be given when selecting positive control cells to ensure that the reagent antiserum can detect weak expressions

of the antigen. An example is anti-C, which often contains mostly anti-Ce (rhi) and may not react with the CcDE phenotype. This phenotype is not uncommon in Mexican-Americans, Native Americans, and Asian-Americans, and a false-negative reaction could lead to an erroneous interpretation of exclusion. Only a few laboratories still include RBC antigen testing in their testing panel. However, on occasion it is still necessary to interpret previous results or perform testing on new individuals, the results of which need to be interpreted in light of previous testing performed with classic systems on persons who are now unavailable for additional testing (e.g., an additional child with a now-deceased but previously tested alleged father). Any blood center or transfusion service associated with a parentage testing laboratory could perform these tests without significant difficulty.

RBC Enzymes and Serum Proteins

RBC enzyme and serum protein genetic systems testing is infrequently performed with only two laboratories currently reporting proficiency testing results. Testing methodology consists of separating the different allelic proteins by electrophoresis, followed by staining. Subtyping of the PGM1 and Gc alleles may be done using isoelectric focusing, which allows separation of molecules of similar size, thereby increasing the extent of polymorphism of the system. When isoelectric focusing is used, it is customary to refer to the system with a subscript letter i (e.g., PGM1i or Gci). Phenotypes are identified by the number and respective position of the bands detected and comparison with control specimens expressing at least two known allotypes that are tested in parallel with the unknown specimens. Interpretation of the band patterns must be performed independently by two observers. Rare variants exist in most systems, and the laboratories should maintain a file of variants to permit identification of rare phenotypes.

Immunoglobulins
Three immunoglobulin chains demonstrate a polymorphism that has been applied to paternity testing. The gamma heavy chain expresses the Gm polymorphism; the kappa light chain expresses Km (also termed Inv); and the alpha heavy chain expresses Am. Determination of the phenotype is done by hemagglutination inhibition using indicator RBCs coated with antibodies of known phenotype and reagent anti-Gm, anti-Km, or anti-Am. Reagents are not widely available; genetic interpretation is complex; and phenotypic interpretation is often not possible in infants younger than 6 months of age because of interference with maternal immunoglobulins. For these reasons, few laboratories in the world now use these genetic systems. They are mentioned solely for the sake of completion and historic perspective.

HLA
The HLA complex represents the most polymorphic genetic system in the human genome. As discussed in the previous chapter, it comprises multiple linked loci expressed as class I and class II antigens. In parentage testing with classic methodology (non-DNA), only antigens expressed by the A and the B loci are considered.

The usual method is termed microlymphocytotoxicity. It depends on the evaluation of the reactions of live lymphocytes with a panel of cytotoxic antibodies of known specificity in the presence of complement. Testing is performed in 60 or 72 microwell trays preloaded with reagent antisera. A sufficient number of antisera should be used so that all HLA-A and HLAB specificities recognized by the 1980 HLA Nomenclature Committee of the World Health Organization can be reliably identified. Additional antigens should be tested, if appropriate antisera can be obtained. The larger the number of antigens that can be defined, the more powerful the system becomes; that is, the better it is able to exclude falsely accused men. Because of the large number of existing antigens, no single tray can identify all relevant antigens. Some antigens occur more frequently in specific racial groups, and specially designed trays are available for African Americans and Asians. Such trays should be selected when appropriate. Antisera are either of human origin or monoclonal, and monospecific sera are not available for a large number of antigens. It is required that each antigen be defined by at least two different operationally monospecific sera, by one monospecific and two multispecific sera, or by three multispecific sera. Phenotypes must be verified by reading two independent trays or tray sets. Each tray or tray set must be read independently. In interpreting phenotypes, a certain amount of expertise is needed, requiring knowledge of cross reactivity between antigens and splits of broader reactivity (e.g., A9 splitting into A23 and A24) and familiarity with the unexpected reactivity of the antisera used (false-negative or extra reactions). Because of the variability of HLA antisera, it is recommended that all individuals in a parentage case be tested in the same laboratory.

DNA Polymorphisms: RFLP

RFLP refers to polymorphisms of the DNA that can be detected by restriction enzymes. These enzymes are endonucleases that recognize specific DNA sequences of 4 to 6 bases and cut the double-stranded DNA at that site. These enzymes have been isolated from bacteria and are named after the bacteria from which they were isolated (e.g., Eco RI from Escherichia coli RY13; Hae III from Haemophilus aegyptius). After the isolated DNA is incubated with the restriction