HLA CLASS II ALLELE TYPING IS 100-FOLD MORE SENSITIVE THAN CLASS I IN DETECTING CHIMERISM STATES.
          JC Delgado, OP Clavijo and EJ Yunis. Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute and Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.
          Umbilical cord blood has become an important alternative for the treatment of blood cell dyscrasias and malignancies. Most transplants using umbilical blood cells have been from HLA-matched donors but there is increasing use of HLA-unmatched donors. Recently, one patient was successfully transplanted with twelve different and unmatched umbilical cord blood cell units. Only one cord blood unit engrafted. This particular cord blood unit was HLA class II identical to the patient but had three mismatches for HLA class I antigens. Sequence-specific primer (SSP) amplification of HLA class I antigens proved to be useful in determining complete chimerism state 10 days after the transplant. No evidence of other umbilical blood cell units was found. The level of sensitivity detection of blood cell chimerism by DNA-based HLA typing methods is unknown. In the present study, an artificial state of chimerism was created in vitro to determine the sensitivity of PCR-SSP for HLA class I and class II alleles. Five different experiments consisting of 6 samples containing a constant number (5x106) of peripheral blood mononuclear cells (recipient cells) and a decreasing number (5x106, 5x105, 5x104, 5x103, 5x102 and 50, respectively) of homozygous cells for different HLA class I and class II (donor cells) were produced. PCR-SSP of the differential HLA-A, B, DR and DQ donor cell alleles were carried out. The results showed that PCR-SSP for class II alleles allowed the detection of 1 donor cell in 104 recipient cells. For class I alleles, 1 donor cell was detected in 100 recipient cells. We conclude that PCR-SSP of donor cell class II alleles are 100-fold more sensitive than PCR-SSP of class I alleles. These results emphasize the limitation of HLA typing for the interpretation of chimerism states in cases of HLA-unmatched umbilical cord blood transplantation.