ABO Blood Group System PDF

120 PART II Blood Groups and Serologic Testing Even today, transfusion of the wrong ABO group remains a cause of death in hemolytic transfusion reaction fatalities BOX 6–1 reported to the FDA; however, transfusion-related acute lung...

120 PART II Blood Groups and Serologic Testing Even today, transfusion of the wrong ABO group remains a cause of death in hemolytic transfusion reaction fatalities BOX 6–1 reported to the FDA; however, transfusion-related acute lung Causes of Fatal Hemolytic Transfusion Reactions Due injury (TRALI) was the most frequent cause of death in fiscal to ABO-Incompatible Blood Transfusions in FY 2015 year (FY) 20151 (Table 6–1). In FY 2015, there were two re- Case 1: Sample collected from improperly identified patient ports of fatal hemolytic transfusion reactions due to ABO- (phlebotomist error) incompatible blood product transfusions (Box 6–1 lists the Case 2: A group B patient received a group A apheresis platelet. causes in each of these two cases).1 This chapter presents the The platelet was later identified with a high anti-B titer of 1:2048. ABO blood group system and discusses the biochemistry, properties, and characteristics of ABO antigens and antibod- Data from 2015, F. R. (n.d.): Vaccines, Blood & Biologics; U.S. Food and Drug ies. In addition, weak subgroups and common discrepancies Administration. Fatalities Reported to FDA Following Blood Collection and Transfusion: Annual Summary for Fiscal Year 2015. Retrieved March 10, 2017, will be introduced to provide a working knowledge for from www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ReportaProblem/ routine ABO testing. TransfusionDonationFatalities/ucm518148.pdf. Historical Perspective and Routine ABO Testing and B cells. Figure 6–2 outlines the steps of performing the reverse ABO grouping (see color insert following page 128), Karl Landsteiner truly opened the doors of blood banking and Table 6–3 summarizes the results of the procedures. with his discovery of the first human blood group system, Table 6–4 lists the characteristics of the routine reagents used ABO. This marked the beginning of the concept of individual for ABO testing in the blood bank laboratory. uniqueness defined by the RBC antigens present on the RBC ABO grouping is the most frequently performed test in membrane. In 1901, Landsteiner drew blood from himself the blood bank. Both ABO forward and reverse grouping and five associates, separated the cells and serum, and then tests must be performed on all donors and patients.3 There mixed each cell sample with each serum.2 He was inadver- is always an inverse reciprocal relationship between the for- tently the first individual to perform forward and reverse ward and reverse type; thus, one serves as a check on the grouping. Forward grouping (front type) is defined as using other. For example, if the individual has A antigens only on known sources of commercial antisera (anti-A, anti-B) to de- their red blood cells, there will be an “expected” naturally tect antigens on an individual’s RBCs. Figure 6–1 outlines occurring anti-B antibody in their serum since they lack the the steps of performing the forward grouping for ABO (see B antigen. color insert following page 128), and Table 6–2 lists the re- It has been postulated that bacteria, pollen particles, and sults of the forward grouping procedure. Reverse grouping other substances present in nature are chemically similar to (back type) is defined as detecting ABO antibodies in the A and B antigens. Bacteria are widespread in the environ- patient’s serum by using known reagent RBCs, namely A1 ment, which constantly exposes individuals to A-like and Table 6–1 Fatality Complication Breakdown by Imputability FY 2015 Doubtful/ Undetermined Definite/ Probable/ Unlikely/ Ruled Out/ Not Assessable Category Certain Likely Possible Improbable Excluded or Evaluable Total TRALI** 5 N/A* 7 1 1 – 14 HTR** (non-ABO) 2 1 1 1 – – 5 HTR (ABO) 2 – – – – – 2 Contamination 3 – 2 – – 5 (Bacterial) TACO** 3 6 2 – – 1 12 Allergy or 2 – – – – – 2 Anaphylaxis Hypotensive – 1 – 1 – – 2 Reaction *Definitions based on the Canadian Consensus Panel on TRALI. ** HTR = hemolytic transfusion reaction; TACO = transfusion-associated circulatory overload; TRALI = transfusion-related acute lung injury Data from 2015, F. R. (n.d.): Vaccines, Blood & Biologics; U.S. Food and Drug Administration. Fatalities Reported to FDA Following Blood Collection and Transfusion: Annual Summary for Fiscal Year 2015. Retrieved March 10, 2017, from www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ReportaProblem/TransfusionDonationFatalities/ucm204763.htm  Chapter 6 The ABO Blood Group System 121 Table 6–2 ABO Forward Grouping: B-like antigens. This exposure serves as a source of stimula- tion of anti-A and anti-B. All other defined blood group sys- Principle—Detection of Antigens tems do not regularly have “naturally occurring” antibodies on Patient’s RBCs With Known expected in their serum to antigens they lack on their RBCs. Commercial Antisera Antibody production in most other blood group systems Patient RBCs Patient RBCs Interpretation requires the introduction of foreign RBCs by either transfu- with Anti-A with Anti-B of Blood Group sion or pregnancy, although some individuals can occasion- ally have antibodies present that are not related to the 0 0 O introduction of foreign RBCs. (These antibodies are usually 4+ 0 A of the IgM type and are not consistently present or expected in everyone’s serum.) Performance of serum grouping is, 0 4+ B therefore, unique to the ABO blood group system. The reg- 4+ 4+ AB ular occurrence of anti-A and/or anti-B in persons lacking the corresponding antigen(s) serves as a confirmation of re- + = visual agglutination; 0 = negative sults in ABO grouping. Table 6–5 summarizes the forward Note: Reaction gradings vary from patient to patient. and reverse grouping for the common ABO blood groups. The frequency of the ABO blood groups differs among Table 6–3 ABO Reverse Grouping: selected populations and ethnic groups (Table 6–6).4 For example, group B is found twice as frequently in blacks and Principle—Detection of ABO Asians as in whites. There is also a significant decrease in Antibodies (Isoagglutinins) in group A distribution in these two ethnic populations com- Serum of Patient With Known pared to whites. It has been reported that subgroup A2 is Commercial RBCs rarely found in Asians.5 Patient Serum Patient Serum with Reagent with Reagent Interpretation ABO Antibodies A1 Cells B Cells of Blood Group Individuals normally produce antibodies directed against the 4+ 4+ O A and/or B antigen(s) absent from their RBCs. These anti- 0 3+ A bodies have been described as naturally occurring because they are produced without any exposure to RBCs. The ABO 3+ 0 B antibodies are predominantly IgM, activate complement, and 0 0 AB react at room temperature or colder.5 ABO antibodies produce strong direct agglutination reactions during ABO + = visual agglutination; 0 = negative testing. ABO antibody production is initiated at birth, Note: Reaction gradings vary from patient to patient. but titers are generally too low for detection until infants are Table 6–4 Characteristics of Routine Reagents Used for ABO Testing Forward Grouping Anti-A Reagent Anti-B Reagent Monoclonal antibody* Monoclonal antibody* Highly specific Highly specific IgM IgM Clear blue-colored reagent Clear yellow-colored reagent (contains an acriflavine dye) Expected 3+ to 4+ reaction Expected 3+ to 4+ reaction Usually use 1–2 drops Usually use 1–2 drops Reverse Grouping Reagent A1 and B Cells Human source 4%–5% RBC suspension Expected 2+ to 4+ reaction usually use 1 drop *General rule: Always drop clear solutions first and RBCs second to make sure you have added both a source of antibody and antigen. 122 PART II Blood Groups and Serologic Testing Table 6–5 Summary of Forward and Reverse Groupings Forward Group Patient’s Cells With Reagents Reverse Group Patient’s Serum With Reagents Blood Antigen(S) Antibody(ies) Group Anti-A Anti-B on RBCS A1 Cells B Cells in Serum O 0 0 No A or B antigen 4+ 4+ A and B A 4+ 0 A 0 2+ B B 0 4+ B 3+ 0 A AB 3+ 3+ A and B 0 0 No A or B antibodies 0 = negative (no agglutination); + = visual agglutination Note: Reaction gradings vary from patient to patient. *Percentages rounded to the nearest whole number Table 6–6 ABO Phenotype Frequencies if group O serum is adsorbed with A or B cells, the antibody eluted will react with both A and B cells.6,7 Anti-A,B antibody of Ethnic Groups in the United is not a combination of anti-A and anti-B but is a separate States “cross-reacting” antibody that is usually IgG in nature.5 U.S. Frequencies (%) (Rounded to the Nearest Knowing the amount of IgG anti-A, anti-B, or anti-A,B in Whole Number) a woman’s serum sometimes allows prediction or diagnosis Phenotype Whites Blacks Hispanic Asian** of hemolytic disease of the fetus and newborn (HDFN) caused by ABO incompatibility.8 (See Chapter 20, O 45 50 56 40 “Hemolytic Disease of the Fetus and Newborn [HDFN]”). A 40 26 31 28 Both immunoglobulin classes of ABO antibodies react preferentially at room temperature (20°C to 24°C) or below B 11 20 10 25 and efficiently activate complement at 37°C.9 AB 4 4 3 7 Anti-A,B reagent is routinely used for performing ABO confirmation of group O donor units simply because it is *Hispanic includes Mexican, Puerto Rican, Cuban, and other Hispanics. more economical to use one reagent instead of both anti-A **Asian includes Chinese, Filipino, Indian, Japanese, Korean, and Vietnamese. and anti-B.3 Use of anti-A,B reagent is not required for Data from Garratty G, Glynn SA, McEntire R. ABO and Rh (D) phenotype frequencies of different racial/ethnic groups in the United States. Transfusion. 2004;44:703–706. routine patient ABO testing.10 Some believe anti-A,B is more effective at detecting weakly expressed A and B antigens than reagent anti-A or anti-B. The production and use of mono- clonal antisera, however, have made anti-A and anti-B 3 to 6 months old.5 Therefore, most antibodies found in cord reagents much more sensitive, to the point where weak A blood serum are of maternal origin. Results of serum ABO and B antigens are detected routinely. Reagent anti-A,B can testing before 3 to 6 months of age cannot be considered be prepared using blended monoclonal anti-A and anti-B; valid because some or all of the antibodies present may polyclonal human anti-A,B; or a blend of monoclonal be IgG maternal antibodies that crossed the placenta. As a anti-A, anti-B, and anti-A,B.3 Always consult the manufac- result, it is logical to perform only forward grouping on cord turer’s product insert to determine if a reagent anti-A,B reacts blood from newborn infants. with a specific weak A phenotype. Antibody production peaks between 5 and 10 years of age and declines later in life.5 Elderly people usually have lower Inheritance of the ABO Blood Groups levels of anti-A and anti-B; therefore, antibodies may be undetectable in the reverse grouping (see the “ABO Discrep- The theory for the inheritance of the ABO blood groups ancies” section later in this chapter). ABO antibodies can was first described in 1924 by indicating that an individual cause rapid intravascular hemolysis if the wrong ABO group inherits one ABO gene from each parent and that these two is transfused, potentially resulting in patient death.1 genes determine which ABO antigens are present on the Although anti-A (from a group B individual) and anti-B RBC membrane. The inheritance of ABO genes, therefore, (from a group A individual) contain predominantly IgM follows simple Mendelian genetics. ABO, like most other antibody, small quantities of IgG may also be present.5 Serum blood group systems, is codominant in expression.9 (For a from group O individuals contains anti-A, anti-B, and review of genetics, see Chapter 2, “Basic Genetics.”) One anti-A,B. Anti-A,B reacts with both A and B cells. Anti-A,B position, or locus, on each chromosome 9 is occupied by antibody activity, originally thought to be just a mixture an A, B, or O gene.11,12 The O gene is considered an of anti-A and anti-B, cannot be separated into a pure speci- amorph, as no detectable antigen is produced in response ficity when adsorbed with either A or B cells.6,7 For example, to the inheritance of this gene. The group O phenotype is  Chapter 6 The ABO Blood Group System 123 an autosomal recessive trait with the inheritance of two Table 6–7 ABO Groups of Offspring from nonfunctional O genes. Various Possible ABO Matings The designations group A and B refer to phenotypes, whereas AA, BO, and OO denote genotypes. In the case of Offspring Mating an O individual, both phenotype and genotype are the same Possible Mating Phenotypes because that individual would have to be homozygous for Phenotypes Genotypes (and Genotypes) the O gene. An individual who has the phenotype A (or B) A×A AA × AA A (AA) can have the genotype AA or AO (or BB or BO). Box 6–2 lists the ABO genotypes and phenotypes. Serologically, it is AA × AO A (AA or AO) not possible to determine the genotype from the phenotype AO × AO A (AA or AO) or O(OO) of an A or B individual. Family studies or molecular assays would need to be performed to determine the exact geno- B×B BB × BB B (BB) type. The phenotype and genotype are the same in an AB BB × BO B (BB or BO) individual because of the inheritance of both the A and B gene. Table 6–7 lists possible ABO phenotypes and geno- BO × BO B (BB or BO) or O (OO) types from various matings. AB × AB AB × AB AB (AB) or A (AA) or B(BB) Formation of A, B, and H Red Blood Cell Antigens O×O OO × OO O (OO) A×B AA × BB AB (AB) The formation of ABH antigens results from the interaction of genes at three separate loci (ABO, Hh, and Se). These AO × BB AB (AB) or B (BO) genes do not actually code for the production of antigens but AA × BO AB (AB) or A (AO) rather produce specific glycosyltransferases that add sugars to a basic precursor substance (Table 6–8). A paragloboside AO × BO AB (AB) or A (AO) or B or glycan is the same basic precursor material from which (BO) or O (OO) A, B, and H antigens all originate. Specific enzyme trans- A×O AA × OO A (AO) ferases elicited by an inherited gene attach sugars to the para- globoside/glycan.11–13 AO × OO A (AO) or O (OO) When the terminal galactose on the precursor substance A × AB AA × AB AB (AB) or A (AA) is attached to the N-acetylglucosamine in a beta 1 → 4 link- age (Fig. 6–3), the precursor substance on erythrocytes is re- AO × AB AB (AB) or A (AA or AO) ferred to as type 2. ABH antigens on the RBC are constructed or B (BO) on oligosaccharide chains of a type 2 precursor substance.14 B×O BB × OO B (BO) A type 1 precursor substance refers to a beta 1 → 3 linkage between galactose and N-acetylglucosamine (see “Formation BO × OO B (BO) or O (OO) of A, B, and H Soluble Antigens” section). B × AB BB × AB AB (AB) or B (BB) The H antigen is the precursor structure on which A and B antigens are made. Inheritance of the H gene results in BO × AB AB (AB) or B (BB or BO) or A (AO) formation of the H antigen. The FUT 1 (H) and FUT 2 (Se) genes are closely linked and located on chromosome 19, AB × O AB × OO A (AO) or B (BO) in contrast to the ABO genes located on chromosome 9. The H and Se genes are not part of the ABO system; yet, their in- heritance influences A and B antigen expression. The H gene BOX 6–2 must be inherited to form ABO antigens on the RBCs, and ABO Genotypes and Phenotypes the Se gene must be inherited to form ABO antigens in Genotype Phenotype secretions. A 1A 1 A1 The ABH antigens develop early in fetal life and do not A 1A 2 A1 increase much in strength during the gestational period. The A 1O A1 RBCs of the newborn have been estimated to carry anywhere A 2A 2 A2 from 25% to 50% of the number of antigenic sites found on A 2O A2 the adult RBC.9 Consequently, reactions of newborn RBCs A 1B A 1B with ABO reagent antisera are frequently weaker than reac- A 2B A 2B tions with adult cells. The expression of A and B antigens on OO O the RBCs is fully developed by 2 to 4 years of age and BB B remains constant throughout life.9 In addition to age, the BO B phenotypic expression of ABH antigens may vary with race, genetic interaction, and disease states.15 124 PART II Blood Groups and Serologic Testing Table 6–8 Glycosyltransferases and Immunodominant Sugars Responsible for H, A, and B Antigen Specificities Gene Glycosyltransferase Immunodominant Sugar Antigen H (FUT1) α-2-L-fucosyltransferase L-fucose H A α-3-N-acetylgalactosaminyltransferase N-acetyl-D-galactosamine A B α-3-D-galactosyltransferase D-galactose B (6) CH2OH Fucose: 5 0 GAL FUC Immunodominant 4 D-galactose 1 GLNAC sugar responsible "H" antigen for "H" specificity 3 2 0 (6) CH2OH GAL "1 4 Linkage" 5 0 4 N-acetylglucosamine 1 GL Protein 3 2 Type-2 precursor chain NHCOCH3 Ceramide D-galactose Glucose Spectrin ( Precursor Structure ) P.S. HH Hh L-fucosyl transferase "H" antigen (genotype inherited) Figure 6–3. Type 2 precursor chain. Figure 6–4. Formation of the H antigen. Interaction of Hh and ABO Genes hh is extremely rare. The term Bombay has been used to refer to the phenotype that lacks normal expression of the Individuals who are blood group O inherit at least one ABH antigens because of the inheritance of the hh geno- FUT 1(H) gene (genotype HH or Hh) and two O genes. The type. The hh genotype does not elicit production of α-2- H gene elicits the production of an enzyme called α-2-L- L-fucosyltransferase. Accordingly, L-fucose is not added to fucosyltransferase that transfers the sugar L-fucose to an the type 2 chain and H substance is not expressed on the oligosaccharide chain on the terminal galactose of type 2 RBC. Even though Bombay (hh) individuals may inherit chains.15 Sugars occupying the terminal positions of this pre- ABO genes, normal expression, as reflected in the formation cursor chain and conferring blood group specificity are called of A, B, or H antigens, does not occur. (See “The Bombay the immunodominant sugars. Therefore, L-fucose is the Phenotypes (Oh)” section.) sugar responsible for H specificity (blood group O; Fig. 6–4). In the formation of blood group A, the A gene (AA or AO) The O gene at the ABO locus is sometimes referred to as an codes for production of α-3-N-acetylgalactosaminyltrans- amorph and does not elicit the production of a catalytically ferase, which transfers an N-acetyl-D-galactosamine(GalNAc) active polypeptide transferase; therefore, the H substance re- sugar to the H substance. This sugar confers A specificity mains unmodified.13 Consequently, the O blood group has (blood group A; Fig. 6–5). The A-specific immunodominant the highest concentration of H antigen. The H substance sugar is linked to a type 2 precursor substance that now con- (L-fucose) must be formed for the other sugars to be attached tains H substance through the action of the H gene. in response to an inherited A and/or B gene. The A gene tends to elicit higher concentrations of trans- The H gene is present in more than 99.99% of the ran- ferase than the B gene, leading to conversion of nearly all of dom population. Its allele, h, is quite rare, and the genotype the H antigen on the RBCs to A antigen sites. As many as 126 PART II Blood Groups and Serologic Testing ar s ug nt ) en ina ne tig mi an d om tosa "A no lac mu l ga i( m et y ac N- e feras ns l tra ny mi to sa ac /AO l gal AA ety ac "H structure" N- "B antigen" Type-2-precursor chain HH / Hh BB/BO (immunodominant (immunodominant (Precursor structure) L-fucosyl transferase D-galactosyl transferase sugar L-fucose) sugar galactose) A an dB N- ge ga ac e ne lac tyl s tos ga y l l ac hh tra tos ns am fer in as yl es an d "A B su an ga tig rs en an N- "( d ac im Precursor structure unchanged ga ety mun lac tos l ga od e) la c om (Bombay phenotype) to s ina am nt ine Figure 6–7. Interaction of the Hh and ABO genes. Formation of A, B, and H Soluble Antigens ABH antigens are integral parts of the membranes of RBCs, endothelial cells, platelets, lymphocytes, and epithelial cells.8 Genotype: Se se water-soluble secretions ABH-soluble antigens can also be found in all body secretions. AB produced by tissue cells Their presence is dependent on the ABO genes inherited and HH on the inheritance of another set of genes called Sese (secretor genes) that regulate their formation. Eighty percent of the ran- dom U.S. population are known as secretors because they have inherited a secretor gene (SeSe or Sese). The inheritance of a FUT 2 (Se) gene codes for the production of the transferase α-2-L-fucosyltransferase that modifies the type 1 precursor substance in secretions to form H substance.22 If the corre- H A B sponding gene is present, this H substance can then be further modified to express A and B substance in secretions such as saliva. For example, a group A individual who is a secretor (SeSe or Sese) will secrete glycoproteins carrying A and H anti- gens. However, the Se gene does not affect the formation of A, = A-Acetylgalactosamine B, or H antigens on the RBC. It is the presence of the Se gene– = D-Galactose specified α-2-L-fucosyltransferase that determines whether = N-Acetylglucosamine ABH-soluble substances will be secreted (Fig. 6–8).22 People = L-fucose who inherit the sese genotype are termed nonsecretors. = Protein backbone Comparison of A, B, and H Antigens on RBCs with A, B, and H Soluble Substances Se AB A, B, H P.S. H antigen soluble H antigens The formation of soluble A, B, and H substances is the same as that described for the formation of A, B, and H antigens Figure 6–8. Secretor ABH glycoprotein substances.  Chapter 6 The ABO Blood Group System 127 on the RBCs, except for a few minor distinctions that are linkage position of galactose (Gal) to N-acetylglucosamine compared in Table 6–9. (GlcNAc); specifically type 1 has a beta 1→3 linkage while In the past, tests for ABH secretion were used to establish type 2 has a beta 1→4 linkage.22 Addition of specific immu- the true ABO group of an individual with poorly developed nodominant sugars to the type 2 and 4 chains leads to for- RBC antigens. The term secretor refers only to secretion mation of A, B, and H antigens on the RBC membrane, with of A, B, and H soluble antigens in body fluids. The demon- the majority being present in the form of type 2 chains. stration of A, B, and H substances in saliva is evidence of the Adding the same immunodominant sugars to type 1 and inheritance of an A gene, B gene, H gene, and Se gene. The 3 chains in the body secretions allow for A, B, and H soluble glycoprotein-soluble substances (or antigens) normally substances to be made in body secretions. Box 6–3 summa- found in the saliva of secretors are listed in Table 6–10. Both rizes the body fluids in which ABH-soluble substances can ABH red blood cell antigens and ABH-soluble substances be found. are formed due to the attachment of an immunodominant The procedure for determining the secretor status sugar to an oligosaccharide chain. Although several types (saliva studies) can be found as Procedure 6-1 on of oligosaccharide chains exist, types 1 and 3 are primarily the textbook’s companion website. associated with body secretions, while types 2 and 4 are associated with the red blood cell membrane.22 Type 1 and ABO Subgroups 2 chains are more abundant, and they differ only in the The original reports of most ABO subgroups were made Table 6–9 Comparison of ABH Antigens before the availability of the monoclonal typing reagents currently used in routine ABO grouping. ABO subgroups on RBCs with A, B, and represent phenotypes showing weaker and variable serologic H Soluble Substances reactivity with the commonly used human polyclonal ABH Antigens A, B, and H Soluble anti-A, anti-B, and anti-A,B reagents. on RBCs Substances Secreted substances are A Subgroups RBC antigens can be glycolipids, glycoproteins, glycoproteins. or glycosphingolipids. In 1911, von Dungern described two different A antigens based on reactions between group A RBCs and anti-A and anti- RBC antigens are synthesized Secreted substances are A1.23 Group A RBCs that react with both anti-A and anti-A1 only on type 2 precursor chains. primarily synthesized on are classified as A1, whereas those that react with anti-A and type 1 precursor chains. not anti-A1 are classified as A2 (Table 6–11 and Figs. 6–9 and Type 2 chain refers to a Type 1 chain refers to a 6–10). RBCs from A1 and A2 individuals react equally strong beta 1→4 linkage in which beta-1→3 linkage in which the number one carbon the number one carbon of the of the galactose is attached to galactose is attached to the the number four carbon of number three carbon of BOX 6–3 the N-acetylglucosamine sugar the N-acetylglucosamine sugar of the precursor substance. of the precursor substance. Fluids in Which A, B, and H Substances Can Be Detected in Secretors The enzyme produced by The enzyme produced by the H (FUT 1) gene (α-2-L- the Se (FUT 2) gene (α-2-L- Saliva fucosyltransferase) acts fucosyltransferase) preferen- Tears primarily on type 2 chains, tially acts on type 1 chains in Urine which are prevalent on the secretory tissues. Digestive juices RBC membrane. Bile Milk Amniotic fluid Table 6–10 ABH Substance in the Saliva Pathological fluids: pleural, peritoneal, pericardial, ovarian cyst of Secretors (SeSe or Sese)* Substances in Saliva ABO Group A B H Table 6–11 A1 Versus A2 Phenotypes O None None ↑↑ Reactions of Patient’s RBCs With A ↑↑ None ↑ Blood Anti-A Reagent Anti-A1 Lectin Group (anti-A plus anti-A1) Reagent B None ↑↑ ↑ A1 + + AB ↑↑ ↑↑ ↑ A2 + 0 * Nonsecretors (sese) have no ABH substances in saliva. ↑↑ and ↑, respectively, represent the concentration of ABH substances in saliva. + = positive (agglutination); 0 = negative (no agglutination) 128 PART II Blood Groups and Serologic Testing A1 A1 A A1 A A A A1 A A A1 A A A A1 A1 A A1 A2 Reactions of Patients’ Red Cells with Anti-A Anti-A1 Blood Group Antigen Present (Anti-A plus Anti-A1) lectin A1 A1 A + + A2 A + 0 Figure 6–9. A1 versus A2 phenotypes. A1 A1 A A A A1 A1 A1 A1 A A A1 A1 A2 Reactions of Patients’ Red Cells with Anti-A Anti-A1 Blood Group Antigen Present (Anti-A plus Anti-A1) lectin A1 A1 + + A + 0 Figure 6–10. A1 versus A2 phenotypes (alternative con- A2 ceptual presentation). with current reagent monoclonal anti-A in ABO forward The production of both types of antigens is a result of an typing tests.2 inherited gene at the ABO locus. Inheritance of an A1 gene The A subgroups are more common than B subgroups. elicits production of high concentrations of the enzyme The weaker serologic reactivity of ABO subgroups is attrib- α-3-N-acetylgalactosaminyltransferase, which converts uted to the decreased number of A and B antigen sites on almost all of the H precursor structure to A1 antigens on the their red blood cells. Classification into A1 and A2 pheno- RBCs. The very potent gene A1 creates between 810,000 and types accounts for 99% of all group A individuals. The cells 1,170,000 antigen sites on the adult A1 RBC, whereas inher- of approximately 80% of all group A (or AB) individuals are iting an A2 gene results in production of only 240,000 to A1 (or A1B), and the remaining 20% are A2 (or A2B) or 290,000 antigen sites on the adult A2 RBC.9 The A2 allele is weaker subgroups. The differences between A1 and A2 are characterized by a single base substitution at nucleotide 467 both quantitative and qualitative (Table 6–12). and a single base deletion at nucleotide 1060 (1060delC) in exon 7.19 These substitutions alter the active site of the coding region and subsequently change the specificity of the A glycosyltransferase. The immunodominant sugar on both Table 6–12 Quantitative and Qualitative A1 and A2 RBCs is N-acetyl-D-galactosamine. Differences of Subgroups A1 Qualitative differences also exist, since 1% to 8% of A2 in- and A2 dividuals produce anti-A1 in their serum and 22% to 35% of Quantitative Qualitative A2B individuals produce anti-A1.9 This antibody can cause discrepancies between forward and reverse ABO testing and ↓ Number of antigen sites Differences in the precursor incompatibilities in crossmatches with A1 or A1B cells. Anti- oligosaccharide chains A1 is a naturally occurring IgM cold-reacting antibody and ↓ Amount of transferase Subtle differences in trans- is unlikely to cause a transfusion reaction because it usually enzyme ferase enzymes reacts only at temperatures well below 37°C. It is considered ↓ Amount of branching Formation of anti-A1, in a per- clinically significant if it is reactive at 37°C. centage of some subgroups The antigens present on the RBCs of A1 and A2 individu- als can be depicted in two ways. Most often, A1 RBCS are 136 PART II Blood Groups and Serologic Testing in the forward grouping. All these disease states previously saline suspension of RBCs can usually resolve the ABO dis- mentioned may result in discrepancies between the forward crepancy. It is important to make sure any and all technical and reverse groupings, indicating that the patient’s red blood factors that may have given rise to the ABO discrepancy are cell group is not what it seems. All ABO discrepancies must reviewed and corrected. It is also essential to obtain adequate be resolved before blood for transfusion is released for that information regarding the patient’s age, diagnosis, transfu- patient. In some cases, secretor or molecular studies may sion history, medications, and history of pregnancy. If help confirm the patient’s true ABO group. the discrepancy persists and appears to be due to an error in specimen collection or identification, a new sample must ABO Discrepancies be drawn from the patient and all RBC and serum testing repeated. ABO discrepancies occur when unexpected reactions are ob- When a discrepancy is encountered, all results must be tained in the forward and/or reverse grouping. These can be recorded, but interpretation of the ABO type must be delayed due to problems with the patient’s serum (reverse grouping), until the discrepancy is resolved. If the blood is from a problems with the patient’s RBCs (forward grouping), or potential transfusion recipient, it may be necessary to admin- problems with both the serum and cells. The unexpected ister group O, Rh-compatible RBCs before the discrepancy is reaction(s) may be due to an extra positive reaction or a resolved. In general, when investigating ABO discrepancies, weak or missing reaction in the forward and reverse group- always remember that RBC and serum grouping reactions ing. All ABO discrepancies must be resolved prior to report- are very strong (3+ to 4+) and the weaker reactions typically ing a patient or donor ABO group. represent the discrepancy. Figure 6–14 shows an algorithm for resolving ABO discrepancies. Technical Errors Categories of ABO Discrepancies Technical errors can also cause ABO discrepancies. Examples include: blood sample and test tube labeling errors, failure to ABO discrepancies may be arbitrarily divided into four add reagents, or the addition of incorrect reagents or sample. major categories: group I, group II, group III, and group IV Serum and antiserum should always be added first, followed discrepancies. by the patient or reagent RBCs to avoid both reagent contam- ination and potential omission of either patient sample or Group I Discrepancies reagent. Results must be recorded immediately after obtaining Group I discrepancies are associated with unexpected reac- them to avoid transcription errors. Always examine reagent tions in the reverse grouping due to weakly reacting or miss- vials concurrently while performing ABO testing and quality ing antibodies. These discrepancies are more common than control testing for possible contamination. Some of the com- those in the other groups listed. When a reaction in the mon causes of technical errors leading to ABO discrepancies serum grouping is weak or missing, a group I discrepancy in the forward and reverse groupings are listed in Box 6–8. should be suspected because, normally, RBC and serum grouping reactions are very strong (4+). One of the reasons Resolution for the missing or weak isoagglutinins is that the patient has depressed antibody production or cannot produce the ABO If initial testing was performed using RBCs suspended in antibodies. Common populations with discrepancies in this serum or plasma, repeat testing of the same sample using a group are: Newborns (ABO antibody production is not detectable until 3 to 6 months of age) BOX 6–8 Elderly patients (production of ABO antibodies is Common Sources of Technical Errors Resulting depressed) in ABO Discrepancies Patients with a leukemia (e.g., chronic lymphocytic leukemia) or lymphoma (e.g., malignant lymphoma) Incorrect or inadequate identification of blood specimens, test tubes, or slides demonstrating hypogammaglobulinemia Cell suspension either too heavy or too light Patients using immunosuppressive drugs that yield Clerical errors or incorrect recording of results hypogammaglobulinemia A mix-up in samples Patients with congenital or acquired agammaglobulinemia Missed observation of hemolysis or immunodeficiency diseases Failure to add reagents Patients with bone marrow or hematopoietic progenitor Failure to add sample stem cell (HPC) transplants (patients develop hypogam- Failure to follow manufacturer’s instructions maglobulinemia from therapy and start producing a Uncalibrated centrifuge different RBC population from that of the transplanted Overcentrifugation or undercentrifugation bone marrow) Contaminated reagents Patients whose existing ABO antibodies may have been Warming during centrifugation diluted by plasma transfusion or exchange transfusion ABO subgroups  Chapter 6 The ABO Blood Group System 137 ABO DISCREPANCY Forward and Reverse testing do not match as expected. Note: The initial testing was performed using patient’s RBCs If an error in specimen collection suspended in serum or plasma. and identification is suspected. Wash patient’s RBCs with saline and repeat Testing. Request a new sample to be drawn from the patient. No Discrepancy Same unexpected Rx between Forward and Reverse testing Repeat Testing Report out ABO group Look up information on Patient: No Discrepancy Age, Diagnosis, Medications,Transfusions, and Pregnancy History Report out ABO group Determine whether the discrepancy is in the Red Cell or Serum results by observing weakest reactivity. Example: Patient Serum Problem Example: Red Cell Problem Example: Both Serum and RBC Problem Weak Reactions in the Weak Reactions in the Weak Reaction in both Reverse grouping Forward grouping Forward and Reverse grouping Anti-A 4 Anti-A 2mf Anti-A 1 Anti-B 4 Anti-B 0 Anti-B 1 A1 Cell 2 A1 Cell 0 A1 Cell 1 B Cell 2 B Cell 4 B Cell 2 Probable Group AB with Probable Group A with Probable Group O with the following possibilities: the following possibilities: the following possibilities: 1. Cold Reacting Alloantibody 1. Out of Group Transfusion 1. Cold Autoantibody (i.e. Anti-M, Anti-P1 most common) (i.e. Group O units 2. Cold Autoantibody and 2. Cold Reacting Autoantibody transfused to an A patient.) Cold Alloantibody (i.e. Anti-I, Anti-H, Anti-IH) 2. Out of Group Bone Marrow/ 3. Out of Group Bone Marrow/ 3. Passively Acquired Antibody Stem Cell Transplantation Stem Cell Transplantation (i.e. plasma exchange, 3. Leukemia/Lymphoma 4. Passively Acquired Antibody mismatched platelets) 4. Fetal-Maternal Bleed 4. Rouleaux 5. A3 Subgroup Resolution: Resolution: 1. Wash patient cells with (Refer to chapter 11) warm saline and retest 1. Run Antibody Screen Resolution: 2. Run DAT and Auto Control 2. Run Auto Control (Refer to chapter 5) (Refer to chapter 5) 3. Run saline replacement 1. Run DAT 3. Run Antibody Screen for rouleaux 2. Run Auto Control (Refer to chapter 11) Figure 6–14. Algorithm for resolving ABO discrepancies. Resolution of Common Group I Discrepancies serum by incubating the patient serum with reagent A1 and Obtaining patient clinical history may immediately resolve B cells at room temperature for approximately 15 to 30 min- this type of discrepancy. If the history indicates the patient utes. If there is still no reaction after centrifugation, the is an elderly individual or has a diagnosis indicative of serum-cell mixtures can be incubated at 4°C for 15 to hypogammaglobulinemia, the best way to resolve this dis- 30 minutes. An auto control and O cell control must always crepancy is to enhance the weak or missing reaction in the be tested concurrently with the reverse typing when trying  Chapter 6 The ABO Blood Group System 139 incompatibility occurs when both a major and minor Table 6–19 shows the ABO testing results of an acquired incompatibility are present (e.g., the donor is group A and B phenomenon. the recipient is group B).56 Since ABO antigens are histo-blood group antigens Resolution of Common Group II Discrepancies present on many tissues, including the lung, pancreas, The agglutination of weakly reactive antigens with the bowel, endothelium, heart, and kidney, recipients of ABO- reagent antisera can be enhanced by incubating the test mix- incompatible bone marrow or hematopoietic progenitor ture at room temperature for up to 30 minutes to increase stem cell transplantation pose distinctive challenges to the association of the antibody with the RBC antigen. If the transfusion service staff when selecting blood components reaction is still negative, incubate the text mixture at 4°C for for transfusion. Following an ABO-incompatible HPC 15 to 30 minutes. Include group O and autologous cells as transplant, the pretransplant ABO type will remain in these controls. RBCs can also be pretreated with enzymes and tissues for the rest of the patient’s life. Accordingly, the retested with reagent antisera. patient will never make antibody against the ABO type the The acquired B antigen arises when bacterial enzymes body still sees as self (e.g., a group B recipient converted modify the immunodominant blood group A sugar (N-acetyl- to group A will never make anti-B). RBCs, platelets, and D-galactosamine) into D-galactosamine, which is sufficiently plasma products must be compatible with both the donor similar to the group B sugar (D-galactose) to cross-react with and recipient blood types (see Chapter 19, “Cellular Ther- anti-B antisera. This pseudo-B antigen is formed at the ex- apy in the Transplant Setting”). pense of the A1 antigen and disappears following the patient’s Group II Discrepancies recovery.55 The reaction of the appropriate antiserum with these acquired antigens demonstrates a weak reaction, often Group II discrepancies are associated with unexpected reac- yielding a mixed-field appearance. tions in the forward grouping due to weakly reacting or miss- Blood group reagents of a monoclonal anti-B clone (ES4) ing antigens. This group of discrepancies is probably the least strongly agglutinate cells with the acquired B antigen. The frequently encountered. The following are some of the pH of reagents containing ES4 has been lowered, and conse- causes of discrepancies in this group: quently, only those cells with the strongest examples of ac- Subgroups of A or B may be present (see the “ABO Sub- quired B antigen react with the antisera. Testing the patient’s groups” section). serum or plasma against autologous RBCs gives a negative re- Leukemias may yield weakened A or B antigens (Table 6–18), action, because the anti-B in the serum does not agglutinate and Hodgkin’s disease has been reported in some cases to the patient’s RBCs with the acquired B antigen. The acquired mimic the depression of antigens found in leukemia. B antigen is also not agglutinated when reacted with anti-B The “acquired B” phenomenon will show weak reactions that has a pH greater than 8.5 or less than 6.28 Secretor studies with anti-B antisera and is most often associated with dis- can be performed when trying to characterize the acquired eases of the digestive tract (e.g., cancer of the colon). B phenomenon. If the patient is in fact a secretor, only the Table 6–18 Serologic Reactions Typical of Leukemia Forward Grouping Reaction of Patient Reverse Grouping Reaction of Patient Cells With Serum With Patient Phenotype Anti-A Anti-B A1 Cells B Cells A +mf 0 0 3+ B 0 ±/+ 4+ 0 Note: Weak reactivity with anti-A and anti-B is because the disease, leukemia, has resulted in the weakened expression of the corresponding antigen. Table 6–19 Example of ABO Discrepancy Caused by an Acquired B Antigen Forward Grouping Reaction of Patient’s Reverse Grouping Reaction of Patient’s Cells With Serum With Anti-A Anti-B A1 Cells B Cells Patient 4+ 2+ 0 4+ Patient’s probable group: A Note: Patient RBCs have acquired a B-like antigen that reacts with reagent anti-B and is associated with cancer of the colon or other diseases of the digestive tract. Resolution: (1) Acidify Anti-B reagent to a pH of 6. (2) Run DAT (refer to Chapter 5, “The Antiglobulin Test”). (3) Run autocontrol.  Chapter 6 The ABO Blood Group System 141 be observed on microscopic examination (Fig. 6–15). Cell grouping can usually be accomplished by washing the patient’s RBCs several times with saline. Performing a saline replacement technique will free the cells in the case of rouleaux formation in the reverse type. In this procedure, serum is removed and replaced by an equal volume of saline. In true agglutination, RBC clumping will still remain after the addition of saline. Rouleaux can be a nuisance in the laboratory, since it is an in vitro problem observed during laboratory testing. It is not an in vivo problem for the patient. The complete procedure for saline replacement can be found in Procedure 11-3 on the textbook’s com- panion website. Cord blood samples can pose a problem in ABO testing, since cord cells may be contaminated with a substance called Wharton’s jelly. This substance is a viscous mucopolysaccha- ride material present on cord blood cells that may cause the Figure 6–16. Autoagglutination in a patient with cold agglutinin disease. red blood cells’ aggregation. Thoroughly washing cord cells six to eight times with saline should alleviate spontaneous rouleaux due to Wharton’s jelly and result in an accurate Resolution of Common Group IV Discrepancies ABO grouping. However, since testing is usually not per- Potent cold autoantibodies can cause spontaneous agglutina- formed on cord serum because the antibodies detected are tion of the patient’s cells. These cells often yield a positive direct usually of maternal origin, reverse grouping may still not Coombs’ or antiglobulin test (see Chapter 21, “Autoimmune correlate with the RBC forward grouping. Hemolytic Anemias”). If the antibody in the serum reacts with Group IV Discrepancies all adult cells, for example, anti-I, the reagent A1 and B cells These discrepancies between forward and reverse groupings used in the reverse grouping also agglutinate. are due to miscellaneous problems that have the following To resolve this discrepancy, the patient’s RBCs could be causes: incubated at 37°C for a short period, then washed with saline at 37°C three times and retyped. If this is not successful in Cold reactive autoantibodies in which RBCs are so heavily resolving the forward type, the patient’s RBCs can be treated coated with antibody that they spontaneously agglutinate, with 0.01 M dithiothreitol (DTT) to disperse IgM-related independent of the specificity of the reagent antibody agglutination. As for the serum, the reagent RBCs and serum (Fig. 6–16 and Table 6–22). can be warmed to 37°C for 10 to 15 minutes, mixed, tested, Circulating RBCs of more than one ABO group due to RBC and read at 37°C. The test can be converted to the antihuman transfusion or marrow/stem cell transplant globulin phase if necessary. Weakly reactive anti-A or anti-B Unexpected ABO isoagglutinins may not react at 37°C, which is outside their optimum Unexpected non-ABO alloantibodies thermal range. If the reverse typing is still negative (and a positive result was expected), a cold autoabsorption (patient cells with patient serum) could be performed to remove the cold autoantibody from the serum. The absorbed serum can then be used to repeat the serum typing at room tempera- ture. (Refer to Chapter 10 for cold autoadsorption and alloadsorption with rabbit erythrocyte stroma [RESt] for the removal of cold autoantibodies.) Unexpected ABO isoagglutinins in the patient’s serum react at room temperature with the corresponding antigen present on the reagent cells (Table 6–23). Examples of this type of ABO discrepancy include A2 and A2B individuals, who can produce naturally occurring anti-A1, or A1 and A1B, individuals who may produce naturally occurring anti-H. (Refer to the previous sections on ABO subgroups.) Serum grouping can be repeated using at least three examples of A1, A2, B cells; O cells; and an autologous control (patient’s serum mixed with patient’s RBCs).3 The specificity of the antibody can be determined by examining the pattern of Figure 6–15. Rouleaux. reactivity (e.g., if the antibody agglutinates only A1 cells, it 144 PART II Blood Groups and Serologic Testing Table 6–25 ABO Discrepancies Between Forward and Reverse Grouping—cont’d Forward Grouping Reverse Grouping Patient Anti-A Anti-B A1 Cells B Cells O Cells Autocontrol Possible Cause Resolution Steps (3) Cold autoantibody with (3) Perform cold panel underlying cold or RT reacting autoabsorb or RESt, alloantibody (probable group and run panel on ab- AB with an auto anti-I and a sorbed serum; select high-frequency cold antibody reverse cells lacking [e.g., anti-P1, anti-M, anti-Leb]) antigen for identified alloantibody; repeat reverse group on absorbed serum to determine true ABO group or at 37°C 4 4+ 4+ 1+ 0 0 0 Subgroup of AB; probable A2B Use anti-A1 lectin, test with anti-A1 serum against addi- tional A1, A2, and O cells 5 4+ 0 0 4+ 3+ 0 A1 with potent anti-H Confirm A1 group with anti-A1 lectin; test additional A2, O, and A1 cells and an Oh if available 6 0 0 4+ 4+ 4+ 0 Oh Bombay Test with anti-H lectin; test Oh cells if available; send to reference labo- ratory for confirmation 7 0 0 2+ 4+ 0 0 Subgroup of A; probable Ax Perform saliva studies or with anti-A1 absorption/elution 8 4+ 2+ 0 4+ 0 0 Group A with an acquired B Check history of patient antigen for lower gastrointesti- nal problem or sep- ticemia; acidify anti-B typing reagent to pH 6.0 by adding 1 or 2 drops of 1 N HCl to 1 mL of anti-B antisera, and measure with a pH meter (this acidified anti-B antisera would agglutinate only true B antigens not acquired B antigens), test serum against autologous cells 9 4+ 4+ 2+ 0 2+ 0 Group AB with cold antibody Perform antibody screen and panel, identify room temperature antibody, repeat serum type with antigen negative reagent cells or perform serum type at 37°C 10 0 4+ 4+ 1+ 1+ 1+ Group B with cold autoantibody Enzyme-treat RBCs and perform autoabsorption at 4°C or perform prewarmed testing *Absorption should not be performed on patient’s cells that have been transfused within the last 3 months RT = room temperature  Chapter 6 The ABO Blood Group System 145 Causes of ABO Discrepancies Forward Reverse (RBCs) (plasma) Missing/weak Extra Mixed Missing/weak Extra Antigens Antigens Field Antibodies Antibodies A or B Acquired B Group O Anti-A1 Newborns, Subgroups (intestinal transfusions Elderly, disease) Immunocom- promised Disease Rouleaux Stem Cell Cold (leukemia) (hyperpro- Transplant Alloanti- teinemia) bodies Cold Auto- A3 or B3 Rouleaux antibodies Phenotype (hyperpro- teinemia) Wharton’s Cold Auto- jelly antibodies Passively Acquired Antibody (i.e. plasma exchange, Figure 6–18. Simplified summary of ABO mismatched discrepancies. platelets) CASE STUDY 1. Where is the discrepancy? 2. What testing would you perform next to resolve the Case 6-1 discrepancy? A 45-year-old woman, who has given birth to three Part 2 children and has a history of five cases of dilation and The patient’s serum was then tested with A2 cells and curettage, is scheduled for a partial hysterectomy at a O cells and the patient’s red blood cells tested with community hospital. Preoperative laboratory tests include Anti-A1 lectin. a type and screen. There is no history of transfusions. A2 Cells O Cells Anti-A1 Lectin Part 1 Patient serum 0 0 Patient RBCs 0 ABO and Rh Typing 3. How would you interpret these results? Anti-A Anti-B Anti-A,B A1Cells B Cells Anti-D 4. Why were O POS RBCs chosen for transfusion? 3+ 0 3+ 2+ 4+ 3+ Antibody Screen 37°C AHG CC SCI 0 0 √ SCII 0 0 √ SCIII 0 0 √