Determination of optimal method for antibody identification in a reference laboratory.

Methods commonly used for antibody identification are hemagglutination (tube), column agglutination (gel), and solid-phase red cell adherence. Our AABB immunohematology reference laboratory (IRL) conducted a study to determine which antibody identification testing method was optimal for detecting all clinically significant antibodies. Patient specimens were sent to our IRL from August 2008 to September 2009. Routine testing was performed by tube method and then by manual gel and manual solid-phase methods. Of the 254 samples tested, 115 showed agreement in antibody identification with all three methods. The tube method identified all but six clinically significant antibodies. The gel method did not identify 59 clinically significant antibodies. Fifty-six clinically significant antibodies were not identified by solid-phase testing. Tube testing identified 27 clinically insignificant antibodies, primarily cold autoantibodies. Gel and solid-phase methodologies identified two and three cold autoantibodies, respectively. Solid-phase testing failed to detect 12 examples of anti-K. No identifiable pattern of reactivity was found in 13 samples using gel testing compared with 6 for solid-phase and none for tube methodologies. Hemagglutination tube method was the best choice for our IRL because it missed the fewest number of clinically significant alloantibodies. Benefits also included the ability to use various potentiating factors, incubation times, and temperature phases to enhance antibody identification. The tube method provided critical data for determining antibody clinical significance.

The Redelberger antigen: a family study, a family story.

The Redelberger antigen (Rba) was first discovered in 1974 on the RBCs of a blood donor who was an employee of the Community Blood Center in Dayton, Ohio. The discovery was made as a result of the investigation of a reagent contamination problem. Two examples of the Rba antigen were subsequently identified in the United Kingdom,but no "new"examples have been identified in the United States or Europe. Anti-Rba is a commonly occurring antibody, often found in combination with other antibody specificities, especially in combination with other antibodies to low-incidence antigens.

Review: monoclonal reagents and detection of unusual or rare phenotypes or antibodies.

Monoclonal antibodies have been used in the formulation of commercially available blood grouping reagents since the early 1990s. It became apparent early on that introducing them into routine use along with, or instead of, human- or animal-derived reagents could and did lead to discrepant reactions. These discrepancies most often came to light when confirming a blood type obtained previously with human- or animal-source reagents or when using two or more sources of a reagent from the same or another manufacturer to perform blood typing or antibody detection or identification testing. A number of factors contribute to differences in reactivity of reagents that are of the same specificity but are from more than one source. One factor is the use of different clones of the same specificity to manufacture blood bank reagents. Another is the effect of the various diluents used by different manufacturers to formulate reagents that contain the same clone(s). In addition, RBCs having unusual or rare phenotypes can cause discrepant reactions when performing phenotyping. Discrepant reactions can also occur because of patient or donor antibodies that react in an unusual manner when antiglobulin tests are performed with monoclonal antihuman globulin (AHG) versus rabbit AHG reagent. It is important to know the identity of the unusual or rare phenotypes and antibodies and to be able to recognize the different types of reactions that will be observed when using more than one reagent of the same specificity. Most importantly, one must be able to interpret reactions correctly and establish the true blood type of the RBCs or specificity of the antibodies. This review will describe situations in which the use of monoclonal reagents from more than one source or manufacturer, or comparison with results of human- and animal-source reagents, resulted in discrepancies with unusual or rare phenotypes or antibodies. Many of the samples described in this review were sent to the reference laboratory at Gamma Biologicals, Inc., in Houston, Texas, which later became ImmucorGamma with sites in Norcross, Georgia, and Houston, Texas.

Case report:DNA testing resolves unusual results in the Dombrock system.

Typing for antigens in the Dombrock blood group system and identifying the corresponding antibodies are notoriously difficult tasks. The reagents are scarce and the antibodies are weakly reactive. When RBCs from family members of a patient with an antibody to a high-prevalence Dombrock antigen were tested for compatibility,an unusual pattern of inheritance was observed:RBCs from the patient's children and one niece, in addition to those from some of the patient's siblings,were compatible. This prompted the performance of DNA-based assays for DO alleles and the results obtained were consistent with and explained the compatibility test results. It was possible to study this large kindred because of the cooperation of family members, hospital personnel, and reference laboratory staff.

Genomic analysis of clinical samples with serologic ABO blood grouping discrepancies: identification of 15 novel A and B subgroup alleles.

Since the cloning in 1990 of complementary DNA corresponding to messenger RNA transcribed at the blood group ABO locus, polymorphisms and phenotype-genotype correlations have been reported by several investigators. Exons 6 and 7, constituting 77% of the gene, have been analyzed previously in samples with variant phenotypes but for many subgroups the molecular basis remains unknown. This study analyzed 324 blood samples involved in ABO grouping discrepancies and determined their ABO genotype. Samples from individuals found to have known subgroup alleles (n = 53), acquired ABO phenotypes associated with different medical conditions (n = 65), probable chimerism (n = 3), and common red blood cell phenotypes (n = 109) were evaluated by ABO genotype screening only. Other samples (n = 94) from apparently healthy donors with weak expression of A or B antigens were considered potential subgroup samples without known molecular background. The full coding region (exons 1-7) and 2 proposed regulatory regions of the ABO gene were sequenced in selected A (n = 22) or B (n = 12) subgroup samples. Fifteen novel ABO subgroup alleles were identified, 2 of which are the first examples of mutations outside exon 7 associated with weak subgroups. Each allele was characterized by a missense or nonsense mutation for which screening by allele-specific primer polymerase chain reaction was performed. The novel mutations were encountered in 28 of the remaining 60 A and B subgroup samples but not among normal donors. As a result of this study, the number of definable alleles associated with weak ABO subgroups has increased from the 14 previously published to 29.

MAM: a "new" high-incidence antigen found on multiple cell lines.

BACKGROUND: Three women have been identified with an antibody to a "new" high-incidence antigen found on multiple cell lines. CASE REPORTS: The proposita, M.A.M., presented during her third pregnancy with an antibody reacting with all RBCs tested except her own. She delivered a thrombocytopenic infant with a 3+ DAT, but without symptoms of HDN. The second example, A.N., presented during her third pregnancy with an antibody reacting with all RBCs tested except her own and those of M.A.M. She delivered a slightly thrombocytopenic but severely anemic infant. The third example, F.K., a sister of A.N., has an antibody reacting with all RBCs tested except her own and those of M.A.M. and A.N. CONCLUSION: This "new" high-incidence antigen has been named MAM and assigned high-incidence antigen number 901016 by the International Society of Blood Transfusion. The corresponding antibody, anti-MAM, has been shown to cause HDN and has the potential to shorten RBC survival after the transfusion of incompatible RBC units, as determined by monocyte monolayer assay. Immunoblotting and flow cytometry show that this new antibody reacts with various WBC lines in addition to RBCs. This antibody also appears to react with platelets in some assays.

The first reported case of anti-Dob causing an acute hemolytic transfusion reaction.

The antibodies of the Dombrock blood group system have only rarely been encountered in transfusion practice, and anti-Dob has not previously been implicated in an acute hemolytic transfusion reaction. We have encountered the first such case involving a chronically transfused black female with hemoglobin SS disease and multiple antibodies in her serum. During a previous admission for sickle cell crisis, the patient received 3 units of compatible blood with no untoward effects. Serum obtained 21 days later contained, in addition to the known antibodies, anti-S plus an unidentified antibody showing characteristics of HTLA. Blood lacking the E, K1, Fy(a), Jk(b) and S antigens was obtained, and 2 least incompatible units were transfused. While administering the second unit, the patient complained of fever and low back pain, and hemoglobinemia was detected. Anti-Dob was identified in the post-reaction samples by absorption-elution tests, and the patient was confirmed to be Do(a+b-). The first unit transfused during this hemolytic episode tested Do (b+). This case, and a similar case involving anti-Doa reported in 1986, strengthens the belief that Dombrock antibodies are clinically significant and illustrates the need for their differentiation, prior to transfusion from less clinically significant HTLA antibodies.

Evidence that several high-frequency human blood group antigens reside on phosphatidylinositol-linked erythrocyte membrane proteins.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder associated with absence of expression of phosphatidylinositol (PI)-linked membrane proteins from circulating hematopoietic cells of multiple lineages. Recent work demonstrated that decay accelerating factor, one such PI-linked protein, bears the Cromer-related blood group antigens. This study demonstrated that other high incidence antigens, including Cartwright (Yta/Ytb), Holley-Gregory (Hy/Gya), John Milton Hagen (JMH), and Dombrock (Doa/Dob), are absent from the complement-sensitive (PNH III) erythrocytes of patients with PNH. The relatively normal, complement-insensitive erythrocytes from the same patients express these antigens normally. Therefore, these antigens most likely reside on PI-linked proteins absent from PNH III, but not PNH I, erythrocytes.

Serological investigation and clinical significance of high-titer, low-avidity (HTLA) antibodies.

High-titer, low-avidity (HTLA) antibodies are frequently described as "reactive weakly by the antiglobulin test." These antibodies include: anti-Chido (Cha) and anti-Rodgers (Rga), and anti-Cost-Stirling (Csa) and anti-York (Yka), anti-Knops (Kna) and anti-McCoy (McCa), and anti-John Milton Hagen (JMH), the majority of which are directed at high-incidence red cell antigens. The classic HTLA antibodies are thought to be incapable of fixing complement or causing in vitro hemolysis. Present data indicate that these antibodies do not cause either increased red cell destruction when incompatible blood is transfused or hemolytic disease of the newborn. Special serological techniques can be used to differentiate the antibodies within the HTLA classification from antibodies not of an HTLA nature.