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Anti‐D reagents should be chosen accordingly to the prevalence of D variants in the obstetric population

Anti‐D reagents should be chosen accordingly to the prevalence of D variants in the obstetric... INTRODUCTIONThe RhD antigen is clinically the most significant blood group antigen for obstetric medicine due to its immunogenicity and the high incidence of RhD‐ individuals. Depending on the population, 3%‐25% of Caucasians are D−, with 15%‐20% prevalence being most commonly reported. Anti‐D is still the leading cause of the hemolytic disease of fetus and newborn (HDFN) in the obstetric population of Split‐Dalmatian County, Croatia.The division between D+ and D− status is not always straightforward, due to over 200 D variant phenotypes having been reported to date. Generally, “weak D” variants are result of mutations in which all D epitopes are present but with reduced expression, while “partial D” represents variants in which the mutations result in the absence of certain polypeptides on the outer RBC surface. Recent research found weak D types to also possess slightly to considerably altered D antigens, with the extent of qualitative changes overlapping with that in partial D. The prevalence of weak D types among Caucasians is reported to be about 0.2%‐1%. Most weak D variants in Caucasians are weak D type 1, type 2 and type 3 (RHD*weak D type 1, RHD*weak D type 2, RHD*weak D type 3, which are not known to form alloanti‐D. The most common partial D variants among Caucasians is DNB. DVI is clinically the most relevant, being the variant most commonly involved in anti‐D immunization and HDFN. Its prevalence is reported to be between 0.02%‐0.05%. The distinction between weak and partial D is crucial since the individuals with partial D may form alloanti‐D, being regarded as D− considering transfusion therapy and perinatal management. Albeit rare, the cases of HDFN in neonates from mothers with D variant antigen had been reported, and fatal outcomes did occur. Differentiation between weak and partial D in routine serologic practice is problematic, since in both weak and partial D variants, the individual's RBC may give weak reactions with some anti‐D reagents, or the reaction with some anti‐D reagents may not exist. Different reagents contain different monoclonal antibody and protein concentrations or agglutination enhancers. Also, the difference between the serologic methods used for blood typing can cause discrepancies in the results. In Europe, anti‐D reagents are selected to deliberately type DVI pregnant women as D− to ensure that those mothers receive Rh immune globulin (RhIG) prophylaxis in their second trimester and/or postpartum. For a period of 5 years, we determined the prevalence of discrepant serologic results in a large obstetric population. We analyzed the incidence of D variants and serologic reactivity of D variants in column technology and tube method. We compared serologic results with the results of RHD genotyping.The aim of this study was to establish selection criteria of anti‐D reagents for our population which would ensure that partial D carriers are not assigned D+ status for transfusion therapy and immunoprophylaxis. This would enable us to evaluate prevention strategy of anti‐D immunizations in pregnant women and patients with partial D variants.MATERIALS AND METHODSThis study was performed at the Department of Transfusion Medicine of the Split University Hospital Center, in Split, Croatia from April 2008 to December 2012 (5 years). The Hospital is a regional institution responsible for the overall pregnancy care, monitoring of RBC alloimmunized women and the management of deliveries. All pregnant women in this region were typed for ABO and RhD blood groups and tested for irregular antibodies at their initial visit. Anti‐D immunoprophylaxis is routinely administered in D− and partial D pregnant women after the delivery of D+ child, except in cases in which anti‐D has been detected.Routine typing of D antigen in column technologyFrom April 10, 2008 to December 31, 2012 RhD typing was performed by automated microcolumn technology using ABO‐DD Grouping Cassette® (Ortho Clinical Diagnostics, Raritan, NJ, USA) which contains two monoclonal IgM anti‐D reagents (clone D7B8, and clone RUM‐1). Samples of women whose D antigen in column method was not strong positive (4+) but graded as 3+ or less were considered to be discrepant and they underwent further serological evaluation by additional RhD typing in direct and indirect agglutination. Figure  shows algorithm of interpretation of D antigen in microcolumn RhD typing.Algorithm of D antigen testing using microcolumn beads technology, from April 10, 2008 to December 31, 2012Additional serologic RhD typingAdditional RhD typing in direct agglutination was done with five monoclonal antibodies in tube testing: Ortho Anti‐D® (P3x61 monoclonal IgM, Clinical Diagnostics, Raritan, NJ, USA); DiaClon® Anti‐D Monoclonal IgG and IgM (MS‐26, TH28, DiaMed GmbH, Cressier, Switzerland); Anti‐DM MonoGnost® (MS‐201, BioGnost, Zagreb, Croatia); NovaClone® Anti‐D IgM+IgG Monoclonal Blend (CI 175‐2, D415, 1E4, Immucor Gamma, Dartmouth, NS, Canada); Anti‐D MG MonoGnost® (RUM‐1 IgM, MS‐26 IgG, BioGnosta).D‐screen kit for partial D identification using indirect agglutination including six different monoclonal D monoclonal antibodies which enables detection of partial D categories II, IV, V, VI, VII, DFR, DBT and RoHar (ID‐Partial RhD Typing Set®, DiaMed AG, Morat, Switzerland) was the second additional typing method used for discrepant results. The test is performed manually in gel microcolumn cards which are contained in the typing set. Phenotype determination of the Rh antigens (C, c, E, e) and Kell antigen was performed in microcolumn method (Rh/K Cassette®; Ortho Clinical Diagnostics).RHD genotypingAll women whose D antigen in column method during routine serologic D typing was considered to be discrepant were, after the additional serologic D typing, forwarded to RHD genotyping. The genotyping was performed in Department of Molecular Diagnostics of Croatian Institute of Transfusion Medicine in Zagreb, Croatia. DNA extraction from EDTA blood samples was done by QIAamp DNA Blood Mini kit® (Qiaqen, Hilden, Germany) or MagNA Pure LC® (Roche Molecular Biochemicals, Mannheim, Germany). Particular segments of the RHD gene sequence are multiplied by RHD genotyping using primers specific for the known mutations characterizing particular weak D types by use of the polymerase chain reaction with sequence specific priming (PCR‐SSP). RHD genotyping was performed by commercial genotyping kits (Ready Gene weak D and Ready gene CDE, Inno‐Train, Kronberg, Germany).Statistical analysisStatistical analyses were performed using computer software MedCalc®, version 12.5 (MedCalc Software, Ostend, Belgium). Relative frequencies and accompanying 95% confidence intervals (95% CI) were used to estimate the prevalence of variants.RESULTSDuring the period of five years we tested 12 689 primiparous women. D antigen status was consistent (D+ or D−) for 12 632 (99.55%) of them, while 57 (0.45%) women gave weak agglutination reactions in routine serologic D typing procedure (Table ).Prevalence of D variants in pregnant womenRhD determined N=12 689NPrevalence in 10 000 (95% CI)Consistent serologic results12 63299.55% (99.43; 99.67) RhD+10 35981.63% (80.96; 82.31) RhD−227317.91% (17.25; 18.58)Discrepant serologic results, resolved by RHD genotyping570.45% (0.33; 0.57) RHD* weak D type 1240.19% (0.11; 0.26) RHD* weak D type 3180.14% (0.08; 0.21) Weak D10.01% (0.00; 0.02) RHD*05.05100.08% (0.03; 0.13) D+40.03% (0.00; 0.06)RHD* weak D type 1/3 heterozygous status.These samples should be sequenced to distinguish a possible D variant and they were not taken into calculation of D variant prevalence.Results of RHD genotypingMolecular analysis of those 57 discrepant cases found 43 women to be weak D variant carriers. Ten of weak D variant carriers were genotyped as partial D, and four cases were resolved as regular D+ (Table ). For these four samples to really distinguish whether they represent a D variant that could not be detected by commercial tests or even a new D variant, RHD sequencing should be performed. The prevalence of D variants detected by column technology in our population of primiparous women was 0.42% (95% CI 0.31; 0.53). The aforementioned four samples that were resolved as D+ were not taken into account.Results of additional serologic analysis of weak DAt the initial routine D typing, the individuals who were later genotyped as weak D gave equivocally weak agglutination reactions in microcolumn beads method with both monoclonal reagents (graded 3+ or less), as shown in Figure . The analysis with direct agglutination in tube method with five monoclonal reagents found cell lines RUM‐1 and CI 175‐2 showed to be the most sensitive with 97.67% reactivity with weak D samples (Table ).Serologic reactivity of weak D (type 1) in microcolumn technologySerologic reactivities of D variants in direct agglutination with six monocolonal anti‐D reagentsD variantsPatientsReagentsabcdeRHD* weak D type 1N=1311111N=701111N=100111N=200011N=100001Total N (%)24 (100)13 (54.1)20 (83.3)21 (87.5)23 (95.8)24 (100)RHD* weak D type 3N=1411111N=111011N=100111N=100011N=100010Total N (%)N=18 (100)15 (83.3)15 (83.3)15 (83.3)18 (100)17 (94.4)Total weak DN=4328 (65.1)35 (81.3)37 (86)42 (97.6)42 (97.6)D category Type VaN=511110N=501110Total partial D N (%)N=10(100)5 (50)10 (100)10 (100)10 (100)01, agglutination; 0, no agglutination.aOrtho IgM P3x61; bDiaMed IgM+IgG MS‐26, TH28; cBioGnost MS‐20; dNovaClone IgM+IgG CI 175‐2, D415, 1E4; eMonoGnost IgM+IgG RUM‐1 MS‐26.One sample of RHD* weak D type 1/3 heterozygous status agglutinated reagents c, d, e.In direct agglutination in ID‐partial set, one weak D type 3 sample and five weak D type 1 samples failed to react with LDM1 clone. They showed positive reactions in indirect agglutination with all other five monoclonal reagents. One weak D type 1 sample failed to show agglutination with cell lines LHM76/55 and LHM59/19 (Table ).Serologic reactions of D variants with six monoclonal antibodies using set for indirect agglutination ID‐Partial (DiaMed)D variantsPatientsReagentsABCDEFRHD* weak D type 1N=18111111N=5111110N=1011011Total N (%)N=24 (100)23 (95.8)24 (100)24 (100)23 (95.8)24 (100)19 (79.1)RHD* weak D type 3N=17111111N=1111110Total N (%)N=18 (100)18 (100)18 (100)18 (100)18 (100)18 (100)17 (94.4)Total weak D N (%)N=43 (100)42 (97.6)43 (100)43 (100)42 (97.6)43 (100)37 (86)D category type VaN=7110101N=3110100Total partial D N (%)N=10 (100)10 (100)10 (100)010 (100)07 (70.00)ALHM76/55 (IgG); BLHM77/64 (IgG); CLHM70/45 (IgG); DLHM59/19 (IgG); ELHM169/80 (IgG); FLDM1 (IgM).One sample of RHD* weak D type 1/3 heterozygous status showed agglutination with all six reagents.All four cases of samples which showed discrepant results in routine RhD typing and were initially presumed to be some D variant type, but could not be distinguished from normal D+ in molecular analysis, showed weak agglutination reactions with all five anti‐D monoclonal antibodies in tube method. This inability to detect some D weak in molecular analysis is due to the fact that all D weak types were not included in commercial assay (possibly could be done by sequencing).Results of additional serologic analysis of partial DAll individuals genotyped as D category type RHD*05.05 (N=10) were correctly recognized as partial D during the routine testing in microcolumn method due to strong positive agglutination reactions with clone D7B8 and weaker reactions (2+ or less) with clone RUM‐1 (Figure ).Serologic reactivity of partial D (RHD*05.05) in microcolumn technologyDirect agglutination reactions with five anti‐D monoclonal reagents of all D category Va samples were clearly positive except with the clone RUM1 (Table ). Indirect agglutination with six anti‐D monoclonal reagents in D category type Va samples consistently failed to show reaction with the clone LHM70/45 and LHM96/80, but reacted with the clones LHM76/55, LHM77/64 and LHM59/19. Reactivity with LDM1 was variable (Table ). A single case of DNB was interpreted as regular D+ in routine typing and was discovered only after the immunization had occurred. As expected, most of weak D cases were CcDee Rh phenotype (88.37%). Three cases of weak D type 3 were CCDee phenotype, and one was CcDEe phenotype. The DNB case was CcDEe phenotype.DISSCUSIONA recent study showed that RHD genotyping of women with serologically inconclusive results, rather than managing them all as D−, would be cost‐saving over long‐time periods. The newest recommendations from a Work Group on RHD genotyping, directed by AABB and CAP (College of American Pathologists) state that RHD genotyping should be performed when discordant RhD typing results are encountered and/or a serologic weak D phenotype is detected in females of childbearing potential, patients, newborns and potential transfusion recipients. The benefit would be fewer unnecessary injections of RhIG and increased availability of D− RBC units.Microcolumn method is known to be highly sensitive for RhD typing. In this study, the criterion for additional RhD analysis was reactivity in direct agglutination 3+ or less (score of not more than 10) with one or both anti‐D reagents. Of 57 women who fell into the criterion, 53 women were found to be either weak or partial D variants. Although other studies recommend the cutoff score of not more than 8 (≤2+) by gel technology for assigning D negative status considering transfusion and immunoprophylaxis, the results or our study shows that the reactivity criterion in direct agglutination of score 10 (3+) or less is acceptable as the cutoff value. The frequency of D variants in our study was in concordance with the findings from other studies, and so was the distribution of Rh phenotypes.We found that the most common partial D variant in our population was D category Va, with prevalence of 0.08%. Therefore, we suggest the choice of anti‐D reagent nonreactive for DVI and DVa category for our patients and obstetric population. Such practice would ensure that those women are typed as D− and that they receive anti‐D immunoprophylaxis. In this study, partial D category VI was not found in direct typing because it is categorized as D− by our serologic selection criteria for routine RhD typing. However, in our previous study on 8670 blood donors in the same population, we used the same serologic methods and reagents to investigate donors which typed D− in direct agglutination but D+ in IAT, and genotyping did not reveal a single case of D category VI. This was a surprising finding, since D category VI is the clinically most relevant partial D type among Caucasians with frequency between 0.02%‐0.05%. Adjusting the reagent choice would be in accordance with suggestions from other authors, stating that the profile of monoclonal antibodies for RhD typing should be chosen individually regarding to the population for which it is used. In our study, DVa category samples could be clearly distinguished from weak D samples by routine serology alone, as partial D consistently showed weak to negative agglutination with one anti‐D monoclonal antibody but reacted with second one in routine testing. All cases failed to react with monoclonal cell line RUM‐1 in tube method. In microcolumn method, they gave weak positive reactions with cell line RUM‐1, in opposite to cell line D7B8, with which they gave strong positive reactions.We noticed that reactions of partial D category Va were not entirely in concordance with manufacturer's worksheet in IAT typing with ID‐partial typing set (Diamed). As expected, the reaction was omitted with cell line LHM70/45, but unexpectedly also with LHM169/80. This is an important finding because it shows that these cell lines should not be used for donor and/or neonate typing as they would type as D−. Other authors did not report this outcome. While weak D type 1 and type 3 were in our study indistinguishable from one another based on serology, weak D type 3 was observed to react generally in higher percentage in both direct and indirect typing. This was an expected outcome since weak D type 3 has been reported by other authors to have one of the highest antigen densities among weak D variants, with mean value of antigen density of 1900 antigens per cell, while for weak D type 1 and 2 that value is 700 and 600, respectively. However, we must emphasize that weak D types susceptible for immunization, such as weak D types 4.0 (RHD*09.03.01), 4.2.1 (RHD*09.01.01) and 15 (RHD*weak partial 15) were not represented in the examined obstetric population. The research on a different population, in which such D variants are present, found that it was not possible to rely on serology only to prevent immunization events in weak D carriers, since they could not be distinguished from other weak D types.All partial DVa cases were CcDee phenotype. Other authors reported DNB to be associated with CcDee or CCDee phenotype.As previously observed by Pirenne et al., our research shows how careful selection of anti‐D reagents and serologic methods can ensure high level of safety in detection of partial D individuals. It is already recommended by multiple authors to implement molecular typing whenever possible to clarify ambiguous D antigen typing results. In the previous study on our obstetric population, anti‐D immunization occurred in pregnant two partial D carriers: type D category Va and one DNB carrier. Those D category Va carriers were assigned as D+ status due to the choice of reagents used at the time. They did not receive immunoprophylaxis and one of them was transfused with D+ RBC unit. The single case of partial DNB category was detected after the immunization had occurred, similarly as previously reported by other authors. We were therefore unable to determine the prevalence of partial D variant category DNB in our population, because it was typed as D+ due to our reagent selection criteria.The follow‐up of anti‐D immunizations in patients with D variants, completed with results of RHD genotyping, was an indicator which enables us to assess the correctness of the choice of reagents for RhD typing for the population in question. The appropriate choice of reagents will enable the serology methods to recognize the cases in which RHD genotyping is required. The shortcoming of this study is the possibility that some D variants might have been recognized as D+ using the current selection of monoclonal reagents, as is the case with DNB. That is where long‐term follow‐up of anti‐D immunizations emerges as the indicator of reagent selection correctness.We could conclude that cell line RUM‐1 should be used for RhD typing of patients in our population, since it gives weak or negative reactions with partial D category Va category, which we found to be the most prevalent partial D variant in our region. Anti‐D reagents should be chosen accordingly to the population's D variants prevalence to recognize D variants which are at risk for immunization.REFERENCESMollison PL, Klein HG, Anstee DJ. Blood Transfusion in Clinical Medicine, 11th ed. Oxford: Blackwell Scientific publications; 2005:163‐208; 496‐545.Urbaniak SJ, Greiss MA. RhD haemolytic disease of the fetus and the newborn. Blood Rev. 2000;14:44‐61.Esteban R, Montero R, Flegel WA, et al. The D category VI type 4 allele is prevalent in the Spanish population. Transfusion. 2006;46:616‐623.Wagner FF, Frohmajer A, Ladewig B, et al. Weak D alleles express distinct phenotypes. Blood. 2000;95:2699‐2708.Wagner FF, Frohmajer A, Flegel WA. RHD positive haplotypes in D negative Europeans. BMC Genet. 2001;2:10.Muller H, Wagner FF, Trockenbacher A, et al. PCR screening for common weak D types shows different distributions in three Central European populations. Transfusion. 2001;41:45‐52.Dajak S, Lukacevic Krstic J, Körmöczi G, Dogic V, Burilovic V. Characteristics and frequency of DEL phenotype detected by indirect antiglobulin test in Dalmatia county of Croatia. Transfus Apher Sci. 2014;50:210‐213.Dajak S, Čulić S, Stefanović V, Lukačević J. Relationship between previous maternal transfusions and haemolytic disease of the foetus and newborn mediated by non‐RhD antibodies. Blood Transfus. 2013;11:528‐532.isbtweb.org [homepage on the internet]. International Society of Blood Transfusion: Red Cell Immunogenetics and Blood Group Terminology. http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/. Cited September 28, 2016.Wagner FF, Gassner C, Muller TH, Schönitzer D, Schunter F, Flegel WA. Molecular basis of weak D phenotypes. Blood. 1999;93:385‐393.Christiansen M, Samuelsen B, Christiansen L, Morbjerg T, Bredahl C, Grunnet N. Correlation between serology and genetics of weak D types in Denmark. Transfusion. 2008;48:187‐193.Wagner FF, Eicher NI, Jørgensen JR, Lonicer CB, Flegel WA. DNB: a partial D with anti‐D frequent in Central Europe. Blood. 2002;100:2252‐2256.Wagner FF, Kasulke D, Kerowgan M, Flegel WA. Frequencies of the blood groups ABO, Rhesus, D category VI, Kell, and of clinically relevant high‐frequency antigens in south‐western Germany. Infusionsther Transfusionsmed 1995;22:285‐290.Cannon M, Pierce R, Taber EB, Schucker J. Fatal hydrops fetalis caused by anti‐D in a mother with partial D. Obstet Gynecol. 2003;102:1143‐1145.Wang D, Lane C, Quillen K. Prevalence of RhD variants, confirmed by molecular genotyping, in a multiethnic prenatal population. Am J Clin Pathol. 2010;134:438‐442.Gardener GJ, Legler TJ, Hyett JA, Liew YW, Flower RL, Hyland CA. Anti‐D in pregnant women with the RHD(IVS3+1G>A)‐associated DEL phenotype. Transfusion. 2012;52:2016‐2019.Lai M, Grasso C, Boschi I, D'Onofrio G, Pascali V, Leone G. Characterization of anti‐D monoclonal antibody reagents based on their reactivity with the weak D phenotype. Transfusion. 2009;49:937‐942.Avent ND, Reid ME. The Rh blood group system: a review. Blood. 2000;95:375‐387.Kacker S, Vassallo R, Keller MA, et al. Financial implications of RHD genotyping of pregnant women with a serologic weak D phenotype. Transfusion. 2015;55:2095‐2103.Sandler SG, Flegel WA, Westhoff CM, et al. College of American Pathologists Transfusion Medicine Resource Committee Work Group: It's time to phase in RHD genotyping for patients with a serologic weak D phenotype. Transfusion. 2015;55:680‐689.Denomme GA, Dake LR, Vilensky D, Ramyar L, Judd WJ. Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques. Transfusion. 2008;48:473‐478.Kulkarni S, Kasiviswanathan V, Ghosh K. A simple diagnostic strategy for RhD typing in discrepant cases in the Indian population. Blood Transfus. 2012;11:37‐42.Abdelrazik AM, Elshafie SM, Ezzat Ahmed GM, Abdelaziz HM. Combining serology and molecular typing of weak D role in improving D typing strategy in Egypt. Transfusion. 2013;53(11 suppl 2):2940‐2944.Pirenne FN, Verdier M, Lejalle A, et al. Weak D phenotypes and transfusion safety; where do we stand in daily practice? Transfusion 2007;47:1616‐1620.Van Sandt VST, Gassner C, Emonds MP, Legler TJ, Mahieu S, Körmöczi GF. RHD variants in Flanders, Belgium. Transfusion. 2015;55:1411‐1417.Lukacevic Krstic J, Dajak S, Bingulac‐Popovic J, Dogic V, Mratinovic‐Mikulandra J. Anti‐D antibodies in pregnant D variant antigen carriers initially typed as RhD+. Transfus Med Hemother 2016;43:419‐424. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Laboratory Analysis Wiley

Anti‐D reagents should be chosen accordingly to the prevalence of D variants in the obstetric population

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Wiley
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Copyright © 2018 Wiley Periodicals, Inc.
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0887-8013
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1098-2825
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10.1002/jcla.22285
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Abstract

INTRODUCTIONThe RhD antigen is clinically the most significant blood group antigen for obstetric medicine due to its immunogenicity and the high incidence of RhD‐ individuals. Depending on the population, 3%‐25% of Caucasians are D−, with 15%‐20% prevalence being most commonly reported. Anti‐D is still the leading cause of the hemolytic disease of fetus and newborn (HDFN) in the obstetric population of Split‐Dalmatian County, Croatia.The division between D+ and D− status is not always straightforward, due to over 200 D variant phenotypes having been reported to date. Generally, “weak D” variants are result of mutations in which all D epitopes are present but with reduced expression, while “partial D” represents variants in which the mutations result in the absence of certain polypeptides on the outer RBC surface. Recent research found weak D types to also possess slightly to considerably altered D antigens, with the extent of qualitative changes overlapping with that in partial D. The prevalence of weak D types among Caucasians is reported to be about 0.2%‐1%. Most weak D variants in Caucasians are weak D type 1, type 2 and type 3 (RHD*weak D type 1, RHD*weak D type 2, RHD*weak D type 3, which are not known to form alloanti‐D. The most common partial D variants among Caucasians is DNB. DVI is clinically the most relevant, being the variant most commonly involved in anti‐D immunization and HDFN. Its prevalence is reported to be between 0.02%‐0.05%. The distinction between weak and partial D is crucial since the individuals with partial D may form alloanti‐D, being regarded as D− considering transfusion therapy and perinatal management. Albeit rare, the cases of HDFN in neonates from mothers with D variant antigen had been reported, and fatal outcomes did occur. Differentiation between weak and partial D in routine serologic practice is problematic, since in both weak and partial D variants, the individual's RBC may give weak reactions with some anti‐D reagents, or the reaction with some anti‐D reagents may not exist. Different reagents contain different monoclonal antibody and protein concentrations or agglutination enhancers. Also, the difference between the serologic methods used for blood typing can cause discrepancies in the results. In Europe, anti‐D reagents are selected to deliberately type DVI pregnant women as D− to ensure that those mothers receive Rh immune globulin (RhIG) prophylaxis in their second trimester and/or postpartum. For a period of 5 years, we determined the prevalence of discrepant serologic results in a large obstetric population. We analyzed the incidence of D variants and serologic reactivity of D variants in column technology and tube method. We compared serologic results with the results of RHD genotyping.The aim of this study was to establish selection criteria of anti‐D reagents for our population which would ensure that partial D carriers are not assigned D+ status for transfusion therapy and immunoprophylaxis. This would enable us to evaluate prevention strategy of anti‐D immunizations in pregnant women and patients with partial D variants.MATERIALS AND METHODSThis study was performed at the Department of Transfusion Medicine of the Split University Hospital Center, in Split, Croatia from April 2008 to December 2012 (5 years). The Hospital is a regional institution responsible for the overall pregnancy care, monitoring of RBC alloimmunized women and the management of deliveries. All pregnant women in this region were typed for ABO and RhD blood groups and tested for irregular antibodies at their initial visit. Anti‐D immunoprophylaxis is routinely administered in D− and partial D pregnant women after the delivery of D+ child, except in cases in which anti‐D has been detected.Routine typing of D antigen in column technologyFrom April 10, 2008 to December 31, 2012 RhD typing was performed by automated microcolumn technology using ABO‐DD Grouping Cassette® (Ortho Clinical Diagnostics, Raritan, NJ, USA) which contains two monoclonal IgM anti‐D reagents (clone D7B8, and clone RUM‐1). Samples of women whose D antigen in column method was not strong positive (4+) but graded as 3+ or less were considered to be discrepant and they underwent further serological evaluation by additional RhD typing in direct and indirect agglutination. Figure  shows algorithm of interpretation of D antigen in microcolumn RhD typing.Algorithm of D antigen testing using microcolumn beads technology, from April 10, 2008 to December 31, 2012Additional serologic RhD typingAdditional RhD typing in direct agglutination was done with five monoclonal antibodies in tube testing: Ortho Anti‐D® (P3x61 monoclonal IgM, Clinical Diagnostics, Raritan, NJ, USA); DiaClon® Anti‐D Monoclonal IgG and IgM (MS‐26, TH28, DiaMed GmbH, Cressier, Switzerland); Anti‐DM MonoGnost® (MS‐201, BioGnost, Zagreb, Croatia); NovaClone® Anti‐D IgM+IgG Monoclonal Blend (CI 175‐2, D415, 1E4, Immucor Gamma, Dartmouth, NS, Canada); Anti‐D MG MonoGnost® (RUM‐1 IgM, MS‐26 IgG, BioGnosta).D‐screen kit for partial D identification using indirect agglutination including six different monoclonal D monoclonal antibodies which enables detection of partial D categories II, IV, V, VI, VII, DFR, DBT and RoHar (ID‐Partial RhD Typing Set®, DiaMed AG, Morat, Switzerland) was the second additional typing method used for discrepant results. The test is performed manually in gel microcolumn cards which are contained in the typing set. Phenotype determination of the Rh antigens (C, c, E, e) and Kell antigen was performed in microcolumn method (Rh/K Cassette®; Ortho Clinical Diagnostics).RHD genotypingAll women whose D antigen in column method during routine serologic D typing was considered to be discrepant were, after the additional serologic D typing, forwarded to RHD genotyping. The genotyping was performed in Department of Molecular Diagnostics of Croatian Institute of Transfusion Medicine in Zagreb, Croatia. DNA extraction from EDTA blood samples was done by QIAamp DNA Blood Mini kit® (Qiaqen, Hilden, Germany) or MagNA Pure LC® (Roche Molecular Biochemicals, Mannheim, Germany). Particular segments of the RHD gene sequence are multiplied by RHD genotyping using primers specific for the known mutations characterizing particular weak D types by use of the polymerase chain reaction with sequence specific priming (PCR‐SSP). RHD genotyping was performed by commercial genotyping kits (Ready Gene weak D and Ready gene CDE, Inno‐Train, Kronberg, Germany).Statistical analysisStatistical analyses were performed using computer software MedCalc®, version 12.5 (MedCalc Software, Ostend, Belgium). Relative frequencies and accompanying 95% confidence intervals (95% CI) were used to estimate the prevalence of variants.RESULTSDuring the period of five years we tested 12 689 primiparous women. D antigen status was consistent (D+ or D−) for 12 632 (99.55%) of them, while 57 (0.45%) women gave weak agglutination reactions in routine serologic D typing procedure (Table ).Prevalence of D variants in pregnant womenRhD determined N=12 689NPrevalence in 10 000 (95% CI)Consistent serologic results12 63299.55% (99.43; 99.67) RhD+10 35981.63% (80.96; 82.31) RhD−227317.91% (17.25; 18.58)Discrepant serologic results, resolved by RHD genotyping570.45% (0.33; 0.57) RHD* weak D type 1240.19% (0.11; 0.26) RHD* weak D type 3180.14% (0.08; 0.21) Weak D10.01% (0.00; 0.02) RHD*05.05100.08% (0.03; 0.13) D+40.03% (0.00; 0.06)RHD* weak D type 1/3 heterozygous status.These samples should be sequenced to distinguish a possible D variant and they were not taken into calculation of D variant prevalence.Results of RHD genotypingMolecular analysis of those 57 discrepant cases found 43 women to be weak D variant carriers. Ten of weak D variant carriers were genotyped as partial D, and four cases were resolved as regular D+ (Table ). For these four samples to really distinguish whether they represent a D variant that could not be detected by commercial tests or even a new D variant, RHD sequencing should be performed. The prevalence of D variants detected by column technology in our population of primiparous women was 0.42% (95% CI 0.31; 0.53). The aforementioned four samples that were resolved as D+ were not taken into account.Results of additional serologic analysis of weak DAt the initial routine D typing, the individuals who were later genotyped as weak D gave equivocally weak agglutination reactions in microcolumn beads method with both monoclonal reagents (graded 3+ or less), as shown in Figure . The analysis with direct agglutination in tube method with five monoclonal reagents found cell lines RUM‐1 and CI 175‐2 showed to be the most sensitive with 97.67% reactivity with weak D samples (Table ).Serologic reactivity of weak D (type 1) in microcolumn technologySerologic reactivities of D variants in direct agglutination with six monocolonal anti‐D reagentsD variantsPatientsReagentsabcdeRHD* weak D type 1N=1311111N=701111N=100111N=200011N=100001Total N (%)24 (100)13 (54.1)20 (83.3)21 (87.5)23 (95.8)24 (100)RHD* weak D type 3N=1411111N=111011N=100111N=100011N=100010Total N (%)N=18 (100)15 (83.3)15 (83.3)15 (83.3)18 (100)17 (94.4)Total weak DN=4328 (65.1)35 (81.3)37 (86)42 (97.6)42 (97.6)D category Type VaN=511110N=501110Total partial D N (%)N=10(100)5 (50)10 (100)10 (100)10 (100)01, agglutination; 0, no agglutination.aOrtho IgM P3x61; bDiaMed IgM+IgG MS‐26, TH28; cBioGnost MS‐20; dNovaClone IgM+IgG CI 175‐2, D415, 1E4; eMonoGnost IgM+IgG RUM‐1 MS‐26.One sample of RHD* weak D type 1/3 heterozygous status agglutinated reagents c, d, e.In direct agglutination in ID‐partial set, one weak D type 3 sample and five weak D type 1 samples failed to react with LDM1 clone. They showed positive reactions in indirect agglutination with all other five monoclonal reagents. One weak D type 1 sample failed to show agglutination with cell lines LHM76/55 and LHM59/19 (Table ).Serologic reactions of D variants with six monoclonal antibodies using set for indirect agglutination ID‐Partial (DiaMed)D variantsPatientsReagentsABCDEFRHD* weak D type 1N=18111111N=5111110N=1011011Total N (%)N=24 (100)23 (95.8)24 (100)24 (100)23 (95.8)24 (100)19 (79.1)RHD* weak D type 3N=17111111N=1111110Total N (%)N=18 (100)18 (100)18 (100)18 (100)18 (100)18 (100)17 (94.4)Total weak D N (%)N=43 (100)42 (97.6)43 (100)43 (100)42 (97.6)43 (100)37 (86)D category type VaN=7110101N=3110100Total partial D N (%)N=10 (100)10 (100)10 (100)010 (100)07 (70.00)ALHM76/55 (IgG); BLHM77/64 (IgG); CLHM70/45 (IgG); DLHM59/19 (IgG); ELHM169/80 (IgG); FLDM1 (IgM).One sample of RHD* weak D type 1/3 heterozygous status showed agglutination with all six reagents.All four cases of samples which showed discrepant results in routine RhD typing and were initially presumed to be some D variant type, but could not be distinguished from normal D+ in molecular analysis, showed weak agglutination reactions with all five anti‐D monoclonal antibodies in tube method. This inability to detect some D weak in molecular analysis is due to the fact that all D weak types were not included in commercial assay (possibly could be done by sequencing).Results of additional serologic analysis of partial DAll individuals genotyped as D category type RHD*05.05 (N=10) were correctly recognized as partial D during the routine testing in microcolumn method due to strong positive agglutination reactions with clone D7B8 and weaker reactions (2+ or less) with clone RUM‐1 (Figure ).Serologic reactivity of partial D (RHD*05.05) in microcolumn technologyDirect agglutination reactions with five anti‐D monoclonal reagents of all D category Va samples were clearly positive except with the clone RUM1 (Table ). Indirect agglutination with six anti‐D monoclonal reagents in D category type Va samples consistently failed to show reaction with the clone LHM70/45 and LHM96/80, but reacted with the clones LHM76/55, LHM77/64 and LHM59/19. Reactivity with LDM1 was variable (Table ). A single case of DNB was interpreted as regular D+ in routine typing and was discovered only after the immunization had occurred. As expected, most of weak D cases were CcDee Rh phenotype (88.37%). Three cases of weak D type 3 were CCDee phenotype, and one was CcDEe phenotype. The DNB case was CcDEe phenotype.DISSCUSIONA recent study showed that RHD genotyping of women with serologically inconclusive results, rather than managing them all as D−, would be cost‐saving over long‐time periods. The newest recommendations from a Work Group on RHD genotyping, directed by AABB and CAP (College of American Pathologists) state that RHD genotyping should be performed when discordant RhD typing results are encountered and/or a serologic weak D phenotype is detected in females of childbearing potential, patients, newborns and potential transfusion recipients. The benefit would be fewer unnecessary injections of RhIG and increased availability of D− RBC units.Microcolumn method is known to be highly sensitive for RhD typing. In this study, the criterion for additional RhD analysis was reactivity in direct agglutination 3+ or less (score of not more than 10) with one or both anti‐D reagents. Of 57 women who fell into the criterion, 53 women were found to be either weak or partial D variants. Although other studies recommend the cutoff score of not more than 8 (≤2+) by gel technology for assigning D negative status considering transfusion and immunoprophylaxis, the results or our study shows that the reactivity criterion in direct agglutination of score 10 (3+) or less is acceptable as the cutoff value. The frequency of D variants in our study was in concordance with the findings from other studies, and so was the distribution of Rh phenotypes.We found that the most common partial D variant in our population was D category Va, with prevalence of 0.08%. Therefore, we suggest the choice of anti‐D reagent nonreactive for DVI and DVa category for our patients and obstetric population. Such practice would ensure that those women are typed as D− and that they receive anti‐D immunoprophylaxis. In this study, partial D category VI was not found in direct typing because it is categorized as D− by our serologic selection criteria for routine RhD typing. However, in our previous study on 8670 blood donors in the same population, we used the same serologic methods and reagents to investigate donors which typed D− in direct agglutination but D+ in IAT, and genotyping did not reveal a single case of D category VI. This was a surprising finding, since D category VI is the clinically most relevant partial D type among Caucasians with frequency between 0.02%‐0.05%. Adjusting the reagent choice would be in accordance with suggestions from other authors, stating that the profile of monoclonal antibodies for RhD typing should be chosen individually regarding to the population for which it is used. In our study, DVa category samples could be clearly distinguished from weak D samples by routine serology alone, as partial D consistently showed weak to negative agglutination with one anti‐D monoclonal antibody but reacted with second one in routine testing. All cases failed to react with monoclonal cell line RUM‐1 in tube method. In microcolumn method, they gave weak positive reactions with cell line RUM‐1, in opposite to cell line D7B8, with which they gave strong positive reactions.We noticed that reactions of partial D category Va were not entirely in concordance with manufacturer's worksheet in IAT typing with ID‐partial typing set (Diamed). As expected, the reaction was omitted with cell line LHM70/45, but unexpectedly also with LHM169/80. This is an important finding because it shows that these cell lines should not be used for donor and/or neonate typing as they would type as D−. Other authors did not report this outcome. While weak D type 1 and type 3 were in our study indistinguishable from one another based on serology, weak D type 3 was observed to react generally in higher percentage in both direct and indirect typing. This was an expected outcome since weak D type 3 has been reported by other authors to have one of the highest antigen densities among weak D variants, with mean value of antigen density of 1900 antigens per cell, while for weak D type 1 and 2 that value is 700 and 600, respectively. However, we must emphasize that weak D types susceptible for immunization, such as weak D types 4.0 (RHD*09.03.01), 4.2.1 (RHD*09.01.01) and 15 (RHD*weak partial 15) were not represented in the examined obstetric population. The research on a different population, in which such D variants are present, found that it was not possible to rely on serology only to prevent immunization events in weak D carriers, since they could not be distinguished from other weak D types.All partial DVa cases were CcDee phenotype. Other authors reported DNB to be associated with CcDee or CCDee phenotype.As previously observed by Pirenne et al., our research shows how careful selection of anti‐D reagents and serologic methods can ensure high level of safety in detection of partial D individuals. It is already recommended by multiple authors to implement molecular typing whenever possible to clarify ambiguous D antigen typing results. In the previous study on our obstetric population, anti‐D immunization occurred in pregnant two partial D carriers: type D category Va and one DNB carrier. Those D category Va carriers were assigned as D+ status due to the choice of reagents used at the time. They did not receive immunoprophylaxis and one of them was transfused with D+ RBC unit. The single case of partial DNB category was detected after the immunization had occurred, similarly as previously reported by other authors. We were therefore unable to determine the prevalence of partial D variant category DNB in our population, because it was typed as D+ due to our reagent selection criteria.The follow‐up of anti‐D immunizations in patients with D variants, completed with results of RHD genotyping, was an indicator which enables us to assess the correctness of the choice of reagents for RhD typing for the population in question. The appropriate choice of reagents will enable the serology methods to recognize the cases in which RHD genotyping is required. The shortcoming of this study is the possibility that some D variants might have been recognized as D+ using the current selection of monoclonal reagents, as is the case with DNB. 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Journal

Journal of Clinical Laboratory AnalysisWiley

Published: Jan 1, 2018

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