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ORIGINAL ARTICLE  
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Serological comparison of weak D versus weakly reacting D by four different methods


1 Department of Transfusion Medicine, JIPMER Blood Centre, JIPMER, Puducherry, India
2 Department of Transfusion Medicine, Institute of Medical Sciences and SUM Hospital, Bhubaneswar, Odisha, India

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Date of Submission18-Mar-2021
Date of Decision10-Aug-2021
Date of Acceptance26-Sep-2021
Date of Web Publication26-Sep-2022
 

   Abstract 

INTRODUCTION: Weak D red cells were defined as having a reduced amount of D antigen (formerly called “Du”) that required an indirect antiglobulin test (IAT) for detection. Weakly reacting D is those which give <2+ reactions on routine methods. The present study is sharing our experience on weak D and weakly positive anti-D in various methods.
MATERIALS AND METHODS: All the blood sample of patients and blood donor, which were RhD negative, were included in the study. Furthermore, RhD positive sample <2+ was included. We repeated blood grouping of all these samples by gel card (Tulip), tube method (two different antisera), slide method, and Solid Phase Red Cell Adherence (SPRCA) (Immucore, USA).
RESULTS: A total number of samples were 27,245. RhD negative found out to be 945 (3.46%). Out of all, 929 (98.3%) samples were Rh D negative in gel card and IAT negative, while 16 (1.7%) were weak D positive. Rh D typing with these samples by different antisera at four platforms showed that 14 were weakly positive (<2+) in any of the four platforms. Similarly, out of 26,300 Rh D Positive samples, 21 samples (0.079%) were serologically weak (<2+). Repeat Rh D typing was done with different antisera in all four platforms. Result showed more than 50% were Rh D negative in any of four platforms.
CONCLUSION: Above observation showed that serological tests at various platforms failed to distinguish weak D from weakly reacting D. Thus, we propose that weakly reacting D should be treated equal as weak D unless they are distinguished by genotyping.

Keywords: Rh D, serological weakly reacting D, weak D


How to cite this URL:
Sahoo D, Kanungo GN, Behera R, Jena PS. Serological comparison of weak D versus weakly reacting D by four different methods. Asian J Transfus Sci [Epub ahead of print] [cited 2022 Dec 4]. Available from: https://www.ajts.org/preprintarticle.asp?id=356882



   Introduction Top


The term Rh refers to a specific red blood cell (RBC) antigen. Currently, Rh blood group system composed of over 61 different antigenic specificities. Rh blood group system is the second most important in terms of transfusion after ABO blood group system. Rh system antigens are highly immunogenic. Rh antibodies are produced only after exposure to foreign RBCs unlike ABO antibodies, which are naturally occurring. Anti-Rh antibodies can produce significant hemolytic disease of the fetus and newborn (HDFN) as well as hemolytic transfusion reactions.

Rh positive indicates that an individual's RBCs possess one particular Rh antigen, the D antigen, on their RBCs. Rh negative indicates that the RBCs lack the D antigen. Traditionally, weak D red cells were defined as having a reduced amount of D antigen (formerly called “Du”) that required an Indirect Antiglobulin Test (IAT) for detection. This is identified in individuals whose D was not detectable at immediate spin. Weak D types can be separated into three categories: position effect, quantitative, and partial-D antigen (missing one or more alleles).[1] It has been shown that up to 2% of individuals with European ancestry possess some altered form of D antigen. Altered D antigen occurs more often in individuals of African descent, but the exact prevalence is not known.

In the United States, a serological weak D phenotype is usually defined as reactivity of RBCs with an anti-D reagent giving no or weak (≤2+) reactivity in initial testing, but agglutinating moderately or strongly with antihuman globulin (a weak D test).[2],[3],[4] In Europe, such a “weak D test” is often understood to be what a reference laboratory does to resolve equivocal serological reactivity, whether by serological or molecular methods.[5] Grading a serological reaction as ≤2+ is often subjective, and there is a lack of consensus for the definition. In India, no such distinct guideline exists, any positive reaction with anti-D antisera considers to be RhD positive. Weakly reacting D is those which give <2+ reactions on routine methods.

Literature search does not show any head-to-head comparison of weak D with weakly reacting D serologically by various anti-D antisera using different platforms. This is going to be study of its first kind. The aim of the present study was to find agreement between various serological tests to distinguish Weak D and weakly reacting D serologically.


   Materials and Methods Top


The present study was a prospective observational study took place in Post Graduate Department of Transfusion Medicine at Institute of Medical Sciences and SUM hospital, Bhubaneswar, Odisha, India, from January 2019 to December 2019.

All the blood sample of patients and blood donor, which were RhD negative, were included in the study. Initial method of testing blood group was Solid Phase Red Cell Adherence (SPRCA) assay (Immucore, USA) using anti–D {IgM (clone RUM-1) antisera} and anti-D {IgM + IgG (clone D175 [IgM] + D415 [IgG]}. All RhD-positive samples <2+ reactions were also included. We repeated Rh D blood grouping of all these samples by other three platforms using

  1. Gel card (Tulip, Goa, India using Anti-D [IgM] [VI-] [Clone P3 × 61 + TH-28]],
  2. Conventional Test Tube method (two different antisera monoclonal antibodies of immunoglobulin class IgM [Clone P3 × 61] and IgM + IgG Blended anti D antisera),
  3. Slide method (using same antisera used in tube method).


Conventional Test Tube method considered the standard assay and compared to SPRCA, Gel card, and slide method results. All the data were recorded, entered into Microsoft Office Excel worksheet, and interpreted.


   Results Top


The total number of sample during the study period was 27,245. The RhD-negative found out to be 945 (3.46%). RhD confirmation test was performed all RhD negative tests. Out of above, 929 samples were negative in IAT. Rest sixteen were found to be IAT positive. All these (1.69%) IAT positive considered as weak D. Rh D typing with these samples by different antisera at four platforms showed that 14 were weakly positive (<2+) in any of the four platforms [Table 1]. On repetition with SPRCA, we got 10 were negative, while six had 1+ reaction with anti-D antisera. In tube method, only two were negative, whereas 14 gave weak positive, similar result found on slide, but the median avidity was 22 Seconds.
Table 1: Comparison of weak D and weakly reacting D by 4 methods

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Similarly, out of 26300 Rh D-Positive samples, 21 samples (0.079%) were serologically weak (<2+). All these samples were tested with different anti-D antisera using four different platforms [Table 1]. All were weakly positive in tube and slide (avidity more than 20 s). In repetition in column agglutination test (gel card), 12 were negative, while 9 showed weak reactive. Same samples in SPRCA, 6 were negative, while 15 gave 1+ reaction. Overall result showed more than 50% were Rh D negative in any of four platforms [Figure 1].
Figure 1: Analysis of RhD grouping results with weak D and weakly reacting D

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   Discussion Top


From a clinical perspective, the Rh blood group system is the most important of the all blood group systems[6] after ABO. Among the all blood group antigens in the Rh system, the RhD antigen is the most immunogenic and important in clinical practice.[7],[8],[9] The consequence of any RhD-negative individual who forms anti-D is that the option of transfusing D + RBCs in an emergency is eliminated, and there is now an absolute lifetime requirement for transfusing only D negative RBCs.[5] The consequence of an RhD-negative woman forming anti-D is that any subsequent pregnancy involving an RhD-positive fetus is at risk for HDFN.

In the USA, serological weak D phenotype is usually defined as reactivity of RBCs with an anti-D reagent giving no or weak (≤2+) reactivity in initial testing, but agglutinating moderately or strongly with antihuman globulin (a weak D test).[2],[3],[4] In Europe, such a “weak D test” is often understood to be what a reference laboratory does to resolve equivocal serological reactivity, whether by serological or molecular methods. Grading a serological reaction as ≤2+ is often subjective and there is a lack of consensus for the definition. In the UK, most clinical laboratories use potent anti-D reagents and few use indirect antiglobulin-reactive anti-D reagents. Any serological reaction of ≤2+ is likely to be referred to a Red Cell Immunohaematology Laboratory.

The first phase of anti-D detection may be too insensitive to agglutinate RBCs with a D variant antigen, but the RBCs will be agglutinated by the second weak D test phase, and therefore, the sample is interpreted to be a weak D phenotype. In contrast, the observed prevalence of weak D phenotypes decreases when the method of RhD typing is more sensitive, for example, an automated gel column or solid-phase analyzer using a blend of potent recombinant monoclonal anti-D reagents. In this situation, the highly sensitive first phase of D antigen testing by anti-D will agglutinate RBCs expressing a weak D antigen, and the sample is interpreted to be a conventional RhD positive. These perhaps explain different results we received using different platforms [Table 1]. Thus, a blood sample from a patient or blood donor with a weak D variant antigen may be interpreted to be RhD positive when tested by a laboratory using a sensitive RhD typing method.[5] However, the same sample may be interpreted to express a serological weak D phenotype if tested by the same or a different laboratory using a less potent anti-D reagent.

The present study showed the prevalence of weak D to be 0.05% of total donors and 1.69% in our RhD-negative population. This result is similar to a study from Makroo et al. from North India who found prevalence to be 0.01% of total donors and 0.12% of Rh-negative donors.[10] An estimated 0.2%–1.0% of Caucasians inherit an RHD genotype that codes for a serological weak D phenotype.[11] In North London, the prevalence of weak D phenotypes was estimated to be 0.3% for white and 1.7% for black blood donors.[12]

In India, molecular confirmation of serological weakly reacting D was usually not done everywhere. Only few centers including Indian Institute of Immunohematology perform molecular typing. Therefore, these groups (serological weakly reacting D) of individual have potential risk of developing Rh D antibodies if they are transfused with Rh D-positive red cells unless they are genotypically RHD. Hence, we here by propose that all serologically weakly reacting D individuals should have molecular typing of RHD before transfusion, if not should receive Rh D-negative blood for transfusion.

Most serological weak D phenotypes are detected when a pregnant woman, potential transfusion recipient, or blood donor has a blood sample routinely typed for RhD, and the grade of RBC agglutination is weaker (≤2+) than expected for RhD typing using potent anti-D reagents (3 + to 4+). Furthermore, serological weak D phenotypes are detected when a clinical laboratory types a blood sample as D+, but the laboratory's record of a prior RhD type is D negative. The discrepancy may reflect an error in patient or sample identification. Alternatively, the discrepancy may reflect the increased potency of new monoclonal RhD typing reagents in the laboratory compared to previously use plasma-derived anti-D reagents that were less effective for detecting weakly expressed D variant antigens. The present study has shown that weak D individuals may be typed as weakly reacting D (≤2+) in different platforms of blood group testing due to improved monoclonal anti-D of the current era. Our study also demonstrated that serological weakly reacting D may be typed as D negative/weak D positive. Therefore, all the precautionary steps for weak D individuals should be applied for serological weakly reacting individuals.


   Conclusion Top


There is a lack of agreement between four different techniques using anti-D antisera to distinguish weak D from weakly reacting D serologically. Above observation showed that serological tests at various platforms failed to distinguish weak D from weakly reacting D always. However, similar type of a study needs to perform to validate our result. Thus, we propose that weakly reacting D should be treated equal as weak D unless they are distinguished by genotyping.

Consent

Written consent was obtained from all donors.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Suwe G, Wiler M. The Rh blood group system. In: Harmening D, editor. Modern Blood Banking and Transfusion Practices. 7th ed. Philadelphia: F A Davis; 2019. p. 149-72.  Back to cited text no. 1
    
2.
Jenkins CM, Johnson ST, Bellissimo DB, Gottschall JL. Incidence of weak D in blood donors typed as D positive by the Olympus PK 7200. Immunohematology 2005;21:152-4.  Back to cited text no. 2
    
3.
Daniels G. Variants of RhD – Current testing and clinical consequences. Br J Haematol 2013;161:461-70.  Back to cited text no. 3
    
4.
Sandler SG, Flegel WA, Westhoff CM, Denomme GA, Delaney M, Keller MA, et al. It's time to phase in RHD genotyping for patients with a serologic weak D phenotype. Transfusion 2015;55:680-9.  Back to cited text no. 4
    
5.
Gerald Sandler S, Leonard Chen, Flegel WA. Serological weak D phenotypes: A review and guidance for interpreting the RhD blood type using the RHD genotype. Br J Haematol 2017;179:10-9.  Back to cited text no. 5
    
6.
Storry JR, Castilho L, Chen Q, Daniels G, Denomme G, Flegel WA, et al. International society of blood transfusion working party on red cell immunogenetics and terminology: Report of the Seoul and London meetings. ISBT Sci Ser 2016;11:118-22.  Back to cited text no. 6
    
7.
Gunson HH, Stratton F, Phillips PK. The primary Rho (D) immune response in male volunteers. Br J Haematol 1976;32:317-29.  Back to cited text no. 7
    
8.
Urbaniak SJ, Robertson AE. A successful program of immunizing Rh-negative male volunteers for anti-D production using frozen/thawed blood. Transfusion 1981;21:64-9.  Back to cited text no. 8
    
9.
Storry JR, Castilho L, Daniels G, Flegel WA, Garratty G, de Haas M, et al. International society of blood transfusion working party on red cell immunogenetics and blood group terminology: Cancun report (2012). Vox Sang 2014;107:90-6.  Back to cited text no. 9
    
10.
Makroo RN, Raina V, Chowdhry M, Bhatia A, Gupta R, Rosamma NL. Weak D prevalence among Indian blood donors. Asian J Transfus Sci 2010;4:137-9.  Back to cited text no. 10
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11.
Garratty G. Do we need to be more concerned about weak D antigens? Transfusion 2005;45:1547-51.  Back to cited text no. 11
    
12.
Contreras M, Knight RC. Controversies in transfusion medicine. Testing for Du: Con. Transfusion 1991;31:270-2.  Back to cited text no. 12
    

Top
Correspondence Address:
Dibyajyoti Sahoo,
Department of Transfusion Medicine, JIPMER Blood Centre, JIPMER, Pondicherry - 605 006
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ajts.AJTS_34_21



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