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Phasing-In RHD Genotyping.

In this issue, Sandler and colleagues (1) report the results of the College of American Pathologists (CAP) J-B Transfusion Medicine (Comprehensive) and Educational Survey, in which more than 3100 institutions describe how they perform Rh typing for blood donors, pregnant women, and hospital patients. In accordance with American Association of Blood Banks (AABB) Standards for Blood Banks and Transfusion Services, (2) most hospital laboratories reported that they do not routinely perform a serologic weak-D test on pregnant women or transfusion recipients. This practice results in most pregnant women and hospital patients with a weak-D phenotype being categorized and managed as Rh- (Table 1). (2,3) In contrast, a weak-D test is performed routinely on blood donors whose red blood cells test [D.sup.-] by direct agglutination, resulting in most blood donors with a weak D being categorized and managed as [Rh.sup.+]. (2) This 50-year practice appears to be relatively safe, (4) and there are only a few published reports of persons with a weak-D phenotype forming anti-D antibodies. (5-8) However, this practice confuses patients, blood donors, and caregivers and uses Rh immune globulin (RhIG) and [Rh.sup.-] red blood cells for many persons with a weak D, who could be safely managed as [Rh.sup.+], if their genotypes were known. (3,9,10) The CAP Transfusion Medicine Resource Committee (TMRC) reviewed this practice in the context of the current state of science for RHD genotyping. (1) The TMRC concluded that selective integration of RHD genotyping of weak D phenotypes could improve the accuracy of Rh typing results, thereby reducing unnecessary administration of RhIG in women with a weak D phenotype, and decrease transfusions of [Rh.sup.-] red blood cells in recipients with a weak D phenotype. (1)

The process of phasing-in RHD genotyping in clinical practice has begun in many hospitals, but as the CAP survey indicates, most pregnant women and hospital patients in the United States continue to have their Rh type determined by dated serologic methods. (1) Those laboratories that do not routinely perform weak-D tests for patients typing [Rh.sup.-] by direct agglutination with anti-D should now begin to introduce Rh typing reagents and procedures selected to detect, not to avoid detection of, weak-D phenotypes.

Also see p. 620.

We recently encountered a 27-year-old North African woman who was designated as [Rh.sup.-] for a cesarean section. Her medical history and laboratory test results are representative of a common subset of patients (11) and illustrate how RHD genotyping can improve the management of patients with a weak-D phenotype. We have summarized recommended guidance for diagnostic testing and clinical decision making in women with a weak-D phenotype after delivery of a [D.sup.+] newborn (Table 2).

The woman's routine postpartum blood sample result was strongly positive by a rosette fetal bleed screening test, suggesting the presence of [D.sup.+] fetal red blood cells in her circulation (fetomaternal hemorrhage). However, a quantitative, acid-elution (Kleihauer-Betke) assay result was negative, indicating that the [D.sup.+] red blood cells in her circulation did not contain a significant amount of hemoglobin F; that is, the red blood cells were not of fetal origin. A weak-D test was positive, confirming the clinical impression that her red blood cells expressed an inherited weak-D phenotype. Red blood cells from approximately 0.2% to 1.0% of white people express a weak-D phenotype. (12) A weak-D phenotype has been reported in 0.1% to 10% of all pregnancies that were initially typed as [D.sup.-]. (13-15) We estimate that approximately 90% of patients in the United States with a weak-D phenotype will have one of the prevalent RHD genotypes (types 1, 2, 3, or 4.1). (5,7,11,16) Women with one of these RHD genotypes may be managed as [Rh.sup.+] and do not require RhIG for prenatal or postpartum Rh immunoprophylaxis. (7,17) However, that decision can only be made by RHD genotyping. Even monoclonal anti-D reagents, which were initially believed to be capable of identifying RHD genotypes, cannot distinguish among the most prevalent weak-D genotypes (Table 3). (6,18,19) We performed molecular testing on our patient (20) and established that she had inherited the uncommon weak-D type 25, (21) which required management as [Rh.sup.-] for purposes of Rh immunoprophylaxis and transfusion of red blood cells.

The second step in phasing-in RHD genotyping will be establishing standardized, cost-effective, RHD genotyping protocols for laboratories. Most hospitals will not have a sufficient volume of patients with a weak-D phenotype to justify establishing in-hospital RHD genotyping services. Hospitals are likely to refer blood samples to regional reference laboratories where high test volumes will support both basic and complex genotyping services. A molecular test in D~ pregnancies may pay for itself by avoiding the costs associated with often unnecessary multiple administrations of RhIG. (4,17,22) Presently, there are no US Food and Drug Administration-approved molecular test kits for determining the Rh type, but several unlicensed products are marketed commercially in the United States. These products use polymerase chain reaction with detection by gels, bioarray chips, or bead chips. Any of these test kits and test platforms can be used for patient care as tests of either "high" or "moderate" under the Clinical Laboratory Improvement Amendments of 1988.

Based on the results of its 2012 survey and review of the science of RHD genotyping, the CAP TMRC has recommended a multiorganizational collaboration among obstetricians, transfusion medicine specialists, serologists, and molecular scientists to update current practice guidelines and establish a nationwide, uniform practice. (1) The CAP and AABB have formed the Work Group on Phasing-In RHD Genotyping. We believe that the time has come to transition from serologic to molecular methods for managing weak-D phenotypes. Our case illustrates how easily this transition can be accomplished. In conclusion, we support the CAP TMRC's initiative.

We thank S. Gerald Sandler, MD, and Harvey G. Klein, MD, for reviewing the manuscript; A. Hallie Lee-Stroka, MT(ASCP)SBB; Neil Bangs, MS MT(ASCP)SBB; Sherry L. Sheldon, MT(ASCP)SBB; and Debrean Ann Loy, MT(ASCP)ASQ, for performing serology; David Allan Stiles, MS, and Supatta Mary Lucas, MLT(ASCP), for performing RHD sequencing; and Kshitij Srivastava, PhD, for nucleotide sequence data entry.


(1.) Sandler SG, Roseff SD, Domen RE, Shaz BH, Gottschall JL. Policies and procedures related to testing for weak D phenotypes and administration of Rh immune globulin. Arch Pathol Lab Med. 2014; 138(5):620-625.

(2.) Carson TH. Standards for Blood Banks and Transfusion Services. 28th ed. Bethesda MD: American Association of Blood Banks; 2012.

(3.) Kennedy MS. Perinatal issues in transfusion practice. In: Roback JD, Grossman BJ, Harris T, Hillyer CD, eds. AABB Technical Manual. 18th ed. Bethesda, MD: American Association of Blood Banks; 2012:637-650.

(4.) Sandler SG, Gottschall JL. Obstetrics and gynecology: ask the experts--postpartum Rh immunoprophylaxis. Washington, DC: The American College of Obstetricians and Gynecologists. Documents/Dec2012_AtE_Sandler.pdf. Accessed October 17, 2013.

(5.) Wagner FF, Gassner C, Muller TH, Schonitzer D, Schunter F, Flegel WA. Molecular basis of weak D phenotypes. Blood. 1999; 93(1):385-393.

(6.) Wagner FF, Frohmajer A, Ladewig B, et al. Weak D alleles express distinct phenotypes. Blood. 2000; 95(8):2699-2708.

(7.) Flegel WA. How I manage donors and patients with a weak D phenotype. Curr Opin Hematol. 2006; 13(6):476-483.

(8.) McGann H, Wenk RE. Alloimmunization to the D antigen by a patient with weak D type 21. immunohematology. 2010; 26(1):27-29.

(9.) Sandler SG, Li W, Langeberg A, Landy HJ. New laboratory procedures and Rh blood type changes in a pregnant woman. Obstet Gynecol. 2012; 119(2, pt 2): 426-428.

(10.) Sandler SG, Gottschall JL. Postpartum Rh immunoprophylaxis. Obstet Gynecol. 2012; 120(6):1428-1438.

(11.) 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; 45(10):1554-1560.

(12.) Garratty G. Do we need to be more concerned about weak D antigens? Transfusion. 2005; 45(10):1547-1551.

(13.) 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(3):438-442.

(14.) Cruz BR, Chiba AK, Moritz E, Bordin JO. RHD alleles in Brazilian blood donors with weak D or D-negative phenotypes. Transfus Med. 2012; 22(2):84-89.

(15.) Moussa H, Tsochandaridis M, Chakroun T, et al. Molecular background of D-negative phenotype in the Tunisian population. Transfus Med. 2012; 22(3):192-198.

(16.) Flegel WA, Denomme GA, Yazer MH. On the complexity of D antigen typing: a handy decision tree in the age of molecular blood group diagnostics. J Obstet Gynaecol Can. 2007; 29(9):746-752.

(17.) Flegel WA, Wagner FF. Molecular genetics of RH. Vox Sang. 2000; 78(suppl 2):109-115.

(18.) Flegel WA, von Zabern I, Doescher A, et al. D variants at the RhD vestibule in the weak D type 4 and Eurasian D clusters. Transfusion. 2009; 49(6):1059-1069.

(19.) Pham BN, Roussel M, Peyrard T, et al. Anti-D investigations in individuals expressing weak D Type 1 or weak D Type 2: allo- or autoantibodies? Transfusion. 2011; 51(12):2679-2685.

(20.) Fasano RM, Monaco A, Meier ER, et al. RH genotyping in a sickle cell disease patient contributing to hematopoietic stem cell transplantation donor selection and management. Blood. 2010; 116(15):2836-2838.

(21.) Schmid P, von Z, I, Scharberg EA, Wagner FF, Flegel WA. Specific amino acid substitutions cause distinct expression of JAL (RH48) and JAHK (RH53) antigens in RhCE and not in RhD. Transfusion. 2010; 50(1):267-269.

(22.) Flegel WA. The genetics of the Rhesus blood group system. Blood Transf. 2007; 5(2):50-57.

Willy A. Flegel, MD; Susan D. Roseff, MD; Ashok Tholpady, MD

Accepted for publication November 25, 2013.

From the Laboratory Services Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland (Drs Flegel and Tholpady); and the Department of Pathology, Virginia Commonwealth University Health System, Richmond, Virginia (Dr Roseff).

doi: 10.5858/2013-0509-ED

Dr Flegel receives royalties from the Deutsches Rotes Kreuz Blutspendedienst Baden-WUrttemberg-Hessen and holds intellectual property rights for RHD genotyping. The other authors have no relevant financial interest in the products or companies described in this article.

The views expressed do not necessarily represent the view of the National Institutes of Health, the Department of Health and Human Services, or the US Federal Government. None of the serologic antiD panels or molecular immunohematology tests discussed here have been licensed or approved by the US Food and Drug Administration.

Reprints: Willy A. Flegel, MD, Laboratory Services Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, 10 Center Dr, Bethesda, MD 20892 (e-mail:
Table 1. Guidance for Weak D Typing in the Clinical Laboratory
(Standards of Care)

Guidance                  Document               Source, y

Requirements      AABB Standards for Blood   Carson, (2) 2012
                  Banks and Transfusion
                  Services, section 5.28.2

                  AABB Standards for Blood   Carson, (2) 2012
                  Banks and Transfusion
                  Services, section 5.8.2
Recommendations   AABB Technical Manual,     Kennedy, (3) 2012
                  Chapter 22

Guidance                                  Text

Requirements      Women who are pregnant or who have been pregnant
                  recently shall be considered for RhIG administration
                  when all of the following apply:
                  1. The woman's test for D antigen is negative. A
                  test for weak D is not required.
                  2. The woman is not known to be actively immunized
                  to the D antigen.
                  3. The Rh type of the fetus-infant is unknown or the
                  type of the fetus-infant is positive when tested for
                  D or weak D. Weak-D testing is required when the
                  test for D is negative.
                  Testing of donor blood: If the initial test with
                  anti-D is negative, the blood shall be tested using
                  a method designed to detect weak D.
Recommendations   Women with red cells that are clearly positive on
                  the weak-D test should be considered D+ and not
                  receive RhIG, although rarely a positive weak-D test
                  can be caused by a partial D antigen.

Abbreviations: AABB, American Association of Blood Banks; RhIG, Rh
immune globulin.

Table 2. Diagnostic Tests Performed for Clinical Decision Making
in the Mother After Delivery of a [D.sup.+] Neonate

Test                 Required by                  Purpose

D typing (a)      AABB Standards for Blood   Determine [D.sup.+]/
                  Banks and Transfusion      [D.sup.-] status of
                  Services                   patient
Rosette fetal     AABB Technical Manual      Detection of D+ RBCs in
bleed screen                                 a [D.sup.-] mother after


Kleihauer-        AABB Technical Manual      Quantification of fetal
Betke test                                   RBCs

Weak-D test (b)   At discretion of medical   Determine whether mother
                  director                   carries a weak-D

RHD genotyping    At discretion of medical   Test for the prevalent
                  director                   weak-D types 1, 2, 3,
                                             and 4.1

Test              Test Result                Conclusion

D typing (a)      Negative      Patient is [D.sup.-]

Rosette fetal     Positive      D+ RBCs are present in mother's
bleed screen                    circulation

Kleihauer-        Negative      1. No fetal RBCs are present
Betke test                      2. Positive rosette test is caused by
                                mother's RBCs
Weak-D test (b)   Positive      1. Mother carries a weak-D phenotype
                                2. Serologic weak-D test is not
                                conclusive to determine anti-D
                                immunization potential
RHD genotyping    Negative      1. Mother carries none of the
                                prevalent weak-D types (which are not
                                prone to anti-D alloimmunization)
                                2. Immunization by the D+ fetus may

Test                    Next step

D typing (a)      Check for fetal blood in
                  mother's circulation

Rosette fetal     1. Determine whether [D.sup.+]
bleed screen      RBCs are fetal in origin
                  2. Quantify [D.sup.+] RBCs for
                  appropriate dose of RhIG
Kleihauer-        Use a more-sensitive test
Betke test        for D typing

Weak-D test (b)   Use a conclusive test to
                  determine whether RhIG
                  is needed

RHD genotyping    Administer RhIG

Abbreviations: Ig, immunoglobulin; RBCs, red blood cell; RhIG, Rh
immune globulin.

(a) Immediate spin (not incubated and without antiglobulin), using 2
different monoclonal anti-D reagents (clones MS201/lgM and MS26/lgG;
and clones MS201/lgM and TH28/lgG).

(b) Incubated with antiglobulin, using monoclonal anti-D and
anti-human globulin (anti-IgG or anti-IgG, anti-C3d).

Table 3. Serologic Reactivity With 21 Monoclonal Anti-D Reagents

                      Monoclonal Anti-D Antibody

Reagen      Clone,            Isotype         Epitope   Patient (b)
              No.                               (a)      CcDee (c)

Panel 1

A         LHM76/58      Ig[G.sub.1[lambda]]     ND      ++++
B         LHM76/59      Ig[G.sub.1]             ND      ++++
C         LHM174/102    Ig[G.sub.3[kappa]]      1.2     + (w)
D         LHM50/2B      Ig[G.sub.1[lambda]]     6.3     ++++
E         LHM169/81     Ig[G.sub.3[kappa]]      1.1     ++++
F         ESD1          Ig[G.sub.1[kappa]]      ND      ++++
G         LHM76/55      Ig[G.sub.1[kappa]]      3.1     ++++
H         LHM77/64      Ig[G.sub.1[kappa]]      9.1     ++++
I         LHM70/45      Ig[G.sub.1[lambda]]     1.2      0
J         LHM59/19      Ig[G.sub.3[kappa]]      8.1     ++++
K         LHM169/80     Ig[G.sub.3[lambda]]     6.3     +++
L         LHM57/17      Ig[G.sub.1[lambda]]     6.3     + (w)

Panel 2

1         HM10          IgM                     6.6       0
2         HM16          IgG                     6.4     ++++
3         P3x61         IgM                     6.1       0
4         P3x35         IgG                     5.4       0
5         P3x21211F1    IgM                     8.2       0
6         P3x21223B10   IgM                     9.1       0
7         P3x241        IgG                     5.4     ++++
8         P3x249        IgG                     2.1     ++++
9         P3x290        IgG                     3.1     ++++

                     Phenotype Controls (b,c)

Reagen    Weak-D Type 1   Weak-D Type 2   Weak-D Type 3
              CcDee           ccDEe           CcDee

Panel 1

A         ++++            +++             ++++
B         ++++            +++             ++++
C         +++             ++              +++
D         ++++            ++++            ++++
E         ++++            ++++            ++++
F         ++++            ++++            ++++
G         ++++            +++             ++++
H         ++++            +++             ++++
I         +++             +               +++
J         +++             +++             ++++
K         ++++            +++             ++++
L         +               ++              ++

Panel 2

1         ++++             0              ++
2         ++++            +++             ++++
3         ++++            ++              ++++
4         ++++            +++             ++++
5         +                0                0
6         ++               0              ++
7         ++++            ++++            ++++
8         ++++            ++++            ++++
9         ++++            ++++            ++++

          Phenotype Controls (b,c)

Reagen    Partial DVII   Normal-D
             CcDee        CcDee

Panel 1

A         ++++           ++++
B         ++++           ++++
C         ++++           ++++
D         ++++           ++++
E         ++++           ++++
F         ++++           ++++
G         ++++           ++++
H         ++++           ++++
I         ++++           ++++
J          0             ++++
K         ++++           ++++
L         ++++           +++

Panel 2

1         ++++           ++++
2         ++++           ++++
3         ++++           ++++
4         ++++           ++++
5          0             ++++
6         ++             +++
7         ++++           ++++
8         ++++           ++++
9         ++++           ++++

Abbreviations: Ig, immunoglobulin; ND, not determined.

(a) Serologic reaction strength: 0, no reactivity; + (w), weak
positive; ++, somewhat positive, +++, positive; ++++, strongly
positive reactivity.

(b) Epitope patterns (epD) as described previously by Flegel et al,
(18) 2009.

(c) GenBank accession number JX495049 (National Center for
Biotechnology Information, Bethesda, Maryland).
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Title Annotation:Editorials
Author:Flegel, Willy A.; Roseff, Susan D.; Tholpady, Ashok
Publication:Archives of Pathology & Laboratory Medicine
Date:May 1, 2014
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