Comparison of prenatal anti-D titration testing by gel and tube methods: A review of the literature.

BACKGROUND: Antenatal titration testing is traditionally performed using a manual tube test. Tube testing has limitations; it is a manual, time-consuming method with wide interobserver variability. Gel-based testing is an attractive alternative because it is more precise and can be automated. This study's objective was to summarize the published literature that assessed the relationship between titrations performed by tube and gel for anti-D alloimmunized pregnancies. STUDY DESIGN AND METHODS: A comprehensive literature search was performed. Articles were selected if research was original and compared at least five pairs of anti-D titration tests performed by gel and tube. Differences in the number of dilutions between gel and tube methods were compared overall by study and cell type using linear models. RESULTS: A total of 512 articles were identified; eight were included, and titer data from 384 tube and gel pairs were abstracted. The median anti-D titer in tube was 8 (range 0-2048) and by gel was 64 (range 0-4096). Anti-D gel titration results were 2.1 (95% CI; 1-3.3) additional dilutions greater than in tube. Most studies utilized double-dose reagent cells for testing. At a tube titer of 16, the sensitivity and specificity of gel titrations is maximal (91% and 94% respectively) at a gel titer of 64. CONCLUSION: Overall, titrations performed by gel were two dilutions higher than the corresponding tube titer. For titrations, double-dose reagent cells should be considered to standardize practice. A rigorous prospective study is needed to compare tube titrations with gel titrations using a standardized process.

Elimination of pretransfusion RhD typing at Mackay Memorial Hospital, Taiwan-30-year experience (1988-2017).

BACKGROUND: The frequency of the RhD negative (D-) phenotype among the population of Taiwan is only 0·34% and so anti-D is a relatively rare antibody. Routine pretransfusion D typing of patients at Mackay Memorial Hospital (MMH) was discontinued in 1988, and this report is a look back and retrospective evaluation over 30-years (1988-2017). STUDY DESIGN AND METHODS: The incidence of anti-D among patients at MMH during the periods 1984-1988 (when D typing was performed) and 1988-2017 (when D typing was not performed) was reviewed. Also, the incidence of anti-D among both MMH patients and voluntary blood donors at the Taiwan Blood Foundation was compared. The importance of anti-'Mia ' in Taiwan is also discussed. RESULTS: The incidence of anti-D relative to other Rh antibodies among MMH patients when D typing was performed and D typing not performed has remained relatively unchanged (5%). The frequencies of anti-D and anti-'Mia ' among 38 537 patients who were transfused at MMH during 2008-2017 were found to be 0·06% and 2·6%, respectively. During the same period, among 3 510 131 blood donors at Taiwan Blood Foundation, the frequencies of anti-D and anti-'Mia ' were 0·004% and 0·2%, respectively. CONCLUSION: The elimination of D typing of patients at MMH has proven to have been a correct and logical decision. D- patients, if they do not carry anti-D, can thus be safely transfused with D+ red cells.

Aggregates in blood filter chambers used from the plasma donations of anti-D donors: evaluation for monoclonal antibody discovery using phage display.

BACKGROUND: RhD-immunoglobulin (RhIg) prevents anti-D alloimmunisation in D-negative pregnant women when the fetus is D-positive, reducing the incidence of haemolytic disease of the fetus and newborn. Manufacturing RhIg is reliant on the limited supply of plasma donations with anti-D antibodies. Monoclonal antibody (mAb) development platforms such as phage display, require blood samples to be collected from anti-D donors, which may be a complicated process. The blood filter chamber (BFC) discarded after an anti-D donor's donation might provide a source of Ig-encoding RNA. This study aims to evaluate whether used BFCs are a suitable source of Ig-encoding RNA for phage display. MATERIAL AND METHODS: Haemonetics PCS2 BFCs were obtained from 10 anti-D donors for total RNA extraction, cDNA synthesis and amplification of VH and VL IgG sequences for assembly of single-chain variable fragments (scFvs). A scFv-phage display library was constructed and 3 rounds of biopanning were performed using D-positive and D-negative red blood cells (RBCs). Positive phage clones were isolated, Sanger sequenced and, where possible, reformatted into full-length human IgGs to define specificity. The BFC aggregates from 2 anti-D donors underwent a Wright-Giemsa stain and hematological cell count. RESULTS: Of 10 BFCs, a sufficient yield of total RNA for library construction was obtained from BFCs containing cellular aggregates (n=5). Aggregate analysis showed lymphocytes were the cellular source of Ig-encoding RNA. From the 5 samples with aggregates, scFvs were assembled from amplified IgG variable regions. The library constructed from 1 of these samples resulted in the isolation of clones binding to D-positive RBCs with IGHV3 gene usage. Of the 4 reformatted IgG, 3 were anti-D and 1 had undefined specificity. DISCUSSION: BFC aggregates are a new and convenient source of Ig-encoding RNA which can be used to construct Ig gene libraries for mAb isolation and discovery via antibody phage display.

Rh immune globulin: an interfering substance in compatibility testing.

CONCLUSIONS: Immunoglobulin therapy that interferes with pretransfusion testing may complicate the interpretation of test results and adversely affect patient management. Rh immune globulin (RhIG) should be considered an interfering immunoglobulin therapy when it is detected in an antibody detection test of a sample from a patient who has been treated with RhIG. Frequently, detection occurs in mother's or newborn's plasma. Because an antenatal injection of RhIG is indicated for pregnant Rh-negative women, anti-D is detected frequently by today's highly sensitive antibody screen methods when the mother's plasma is tested subsequently at delivery. Ascertaining the source of anti-D is complicated by the inability of routine clinical laboratory methods to distinguish anti-D due to RhIG from alloimmune anti-D. A combination of qualitative and quantitative test methods, as well as a complete clinical history, is necessary for accurate diagnosis and patient management.

Maternal hepatitis C (HCV) infection and Anti-D immunoglobulin therapy: study testing antibodies, RNA and Genotype of HCV in Baghdad.

Introduction: Hepatitis C virus (HCV) infection is a serious health problem. It is a major contributor to end-stage liver disease. Worldwide, 1-8% of all pregnant women were infected. Women with viral hepatitis may be at an increased risk of pregnancy complications. There are several obstetrics intervention acts as risk factors, which are specific to women pertaining the HCV infection; anti-D immunoglobulin (Ig) therapy may be one of them. Our objectives were to estimate the prevalence of HCV antibodies (anti-HCV), RNA, and genotype distribution among women with anti-D Ig therapy. Materials and methods: A cross sectional study was conducted. A sample of 154 Rhesus negative (Rh - ve) pregnant women regardless of the anti-D Ig therapy was collected. Anti-HCV were tested using third generation enzyme immunoassay (EIA-3) and immunoblot assay (Lia Tek-111), subsequently. In addition, 89 serum samples were subjected to molecular analysis using RT-PCR and DNA enzyme immunoassay (DEIA) method for the detection of HCV-RNA and genotypes. Results: Anti-HCV, and HCV-RNA seroprevalence were significantly higher (17.1, 35.5%) among women with anti-D Ig than their counter group (6.4, 13.16%), p = .038, .018, respectively. Significant direct positive dose response correlation (r = 0.78, p = .005) had been seen between number of anti-D Ig therapy and anti-HCV seropositive rate. Anti-D Ig therapy act as a risk factor (odds ratio (OR) = 3.01, 95%CI: 1.01-8.9) especially from the third dose onward. Women with anti-D Ig therapy were at higher risk (3.6 times more) of positive HCV-RNA (OR =3.6, 95%CI =1.19-10.837). Genotype HCV-1b showed higher prevalent (52.9%) among the recipients of anti-D Ig therapy while genotype HCV-3a (6.6%) was the lowest. Conclusions: Our study showed that Anti-D immunoglobulin therapy acts as a risk factor for acquiring HCV infection. Screening for HCV should be recommended for all recipients of anti-D Ig. Not only HCV antibodies but HCV-RNA detection being recommended for the diagnosis of HCV infection. A brief rational: Pregnant women with HCV infection are at risk of adverse obstetric outcome. Anti-D Ig therapy may be a risk factor for HCV infection. Hence, we conducted a cross sectional study with the objectives to estimate the prevalence of HCV antibodies (anti-HCV), RNA, and genotype distribution among women with anti-D Ig therapy. We found that anti-HCV and HCV-RNA seroprevalence were significantly higher in women with anti-D Ig. In addition, women with anti-D Ig therapy were 3.6 times more at risk of positive HCV-RNA with genotype HCV-1b showed higher prevalence. Therefore, anti-D Ig therapy is a risk factor for acquiring HCV infection and we recommend screening for HCV for all recipients of anti-D Ig. In addition, the diagnosis of HCV infection, should be made with HCV antibodies and HCV-RNA detection.

Persistent hemolytic disease of the fetus and newborn (HDFN) associated with passive acquisition of anti-D in maternal breast milk.

BACKGROUND: Anti-D is a well-documented, significant cause of hemolytic disease of the fetus and newborn (HDFN), but its presence in breast milk is not routinely described. Theoretically, breast milk containing anti-D could have the potential to exacerbate HDFN if ingested by the affected infant. STUDY DESIGN AND METHODS: This is a case report of a 28-week premature male neonate with hydrops fetalis born to a 32-year-old woman (gravidity 3/parity 3) with anti-D and anti-G. The male neonate experienced prolonged HDFN due to passive acquisition of anti-D in the mother's breast milk. RESULTS: The mother's breast milk reacted strongly (4+) with the D-positive cells in the antibody screen test. Discontinuation of breast milk feeding and addition of total parenteral nutrition led to the cessation of clinically significant HDFN. CONCLUSION: Although anti-D is a significant cause of HDFN through placental transfer of antibody, exacerbation of the condition through breast milk antibodies is rarely described. The current case highlights the possibility of this occurring. Discontinuation of maternal breast milk feedings should be considered in infants with HDFN who do not respond to standard treatment.

Impact of Uniform Methods on Interlaboratory Antibody Titration Variability: Antibody Titration and Uniform Methods.

CONTEXT: -Substantial variability between different antibody titration methods prompted development and introduction of uniform methods in 2008. OBJECTIVE: -To determine whether uniform methods consistently decrease interlaboratory variation in proficiency testing. DESIGN: -Proficiency testing data for antibody titration between 2009 and 2013 were obtained from the College of American Pathologists. Each laboratory was supplied plasma and red cells to determine anti-A and anti-D antibody titers by their standard method: gel or tube by uniform or other methods at different testing phases (immediate spin and/or room temperature [anti-A], and/or anti-human globulin [AHG: anti-A and anti-D]) with different additives. Interlaboratory variations were compared by analyzing the distribution of titer results by method and phase. RESULTS: -A median of 574 and 1100 responses were reported for anti-A and anti-D antibody titers, respectively, during a 5-year period. The 3 most frequent (median) methods performed for anti-A antibody were uniform tube room temperature (147.5; range, 119-159), uniform tube AHG (143.5; range, 134-150), and other tube AHG (97; range, 82-116); for anti-D antibody, the methods were other tube (451; range, 431-465), uniform tube (404; range, 382-462), and uniform gel (137; range, 121-153). Of the larger reported methods, uniform gel AHG phase for anti-A and anti-D antibodies had the most participants with the same result (mode). For anti-A antibody, 0 of 8 (uniform versus other tube room temperature) and 1 of 8 (uniform versus other tube AHG), and for anti-D antibody, 0 of 8 (uniform versus other tube) and 0 of 8 (uniform versus other gel) proficiency tests showed significant titer variability reduction. CONCLUSION: -Uniform methods harmonize laboratory techniques but rarely reduce interlaboratory titer variance in comparison with other methods.

Resolving variable maternal D typing using serology and genotyping in selected prenatal patients.

BACKGROUND: RhIG prophylaxis for D- pregnant women prevents hemolytic disease of the newborn and typically depends on results of serologic D typing. Interpretation and follow-up of weak D serology is variable. Recent recommendations promote genotyping for RHD status determination in those with weak D serology. Canadian Blood Services performs comprehensive serologic prenatal testing in four provinces. Genotyping is used to determine D typing in patients with weak D. STUDY DESIGN AND METHODS: A serologic algorithm identified which patients require genotyping for RHD determination. Genotyping was performed on one of two commercially available platforms. RESULTS: Only 0.4% of D- patients met criteria for genotyping. Sixty-one percent were weak D Type 1, 2, or 3. Thirty percent had a partial or weak D other than Type 1, 2, or 3. Eleven had variants which remained unresolved. Seventeen were D+ and four were D-. CONCLUSIONS: Genotyping of patients with weak D serology led to an identified genotype in most patients. RhIG administration was avoided in 66% who were weak D Type 1, 2, or 3 or were D+. The use of a serologic algorithm to select patients for RHD genotyping identifies a majority of patients with weak D types not at risk for alloimmunization. This approach limits the number of genotyping investigations and the cost of providing classification for weak D types.

Evaluation of different testing methods for identification of RhIG in red blood cell antibody detection.

BACKGROUND: RhIG has had great success in protecting fetuses from potential harm; however, little work has been done to demonstrate how long RhIG reactivity is detected in the mother after administration when using common red blood cell antibody detection methods. STUDY DESIGN AND METHODS: A retrospective investigation was performed examining positive antibody identification panels due to RhIG. These panels were run on solid-phase (SP) testing. The time to a positive result, length of detection, and positive strength of reactivity (PSR) were evaluated. Additionally, a comparative study was performed evaluating how sensitive SP, gel (GT), and tube testing (TT) were at detecting RhIG using serially diluted plasma samples spiked with different RhIG formulas. RESULTS: Retrospectively, most antibody identification panels by SP were positive 3.5 months after RhIG administration and demonstrated a strong PSR. The longest recorded positive panel was present at 4.5 months. RhIG administered intramuscularly could not be detected until several hours after injection. The comparative study showed that SP was the most sensitive method while GT and TT were comparable to one another in detecting RhIG. SP also recorded strong PSR at very low concentrations of RhIG. GT and TT recorded weak PSR even with higher concentrations of RhIG. CONCLUSION: SP is the most sensitive testing method and has the ability to detect RhIG 4 to 5 months after administration. TT and GT have the ability to detect RhIG up to 3 to 4 months after administration. Different RhIG formulas may show slightly different lengths of detection.

Identification of RHD alleles with the potential of anti-D immunization among seemingly D- blood donors in Upper Austria.

BACKGROUND: Aberrant RHD alleles leading to a reduced expression of D antigen on the red blood cell (RBC) surface may be mistyped as D- by serology. To quantify the occurrence of weak D, DEL, and D+/- chimera among apparent D- first-time blood donors, polymerase chain reaction (PCR) screening was implemented as a routine service. STUDY DESIGN AND METHODS: A total of 23,330 pretyped D- samples were tested for RHD markers in Exons 4, 7, and 10 in pools of 20 by PCR. Samples with positive results in PCR were reevaluated by exon-specific PCRs, DNA sequencing, and serologic methods. RESULTS: Among 94 PCR-positive samples, 74 exhibited a weak D or DEL phenotype, dubbed weak D type 1, weak D type 2, weak D type 5, weak D type 32, weak D type 4.3, RHD(M295I), RHD(del147), and RHD(1227G>A). The most prevalent alleles were weak D type 4.3 (n = 31) and RHD(IVS3+1G>A) (n = 24). CONCLUSIONS: As a clinical consequence, 74 blood donor samples carrying weak D and DEL phenotypes with the potential of causing secondary immunizations in recipients were reclassified as D+. Those samples were reliably amplified by RHD Exon 7 PCR; therefore, its usage in the Upper Austrian population is recommended. The association of the weak D type 4.3 samples with a ce leads to the policy that all apparently D- donors should be tested with genotyping methods; otherwise, potentially immunogenic RHD alleles may be overseen.

In vitro functional test of two subclasses of an anti-RhD antibody produced by transient expression in COS cells.

For over 35 years hemolytic disease of the fetus and newborn (HDFN) due to RhD has been effectively prevented by anti-RhD antibodies obtained from alloimmunized women or deliberately immunized men. However, due to the reduced number of immunized women and for ethical reasons it is foreseen that other sources of anti-RhD will be needed. One such source is recombinant human antibodies. Here we describe the construction of plasmids encoding two subclasses (IgG1 and IgG3) of an anti-RhD antibody, their transient expression in COS cells, and subsequent functional characterization of the antibodies with regard to specificity and ability to mediate a respiratory burst. The recombinant anti-RhD antibodies were specific for the RhD antigen and were able to mediate a respiratory burst. Thus these antibodies might be of use as future rhesus prophylaxis.

International collaborative study to evaluate a candidate reference preparation to define an appropriate specified limit of anti-D in intravenous immunoglobulin products.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate a lyophilized intravenous immunoglobulin (IVIG) preparation containing anti-D (02/228; nominal reciprocal titre of 8) for its suitability to define the maximum limit of anti-D in IVIG products when used in a proposed reference method of direct haemagglutination of papain-treated erythrocytes, in an international collaborative study. MATERIALS AND METHODS: Twenty laboratories tested 02/228 along with a negative control IVIG preparation and four IVIG samples containing different levels of anti-D. Nineteen laboratories performed direct haemagglutination methodology using papain-treated erythrocytes; five of these laboratories and one additional laboratory performed their in-house haemagglutination methodology (all indirect antiglobulin tests). RESULTS: The mode titre of 02/228, obtained by using the proposed reference method, was 8 (62.5% of tests). However, there was wide variation in haemagglutination titres between laboratories for three of the four samples. Correcting the titres of the samples relative to those of the proposed reference preparation reduced the interlaboratory variability and increased the frequency of the mode titres in three out of four samples. The indirect antiglobulin tests also showed wide interlaboratory variability and were less sensitive than the direct method in four laboratories. Eleven of the 14 laboratories that expressed an opinion considered that the level of anti-D in 02/228 was appropriate to define a specified limit. CONCLUSIONS: Our results demonstrate the necessity of using a reference preparation to define the maximum level of anti-D in IVIG products and ensure sufficient sensitivity in haemagglutination testing methodology. On the basis of these results, members of the European Pharmacopoeia Expert Group 6B recommended revision of the appropriate monograph to include this new specification and test. The Food and Drug Administration in the USA intends to adopt the same maximal specification defined by the reference preparation and to recommend the same test for the safety of IVIG products. Preparations 02/228 and 02/226 were also established by the World Health Organization as International Reference Reagents to standardize haemagglutination testing for anti-D in normal IVIG products.

Collaborative study for establishment of a global standard for the potency assay of human anti-D immunoglobulin.

An international collaborative study aimed at establishing a global standard for the potency assay of anti-D immunoglobulin was started in 2002. 25 laboratories participated in this study run under the common aegis of the World Health Organization, the United States Food and Drug Administration (US-FDA) and the European Directorate for the Quality of Medicines (EDQM). The potencies of three candidate materials and the US-FDA standard (lot 3) included for comparison were evaluated using AutoAnalyzer, competitive enzyme-linked immunoassay (competitive EIA), flow cytometric methods or own "in-house" methods. Critical reagent, standardised procedures and standardised assay design were provided for either method, where appropriate. Central statistical evaluation of the potency data submitted by the participants was performed using a parallel line model. Agreement between laboratories and assay methods for all samples was observed. Intra-laboratory variability was lowest for laboratories performing flow cytometry and highest for laboratories that performed their in-house methods. Inter-laboratory variability was acceptable for all samples when assayed by AutoAnalyzer, competitive (EIA) and flow cytometric methods. It was concluded that sample A is most suitable to serve as a global standard and that sample C could serve as a reserve European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch provided that suitable stability is demonstrated. Sample A was adopted by the Ph. Eur. Commission at its 115th session (March 2003) as the first Ph. Eur. BRP (available from the EDQM: catalog number Y0000219) with the assigned potency of 285 IU/ampoule.

A global standard for anti-D immunoglobulin: international collaborative study to evaluate a candidate preparation.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate a lyophilized anti-D immunoglobulin preparation to serve as a global standard for potency assays of anti-D immunoglobulin products. MATERIALS AND METHODS: The candidate global standard, 01/572, was calibrated against the World Health Organization (WHO) International Reference Preparation (IRP) for anti-D immunoglobulin, human (68/419), along with two reserve candidate reference preparations, in an international collaborative study involving 25 laboratories in 15 countries. The United States Food and Drug Administration (US-FDA) Center for Biologics Evaluation and Research (CBER) Standard for anti-D immunoglobulin, Lot 3, was included for comparison. Most laboratories (20/25) performed AutoAnalyser methodology, competitive enzyme-linked immunoassay (EIA) and/or flow cytometry. RESULTS: The overall mean potency of the candidate global standard, 01/572, was 284.5 international units (IU)/ampoule, with an interlaboratory variability, expressed as a percentage geometric coefficient of variation (% gcv), of 9.7. The mean potency of the US Standard was 859.4 IU/ml with an interlaboratory variability of 9.5% gcv, excluding an outlier. The mean potencies of the reserve preparations per ampoule/vial were 110.6 IU and 106.7 IU when calibrated against the IRP, and 112.2 IU and 106.6 IU when calibrated against 01/572, respectively, with interlaboratory % gcv values of 9.6-18.3 (excluding outliers). CONCLUSIONS: Preparation 01/572 proved more suitable for use as a global standard than the reserve candidate preparations and was established, with an assigned potency of 285 IU/ampoule, by the WHO as the 2nd International Standard for anti-D immunoglobulin; by FDA-CBER as the Standard for anti-D immunoglobulin, Lot 4; and by the European Directorate for the Quality of Medicines (EDQM) as the 1st Biological Reference Preparation for anti-D immunoglobulin.

A stable lyophilized reagent for use in a potential reference assay for quantitation of anti-D in immunoglobulin products.

Quality control of anti-D immunoglobulins intended for in vivo clinical use requires in vitro assay of potency. A lyophilized biotinylated monoclonal anti-D (biotinylated Brad-5; 99/698) has been evaluated for its suitability to serve as a working reagent in a competitive enzyme-linked immunoassay (EIA) for anti-D quantitation. The reagent demonstrated acceptable stability in accelerated degradation tests and following reconstitution. Twelve international laboratories obtained comparable potencies for each of nine anti-D samples using 99/698 in a standardized assay procedure using erythrocytes fixed to microtitre plates. We also describe the use of trehalose for stabilization of dried erythrocyte-coated microtitre plates.

Non-invasive RNA-based determination of fetal Rhesus D type: a prospective study based on 96 pregnancies.

OBJECTIVES: To develop a non-invasive method for determining fetal RhD status in order to provide improved care for women most at risk. DESIGN: A prospective study. METHODS: Fetal erythroblasts were enriched from the peripheral circulation of 96 RhD negative women with pregnancies at various stages in gestation using discontinuous density gradients. Amplification of RhD-specific mRNAs was carried out by reverse transcription-polymerase chain reaction assay. RNA, rather than DNA, was selected for amplification because it rarely contaminates samples, thus resulting in fewer false positives; moreover, its presence in multiple copies per cell should enhance the sensitivity of the assay, resulting in fewer false negatives. The study was prospective, relying on postnatal serological confirmation of RhD phenotype. RESULTS: The assay was 75% accurate at predicting fetal RhD status, comparing favourably with standard genomic DNA-based assays. However, we found that accuracy dropped from 85% (29/34) in the third trimester of pregnancy, to 82% (32/39) in the second and 48% (11/23) in the first trimester. Discordant data were due to false negatives in the majority (78%) of cases. CONCLUSIONS: We suggest that reverse transcription may be a useful and perhaps more sensitive alternative to standard genomic polymerase chain reaction in the majority of cases. However, under certain circumstances the absence or reduction of fetal erythroblasts or possibly RhD mRNA in some preparations may compromise the accuracy of the assay.

Detection of weak D and D(VI) red cells in D-negative mixtures by flow cytometry: implications for feto-maternal haemorrhage quantification and D typing policies for newborns.

Quantitation of feto-maternal haemorrhage (FMH) by flow cytometry (FC) has been shown to be more accurate than the Kleihauer-Bekte test. Fetal cells will be predominately of R1r or R2r phenotype, with antigen site numbers per cell (SPC) of between 9900 and 16000. If the fetus is of weak D or partial D(VI) phenotype, fewer SPC will be present. Red cells from 20 adult weak D samples were mixed with rr red cells to give 1% mixes. Mixtures were stained and analysed by FC, using two different monoclonal reagents. The SPC of each sample was measured using SOL-ELSA with Scatchard plot analysis. 18 samples could not be distinguished and had <1000 SPC. Two samples that could be distinguished had 1350 and 3000 SPC. Red cells from seven samples of D(VI) were also analysed. None of these samples could be distinguished: SPC were all <1000. Although one of the reagents used reacts with D(VI) cells, quantitation of a D(VI) FMH would not be possible due to low SPC. The ability of fetal red cells with low Rh D SPC to cause immunization is questionable; failure to measure FMH in these cases is unlikely to cause clinical problems, as long as suitably sensitive serological reagents and techniques are used to type all weak D and D variant babies as Rh D positive, and thus ensure that the mother is given the appropriate dose of anti-D.