HLA-A, -B positive sera of south Indian parous women were screened for the

presence of HLA-DR and -DQ antibodies. The sera were absorbed with pooled

platelets in a micro-method and screened against HLA-DR and -DQ typed B-lymphocytes

panel. Out of fifty two sera, twenty were found to contain HLA-DR and -DQ

antibodies. Seven sera were demonstrated to be specific for well defined DR and DQ antigen

Chandrasekaran, P

Damodaran, C

Narayanan, P R

Prabhakar, R

Selvaraj, P

Subramanian, V S

NIRT Institutional Repository

V.S Subramanian1. P. Selvaraj2, P.R. NaraC. Damodaran1* and P. Chandrayanan2 R. Prabhakar2,Sekharan1HLA-A, -B positive sera of south Indian parous women were screened for thepresence of HLA-DR and -DQ antibodies. The sera were absorbed with pooledplatelets in a micro-method and screened against HLA-DR and -DQ typed B-lym-phocytes panel.Out of fifty two sera, twenty were found to contain HLA-DR and -DQ   antibodies. Seven sera were demonstrated to be specific for well defined DR andThe HLA (Human Leucocyte Antigen) com-plex has been localized to the short arm of thechromosome number 6 in the distal portion of 6p21.3 band. This complex consists of at leastseven well estimated loci which are designatedas HLA-A, -B, -C, -D, -DR, -DQ and -DP. HLA-A,-B and -C antigens are known as class I antigensand HLA-D, -DR, -DQ and -DP antigens areknown as class II antigens (1).HLA-phenotyping of class I and some classII antigens is being carried out by serologicaltyping method using allospecific human antiseraand HLA-genotyping by restriction fragmentlength polymorphism (RFLP) using DNA probesfor the HLA region of the human genome (2)and/or polymerase chain reaction (PCR)amplified human DNA (HLA genome) with se-quence specific oligonucleotide (SSO) probes (3).It has been well established that the sera ofpregnant women are the most suitable and easilyobtainable source for HLA antibodies. Further,it is evident that HLA-A, -B, -C antisera alsofrequently contain anti-HLA -DR (and -DQ) an-tibodies; specific -DR (and -DQ) antisera withoutMA-ABC antibodies have also been observedthough rarely (4 - 6). In our earlier reports (7, 8),data on the screening for HLA-A, -B (class I)antibodies in the sera of South Indian parouswomen were presented. In the present studyHLA-A, -B (class I) positive sera of the previoustudies were screened for the presence of HLA-DR and -DQ (class- II) antibodies and the resultsare discussed.Materials and Methods(i) Collection of parous women seraPostpartum Mood dots were collected im-mediately after delivery (primiparous and multi-parous) and the sera were separated and storedat -90ºC as described elsewhere (7,9).(ii) Screening of sera for HLA-A. -BantibodiesA total number of 1161 sera were screenedagainst HLA-A, -B typed cell panel members andthe results have been published in our earlierreports (7,8).(iii) Screening of sera for HLA-DRand -DQ antibodiesThe HLA-A, -B positive sera were screenedfor the presence of HLA-DR, and -DQ antibodiesagainst 32 normal individuals.110 lndian Journal of Forensic Sciences (Vol. 6) 1992(iii)a. Elimination of HLA-A, -B, -CantibodiesIt is well established that platelets containHLA-A, -B and -C antigens on their surface (ad-sorbed antigens). Platelets from 20 differentindividuals were separated and pooled. Thepooled platelets were used to absorb the anti-HLA antibodies from the HLA-A, -B positive sera.The method described by Cambon- Thomsen etal (9) was followed. In brief, all the HLA-A, -Bpositive sera (1µl) were plated in 60 well Terasakiplates. The pre-dotted walls were added with 1.5x 106 pooled platelets (10.5 x 109/ml) in 1 µlsaline and stored at -20°C until use.(iii)b. T and B cell separationT and B cells from peripheral bloodmononuclear cells were separated by nylonwooI column adherence method (10). In brief, plastic drinking straws approximately 8cmheight were packed with 75 to 100 mg of nylonwool for 4 to 5 cm height. The columns weresterilized (10 psi) and used for T and B-cellseparation. The nylon wool columns wererinsed with TC 199 medium containing 10% foe-ial calf serum (FCS) and antibiotics. Ap-proximately 10 - 12 x 106 peripheral Moodmononulear cells (Ficoll-Hypaque s parated)in 1 ml medium containing 10% FCS was loadedon the top of the column and incubated for 45minutes. Afterwards the nylon wool nonad-herent cells (enriched T-cell population) wereeluted using 7ml of warm medium. Nylon wooladherent cells (enriched B-cell population) wereeluted with 5 ml medium by pressing andsqueezing the nylon wool column and used forHLA-DR and -DQ serological typing.(iii)c. HLA-DR and -DQ typing in nor-mal volunteers (for cell panel)HLA-DR and -DQ typing of normal volunteers(32 individuals) was performed by two stage NIHstandard microlymphocytotoxicity (antibody de-pendent complement mediated cytotoxicity)assay (11). A total number of 38 well definedHLA-DR and -DQ sera (Biotest, West Germany)covering DR 1 to DR 9 and DQ 1 to DQ 3 antigenspecificities were used (Table 1).(iii)d. Screening of sera for HLA-DRand -DQ antibodiesThe plates containing the HLA-A, -B positivesera (pre- dotted) and pooled platelets wereincubated at room temperature for 1 h with twoor 3 times vortexing; and then the plates wereused for -DR and -DQ antibody screening. Forscreening procedure the two stage NIH standardmicrolymphocytotoxicity (antibody dependentcomplement mediated microlym-phocytotoxicity) assay was followed (11). Inbrief, one µI aliquot containing 2000-3000 (nylonwool column separated enriched) B-lym-phocytes was added to each well. The plateswere incubated for 1 h at 25°C; 5 µI of rabbitcomplement (absorbed with white cells) wasadded and incubated further for another 2 h.Then the reaction was arrested by adding 5 µl of3% eosin and neutralized formaldehyde. Theplates were read under phase contrast micro-scope.ResultsOur earlier reports on the screening of parouswomen sera for HLA-A, -B antibodies revealed59 sera out of the total 1161 to be positive (Table2) (7,8).Out of these 59 HLA-A, -B positive sera, 52were screened presently for HLA-DR and -DQantibodies. Out of the 52, only 20 exhibitedpositive reaction on B lymphocytes when testedn the HLA-DR and -DQ typed B cdl panel (32individuals) (Table 1). Eighteen sera were foundnegative and 14 sera still elude exact assignmentwarranting further analysis (Table 3).Out of the 20 HLA-DR and -DQ positive sera,7 were found to be either monospecific orduospecific to well defined DR and DQ antigens;the rest of the sera were multispecific (Table 4).Indian Journal of Forensic Sciences (Vol. 6) 1992 111DiscussionThe present study reveals that the frequencyof HLA-DR and -DQ antibodies is less(20 + 14 =34, out of 1161 sera) (2.9%) (Table 3)when compared to the frequency of HLA-ABCantibodies (5.08%) (7,8) (Table 2).Further, it also indicates that anti kHIA-DRand -DC typing reagents can be expected frommultiparous sera (from post- partum blood clots)containing anti HLA-A or -B antibodies (38.5%;Table 3). Sera without ABC antibodies have alsobeen reported to contain HLA-DR and -DR an-tibodies; however, the frequency of class II an-tibodies is less in these sera when compared to thesera that are positive for HLA-ABC antigens (12).AcknowledgementThe authors thank the Medical Superinten-dent, Government Kasturba Gandhi Hospital forWomen and Children and the Superintendent ofKalyani General Hospital, Madras and their staffFor permitting and for assisting in the collectionof samp!es.References1. Crumpton MJ (1987); HLA in medicine, In:British Medical Bulletin, Vol. 43, pp i-vi.Churchill Livingstone, London.2. Bidwell JL and Bignon JD (1991); DNA-RFLP methods and interpretation schemefor HLA-DR and DQ typing. EuropeanJournal of Immunogenetics 18, 5-22.3. Baxter-Lowe LA, Hunter JB, Casper JT andGorski J (1989); HLA gene amplificationand hybridization analysis of polymor-phism. HLA matching for bone marrowtransplantation of a patient with HLA-deficientsevere combined immunodeficiencysyndrome. J. Clin. Investigation 84, 613-618. 4. Jones EA, Goodfellow PN, Bodmer JG andBodmer WF (1975); Serological identifica-tion of HL-A linked human "la-type" an-tigens Nature 256, 650-6525. Baricordi OR, Belvedere M, Fagiolo U, NeriTM and Trabace S (1977); Screening forsera with B cell antibodies in Region Italy.Tissue Antigens 10, 130.6. Stem P and Rittner Ch (1981); Ein HLA-DRscreening programme. Arztl. Lab 27, 209-214.7.8.9.10.11.12.Selvakumar A, Selvaraj P, Damodaran Cand Chandra Sekharan P (1987); Screen-ing of sera from South Indian pregnantwomen for the presence of HLA an-tibodies; In: Proceedings of the first Inter-national Conference on Forensic Science(Madras. 1985) pp 285-287.Subramanian VS, Gunachandran N,Damodaran C and Chandra Sekharan P(1992) ; Screening of parous women’s serafor the presence of HLA antibodies. IndianJournal of Forensic Sciences 6, 17 - 19.Cambon-Thomsen A, Sommer E, DemurC, Cabot M and Chayon E (1986); Plateletabsorption on the test tray (PATT): A rapidmethod for the screening of HLA class IIantibodies using the two-colour fluores-cence method. Tissue Antigens 26, 193-200.Manickasundari M, Selvaraj P andPitchappan RM (1984); Studies on T-cellsof the lizard, Calotes versicolor: Adherentand nonadherent populations of thespleen. Developmental and ComparativeImmunology 8, 367-374.Terasaki PI and Park MS (1976);Microdroplet lymphocytotoxicity test, In:NlAlD Manual of Tissue Typing Techni-ques. (Eds) Ray JG, Hare DB, PedersenPD and Mullaly Dl; DHEW Publication(NIH), No.76-545;. pp 69-80.Vassalli P, Jeannet M, de Moerloose P andChardonnens X (1979); A screening pro-gram for anti-DR typing reagents. TissueAntigens 13, 77-89.Indian Journal of Forensic Sciences (Vol. 6) 19Table IScreening of cells (thirty two) for HLA-DR and -DQ antigens (cell panel)Specificity (number of cells positive)HLA-DR DR 1 (1); DR 2 (17); DR 3 (4); DR 4 (8); DR 5 (5); DR 6 (2);DR 7 (12); DR 8 (4); DR blank (11)HLA-DQ DQ 1 (25); DQ 2 (12); DQ 3 (14); DQ blank (12)Table 2Frequency of HLA - A, - B antibodies in parous women sera*Sera screened for HLA-A, -B, -C antibodiesPositive seraa) Monospecific sera   b) Multispecific sera  Negative sera*Based on our earlier reports (7,8)Total sera11615916431102Percentage–5.08%1.38%3.7%94.9%Indian Journal of Forensic Science (Vol. 5) 1992 113Table 3Frequency of HLA-DR and -DQ antibodies in HLA-A, -B positive sera of parous womenSera screened for HLA-DR and -DQ antibodiesPositive seraNegative seraStill under analysis for assignment* HLA-A, -B positive sera of parous womenTotal sera Percentage52* –20 38.5%18 34.6%14 26.9%Table 4Analysis on the behaviour of HLA-DR and -DQ positive sera of parous womenSerum Specificity Positive False False Negative r-valuenumber positive negativeCDM 741 DR 2 15 2 2 11 0.73CDM 1111 DR3 3 0 1 26 0.84CDM 1217 DR3 3 0 1 26 0.84CDM 1501 DR2 13 7 4 6 0.24CDM 1521 DR 8 2 0 2 26 0.68CDM 1593 DR 7, DQ 2 8 7 4 11 0.27CDM 1622 DQ2 6 2 6 16 0.43

HLA-DR and -DQ antibodies in the sera of South India Parous women

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HLA-DR and -DQ antibodies in the sera of South India Parous women

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