Phasing-in RHD genotyping

In this issue, Sandler and colleagues¹ 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 AABB Standards,² most hospital laboratories reported that they do not routinely perform a serological 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 negative (Table 1).^{2, 3} In contrast, a weak D test is performed routinely on blood donors whose red blood cells test D-negative by direct agglutination, resulting in most blood donors with a weak D being categorized and managed as Rh positive.² This 50-year-old practice appears to be relatively safe⁴ and there are only a few published reports of persons with a weak D phenotype forming anti-D.^5–8 However, it confuses patients, blood donors and caregivers, and inappropriately utilizes Rh immune globulin and Rh negative red blood cells for many persons with a weak D who could be safely managed as Rh positive, 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 Rh immune globulin in women with a weak D, and decrease transfusion of Rh negative red blood cells in recipients with a weak D phenotype.¹

Table 1.

Guidance for weak D typing in the clinical laboratory (standard of care)

Guidance	Document	Text
Requirements	AABB Standards section 5.28.2	Women who are pregnant or who have been pregnant recently shall be considered for Rh Immune Globulin administration when all of the following apply: The woman’s test for D antigen is negative. A test for weak D is not required. The woman is not known to be actively immunized to the D antigen. 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.2
	AABB Standards section 5.8.2	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.2
Recommendations	AABB Technical Manual Chapter 22	Women with red cells that are clearly positive on the weak D test should be considered D positive and not receive RhIG, although rarely a positive weak D test can be caused by a partial D antigen.3

The process of phasing-in RHD genotyping in clinical practice has begun in many hospitals, but as the CAP survey indicates, the majority of pregnant women and hospital patients in the United States continue to have their Rh type determined by outdated serological methods.¹ The first step in phasing-in RHD genotyping needs to begin in hospital laboratories. Those laboratories that do not routinely perform weak D tests for patients typing Rh negative 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.

We recently encountered a 27-year-old North African woman who was designated as Rh negative for a Caesarean section. Her medical history and laboratory test results are representative of a common subset of patients¹¹ 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-positive newborn (Table 2).

Table 1.

Diagnostic tests performed for clinical decision making in the mother after delivery of a D positive neonate

Test	Required per	Purpose	Result	Conclusion	Next step
D typing*	AABB Standards	Determine D positive/D negative status of patient	Negative	Patient is D negative	Check for fetal blood in mother’s circulation
Rosette fetal bleed screen†	AABB Technical Manual	Detection of D positive RBCs in a D negative mother after delivery	Positive	D positive RBCs are present in mother’s circulation	Determine if D positive RBCs are fetal in origin Quantify D positive RBCs to appropriately dose RhIG
Kleihauer- Betke test‡	AABB Technical Manual	Quantification of fetal RBCs	Negative	No fetal RBCs are present Positive rosette test is caused by mother’s RBCs	Use a more sensitive test for D typing
Weak D test§	At discretion of Medical Director	Determine if mother carries a weak D phenotype	Positive	Mother carries a weak D phenotype Serologic weak D test is not conclusive to determine anti-D immunization potential	Use a conclusive test to determine if RhIG is needed
RHD genotyping∥	At discretion of Medical Director	Test for the prevalent weak D types 1, 2, 3 and 4.1	Negative	Mother carries none of the prevalent weak D types (which are not prone to anti-D alloimmunization) Immunization by the D positive fetus may occur	Administer RhIG

^*immediate spin (not incubated and without antiglobulin): using 2 different monoclonal anti-D reagents (clones MS201/IgM and MS26/IgG, Series 4; and clones MS201/IgM and TH28/IgG, Series 5; Immucor, Norcross, GA)

^†Fetal Bleed Screening Test (Immucor). This kit was phased out in 2013 and replaced by a method with a shorter incubation time.

^‡Fetal Cell Stain Kit (Simmler, High Ridge, MO)

^§incubated and with antiglobulin: using monoclonal anti-D (clones GAMA 401/ IgM and F8D8/IgG, Gamma-clone; Immucor) and anti-human globulin (anti-IgG or anti-IgG, -C3d; polyspecific; Immucor)

^∥BAGene DNA-SSP WEAK D-Type Kit (BAG Health Care, Lich, Germany)

The woman’s routine postpartum blood sample was strongly positive by a rosette fetal bleed screen, suggesting the presence of D+ fetal red blood cells in her circulation (fetomaternal hemorrhage). However, a quantitative acid-elution (Kleihauer-Betke) assay was negative, indicating that the D+ red blood cells in her circulation did not contain a significant amount of hemoglobin F, i.e., 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% – 1.0% of Caucasians express a weak D phenotype.¹² A weak D phenotype has been reported in 0.1 – 10% of all pregnancies that initially typed as D-negative.^13–15 We estimate that approximately 95% of patients in the United States with a weak D phenotype will have one of the RHD genotypes that is prevalent in Caucasians (types 1, 2, 3, or 4.1).^{5, 7, 11, 16} Women with one of these prevalent RHD genotypes may be managed as Rh positive and do not require Rh immune globulin 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 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 patient²⁰ and established that she had inherited the uncommon weak D type 25,²¹ which requires management as Rh negative for purposes of Rh immunoprophylaxis and transfusion of red blood cells.

Table 3.

Serologic reactivity with 21 monoclonal anti-D reagents

				Patient CcDee †	Controls
Monoclonal Anti-D					Weak D type 1 CcDee	Weak D type 2 ccDEe	Weak D type 3 CcDee	Partial DVII CcDee	Normal D CcDee
No.	Clone	Isotype	Epitope*
Panel 1‡
A	LHM76/58	IgG1λ	ND	++++	++++	+++	++++	++++	++++
B	LHM76/59	IgG1	ND	++++	++++	+++	++++	++++	++++
C	LHM174/102	IgG3κ	1.2	+ (w)	+++	++	+++	++++	++++
D	LHM50/2B	IgG1λ	6.3	++++	++++	++++	++++	++++	++++
E	LHM169/81	IgG3κ	1.1	++++	++++	++++	++++	++++	++++
F	ESD1	IgG1κ	ND	++++	++++	++++	++++	++++	++++
G	LHM76/55	IgG1κ	3.1	++++	++++	+++	++++	++++	++++
H	LHM77/64	IgG1κ	9.1	++++	++++	+++	++++	++++	++++
I	LHM70/45	IgG1λ	1.2	0	+++	+	+++	++++	++++
J	LHM59/19	IgG3κ	8.1	++++	+++	+++	++++	0	++++
K	LHM169/80	IgG3λ	6.3	+++	++++	+++	++++	++++	++++
L	LHM57/17	IgG1λ	6.3	+ (w)	+	++	++	++++	+++
Panel 2¶
1	HM10	IgM	6.6	0	++++	0	++	++++	++++
2	HM16	IgG	6.4	++++	++++	+++	++++	++++	++++
3	P3x61	IgM	6.1	0	++++	++	++++	++++	++++
4	P3x35	IgG	5.4	0	++++	+++	++++	++++	++++
5	P3x21211F1	IgM	8.2	0	+	0	0	0	++++
6	P3x21223B10	IgM	9.1	0	++	0	++	++	+++
7	P3x241	IgG	5.4	++++	++++	++++	++++	++++	++++
8	P3x249	IgG	2.1	++++	++++	++++	++++	++++	++++
9	P3x290	IgG	3.1	++++	++++	++++	++++	++++	++++

^*Epitope patterns (epD) as described previously¹⁸

^†GenBank accession number JX495049

^‡Advanced partial RhD typing kit (Alba Bioscience, Edinburgh, UK)

^¶D-Screen (Diagast, Loos, France)

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-negative pregnancies may pay for itself by avoiding the costs associated with often unnecessary multiple administrations of RhIG.^{4, 17, 22} Presently, there are no FDA-approved molecular test kits for determining the Rh type, but several unlicensed commercial kits are marketed commercially in the United States. Products utilizing PCR with sequence-specific primers (PCR-SSP) include BAGene Weak D-TYPE and LIFECODES Red Cell EZ Type Weak D (GTI Diagnostics, Waukesha, WI). High throughput methods utilizing multiplex PCR techniques include the BLOODchip v2.0 (Progenika; Balboa, Spain) and the BioArray RHD Beadchip (Immucor; Norcross, GA). Any of these test kits can be used for patient care as “tests of high complexity” under the Clinical Laboratory Improvement Act (CLIA).

Based on the results of their 2012 survey and review of the science of RHD genotyping, the CAP TMRC has recommended a multi-organizational 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 a Work Group on Phasing-In RHD Genotyping. We believe that the time has come to transition from serological to molecular methods for managing weak D phenotypes. Our case illustrates how easily this transition can be accomplished. We support the CAP TMRC’s initiative.