT ANTIGLOBULIN TESTING

  • DAT detects in vivo sensitization of RBCs by IgG or complement.
  • Procedure: wash patient RBCs, add AHG; agglutination indicates bound antibodies/complement.
  • Used for autoimmune hemolytic anemia, HDN, drug-induced mechanisms, transfusion reactions.

VIII. IDENTIFICATION OF UNEXPECTED ALLOANTIBODIES

  • Antibody screening and identification:
    • Screen with two-three group O cells; identify with panel (10–20 cells).
    • Autocontrol to distinguish autoantibodies from alloantibodies.
    • Use potentiators to enhance detection; patient history important.
  • Antibody panel interpretation:
    • Autocontrol helps define autoantibody vs alloantibody.
    • Reaction phase helps differentiate IgG vs IgM.
    • Strength of reactions informs number of antibodies and dosage effects.
    • Rule of Three for identification confidence.
    • Phenotype confirmation of patient antigens to support identification.
  • High-frequency antigen antibodies and low-frequency antigen antibodies considerations.
  • Cold antibodies and autoantibodies:
    • Cold antibodies may complicate testing; prewarming or adsorption can aid detection of clinically significant alloantibodies.
  • Elution techniques to identify antibodies bound to RBCs.
  • Techniques to resolve multiple antibodies and use of enzyme-treated cells to reveal hidden specificities.
  • Special testing for autoantibodies and cold panels.

IX. PRETRANSFUSION TESTING

  • Compatibility testing scope: recipient identification, specimen handling, ABO/Rh typing, antibody screen/identification, donor ABO/Rh confirmation, crossmatching, and antigen screening for recipient antibodies.
  • Types of crossmatch:
    • Full (complete) crossmatch: donor cells + recipient serum through all phases including AHG.
    • IS crossmatch: performed at room temperature to detect ABO incompatibility first.
    • Electronic crossmatch: computer-assisted crossmatching based on recipient history and negative antibody screen; used when no antibodies are detected in the current and historic screens.
  • Crossmatch limitations:
    • Does not guarantee in vivo survival of RBCs; does not detect bacteria, viruses, or parasites; does not detect WBC antigens; does not prevent alloantibody formation to donor RBCs; does not prevent delayed transfusion reactions.
  • Sample collection and tube labeling:
    • Acceptable tubes: red top, ACD, EDTA, citrate; avoid hemolyzed samples; tube data must match requisition and patient identity band.
  • Crossmatch workflow and documentation requirements, including tagging/inspection/issuance of blood products.
  • Emergency release of uncrossmatched blood: signed by physician; unit tagged; segments removed for later crossmatching.
  • Massive transfusion policies and MSBOS (Maximum Surgical Blood Order Schedule).
  • Crossmatching autologous units; pediatric considerations; neonates less than 4 months old.
  • Pretransfusion testing for non-RBC products (FFP, platelets, cryoprecipitate, granulocytes) is ABO group-based or compatible.

X. HEMOLYTIC DISEASES OF THE NEWBORN

  • Etiology: maternal IgG antibodies cross placenta causing fetal RBC destruction; bilirubin metabolism and risk of kernicterus.
  • Rh HDN: most severe; maternal anti-D after first pregnancy with D-positive fetus; RhIG given to mothers to prevent sensitization.
  • ABO HDN: most common form; usually mild; treated with phototherapy; may require transfusion in rare cases.
  • HDN from other IgG antibodies (Kidd, Kell, etc.): can be severe; antibody titration used to predict severity; amniocentesis for bilirubin measurement; fetal monitoring.
  • Prevention: RhIG prophylaxis at 28 weeks and after delivery for D-negative mothers; Kleihauer-Betke to quantify fetomaternal bleed; fetal screens to guide RhIG dosing.
  • Exchange transfusion and management in severe cases.

XI. BLOOD COLLECTION

  • Donor selection and screening:
    • Registration data, consent, photo ID, and donor history questions (stringent criteria for deferral).
  • Donor questionnaires cover travel history, risk behaviors, major illnesses, medications, pregnancy status in females, high-risk exposures, and more.
  • Donor deferral criteria (Table 5-5): includes infectious disease exposures, high-risk behaviors, certain medications, pregnancy status, tattoo/piercing timelines, and other risk factors.
  • Donor physical exam and minimum criteria.

XII. BLOOD COMPONENTS: PREPARATION, STORAGE, AND SHIPMENT

  • Definitions:
    • Whole blood vs components (RBCs, plasma, platelets, cryoprecipitated factor).
  • Blood collection bags and anticoagulant-preservative solutions: standard volumes, volume adjustments for autologous donations.
  • Storage concepts:
    • Shelf life varies by component and anticoagulant/additive; components experience metabolic changes during storage (ATP, 2,3-BPG, potassium, etc.).
  • Additive solutions (AS-1, AS-3, AS-5) extend shelf life and reduce viscosity; rejuvenation solutions restore 2,3-BPG/ATP prior to transfusion.
  • Component preparation steps from whole blood: separation into RBCs, platelets, plasma, FFP, and cryoprecipitate; pooling and pooling guidelines; cryoprecipitate processing.
  • Storage conditions and expiration dates for different components (e.g., RBCs 1–6°C up to 42 days with additives; platelets 20–24°C for up to 5 days; FFP frozen and thawed; cryoprecipitate handling; irradiated products).
  • Transportation and handling: temperature controls, dry ice for frozen components, room temperature for platelets, temperature monitoring, and discarding out-of-range units.
  • Administration of components: positive patient ID, saline infusion only, 170-micron filter, leukoreduction considerations, maximum transfusion time 4 hours, and thorough documentation.

XIII. BLOOD COMPONENT THERAPY

  • Whole blood: infrequently used; alternative is reconstituted whole blood for massive loss.
  • RBCs: used in various anemias and post-therapy settings; typical rule-of-thumb: 1 unit raises Hb by ~1 g/dL and Hct by ~3 percentage points.
  • Leukocyte-reduced RBCs: reduce febrile/non-hemolytic reactions; leukoreduction can occur during manufacturing or via bedside filtration; target ≤5×10^6 WBC per unit.
  • Frozen vs deglycerolized RBCs: glycerol-based freezing, long-term storage at −65°C; deglycerolization to remove glycerol; deglycerolized RBCs expire 24 hours after thaw.
  • Washed RBCs: for patients with plasma protein hypersensitivity; also used in infants or intrauterine transfusions; some RBC loss occurs during washing.
  • Irradiated RBCs/platelets: to prevent transfusion-associated graft-versus-host disease; recommended minimum dose and indications (e.g., relatives, matched donors, intrauterine transfusions).
  • Platelets: used to prevent/control bleeding; ABO compatibility preferred; platelets can be pooled or prepared by apheresis; platelet lifespan ~3–4 days; leukoreduced platelets reduce febrile reactions; HLA-matched platelets for highly alloimmunized patients.
  • Fresh-Frozen Plasma (FFP): contains coagulation factors; indications include massive transfusion, coagulopathy, liver disease, anticoagulation reversal, TTP/HUS, DIC scenarios, etc.; thawed plasma viability and handling.
  • Cryoprecipitated antihemophiliac factor: concentrates factor VIII, fibrinogen, factor XIII, and von Willebrand factor; used for factor deficiencies; dosing calculations to achieve fibrinogen targets; fibrin glue applications.
  • Granulocyte pheresis: rare; used for severe neutropenia/sepsis; rapid cell degradation; strict storage and usage timelines.
  • Labeling and ISBT 128 labeling standards for products and pools; regulatory labeling requirements.

XIV. TRANSFUSION THERAPY

  • Emergency transfusions:
    • Rapid blood loss management; massive transfusion defined as total blood volume replacement within 24 hours (varies by facility).
    • Emergency transfusions may use type-specific blood when possible; if not, O negative or D-negative as appropriate, with crossmatch performed during/after transfusion.
  • Transfusion indications across contexts: neonatal/pediatric, transplantation, oncology, chronic kidney disease, sickle cell disease, thalassemia, aplastic anemia, etc.
  • Transfusion strategies vary by patient condition and underlying disease; key considerations include alloantibody profiles, antigen matching, leukocyte reduction, infection risk minimization, and CMV status where relevant.

XV. TRANSFUSION REACTIONS

  • Types: Hemolytic (intravascular or extravascular) and nonhemolytic (febrile, allergic).
  • Acute vs delayed reactions:
    • Acute occur within hours; delayed occur days to weeks after transfusion.
  • Immune-mediated mechanisms include antigen–antibody reactions leading to complement activation, cytokine release, and potential DIC or organ injury.
  • Non-immune mediated causes include bacterial contamination, equipment issues, and physical destruction of RBCs.
  • Hemolytic transfusion reactions (HTR): clinical signs include fever, chills, back pain, hemoglobinemia/hemoglobinuria; major sequelae include DIC, renal failure, shock, death; common cause is identification error.
  • Transfusion reactions are managed by stopping transfusion, notifying the physician/lab, patient workup (DAT, hemoglobin, bilirubin, haptoglobin, etc.), and documentation.
  • Other complications: transfusion-associated graft-versus-host disease, bacterial contamination, circulatory overload, hemosiderosis, citrate toxicity, and other rare events.

XVI. TRANSFUSION-TRANSMITTED DISEASES

  • Donor infectious disease testing history and timeline (HBsAg, anti-HBc, HCV, HIV, HTLV, syphilis, CMV, Trypanosoma cruzi, West Nile, Zika).
  • Look-back studies: process to notify recipients of potentially exposed units from donors who later test positive for infectious agents.
  • Safety and quality assurance: FDA, OSHA, CDC roles; universal precautions; standard precautions; PPE and vaccine provisions; regulatory compliance.

XVII. SAFETY AND QUALITY ASSURANCE

  • FDA regulations: Good Manufacturing Practice; procedures, records, personnel qualification, facilities, validation, and quality control.
  • Records: stringent retention, permanent ink, correction practices, and data integrity.
  • Documentation and traceability: donor and recipient records; unit disposition; deferrals; infectious disease testing histories.
  • Validation and supplier management:
    • Validate blood bank information systems prior to use; supplier qualifications.
  • Safety governance: standard precautions, immunization policies, reporting of adverse events, and compliance with federal/state/local safety requirements.

XVIII. BLOOD USAGE REVIEW

  • Peer review and hospital transfusion committee oversight per Joint Commission, CFR, CAP, and AABB standards.
  • Quarterly review of transfusion appropriateness, reaction evaluation, policy development, service adequacy, and ordering practices.
  • Data-driven monitoring of transfusion practices to optimize patient safety and resource use.

SUMMARY OF KEY FORMULAS AND QUANTITIES

  • Punnett square example (ABO inheritance): when a mother with genotype A and a father with genotype B cross to yield offspring with AB, BB, AO, BO, each with probability 25%:
    • Probabilities: P(AB)=P(BB)=P(AO)=P(BO)=0.25
  • Population genetics example: predicting compatible units when multiple antibodies are present
    • Probability of compatible units = product of individual antigen frequencies: ext{Compatibility} = igl(pEigr) imesigl(pMigr) imesigl(p_Cigr) ext{, etc.}
    • Example given: if 30% are E-positive, 78% are M-positive, and 80% are c-positive, compatible units = 0.70 imes 0.22 imes 0.20 = 0.03 ext{ (3%)}.
  • Reduced volume factor for autologous donation anticoagulant calculation:
    • Let A= rac{W}{110} where W is weight in pounds.
    • Anticoagulant needed: B = 70 imes A
    • Anticoagulant to remove: 70-B
    • Blood to collect: 500 imes A
  • RhIG dosing after potential fetomaternal bleed (Kleihauer-Betke):
    • Fetal bleed volume in mL: ext{Bleed} = ext{percent fetal cells} imes 50
    • RhIG doses required: ext{Doses} = rac{ ext{Bleed}}{30}
  • Cryoprecipitate factor VIII dosing (illustrative formula):
    • #units of cryoprecipitate needed = ext{plasma volume} imes rac{ ext{desired level} - ext{initial level}}{80} ext{ IU per bag}

Notes and practical takeaways:

  • ABO/Rh typing and crossmatching remain foundational to safe transfusion practice; always verify patient identity and unit compatibility.
  • Be mindful of discrepancies in forward vs reverse typing (subgroups, Bombay phenotype, secretor status) and pursue confirmatory testing.
  • Non-ABO antibodies require systematic antibody screening and identification to prevent transfusion reactions and HDN.
  • Storage, handling, and transportation of blood products follow strict temperature controls and documentation for safety and traceability.
  • Regulatory frameworks (FDA, AABB, CFR, ISBT 128) govern reagents, labeling, and transfusion practices to ensure quality and patient safety.
  • Ethical and practical considerations in blood usage include minimizing unnecessary transfusions, using compatible and safe products, and maintaining robust transfusion safety cultures across healthcare systems.