Abstracts 12 3

C-7.1 #233

A CLOSER LOOK AT HLA-DR "BLANKS" AND TROUBLESOME VARIANTS WITH THE AID OF SSOP DNA. LF Serci,3, AR Smerglia, GA Teresi, TC Li, DJ Mancuso and DJ Cook. The Cleveland Clinic Foundation Transplant Center, Cleveland, OH.

DNA class II typing has great ly expanded the HLA nomenclature and seems to be leaving the HLA serologist lost in the pack. Should we abandon our quest for antisera to better define DR antigens and their variants? In the process of performing more than 3000 serologic class II typings, 79 samples demonstrated additional serologic reactivi ty or fami ly data that suggested but did not clearly define a second DR antigen. Of these, 19 were included as a family study addressing serologic definit ion of DRBI *0103 . Addit ional serologic react iv i ty included l imited key sera react iv i ty and/or the presence of a DQ or DR52/53 that was not commonly associated wi th the clearly identif ied DR antigen.

SECOND n = 7 9

DR ALLELE

0103

ADDITIONAL REACTIONS

DQ

29 21 19 10

18

DR52 OR OTHER DR53 DR SERA

0 15

FAMILY DATA

19 1301/02 21 4 7 0

14 12 6 5 0 Others 0 2 2

In the DRB1 " 0 1 0 3 group, 15 DQ associations were wi th DQ1 and 3 were wi th DQ7. The DRB1 " 1 3 0 1 / 0 2 group involved 20 DQ1 and 1 DQ3 associations. The DR14 group involved 10 DQ1 associations. One signif icant benefit of this act iv i ty is the fact that our on-going typing t ray analysis now al lows us to ident i fy key antisera that can differentiate DR alleles (i.e. 0103, 1303). DNA class II typing when used in parallel wi th serologic typing can assist in t roubleshoot ing questionable serologic results and, in turn, improve the identif ication and characterization of key, class II reagents. Ult imately, the number of serologic results requiring clarif ication should diminish.

C-7.1 #234

EFFECTS OF HEAT INACTIVATION AND DTT TREATMENT OF SERUM ON IMMUNOGLOBULIN BINDING. Thorne N. Klingman LL, Teresi GA and Cook DJ. The Cleveland Clinic Foundation, Transplant Center, Cleveland, OH.

A positive crossmatch due to IgM antibodies is generally believed to be less detrimental in organ transplantation than one due to IgG. Both heat inactivation (HI) and dithiothreitol (DTT) treatment of serum are utilized by many laboratories to reduce IgM antibodies. Here we report the results of testing 8 samples against a common target cell, by both CDC and flow cytometry (FC), in order to ascertain the effects of HI and DTT treatment on immunoglobulin binding. Samples were examined untreated, heat inactivated at 63 ° for 10 minutes and DTT treated at 37 ° for 30 minutes with 0.01M DTT. The results of CDC reactions (titer) and FC analysis of IgG and IgM binding, expressed as percent fluorescence of the untreated sample are shown.

CYTOTOXICITY FLOW-I FLOW-I M SAMPLE UNT HI DTT HI D'TT HI I~TT

NHS NEG NEG NEG 103% 94% 101% 96% PHS 1:64 1:16 1:32 77% 72% 47% 54%

BBM.1 1:256 1:32 1:256 2% 64% CP-1 >1:512 NEG 1:64 <1% 13%

PATEG 1:256 1:256 1:256 88% 98% 19% 17% PATEL 1:256 1:128 1:256 89% 48% 30% 12% PAT TS 1:32 NEG NEG NEG NEG 21% 19% PAT LI 1:2 NEG NEG NEG NEG < 1% 2%

Normal AB human serum (NHS) and a pool of 5 highly sensitized patient's sera (PHS) served as negative and positive controls, respectively. Two monoclonal antibodies, BBM.1 (IgG) and Campath- 1 (IgM) were also examined. HI had a dramatic effect on both monoclonal controls, whereas DTT primarily effected CP-1. Two highly sensitized patient sera (EG + EL), and the PHS exhibited strong IgG binding that was variably diminished by both techniques, while 2 auto-antibody sera (TS, LI), demonstrated IgM reactivity that was greatly reduced by both treatments. In summary, it appears as expected that both HI and DTT treatment of serum are effective means of reducing IgM auto- antibodies. However, it should be noted that both HI and DTT can influence IgG binding, a fact that should be considered when crossmatching patients using these methods.