Treatment of pregnant women by RhD type

  • Janja Jerina Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia
  • Klara Železnik Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia
  • Tadeja Dovč Drnovšek Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia
  • Irena Bricl Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia
Keywords: variants D, serologic technicques, molecular-biologic methods (PCR-SSP), treatment algorithm


An accurate determination of RhD (D) antigen (Ag) is important for pregnant women, transfusion recipients and blood donors. The decision to receive the pre- and postnatal prophylaxis with anti-D immunoglobulin (RhIG) is based on the D-type. Individuals can be classified as D-positive (D-pos) or D-negative (D-neg) based on the determination of Ag D but there are numerous other versions of Ag D as well (variants D, D-var). Allele variants of the RHD (D) gene can encode different forms of protein D. They are divided into weak, partial and Del. Ag D are determined on the basis of serological techniques. If in doubt, we use the molecular-biological methods for determining the D gene. The definitive definition of D-var is given on the basis of the molecular-biological methods.
From the peripheral venous blood samples taken from pregnant women, we roughly determined Ag D on plates (the Seraclone anti-D reagent) and on gel cards (the DiaClon ABO / D + Reverse Grouping test system (monoclonal antibodies) (Bio-Rad, Germany), with which we do not detect D category VI blood group). For 49 pregnant women we performed extended serological tests of D blood group (commercial ID-Partial RhD Typing Set (Bio-Rad, Germany)). We proceeded with molecular-biological methods: DNA isolation (utilisation of the BioRobot EZ1 and the commercial set EZ1 DNA Blood 350 μl Kit (Qiagen, Germany)), determination of the genotype D with the PCR-SSP method (commercial RBC-Ready Gene CDE, RBC -Ready Gene D weak and RBC-Ready Gene D AddOn (Inno-Train; Germany)), amplification (Veriti apparatus (Apllied BioSystems, USA)), product separation (electrophoretic system (BioRad, USA) on agarose gel (Sigma, Germany).
Our results showed that weak D forms represented 41 cases (83.7 %) and partial forms 8 cases (16.3 %) of all D-var. The most common D-var in pregnant women was weak D type 1 with 17 cases (34.7 %), followed by weak D type 3 with 13 cases (26.5 %) and weak D type 2 with 10 cases (20.4 %). The most common partial D form was D category VII with 5 cases (10.2 %).
Pregnant women and transfusion recipients who are carriers of weak D types 1, 2 or 3 can be safely treated as D-pos, while carriers of all other D-var can be treated as D-neg. Blood donors with D-var are treated as D-pos. By means of this algorithm, approximately 172 unnecessary RhIG applications can be prevented annually and adequate supplies of D-neg erythrocyte blood components can be maintained.


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Investigation strategy for RhD typing discrepancies using a combination of PCR-SSP and serological technicques [cited 2017 Sept 3]. Available from:

Sandler SG, Queenan JT. A Guide to Terminology for Rh Immunoprophylaxis. Obstet Gynecol. 2017 Sep;130(3):633–5. PMID:28796682

Koelewijn JM, Vrijkotte TG, van der Schoot CE, Bonsel GJ, de Haas M. Effect of screening for red cell antibodies, other than anti-D, to detect hemolytic disease of the fetus and newborn: a population study in Neetherlands. Trasfusion. 2008;48(5):941–52.

Dovč T. Določanje plodovega genotipa RHD iz plazme RhD-negativnih nosečnic z metodo PCR v realnem času. [PhD Thesis]. Ljubljana: T. Dovč; 2013.

Westhoff CM. The Rh System. In: Roback JD, Combs MR, Grossman BJ, Hillyer CD, editors. AABB Technical Manual. 16th ed. 2005. pp. 315–32.

Flegl WA, Castilho SL, Keller MA, Klapper EB, Moulds JM, Noizat-Pirenne F, et al. Molecular immunohaematology round table discussions at the AABB Annual Meeting, Philadelphia 2014. Blood Trasfusion 2015;1–9.

Yousuf R, Mustafa AN, Ho SL, Tang YL, Leong CF. Anti-G with concomitant anti-C and anti-D: A case report in a pregnant woman. Asian J Transfus Sci. 2017 Jan-Jun;11(1):62–4. PMID:28316444

Bowman J. Thirty-five years of Rh prophylaxis. Transfusion. 2003 Dec;43(12):1661–6. PMID:14641860

Koelewijn JM, de Haas M, Vrijkotte TG, van der Schoot CE, Bonsel GJ. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis. BJOG. 2009 Sep;116(10):1307–14. PMID:19538414

van der Schoot C, deHaas M, Clausen FB. Genotyping to prevent Rh disease: has the time come? Transfusion medicine and immunohematology 2017;24:544–50.

Pravilnik o transfuzijskih preiskavah in postopkih ob transfuziji (Ur. l. RS, št. 9/07 in 32/18) [cited 2018 Jun 8]. Available from:

Pravilnik o spremembah Pravilnika o transfuzijskih preiskavah in postopkih ob transfuziji. (Ur. l. RS, št. 32/18).

Bricl I. Ciljana zaščita z IgG anti-D. Neinvazivna določitev plodovega genotipa RhD iz periferne krvi RhD-negativnih nosečnic. In: Stezinar SL, ur. Zbornik predavanj strokovnih srečanj Združenja za transfuzijsko medicino Slovenije; 2017; Zreče, Slovenija. V Ljubljani: Slovensko zdravniško društvo; 2017. p. 31.

Koelewijn JM, de Haas M, Vrijkotte TG, Bonsel GJ, van der Schoot CE. One single dose of 200 microg of antenatal RhIG halves the risk of anti-D immunization and hemolytic disease of the fetus and newborn in the next pregnancy. Transfusion. 2008 Aug;48(8):1721–9. PMID:18507749

Daniels G. Variants of RhD—current testing and clinical consequences. Br J Haematol. 2013 May;161(4):461–70. PMID:23432139

Wagner FF, Gassner C, Müller TH, Schönitzer D, Schunter F, Flegel WA. Molecular basis of weak D phenotypes. Blood. 1999 Jan;93(1):385–93. PMID:9864185

Wagner FF, Flegel WA. The Rhesus site. Transfus Med Hemother. 2014 Oct;41(5):357–63. PMID:25538538

Wagner FF, Frohmajer A, Ladewig B, Eicher NI, Lonicer CB, Müller TH, et al. Weak D alleles express distinct phenotypes. Blood. 2000 Apr;95(8):2699–708. PMID:10753853

Li Q, Hou L, Guo ZH, Ye LY, Yue DQ, Zhu ZY. Molecular basis of the RHD gene in blood donors with DEL phenotypes in Shanghai. Vox Sang. 2009 Aug;97(2):139–46. PMID:19490579

Sandler SG, Flegel WA, Westhoff CM, Denomme GA, Delaney M, Keller MA, 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 Mar;55(3):680–9. PMID:25438646

Horn T. Use of RHD Genotyping for Serologic Weak D Phenotypes. American Red cross. [cited 2018 Jun 8]. Available from:

Haspel RL, Westhoff CM. How do I manage Rh typing in obstetric patients? Transfusion. 2015 Mar;55(3):470–4. PMID:25647404

Sandler SG, Roseff SD, Domen RE, Shaz B, Gottschall JL. Policies and procedures related to testing for weak D phenotypes and administration of Rh immune globulin: results and recommendations related to supplemental questions in the Comprehensive Transfusion Medicine survey of the College of American Pathologists. Arch Pathol Lab Med. 2014 May;138(5):620–5. PMID:24786120

Müller TH, Wagner FF, Trockenbacher A, Eicher NI, Flegel WA, Schönitzer D, et al. PCR screening for common weak D types shows different distributions in three Central European populations. Transfusion. 2001 Jan;41(1):45–52. PMID:11161244

Dovc Drnovsek T, Rozman P. The distribution of different RHD variants in Slovenian population [abstract]. Transfus Clin Biol. 2006;13:174–5.

Ansart-Pirenne H, Asso-Bonnet M, Le Pennec PY, Roussel M, Patereau C, Noizat-Pirenne F. RhD variants in Caucasians: consequences for checking clinically relevant alleles. Transfusion. 2004 Sep;44(9):1282–6. PMID:15318849

Araújo F, Rodrigues MJ, Monteiro F, Chabert T, Tavares G, Sousa G, et al. Weak D type 2 is the most prevalent weak D type in Portugal. Transfus Med. 2006 Feb;16(1):63–7. PMID:16480441

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 Oct;22(5):285–90. PMID:8924742

Leader KA, Kumpel BM, Poole GD, Kirkwood JT, Merry AH, Bradley BA. Human monoclonal anti-D with reactivity against category DVI cells used in blood grouping and determination of the incidence of the category DVI phenotype in the DU population. Vox Sang. 1990;58(2):106–11. PMID:2111059

van Rhenen DJ, Thijssen PM, Overbeeke MA. Serological characteristics of partial D antigen category VI in 8 unrelated blood donors. Vox Sang. 1994;66(2):133–6. PMID:8184595

Rupreht RR, Pretnar Hartman K, Galvani V, Rožman P, Čurin Šerbec V. Weak D and partial D in Slovenian population through serology and genotyping. Pflugers Arch. 2000;440 S1:R195–6.

Wagner FF, Frohmajer A, Flegel WA. RHD positive haplotypes in D negative Europeans. BMC Genet. 2001;2:10. PMID:11495631

Wagner FF, Gassner C, Muller TH, Schonitzer D, Schunter F, Flegel WA. Three molecular structures cause rhesus D category VI phenotypes with distinct immunohematologic features. Blood. 1998 Mar;91(6):2157–68. PMID:9490704

Avent ND. RHD genotyping from maternal plasma: guidelines and technical challenges. Methods Mol Biol. 2008;444:185–201. PMID:18425481

Clarke G, Hannon J, Berardi P, Barr G, Cote J, Fallis R, et al. Resolving variable maternal D typing using serology and genotyping in selected prenatal patients. Transfusion. 2016 Dec;56(12):2980–5. PMID:27611891

Garratty G. Do we need to be more concerned about weak D antigens? Transfusion. 2005 Oct;45(10):1547–51. PMID:16181202

Kumpel BM. Efficacy of RhD monoclonal antibodies in clinical trials as replacement therapy for prophylactic anti-D immunoglobulin: more questions than answers. Vox Sang. 2007 Aug;93(2):99–111. PMID:17683353

Overview of comments received on ‘Draft Guideline on the core SmPC for human Anti-D immunoglobulin for intravenous use’ (EMA/CHMP/BPWP/319619/2005 Rev. 2). [cited 2018 Jun 8]. Available from:

Statistični urad republike Slovenije, SURS. [cited 2018 Jun 8]. Available from:

Daniels G. Rh blood group system. In: Daniels G, editor. Human Blood Groups. Oxford: Blackwell Science Ltd; 2002. pp. 195–274.

Dovč-Drnovšek T, Klemenc P, Toplak N, Blejec T, Bricl I, Rožman P. Reliable Determination of Fetal RhD Status by RHD Genotyping from Maternal Plasma. Transfus Med Hemother. 2013 Feb;40(1):37–43. PMID:23637648

Avent ND, Reid ME. The Rh blood group system: a review. Blood. 2000 Jan;95(2):375–87. PMID:10627438

Kacker S, Vassallo R, Keller MA, Westhoff CM, Frick KD, Sandler SG, et al. Financial implications of RHD genotyping of pregnant women with a serologic weak D phenotype. Transfusion. 2015 Sep;55(9):2095–103. PMID:25808011

Flegel WA, Roseff SD, Tholpady A. Phasing-in RHD genotyping. Arch Pathol Lab Med. 2014 May;138(5):585–8. PMID:24786114

Denomme GA, Wagner FF, Fernandes BJ, Li W, Flegel WA. Partial D, weak D types, and novel RHD alleles among 33,864 multiethnic patients: implications for anti-D alloimmunization and prevention. Transfusion. 2005 Oct;45(10):1554–60. PMID:16181204

Drnovšek DT. Molekularno-biološke preiskave za določanje krvnih skupin na Zavodu RS za transfuzijsko medicino. In: Stezinar SL, ur. Zbornik predavanj strokovnih srečanj Združenja za transfuzijsko medicino Slovenije; 2017 Apr 7-8; Zreče, Slovenija. V Ljubljani: Slovensko zdravniško društvo; 2017. p. 28–30.

How to Cite
Jerina J, Železnik K, Dovč Drnovšek T, Bricl I. Treatment of pregnant women by RhD type. ZdravVestn [Internet]. 9Jan.2020 [cited 27Feb.2020];88(11-12):582-9. Available from:
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