Preparation of human monoclonal anti-C cell line from peripheral blood B lymphocytes of D--donor / 中国输血杂志

【Objective】 To establish human hybridoma cell lines, secreting monoclonal antibody against antigens of Rh blood system, from a donor with rare D--phenotype. 【Methods】 Peripheral blood B lymphocytes of an O type female donor, lacking C/c/E/e antigens on her erythrocyte, were transformed with Epstein-Barr virus (EBVs). EBVs were harvested from the cultural supernatant of B95-8 cells. The transformed lymphoblastoid cell line (LCL) secreting antibodies to C antigens were picked up and then hybridized with the myeloma SHM-D33 using electric fusion technique. Hybridoma cells were selected by HATD-Ouabain(HOTD)(Hypoxantine, Aminopterin, Thymidine, 2-Deoxycytide, and Ouabain)culture medium, microplate antibody screening and limited dilution subcloning. The monoclonal antibody was assayed by serological test and was confirmed by flow cytometry (FCM). 【Results】 From the cultural supernatant of D--peripheral blood transformed B lymphocytes, 3A6-C6, which agglutinated with R1R1(DCe/DCe)O-type RBCs but not with R2R2(DcE/DcE)O-type RBCs, was screened and preliminarily identified as anti-C. We established a hybridoma cell line secreting anti-C immunoglobulin M from B cells of D--individual successfully after hybridization with SHM-D33 myeloma cells. 【Conclusion】 The study had laid the groundwork for future research and development of human monoclonal antibodies against Rh antigens of RBC in future for diagnosis and screening purpose.

-D- phenotype screening by polybrene method / 中国输血杂志

【Objective】 To develop a novel screening reagent for -D- phenotype preliminary screening based on the difference in RhD antigen expression level of -D- phenotype and normal RhD phenotype. 【Methods】 RhD antigen expression of -D-phenotype and Rh D-- gene carrier were detected by flow cytometry. By adjusting the concentration of polybrene in the screening system, the red blood cells with high RhD antigen expression level agglutinated, and the preliminary screening of the -D-phenotype and its gene carriers was realized. 【Results】 According to the quantitative results of immunofluorescence intensity (MFI) analysis by flow cytometry, the expression level of RhD antigen in -D- phenotype cells (284 360±16 698, n=3) was about 3 times normal RhD positive cells (98 642±35 908, n=9)(P<0.01), while RhD antigen expression level of RhD-- gene carrier (181 109±39 455, n=4) was about 2 times normal RhD positive cells(P<0.01). RhD antigen expression (144 538±227 445, n=7) of the positive cells screened by 15 μL 3% fresh red blood cell suspension and screening system 35 μL (1 μL IgG anti-D, 29 μL polybrene polybrene, and 5 μL low ionic strength solution) was about 1.5 times normal RhD positive cells. 【Conclusion】 The polybrene preliminary screening system, which can be used for high-throughput screening of -D- phenotype, is a reliable technical method for frequency study of this phenotype.

Investigation on the distribution of weak D and Del phenotype in unrelated blood donors with negative Rh(D) / 实用医学杂志

Objective To investigate the distribution of weak D and Del phenotype in unrelated blood donors with negative Rh (D). Methods Four hundred and nineRhD (-) unrelated donors were screened by the saline agglutination method. Type weak D was detected by the indirect antiglobulin test, and type Del was detected by absorb radiation method. Results In 409 RhD (-) samples, 27 cases (6.61%) of type weak D were checked out, and 61 cases (14.91%) were type Del and 321 cases (78.48%) were corroborate for RhD (-), In the confirmed RhD (-) blood donors, phenotype ccee was most (49.14%), followed by Ccee (23.47%), People with phenotype ccEe accounted for 4.16%in type weak D, followed by Ccee (1.71%). Ccee accounted for 10.02%in type Del, and Ccee accounted for 1.71%. Conclusion RhD (-) donors screened by regular testing should adopt a more sensitive test for verifing type weak D or Del. In order to ensure the security of blood transfusion, people with type weak D and Del should be regarded as RhD positive blood donors, and the RhD negative people deemed to be recipients.

A Case of D--/D-- Phenotype Associated with Moderate Hemolytic Disease of the Newborn / 대한수혈학회지

The very rare D--/D-- phenotype lacks C, c, E, e antigens with strong expression of the D antigen. A 31-year-old woman delivered her second baby, 3.6 kg girl at 38+4 weeks' gestation through repeat-Cesarean section. Her parents were not consanguineous. She had one artificial abortion, one Cesarean section with red blood cell transfusion and two spontaneous abortions. Her red cells were typed as O, D+C-c-E-e- and did not react with anti-Hr(o) (Rh 17). Her serum reacted with all of the screening cells and identification panel cells with strength of (++)~(+++). The baby was mildly jaundiced 12 hours after delivery. At 1 day after delivery, total bilirubin was 17.7 mg/dL, and direct and indirect antiglobulin tests were both positive. Phototherapy was immediately given for the baby but jaundice and anemia were worsened. Twenty six milliliter of the mother's whole blood was given twice to the baby after plasma depletion and leukocyte reduction. The baby showed improvement of jaundice and anemia, and discharged at hospital day 14. As far as we know, this is the third reported case of hemolytic disease of the newborn occurred in the D--/D-- mother with anti-Hr(o) in Korea, and the first case that was neither fatal nor treated with intensive medical care.