SSP-DNA typing using beta-actin internal controls

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Abstracts

7.4 #218

HLA DR, DQ BY SEROLOGY AND peR/SsP. LA Nonn, RM Radvany, Department of Pathology, Loyola University Medical Center, Maywood, IL. We compared PCRlSSP (SSP mixes from Biosynthesis Inc) typing of HLA- DR, DQ antigens with serologic testing. A total of 180 samples were compared. The findings are as follows Serological

PCR/SSP

Quantity not sufficient

19 (10.6%)

0(0%)

Poor cell viability

1

(0.5%)

0(0%)

DQ antigens not detected

17 (9.4%)

0(0%)

Second antigen not detected

13 (7.2%)

0(0%)

n addition, additional HLA-DR antigen splits were definel by PCR/SSP In 46 (25.! %) cases and a questionable serological result was confirmed by PCRI SSP in (3.9%) of all cases. Probably for lack of monospecific antisera the most frequently missed antigen by serology was DR13 (4.4% of all cases), and most frequently unidentified splits were DR17 and DR18. As already reported by Lucas et al (Human Immunology 40(1):154) we also found false positive amplification in DR12 PCR/SSP primer mixes when the template DNA had either DRB1*08, or *14. In conclusion we found HLA-DR DQ typing by PCR/SSP to be a reproducible, reliable and a very useful method for identification of Class II antigens especially in cases of poor cell viability or small sample size.

7.4 #219

SSP-DNA TYPING USING BETA-ACTIN INTERNAL CONTROLS. M. Castro, R. Chen and *R. Vela. Bio-Synthesis, Inc. Lewisville, TX; *University of North Texas, Denton, TX. The use of internal controls during the PCR reaction is useful to confirm true negative results after PCR amplification. Beta-actin is a constitutively expressed human gene product; we designed a primer pair that amplifies a 600bp gene fragment far above from all specific allele bands (200-300bp), which allows for easier identification of control versus specific allele bands. Results of 20 random human DNA samples are shown to illustrate our method.