Immunohematology - 03 ABO Blood Group System

IMMUNOHEMATOLOGY THE ABO BLOOD GROUP SYSTEM ABO BLOOD GROUP SYSTEM • OUTLINE • Introduction • o Functional Roles of Blood Group System o Pre-transfusion Testing • The ABO Blood Group System o Genetic Concept in ABO Inheritance o ABH Antigens o ABO Antibodies o ABH Soluble Substances o ABO Subgroups o Weak Subgroups of A o B Subgroup o Bombay Phenotype o ABO Discrepancies o Four Groups of ABO Discrepancies Discoveries, Laws, Properties, Characteristics, and Inheritance of ABO Blood Group System • • GENETIC CONCEPT IN ABO INHERITANCE • • THE ABO BLOOD GROUP SYSTEM • The ABO system is the most important of all blood groups in transfusion practice. It is clinically significant and this blood group remains the leading cause of death in hemolytic transfusion reaction fatalities reported to the FDA. This chapter presents the ABO blood group system and discusses the biochemistry, properties, and characteristics of ABO antigens and antibodies also their subgroups and discrepancies. • FUNCTIONAL ROLES OF BLOOD GROUP SYSTEMS • • • • • • • Glycosyltransferase - ABO, Lewis, H o H - important for ABO & Lewis Structural Relationship to Red Cell - MNS, Diego, Gerbich o Some blood groups react with enzyme Transport Proteins – Rh, Kidd, Diego, Colton Complement Pathway Molecule – Chido/Rogers, Cromer, Knops Adhesion Molecules – Lutheran, Xg, LW (LandsteinerWeiner), Indian Microbial Receptors – MNS, Duffy, P, Lewis, Cromer Biologic Receptors - Duffy, Knops, Indian PRE-TRANSFUSION TESTING • • • ABO Blood Typing o Routine testing o Due to type-specific blood transfusion o Type-specific is first choice (A = A) Rh typing o Rh +/o Rh antibodies (IgG) Antibody Screening and Identification o To ensure match with antibodies outside ABO and Rh systems o Identification - panels Most clinically significant blood group system in transfusion and transplantation therapy o Blood typing is required prior to transfusion & transplant; routinely tested o Small incompatibility in ABO blood group system may cause immediate hemolytic transfusion reaction (<24h) leading to severe rxn & eventual death of recipient within minutes to hours It is a unique blood group system because it is the only blood group system in which Ab is formed by the individual against Ag that is not found on the RBC The only system with reverse/indirect/backward typing • ABO genes are located in long arm of chromosome 9 Bernstein, 1924 o He explained the manner of inheritance of the ABO genes in the o According to him, each parent contributes half of the genetic information to the child in the unit called “gene.” o 50% haplotype comes from the mother and the other 50% comes from the father. This manner of inheritance is called “codominant manner of inheritance Landsteiner Law o Karl Landsteiner (1901) - discovered the AB genes o Established 3 laws for blood typing 1. The antigen on the RBC determines the blood type of the individual. (surface of RBC) 2. The corresponding antibody is never found on the individual’s serum. • Ag + Ab = agglutination 3. The opposite antibody will always be present in the individual’s serum. o Example: Blood type “ B” ▪ B antigen ▪ Anti-A antibody on serum Blood type/Phenotype & Genotype Blood Type/Phenotype Genotype O OO A AA, AO B BB, BO AB AB NOMENCLATURE • • • Landsteiner Jansky Moss O I IV A II II B III III AB IV I Landsteiner - name is based on antigen present Jansky & Moss - name is based roman numerals o Not routinely used International Society of Blood Transfusion (ISBT) o This system is created which aims to unify and standardize the manner of reporting of RBC Ag. Machine and IO readable system of reporting red cell Ag was used o o ABO Blood Group system: 001:ABO ▪ For the 4 major AG • Blood type A = 001/ABO1 • Blood type B = 002/ABO2 • Blood type AB = 003/ABO3 • Blood type O = 004/ABO4 Numerical system of ISBT - composed of 6 digits ▪ First 3 digits - ABO System; Last 3 digits - Ag • A = 001001 • B = 001002 • AB = 001003 • O = 001004 Immunodominant Locus sugar H gene L-fucosyltransferase L-fucose Chromosome 19 (secretor locus) A gene NN-acetyl-D Long arm of acetylgalactosaminyl galactosamine Chromosome 9 transferase B gene DD-galactose Long arm of galactosyltransferase Chromosome 9 Gene • PUNNET SQUARE Parent alleles A B O A AA “A” AB “AB” AO “A” B “AB” BB “B” BO “B” O AO “A” BO “B” OO “O” • Example 1: Parent blood type: OO x AO o Possible blood type of children: 50% A; 50% O o Exclusion: B, AB • Example 2: Parent blood type: BO x AB o Possible blood type of children: 25% A, 25% AB, 50% B ▪ 25% HOMO, 75% HETERO • Example 3: Parent blood type: AA x BB o Possible blood type: 100% AB, 100% hetero • Example 4: Parent blood type: AO x BO o Possible blood type: 25% A, 25% AB, 25% B, 25% O ▪ 25% Homo, 75% Hetero • ABO BLOOD TYPE FREQUENCIES Blood Type (%) Asians O 40 A 28 B 25 AB 7 Whites 45 40 11 4 Blacks 50 26 20 4 Hispanics 56 31 10 7 ABH ANTIGENS • • • • • The formation of ABH antigens results from the interaction of genes at 3 separate loci (ABO, Hh, Se) In as early as 37th of fetal life, red cell antigens are already expressed on the RBC. Antibodies are developed later ABO genes code not for the Ag themselves but for the production of glycosyltransferases that add immunodominant sugars to a basic precursor substance Glycosyltransferases catalyze transfer immunodominant sugar from the plasma to a basic precursor substance o Catalyze formation of glycosidic bonds that link monosaccharide to form larger polysaccharide Basic Precursor Substance (BPS) is also known as paragloboside/glycan which is an oligosaccharide structure composed of 4 interlinked sugars. o It has 2 types: ▪ Type 1 - precursor substance for glycoprotein ▪ Type 2 - precursor substance for glycolipids o 4 interlinked sugars: glucose, galactose, Nacetylglucosamine, galactose o Basic Precursor Substance is the acceptors structure on RBC for sugars L-fucose (important for the expression of H antigen specificity) o H antigen - acceptor of A & B gene to produce A/B ag • • • • • • Glycosyltransferases H gene is present in chromosome 19 (secretor locus) o L-fucosyltransferase would catalyze the transfer of from the plasma to the Basic Precursor Substance (Lfucose is accepted by the basic precursor substance) o L-fucose is the immunodominant sugar responsible for H antigen specificity. ▪ When attached to BPS, H antigens structure is formed due to L-fucose specificity to H antigen o When L-fucose is attached to the basic precursor substance, H antigen structure is formed because Lfucose is specific for the H. o NOTE: H antigen structure is present in all blood groups regardless of the blood type A,B or AB and O. A gene – present in the long arm of chromosome 9 o N-acetylgalactosaminyltransferase - catalyzes the transfer of immunodominant sugar N-acetyl-Dgalactosamine from the plasma to the acceptor structure of the RBC o The acceptor structure for N-acetyl-D-galactosamine on RBC is H antigen structure. ▪ When the sugar is accepted, the H antigen structure will be modified to become A antigen ▪ N-acetyl-D-galactosamine - responsible for A antigen specificity B gene –present in the long arm of chromosome o D-galactosyltransferase - responsible for inheritance of B antigen ▪ Catalyzes transfer of D-galactose from plasma to the acceptor structure (H antigen) ▪ When accepted, H antigen is modified to become B antigen o D-galactose - responsible for B antigen specificty For blood type AB, the 2 genes are present (A & B gene) o H antigen is also present (as acceptor gene) For blood type O, O antigen is no formed. The H antigen structure is unmodified because it has no A and B antigen. It only possesses the unmodified L-fucose. Without the H structure, the A and B antigen are not present or expressed. Rare blood group that doesn’t have H antigen are Bombay (Oh) phenotype. o Possess an recessive geen H gene is a high frequency gene. This is present in 99.99% of all individual in the population. About 0.01% doesn’t have H gene which is known as H null (hh)/H recessive type/Bombay (Oh) phenotype. GENERAL CHARACTERISTIC OF ABH ANTIGENS • • On the 37th day of fetal life, attachment of immunodominant sugars occurs on the RBC membrane and it is dependent on ABH genes inherited. A antigen is weakly expressed on RBC during fetal life. The A antigens only become strongly expressed a year after birth. • • • • • • B and H antigen are strongly expressed on RBC during development. ABH antigens may be found in secretions of people who are secretors. Secretor individuals are individuals who inherit the so called “Secretor Gene”. A secretor individual (Sese+) can form the ABH soluble substances. These soluble substances are present in body secretion (particularly saliva) except in CSF o CSF contains BBB, any large molecules cannot pass through the Blood-Brain Barrier For nonsecretors (sese-), They do not form ABH soluble substances These antigens could also be found in bacteria and other species. The ABH-like antigens maybe found on the cell wall/surface of certain organism. GROUP ANTIGEN O H • • • • • • • B, H A,B,H These antibodies are not normally present at birth because they are developed 3-6 months after birth In the neonatal serum, if ABO antibodies are detected these antibodies present are not considered to be the newborn’s ab, it is maternally derived IgG abs. these ab were able to cross the placenta during pregnancy so these ab coming from the mother can present in the newborn serum For the newborn, the only blood typing that should be done is forward typing. Due to late development of abs. They occur in 2 forms: o Naturally occurring - normally present o Immune antibodies - present in individual o For the immune antibodies, they are present in individuals of the following condition: ▪ Patient with previous transfusion with mild ABO incompatibility (past interaction) ▪ Female patient with previous pregnancy with mild ABO incompatibility • The incompatibility is between mother and newborn due to the blood type of father These ab are present in some plants and animals They are present in low titer or even absent in some conditions such as: o Patient with AIDS because these patient develop immunocompromised state and therefore, you will have hypogammaglobulinemia o Autoimmune disorder - cells are counteracting own cells that reduces ab production o Active immune system (SLE, RA) ▪ The ab of these people with this disease produces autoantibodies, the remedy to this is immunosuppression to lower the ab production o Patient with Bruton’s disease (Congenital Gammaglobulinemia ▪ They die of overwhelming infection because abs are not formed ABO TYPING ABO ANTIBODIES Are mostly naturally occurring antibodies that are detectable 3-6 months after birth following exposure to ABO-like antigens in the environment Are mostly IgM (cold reacting - 4C) ab and react at room temperature (25C) or below B GENERAL CHARACTERISTICS OF ABO ANTIBODY • • A, H AB INTERACTION OF THE Sese, Zz, AND ABH GENE Secretor gene system (Sese) regulates the formation of H antigen and subsequently, of A and B antigens in secretory cells. • Therefore, it regulates the formation of glycoproteins (because of antigen B present in the secretion) • Zz system regulates production of H antigens on erythrocytes/RBC (glycolipids) ABH ABH soluble Antigens substance Location In RBCs, epithelial In all body fluids except tissues, bone CSF (BBB) (increased marrow, other cells protein in CSF is abnormal) Secreted Glycolipid Glycoprotein substances 1st sugar in the Glucose N-acetylgalactosamine precursor substance (paragloboside) Precursor chain Type 2 Type 1 and 2 Type 1 - predominant paragloboside for the glycoproteins Linkage • 1→4 linkage • Carbon of D-galactose to the carbon of Nacetylglucosamine • Type 2 precursor (1-4), first carbon of galactose to the 4th carbon of Nacetylglucossamine • For 1-3, first carbon of galactose to the 3rd carbon of N-acetylglucosamine (for ABH soluble substances Regulating gene Zz gene Se gene • Linkages - interaction of carbon atom between the sugars such as the carbon of D-galactose to the carbon of Nacetylglucosamine A ANTIBODIES Anti-A, Anti-B, Anti-AB (these 3 abs are mostly of the IgG type, these are high tittered clinically significant) Anti-B (mostly IgM high tittered clinically significant) Anti-A (mostly IgM high tittered clinically significant) None (AB is universal recipient) • • • BLOOD FORWARD TYPING TYPE ANTI A ANTI B ANTI AB O A + (↑↑ 3-4+) + (↑ 2+) B + (↑↑ 3-4+) + (↑ 2+) AB + + + Commonly used, used as screening procedure Uses commercially prepared typing sera Sample: washed RBC (for increased accuracy) BLOOD TYPE A B AB O BLUE TYPING SERA (Anti-A) + + - YELLOW TYPING SERA (Anti-B) + + - • • • • • • o • Saliva must be diluted using NSS to reduce non-ABH glycoproteins present in the sample which may cause postzone phenomenon (excess antigen) ▪ Add 1 mL of NSS to 1mL of saliva o Antisera must also be diluted using NSS Results Step 1 BLOOD REVERSE TYPING TYPE A CELLS B CELLS O CELLS O + + A + B + AB Confirmatory procedure Detects unknown antibodies Sample: serum (plasma contains fibrinogen that can cause reactions affecting testing) Known sample: laboratory prepared red cell suspension O cells are always negative (positive - Bombay phenotype) Saliva + Anti A Saliva + Anti B Saliva + Anti H o o o • • B cell O cell Present O O O O O O O O O A subs. B subs. H subs. E.g. Patient B Secretor (B, H genes) A cell B cell O cell Present 4+ O O O O O O O O None B subs. H subs. ABO SUBGROUPS • These are antigens under the ABO system which reacts less strongly with commercially prepared typing reagents/sera A SUBGROUP • • • Principle: hemagglutination inhibition o There is prevention of RBC clumping to occur o (+) = absence of agglutination o (-) = presence of agglutination 2-Step Procedure o Materials ▪ Saliva ▪ Known RCS ▪ Antisera / Lectin o Step 1: Saliva + Source of Antibody (antisera/lectin) o Step 2: Step 1 Mixture + Known RCS o In step 1, if there is neutralization, agglutination will be inhibited in step 2 Protocols in Saliva Neutralization Test o 2mL of saliva ▪ Check saliva for presence of blood • Presence of blood may affect results ▪ Filter saliva before use to remove impurities ▪ Saliva must be heated at 56C for 10 mins (to inactivate enzymes - salivary amylase) • Clear sample: complete heating • Hazy: incomplete heating; extend heating A cell • TEST FOR ABH SOLUBLE SUBSTANCES: SALIVA NEUTRALIZATION TEST • Soluble Substance Present A substance in saliva - no agglutination occurred due to neutralization of anti A by saliva in step 1 E.g. Patient AB Secretor (A, B, H genes) Saliva + Antisera Anti A (A sub) Anti B (B sub) Anti H (H sub) • Reaction Known A RCS + No A substance in saliva Step 1 Mixture agglutination Known B RCS + No B substance in saliva Step 1 Mixture agglutination Known O RCS + No H substance in saliva Step 1 Mixture agglutination Saliva + Antisera Anti A (A sub) Anti B (B sub) Anti H (H sub) ABH SOLUBLE SUBSTANCES These are glycoproteins found in the secretion of secretor individuals (Sese) except in CSF • These are absent among nonsecretor (sese) individual • (+) secretors - inherit homozygous or heterozygous Secretor genes (SeSe or Sese) • (-) secretors - inherit homozygous recessive secretors (sese); nonsecretors • 3 Regulating Gene: o ABO gene o H gene o Secretor genes Genes BT/ secretor stat A sub B sub H sub A,H,Se “A” type, secretor ↑↑ None ↑ B,H,Se “B” type, secretor None ↑↑ ↑ AB, H, Se “AB” type, secretor ↑ ↑ ↑ H,Se “O” type, secretor None None ↑↑ Step 2 • • • • First reported in 1911 by Sir Von Dungern He based his findings on the blood type “A” red cells with anti-A1 lectin. Anti-A1 lectin which derived from Dolichos biflorus is tested with blood type “A’’ red cells resulting to a positive agglutination result. Blood type A whose red cells reacted positive to anti-A1 is reported as subgroup A1 and those that resulted negative from anti-A1 lectin is reported as subgroup A2 What antigen is present on A1 individuals that are absent in A2 individuals? A1 antigen is present on blood type A RBC The subgroups of A first is differentiated based on the ff: o antigens o antibodies The antigens of subgroup A1 and A2 can be differentiated based from Qualitative and Quantitative differentiation. Subgroup A1 Subgroup A2 The ag are highly The ag are linear Qualitative branching complex ags simple structure The ag are numerous in The ag are few in number (due to Quantitative number (cause 2+ increased production; reaction) may cause 4+ rxn) • o o Subgroup Antigen Antibody A1 H, A, A1 Anti-B (IgM) A2 H, A Anti-B, Anti-A1 Anti-B - high tittered clinically significant Anti-A1 - low tittered clinically insignificant; clinically significant which may cause ABO discrepancy • In the population of about 1-8% of A2 individuals can form anti-A1. • A2B (Blood type AB with subgroup A2) population, about 22-25% can form Anti A1. Forward Blood Group Frequency Anti-A Anti-B Anti-A1 Anti-H A1 Approx. 80% ++++ ++ Wk+ to A2 <20% +++ ++ REACTIVITY OF ANTI-H ANTISERA OR ANTI-H LECTIN WITH ABO BLOOD GROUP • • • Greatest Amount of H Ag - Least Amount of H Ag O > A2 > B > A2B > A1 > A1B Blood type O has the greatest H antigen on the RBC surface followed by A2, etc. The greater the H antigen, the stronger reaction to Anti-H WEAK SUBGROUPS OF A • • Characteristic of Weak Subgroups of A o Decrease number in A antigen site per RBC resulting to weak or no reaction with Anti-A and Anti-AB typing serum (0 or mf+) o Varying degree of agglutination with human anti-AB o Increase variability in the detectability of H antigen on RBC resulting to stronger anti-H reaction o Presence or absence of Anti-A1 in the serum Weak Subgroup of A o A3: demonstrate “mf” rxn with anti-An and most antiA,B o Ax: RBCs do not agglutinate with anti-A but do not agglutinate with anti-A,B o Aend: demonstrate “mf” rxn but only small amounts of RBCs agglutinate, needs microscope to confirm agg o Am: RBCs are not agglutinates, weakly agglutinated by Anti A or Anti A, B o Ay: RBCs are not agglutinated by either Anti-A or AntiA,B o Ael: typically is unagglutinated by anti-A or anti-A,B o Adsorption and elution confirms presence of weak Ay and Ael antigens B SUBGROUP • • • • • This subgroup is infrequent and therefore, it is considered as clinically insignificant The lectin used in this subgroup is Bandareae simplicifolia (known now as Griffonia simplicifolia Characteristic of Weak B Subgroups: o Strength and type of reaction with anti-B o Presence or absence of ABO isoagglutinin o Adsorption elution studies with anti-B o Presence of B substance in the saliva Adsorption technique - confirmatory procedure to determine the presence of weak subgroups A and B Weak B Subgroup o B3: mf pattern in anti-B and anti-A,B o Bx: demonstrate weak agglutination with anti-B & anti-A,B o Bm: characteristically unagglutinated by anti-B or anti-A,B o Bel: extremely rare subgroups, unagglutinated by both anti-B or anti-A,B o These subgroups are prone to discrepancies due to weak reactions (1+, 2+). BOMBAY PHENOTYPE (Oh) • The allele H is very rare and does not produce the Lfucose necessary for the formation of H structure (acceptor to modify H ag to become A/B ag). • The genotype hh or Hnull is also known as the Bombay phenotype (Oh) o Oh - do not react with anti-H lectin o Normal O - OH • 2 types of Bombay Phenotype: o Classical Bombay ▪ Designated as “Oh” ▪ H gene is absent thus, H antigen is absent ▪ First reported in India by Mumbai, 1952. o Parabombay ▪ Designated as Ah, Bh, ABh ▪ H gene is present but weak ▪ A and B antigens are demonstrated on RBC but not H antigen ▪ First identified in a Caucasian patient • Characterisitcs of Bombay Phenotype o Absence of H, A, and B antigens o Presence of Anti-H, Anti-A, Anti-B in serum (clinically significant) ▪ Anti-H - hemolytic type of antibody, reacts in a wide thermal range ▪ O - do not become universal donor, will countereact with the receiver ▪ Bombay is the only donor and receiver o Strong reactivity with Anti-I reagents ▪ *On the RBC of the Classical Bombay, I-antigen like structures are present. I-antigen-like structures is responsible for the strong reactivity with the reagent (hh, Hnull) o A recessive mode of inheritance (hh, Hnull) o No reaction with Anti-H lectin (Ulex europeus) Forward Typing Reverse Anti Anti Anti H derived A B O A B from U. europeus cell cell cell OH + + + (Type “O”) (4+) **w/o anti-H Oh + + + (bombay) **w/o anti-H • Anti-H - important in confirming Bombay phenotypes ABO DISCREPANCIES • • • Screening (forward) and confirmatory (reverse) results must be of the similar blood group. If it’s different to each other there is discrepancy. Any deviation from the expected of antigen on the cell and the opposite antibody in serum is defined as discrepancy Technical Errors Resulting to ABO Discrepancies o Inadequate identification of blood samples, test tubes or slides (always label) o Cell suspensions either too heavy ot too light due to zonal phenomenon o Clerical errors (Most common error in clinical laboratory) o Mix up of samples o Failure to add reagents/failure to follow manufacturer’s instructions (usage, storage, etc.) o Contaminated reagents o Uncalibrated centrifuge • • General Rules to Resolve Discrepancies o Always re-test first ▪ It is performed to confirm the presence the so called procedural error ▪ Procedural error - caused by MT ▪ Check the diagnosis o Check for clerical/ technical error ▪ NOTE: check for errors such as double addition of reagents, uncalibrated centrifuge, clerical errors o Weakest reaction is usually the one in doubt ▪ 3+/4+ - Considered strong reaction/ true reaction ▪ 1+/2+/mf/+ - Doubtful reaction ▪ Weak reactions must be verified o Check results of the screening cells ▪ Screening cells are used to detect alloantibodies ▪ While Autocontrol is used to detect autoantibodies ▪ If the screening cells and autocontrol are positive this indicates presence of unexpected antibodies o Check the patient’s age ▪ Patient’s age is useful for further assess the possible cause of discrepancies. ▪ Newborns <3mos have under/undeveloped abs ▪ For adults >65 y.o, abs are weakly reacting due to decreasing ab titer due to declining immunity o Check the diagnosis ▪ Cancer, autoimmune disease can cause discrepancys o Check the transfusion history ▪ If the patient had been transfused blood in the past, you must consider the presence of Alloantibodies. ▪ The RBC of donor can possibly stimulate the patient to produce antibodies which is alloantibodies. Steps in Solving Discrepancy Problems o Check the result of Autocontrol (AC) ▪ Normal result is negative (O) ▪ Autocontrol contains patient’s serum and RBC ▪ Autocontrol result must be negative. If it’s positive the patient’s serum contains autoantibodies that reacted on its own RBC o Check the result of anti-AB and O cell control ▪ Anti-AB - control in forward • Result: (+) - anti-A,B,AB = (+) anti-AB • Result: (-) - anti-A,B = (-) AB ▪ O-cell - control in reverse • Result: all negative o Determine the forward blood type o Determine the reverse blood type o Determine if there is discrepancy • Forward Reverse Anti A Anti B Anti A cells B cells O cell AB 2+ mf neg 2+ mf neg 4+ neg • FOUR GROUPS OF ABO DISCREPANCIES GROUP 1 • • • Due to weakly reacting or missing antibodies, most common encountered Forward typing is correct, reverse typing is discrepant Usual blood type AB o A cell= false neg o B cell= false neg o O cell= neg It is found in the following: o Newborn (<3 months) o Elderly patients (>65 y.o.) - due to decreasing ab titer o Leukemic patients demonstrating hypogammaglobulinemia such as chronic lymphocytic leukemia (CLL) o Patients with lymphoma o Patients using immunosuppressive drugs ▪ Patient with abnormal hyperactive immune system needs immunosuppressive drugs. ▪ In autoimmune disorder, the immune system is active but the problem is the antibodies produced is Autoantibodies which cannot discriminate self cell to nonself cell o Patient with congenital agammaglobulinemia ▪ Bruton’s disease - antibodies are not formed o Patients with immunodeficiency o Patients ho undergone bone marrow transplant who developed hypogammaglobulinemia (low ab production) from therapy o Chimerism ▪ a rare group 1 discrepancy ▪ Part of group 1 and group 2 discrepancy • Considered as group 2 in Harmenning ▪ Presence of 2 cell population in single individual’s circulation (ex. Fraternal twin ▪ Fraternal twins (true chimerism) ▪ Artificial Chimera • Blood transfusion (O to A or B) • BM transplant • Exchange transfusion • Fetal-maternal bleeding ▪ Example of Chimerism • Baby 1: Type A • AC 0 o Forward blood type: A o Reverse blood type: A o Antigens: A o Antibody: anti-B o No discrepancy Baby 2: Blood type Forward Reverse Anti A Anti B Anti A1 B cells O cell AC AB cells +wk neg +wk neg neg neg 0 o Forward blood type: A (doubtful) o Reverse blood type: AB o Antigens: o Antibodies: missing antibody o Maybe due to newborn status o When immune system is capable of producing abs, ags will recognize abs as self in a mechanism called immunetolerance Resolution o In group 1 discrepancy, reverse typing must be enhanced by reverse typing incubation must be extended at room temp at 15-30 mins. o If cryoglobulin/cold agglutinins are suspected, incubate the serum sample at ref temperature because they react optimally at cooled temp (4C = IgM) GROUP 2 • • • • • Due to missing antigens/weakly reacting antigen and is the least frequently encountered Forward typing is discrepant, Reverse is correct It is found in: o Subgroups of A or B o Leukemia may yield weakened A or B antigens o Hodgkin’s disease o Acquired B (in GIT obstruction, colon, or rectum CA, comatose) EPIC (cause of Group 2 phenomenon) o E - E. coli O86 o P - Proteus spp. o I - intestinal obstruction o C - colorectal cancer Acquired B Phenomenon o EPIC (cause of Acquire B phenomenon) ▪ E ▪ P ▪ I ▪ C • GROUP 3 • • o • When bacterial enzyme of Proteus vulgaris (??) modify N-acetylgalactosamine into D-galactose) – most common cause of bacterial enzyme disease caused by production of D-acetylase ▪ Cause increased permeability of the intestinal wall from absorption of bacterial polysaccharides into the RBC o In Acquired B phenomenon, blood type A is affected. o Patient is naturally blood A o NOTE: Not all red cells can modify the antigens present in the individual, those that red cells only that are exposed or infected by bacterial enzymes. o Blood type: A (bacterially infected) Forward Reverse Anti A Anti B A cell B cells 4+ 1+ Neg 4+ o Resolution ▪ Perform typing sera acidification 1. 1-2 drops of 1N HCl in 1mL of antiserum to maintain pH 6.0 2. After acidifying the typing sera, react forward typing serum • Acidified typing reagent can only react with true antigens. I will not react with modified/acquired antigens Acquired A Phenomenon o Proteus mirabilis is responsible for acquired A phenomenon o Increased permeability of the intestinal wall causes adsorption of B-like polysaccharide onto red cells of A or B patients (P. vulgaris or E coli O86) Other Causes of Group 2 Discrepancies o Antibody low incidence antigen (may be present in reagent antisera) o Excess Blood Group Specific Soluble Substances (BGSS) ▪ Released in cases of stomach or pancreas CA ▪ Secretory cells are seen in stomach and pancreas. If there’s malignancy in pancreas and stomach, there will be hypersecretion of digestive fluid, pancreatic fluids inclusing soluble BGSS ▪ If these 2 are the diagnosis, BGSS could promote dilution effect ▪ *Dilution effect - the excess BGSS could dilute the ags present on the RBC surface affecting the reaction of the sample in the antisera making in weaker to the point there will be negative reaction ▪ Resolution: cell washing (enhance reaction) • • Caused by protein or abnormalities resulting to rouleaux formation (stacking of RBCs due to increased abnormal proteins will cause pseudoagglutination It is found in: o Increased globulin (eg Multiple myeloma (excess IgG), Waldenstrom macroglobulinemia (excess IgM)) o Increased fibrinogen o Presence of plasma expanders (e.g. dextran, PVP (Polyvinyl pyrolidone)) ▪ Plasma expanders - synthetic volume expander o Wharton’s Jelly (present on cord bloods) - a mucopolysaccharide substance found on cord blood. Number of RBC washing must be increased about minimum 6-8x (10x) Sample Forward Reverse CASE Anti Anti Anti A1 B O AC A B A,B cell cell cell 4+ 4+ 4+ 2+ 2+ 2+ 2+ o Forward: AB o Reverse: cannot be determined (spontaneous rxns) o Invalid due to 2+ autocontrol reaction o Possible Cause: ▪ In the serum of the patient, proteins may be increased resulting to rouleaux formation (multiple myeloma) ▪ Cold autoab (possible type: AB w/ autoanti-I) Resolution: o Wash red cell o If cold ab, prewarm technique (IgM in the patient’s serum would be inactive), adsorption technique, treatment with sulfhydryl compounds; rabbit erythrocyte stroma (REST) GROUP 4 • due to miscellaneous problems seen in: o Polyagglutination (Hubener-Thomsen Friedenreich phenomenon – spontaneous red cell agglutination by most normal human serum) ▪ RBC agglutinates nonspecifically and spontaneously to most human sera ▪ Exposure of the hidden antigen/crypt antigen on the RBC surface make the RBC polyagglutinable ▪ o o o o o 3 Types of Polyagglutinable RBC: • Microbially-associated type (acquired) o Most common cause of polyagglutination o When patient is infected with neuraminidase-producing microbe/virus o This enzyme can expose the hidden antigen o Microbes that can form neuraminidase: ▪ Clostridium spp ▪ Vibrio spp. ▪ Pneumococci ▪ influenza virus (can damage respiratory tissue of infected) • Non-microbially associated type o Mutations/ alterations in the hematopoietic tissues leading to the exposure of the hidden antigen in RBC o Less common cause of polyagglutination • Inherited type o Transmission of gene responsible for the exposure of crypt antigen Occurs owing to exposure of a hidden RBC antigen in patients with bacterial and viral infections Cold reacting abs (allo and auto) Unexpected ABO isoagglutinins Warm autoantibodies RBCs with cis AB phenotype (mutation) ▪ Results in the formation of weak RBC antigens ▪ Normally, father and mother gives 1 allele each but in this case, 3 genes are passed down to offspring (2 genes and 1 gene (O gene)) producing weak antigen expression OTHER CASE SAMPLES OF ABO DISCREPANCIES Anti Anti Anti A1 B O AC A B A,B cell cell cell • Forward: O • Reverse: AB • Possible Cause: Group 1 discrepancy (missing ab) o Type O Newborn or geriatric patient (weak reacting) • Resolution: o Check age (newborn/geriatric) o To enhance ab, enhance reverse typing procedure CASE Anti Anti Anti A1 B O AC 2 A B A,B cell cell cell 4+ 4+ 1+ 4+ • Forward: A • Reverse: O • Possible Cause: Antibody against A1 antigen (type A2) • Resolution: Use anti-A1 Lectin to A subgroup type CASE Anti Anti Anti A1 B O AC 3 A B A,B cell cell cell 4+ 4+ 4+ • Forward: O • Reverse: O • Possible Cause: Bombay phenotype (anti-H) • Resolution: Confirm type with anti-H Lectin o Bombay phenotype = (-) anti-H Lectin CASE 4 Anti Anti Anti A1 B O AC A B A,B cell cell cell 4+ 1+ 4+ 4+ neg neg • Forward: AB (anti-B rxn doubt) • Reverse: A • Possible Cause: Group 2 discrepancy o Weak anti-B rxn - acquired B phenomenon o Low incidence antibody • Resolution: o Check/verify diagnosis & patient history o Acidify anti-B TS for forward typing ▪ Retest forward typing o Check viability of antisera ▪ Use antiserum on different lot number CASE Anti Anti Anti A1 B O AC 5 A B A,B cell cell cell -/ +wk -/ +wk 4+ • Forward: A • Reverse: A • Possible Cause: Group 2 discrepancy o Patient may have pancreatic cancer (may cause excess BGSS = dilutional effect) • Resolution: o Cell wash (3x - venous blood; 6x - cord blood) o Retest after cell wash DISCOVERY, LAWS, PROPERTIES, CHARACTERISTICS, AND INHERITANCE OF ABO BLOOD GROUP SYSTEM DISCOVERIES • • CASE 1 • Karl Landsteiner: discovered the first human blood group system (ABO). o In 1910: He is the first individual to perform forward and reverse typing from drawing blood from himself and five associates. Forward Grouping (front type): defined as using known sources of commercial antisera, anti-A & anti-B, to detect antigens on an individual’s RBC. Reverse Grouping (back type): defined as detecting ABO antibodies in the patient’s serum by using known reagent RBCs, namely A1and B cells. LAWS, PROPERTIES & CHARACTERISTICS • There is always an inverse reciprocal relationship between the forward and reverse type. For example, if the individual has A antigens only on their red blood cells, there will be an “expected” naturally occurring anti-B antibody in their serum since they lack the B antigen. Blood type A B AB O Anti-A 4+ 0 4+ 0 Anti-B 0 4+ 4+ 4+ Anti-A, Anti-B 4+ 4+ 4+ 0 A cells 0 4+ 0 4+ B cells 4+ 0 0 4+ O cells 0 0 0 0 • The frequency of the ABO blood groups differs among selected populations and ethnic groups (e.g. group B is found twice as frequently in blacks and Asians as in whites or subgroup A2 is rarely found in Asians) • • • • • • • ABO Antibodies o Naturally occurring because they are produced without any exposure to RBCs o Predominantly IgM, activate complement, and react at room temperature or colder o Produce strong direct agglutination reactions during ABO testing o ABO antibody production is initiated at birth, but titers are generally too low for detection until infants are 3 to 6 months old o Antibody production peaks between 5 and 10 years of age and declines later in life o ABO antibodies can cause rapid intravascular hemolysis if the wrong ABO group is transfused, potentially resulting in patient death ABO Antigens o Develop early in fetal life and do not increase much in strength during the gestational period o RBCs of the newborn have been estimated to carry anywhere from 25% to 50% of the number of antigenic sites found on the adult RBC o Formation of ABH antigens results from the interaction of genes at three separate loci (ABO, Hh, and Se). these genes produce glycosyltransferases that add sugars to a basic precursor substance/ o ABH antigens on the RBC are constructed on oligosaccharide chains of a type 2 precursor substance o Antigens can be glycolipids, glycoproteins, or glycosphingolipids o It has been postulated that bacteria, pollen particles, and other substances present in nature are chemically similar to A and B antigens The formation of ABH antigens results from the interaction of genes at three separate loci (ABO, Hh, and Se). These do not produce antigens but produces specific glycosyltransferases that add sugar to BPS. Basic precursor is named as paragloboside/ceramide. When the terminal galactose on the precursor substance is attached to the N-acetylglucosamine in a beta 1 → 4 linkage, the precursor substance on erythrocytes is referred to as type 2. ABH antigens on the RBC are constructed on oligosaccharide chains of a type 2 precursor substance. A type 1 precursor substance refers to a beta 1 → 3 linkage between galactose and N-acetylglucosamine. INHERITANCE • • • • • • In 1924, the Theory of inheritance for ABO blood groups is first described by indicating that an individual inherits one ABO gene from each parent and that these two genes determine which ABO antigens are present on the RBC membrane. These follows the simple Mendelian genetics. The O gene is considered an amorph. The group O phenotype is an autosomal recessive trait with the inheritance of two nonfunctional O genes. A & B: Inherited as autosomal codominant 50% haplotype from mother, 50% haplotype from father For example: Father is heterozygous B; Mother is AB A B B AB BB O AO BO o Offsprings: 25% - AB, 25% - A, 50% - B