Analysis of factors affecting quantification of fetomaternal hemorrhage by flow cytometry

BACKGROUND: An analysis was carried out to determine the sources and extent of errors encountered in the quantitation of the volume of fetomaternal hemorrhage (FMH) by flow cytometry. Different assay conditions were compared, to define the simplest, most accurate protocol. STUDY DESIGN AND METHODS: D-, D+, and artificial FMH (mixtures of D+ and D- RBCs) were stained either by a direct method (using FITC-conjugated IgG3 D MoAb [BRAD-3]), with or without dual labeling with PE-conjugated anti-GPA, or by indirect methods (using polyclonal anti-D followed by FITC- or biotin-conjugated anti-IgG reagents). Cells were selected for flow cytometric analysis on the basis of either forward or side scatter (log FSC/log SSC) characteristics or of GPA+ labeling or were unselected. The numbers of events labeled with anti-D were determined from histograms. For some samples, 10 replicates of 500,000 events each were analyzed. RESULTS: Background fluorescent events in 10 directly labeled gated D- samples ranged from 0.007 to 0.023 percent, equivalent to 0.15- to 0.51-mL FMH. Both the use of a gate on log FSC/SSC or the selection of GPA+ events only resulted in a reduction in FMH of 0.3 mL or less. The intra-assay variation in FMH, or sampling error, was found to be approximately 10 percent at low artificial FMH (<10 mL) but greater (< or =50% with a CV of 15%) with D- samples. Direct staining was quicker and produced a lower background than indirect staining. CONCLUSION: The inherent sampling error that is due to the random distribution of rare events throughout the blood sample contributed greatly to the variation in the volume of FMH calculated by flow cytometry. The FMH should not be underestimated. For a routine assay, a simplified protocol and calculation will be sufficiently accurate to determine the dose of prophylactic anti-D that should be given to the patient.     

Massive fetomaternal hemorrhage: clearance of fetal red blood cells after intravenous anti-D prophylaxis monitored by flow cytometry

BACKGROUND: The clearance of D+ red blood cells (RBCs) from the circulation in D- individuals mediated by passively administered anti-D occurs by opsonization with the antibody and subsequent removal in the spleen. Few data exist on the kinetics of clearance of large volumes of D+ RBCs from the maternal circulation by anti-D in clinical cases of massive fetomaternal hemorrhage (FMH). CASE REPORT: A 33-year-old D- woman delivered a D+ female infant by emergency cesarean section for suspected fetal anemia. A massive FMH, initially estimated to be approximately 142 mL of RBCs, was found. In addition to the standard dose of intramuscular (IM) anti-D (300 microg) given immediately after delivery, 2700 microg of anti-D was administered intravenously (IV). The clearance of D+ fetal cells from the maternal circulation was monitored by flow cytometry in samples obtained on a daily basis using anti-D. The mother had no detectable anti-D 6 months after delivery. RESULTS: No clearance of fetal cells was apparent after the insufficient dose of IM anti-D. The IV administration of anti-D caused accelerated clearance of D+ fetal RBCs with a t1/2 of 24.5 hours. D+ reticulocytes comprised 4.2 percent of all D+ cells in the maternal circulation at delivery suggesting acute fetal blood loss. CONCLUSIONS: The approach used in this report allowed a detailed analysis of the kinetics related to the clearance of fetal D+ RBCs. Simultaneous measurements of fetal reticulocytes and fetal RBCs in maternal blood may establish the timing of an FMH.     

Primary anti-D immunization by weak D type 2 RBCs

BACKGROUND: D is the most immunogenic blood group antigen. In about 0.4 percent of whites, D is expressed on RBCs in a weak form. Recently, it was found that the weak D phenotypes are caused by a large number of distinct RHD alleles generally encoding altered D proteins. No particular molecular weak D type has yet been shown to induce anti-D. The threshold of D antigen density required for anti-D immunization is not known. CASE REPORT: A 72-year-old D- white man received apparently D- RBCs. Nineteen days later, he developed a positive DAT, and anti-D was found in his serum and an eluate from his RBCs. One donor was found to be D+ with a weak D type. The weak D type was determined by RHD exon 9-specific nucleotide sequencing from genomic DNA. The transfusion recipient showed alloanti-D. Ten months later, anti-D but no other antibody was detectable; the DAT was negative and the eluate was nonreactive. The donor of the incriminated unit was D+ (ccDEe) with weak D due to the weak D type 2 allele, expressing about 450 D antigens per RBC. CONCLUSION: This case provides formal proof that RBCs of weak D type 2 phenotype may cause alloanti-D immunization. Among the more prevalent weak D types in whites, weak D type 2 has the lowest D antigen density. Thus, units of blood from donors of the weak D type 2 phenotype should be labeled D+; the weak D type 2 phenotype may be useful for quality assurance.     

Use of a directly conjugated monoclonal anti-D (BRAD-3) for quantification of fetomaternal hemorrhage by flow cytometry

BACKGROUND: Determination of the volume of fetal D-positive cells in the circulation of D-negative women after delivery is carried out to determine whether additional prophylactic anti-D should be given to the mother. Although the Kleihauer-Betke test is still widely used to calculate the fetomaternal hemorrhage, increasing use is being made of flow cytometry. STUDY DESIGN AND METHODS: A conjugated monoclonal anti- D was prepared by labeling purified BRAD-3 (IgG3) with fluorescein isothiocyanate (FITC-BRAD-3). This reagent was used to label D-positive red cells by a one-step procedure: 5 microL of washed cells were incubated with 50 microL of FITC-BRAD-3 (50 micrograms/mL) at 37 degrees C for 30 minutes; then the cells were washed and 500,000 events were analyzed by flow cytometry. RESULTS: The FITC-BRAD-3 reagent effectively labeled D-positive cells. The percentage of D-positive cells in mixtures containing more than 0.04 percent D-positive cells in D-negative cells was accurately determined by using this reagent and flow cytometry. Although the Kleihauer-Betke test was more accurate than this one-step flow cytometric method at quantifying fetomaternal hemorrhage of < 1 mL, the flow cytometric method was more accurate in the 1- to 7-mL fetomaternal hemorrhage range of 1 to 7 mL (whole-blood equivalents). Analysis of 175 clinical samples for fetomaternal hemorrhage gave consistent quantification results with the three methods used: the Kleihauer-Betke test, flow cytometry with FITC-BRAD- 3, and flow cytometry with polyclonal anti-D followed by FITC-anti-IgG. CONCLUSION: Labeling of samples with FITC-BRAD-3 was simple and rapid. By flow cytometric analysis, good separation of D-positive from D- negative cells was obtained, and fetomaternal hemorrhage of > 1 mL was quantified accurately.     

Quantification of fetomaternal hemorrhage by fluorescence microscopy is equivalent to flow cytometry

BACKGROUND: The quantification of fetal cells in the maternal circulation remains an important goal to determine the amount of anti-D necessary to prevent active immunization of a D- mother giving birth to a D+ baby. Underestimation of fetomaternal hemorrhage (FMH) results in inefficient anti-D prophylaxis and maternal immunization; overestimation of FMH results in higher doses of passively transferred anti-D, higher costs, and the risk of disease transmission. Thus, a reliable method to quantitatively assess FMH is necessary. STUDY DESIGN AND METHODS: Serial dilutions of artificial FMH were quantitatively measured by three different methods: flow cytometry, fluorescence microscopy (each after anti-D staining), and by the Kleihauer-Betke test. The accuracy and precision of the three methods were compared by statistical analysis. RESULTS: Fluorescence microscopy and flow cytometry were comparably accurate and precise in quantifying FMH. In contrast, the accuracy of the Kleihauer-Betke test was poor, resulting in substantial overestimation of FMH in the samples with lower fetal cell concentrations. CONCLUSION: Anti-D flow cytometry and fluorescence microscopy for detection of fetal cells offer equally reliable and precise methods in contrast to the Kleihauer-Betke test. Fluorescence microscopy may be established as standard to quantify FMH in clinical practice because it is comparable to flow cytometry; in addition, it is time saving and is less expensive.     

Absence of D- alloimmunization in AIDS patients receiving D-mismatched RBCs

BACKGROUND: More than 80 percent of D- patients who receive D+ blood become alloimmunized to the D antigen. Anemia occurs in most AIDS patients at some point in the disease. D- patients with AIDS may require blood transfusion and, during times of blood shortage, may receive D+ RBCs. They would be expected to become alloimmunized to the d antigen. STUDY DESIGN AND METHODS: The records of the transfusion service between January 1996 and July 2000 were reviewed for D- patients who received D+ blood. IATs were performed before the initial transfusion and subsequently when the patient required further RBC transfusion. RESULTS: Eight D- AIDS patients who received multiple transfusions (three women and five men; age range, 31-44 years; mean, 44 years) who received between 2 and 11 units (mean, 6.25) of D+ RBCs were identified. Antibody screens were performed at 8 to 65 weeks after transfusion. It was found that none of the eight D- AIDS patients developed anti-D. ABO antibodies were found as expected. During the same period, it was found that six D- patients admitted with other diagnoses who received 1 to 9 units of D+ RBCs, all developed anti-D within 7 to 19 weeks of transfusion. CONCLUSION: Patients with AIDS may not form alloantibodies to the D antigen. This may be attributable to their immunodepressed state, particularly to the decrease in CD4+ T lymphocytes. Therefore, during blood shortages, transfusion of D+ blood to D- AIDS patients may be without any subsequent consequence.     

Prevention of immunization to D+ red blood cells with red blood cell exchange and intravenous Rh immune globulin

BACKGROUND: Although young women who are D- occasionally receive unintentional transfusions with D+ red blood cells (RBCs), there are little data to assist with management of such an event. Two cases of D- girls transfused with D+ RBCs are reported. In an effort to prevent formation of anti-D, RBC exchange followed by administration of intravenous (IV) Rh immune globulin (RhIg) was used. CASE REPORTS: Patient 1, a 56-kg, 16-year-old D- girl, was involved in a motor vehicle crash. She received 4 units of Group O uncrossmatched D+ RBCs. Thirty-six hours after admission, she underwent RBC exchange with 10 units of D- RBCs, followed by a total of 2718 microg of IV RhIg over 32 hours. Six months later, her antibody screen was negative. Patient 2, a 39-kg, 10-year-old D- girl with aplastic anemia, received 1 unit of D+ RBCs. She underwent RBC exchange on the same day with 5 units of D- RBCs, followed by a total of 900 microg of IV RhIg over 8 hours. Six months later her antibody screen was negative. CONCLUSION: RBC exchange followed by a calculated dose of IV RhIg was successful in preventing allo-immunization to D. Several small studies suggest that both trauma and hematology patients may be less capable of becoming immunized with the transfusion of D+ blood components. Until these findings are more clearly defined, there will be times when prevention of immunization of any D- girl is desired. RBC exchange followed by RhIg appears to be one way to achieve this goal.     

Use of a phycoerythrin-conjugated anti-glycophorin A monoclonal antibody as a double label to improve the accuracy of FMH quantification by flow cytometry

The use of flow cytometry for quantifying fetomaternal haemorrhage is increasing, and has been shown to be more accurate than the Kleihauer-Betke test for evaluating larger bleeds of over 4 mL in volume. Red cells are stained with fluorescently labelled monoclonal anti-D. Cells for analysis are normally gated manually on the basis of forward and side scatter. We investigated whether the use of an antiglycophorin A monoclonal antibody conjugate (red cell specific) in a dual labelling technique would improve the gating of RBC and FMH quantification. Mixes of adult rr and cord R1r RBC were prepared to simulate 1, 0.5, 0.25, 0.12 and 0.06% fetal bleeds. Phycoerythrin-conjugated BRIC 256 (mouse monoclonal antiglycophorin A) was used to label all RBC, and FITC-BRAD-3 monoclonal anti-D was used to determine the proportion of D-positive cells. Results from the dual labelling experiments were compared to those from single labelling of the same mixtures with FITC-BRAD-3 alone, using gated and ungated data. The results showed that single labelling with manual gating gave falsely low FMH estimates. We conclude that use of a fluorescently labelled antiglycophorin A antibody improves the accuracy of the FMH measurement by flow cytometry, as manual subjective gating of RBC excludes a higher proportion of fetal than of adult RBC.     

Semiautomated data analysis of flow cytometric estimation of fetomaternal hemorrhage in D- women

BACKGROUND: Accurate and reliable measurement of the volume of fetal D+ cells in D- women is required for adequate anti-D prophylaxis. A semiautomated flow cytometry assay based on a standardized calibration curve that was created with simulated fetomatemal hemorrhage (FMH) mixtures was developed. STUDY DESIGN AND METHODS: A calibration range of 0.083- to 2-percent D+ cells in the D-RBC mixtures (2-44 mL calculated FMH) was analyzed by use of a flow cytometer (XL-MCL, Coulter Electronics Ltd). Linear regression analysis of the calibration curve data with computer software (Excel, Microsoft) allowed semiautomated determination of the FMH volume. To optimize the assay, fresh versus frozen and thawed RBCs, RBCs from adults who are heterozygous for D or cord RBCs, and indirect- or direct-labeling techniques were evaluated by use of MoAbs. RESULTS: Fresh RBCs from adults heterozygous for D were chosen for routine use, although equivalent calibration curves were obtained with all cells tested (n = 12 calibration assays; r2 = 0.999; mean SD, 14%). A monoclonal anti-D reagent (Therad 10, Diagnostics Scotland) worked well in both indirect-(anti-IgG F(ab)-FITC) and direct-(anti-D-FITC) labeling methods compared to the use of BRAD-3 FITC. In routine practice, the FMH volumes obtained were mainly lower than those obtained in the Kleihauer Betke test when there was less than 4 mL of FMH. CONCLUSION: Semiautomated data acquisition and calibration curve analysis represents a further step toward standardization of flow cytometry for accurate FMH quantification and facilitates evaluation and control of day-to-day variations between laboratories, flow cytometers, and operators.     

False-positive eluate reactivity due to the low-ionic wash solution used with commercial acid-elution kits

BACKGROUND: During the use of commercial red cell (RBC) acid-elution kits for adsorption and elution (adsorption/elution) studies with anti- D, unexpected reactive eluates (anti-D) were obtained from D- RBCs. Such results were not obtained with a parallel xylene method or, historically, with heat and ether methods. STUDY DESIGN AND METHODS: Single-donor and commercial polyclonal anti-D samples were incubated with D+ and D- RBCs. Acid eluates were prepared by the manufacturers' directions. Variations in the wash step of the eluate preparation included the use of commercial kit wash solution versus phosphate- buffered saline versus solutions of various ionic strengths. RESULTS: Anti-D was eluted from 20 of 22 samples of D- RBCs after incubation with commercial polyclonal anti-D (titer 512) and from 2 of 3 samples of D- RBCs incubated with single-donor anti-D (titer 256). With a low- titer (16) single-donor anti-D, 0 of 4 eluates from D- RBCs reacted. When phosphate-buffered saline was substituted for the commercial wash solution, 0 of 11 D- RBC eluates reacted, as compared with 9 of 11 D- RBCs that yielded positive 1+(-)2+ eluates with the commercial wash solution. If the recommended initial phosphate-buffered saline wash was omitted before the use of the commercial wash solution, the eluate reactivity was stronger (2+(-)3+). When low-ionic-strength (< 0.03 M) saline was substituted, anti-D was eluted from D- RBCs. All last washes were nonreactive. Antiglobulin tests on all adsorbing D- were negative. CONCLUSION: Commercial wash solutions used for acid elution are at low ionic strength and commonly yield superior eluates, but in the presence of high-titer antibodies, false-positive eluates can result. It is our belief that the low-ionic-strength wash solution caused aggregation of IgG and nonspecific attachment of IgG on RBCs. Aggregates will contain IgG serum antibodies in proportion to the titer of the antibody. It is this nonspecifically bound antibody that is eluted from antigen- negative RBCs.     

Probability of anti-D development in D- patients receiving D+ RBCs

BACKGROUND: In some situations, the administration of D+ RBCs to D- patients is necessary. The probability of a subsequent anti-D formation is assumed to be around 80 percent, a figure based primarily on studies in healthy volunteers. It was hypothesized that patients requiring blood transfusion have a much lower probability of developing antibodies. STUDY DESIGN AND METHODS: A retrospective analysis was performed whereby 78 D- patients were evaluated for the development of RBC antibodies after administration of D+ RBCs. For the analysis of the cross-sectional observations, parametric models were used for interval-censored data. RESULTS: Anti-D was detected in 16 of 78 patients. Considering the individual patient's inspection times, the calculated probability of developing antibody following D+ RBC supply was shown to be below 41.7 percent (upper 95% confidence bound) and estimated as 30.4 percent. The data hinted toward an inverse correlation between the number of transfused units and the probability of antibody formation. Interestingly, 6 of these 16 patients developed additional IgG autoantibody. In 3 of those cases, evidence for prolonged hemolysis was found. CONCLUSION: The actual frequency of antibody formation in our patients is much lower than assumed. On the other hand, prolonged hemolysis probably induced by additional autoreactive antibodies might occur. This possible complication has not yet been addressed. Further studies might reveal whether a less restricted transfusion policy with respect to D matching is justified in selected patients.     

Comparison of flow cytometric assays with isotopic assays of (51)chromium-labeled cells for estimation of red cell clearance or survival in vivo

BACKGROUND: A comparison was made between flow cytometric and conventional radioisotopic assays in the determination of the clearance or survival of small volumes of (51)chromium-labeled D+ red cells after injection into volunteers. STUDY DESIGN AND METHODS: Four clearance studies were performed using 4 mL of autologous D+ cells coated with anti-D at two concentrations (5 or 10 microg anti-D/mL red cells) transfused to two subjects at separate times. Five survival studies were carried out using 5 mL of frozen-thawed D+ cells transfused to five D- subjects with no detectable anti-D. Sequential blood samples were taken for gamma counting and flow cytometry. Several methods were used to stain the transfused red cells, and the data were analyzed by using three flow cytometers. RESULTS: The determination of red cell clearance or survival by radioactivity measurements gave results consistent with published data. However, none of the flow cytometric assays exhibited the necessary sensitivity or accuracy in quantitation of the rare events to provide reliable data for the calculation of the initial clearance rate, the red cell half-life, or the mean cell lifespan, although rough estimates of red cell clearance were obtained in some subjects. This inability to accurately enumerate rare fluorescence-labeled cells was due mainly to the presence of "background" events, which were a considerable problem in some samples, when the coating level of anti-D was less than 3000 molecules of IgG per cell. CONCLUSION: Flow cytometry may enable the crude estimation of the percentage of small volumes (<5 mL) of transfused D+ red cells, but in this study it was found that this method was not sufficiently accurate to determine the initial clearance rate, red cell half-life, or mean cell lifespan. If the proportion of transfused cells in the recipient is about 0.2 percent or less, the use of radioisotopes for labeling cells for quantitative in vivo red cell clearance or survival data should remain the method of choice.     

Detection of multiple passively acquired alloantibodies following infusions of IV Rh immune globulin

BACKGROUND: Passively acquired blood group alloantibodies are detected regularly after infusions of IV Rh immune globulin (RhIG) for the treatment of immune thrombocytopenic purpura (ITP) in D+ patients. STUDY DESIGN AND METHODS: Blood samples from 16 D+ patients with ITP were tested after treatment with IV RhIG for the presence of passively acquired alloantibodies. Similar studies were conducted for three D- patients after injections of IM RhIG for Rh immunoprophyl-axis. Four production lots of IV RhIG and 2 lots of IM RhIG were tested for the presence of alloantibodies. RESULTS: All 16 D+ patients with ITP developed a positive DAT, as well as positive antibody detection test results, after infusions of IV RhIG. All postinfusion plasma samples contained anti-D, as well as one or more additional antibodies, usually anti-C, -E, -G, -V, or -Fy(a). Eluates from patients' RBCs with positive DAT results contained multiple passively acquired alloantibodies. Multiple alloantibodies were detected in samples of different production lots of IV RhIG or IM RhIG. No acute transfusion reactions were observed in five D+ patients with ITP who had been treated with IV RhIG and had been given serologically incompatible D+ RBCs. After injections of IM RhIG, the only passively acquired alloantibody detected was anti-D. CONCLUSION: Plasma samples from D+ patients with ITP treated with IV RhIG regularly contained anti-D and multiple other passively acquired Rh, Duffy, or Kidd system alloantibodies. Postinfusion RBC samples all had positive DAT results with eluates containing anti-D and multiple other Rh, Duffy, or Kidd system antibodies. The consistent detection of multiple passively acquired alloantibodies after IV RhIG, in contrast to the detection of anti-D only after IM RhIG, reflects the immediate effect of the entire (bolus) dose of RhIG by the IV route, the dose for treating ITP that is approximately 10 times the dose for Rh immunoprophylaxis, and the expected serologic incompatibility with recipients' D+ RBCs.     

Anti-D alloimmunization after D-mismatched allogeneic hematopoietic stem cell transplantation in patients with hematologic diseases

BACKGROUND: The de novo development of anti-D after D-mismatched allogeneic hematopoietic stem cell transplantation (AHSCT) is a possibility that must be considered. The transfusion of D- blood components after AHSCT has been recommended but anti-D alloimmunization in this setting has been studied little. Thus, the aim of this study was to analyze anti-D formation after D-mismatched AHSCT. STUDY DESIGN AND METHODS: Thirty patients with a hematologic disease who underwent D-mismatched AHSCT were retrospectively studied. Support therapy included red blood cells (RBCs) and platelet (PLT) concentrates (PCs) from whole-blood donations and PLTs from apheresis. After AHSCT, patients received D+ PCs without administering Rh immunoglobulin (RhIG). An antibody screening to detect anti-D was performed by low-ionic-strength saline-indirect antiglobulin test with the tube test. RESULTS: Fifteen D+ patients received stem cells (SCs) of D- donors and 15 D- patients received SCs of D+ donors. After AHSCT, patients received a median of 11.5 (range, 0-32) D- RBC units. D+ patients received 682 (83%) of 825 PLT units from D+ donors, and D- patients received 573 (85%) of 678 PLT units from D+ donors. None of the 30 patients developed anti-D after a median follow-up of 32 weeks (range, 4-310 weeks). CONCLUSION: Anti-D alloimmunization after performing a D-mismatched AHSCT is infrequent in patients with hematologic diseases although patients receive D-mismatched PLT transfusions without RhIG administration.     

Evaluation of a panel of human monoclonal antibodies to D and exploration of the synergistic effects of blending IgG1 and IgG3 antibodies on their in vitro biologic function

BACKGROUND: The D immunoprophylaxis program has successfully reduced the incidence of Rh hemolytic disease of the newborn (HDN), but it has also reduced the availability of plasma-derived polyclonal anti-D, which constitutes the current therapeutic product. Human monoclonal anti-D from hybridoma cell lines may be an acceptable alternative, and clinical efficacy of each anti-D is being evaluated in several centers. STUDY DESIGN AND METHODS: This study represents the largest assessment (outside of the International Workshops) of human D monoclonal antibodies for potential therapeutic use. The in vitro biologic activity and immunologic and serologic reactivity of a coded panel of 20 D antibodies (THERAD) was investigated. The bioassays used were lymphocyte (K-cell) antibody-dependent cell-mediated cytotoxicity (ADCC), monocyte ADCC, and monocyte chemiluminescence, which together reflect the processes involved in antibody-coated red cell destruction in vivo. From this panel, six antibodies (THERADs 14, 19, 22, 23, 27, and 28, comprising 3 IgG1 and 3 IgG3 D monoclonal antibodies) were further selected to investigate the effects of blending in the three bioassays. RESULTS: Several THERAD blends displayed greater activity than their component parts, in the range of 6 to 124 percent. There was no evidence to suggest functional blocking effects with this restricted panel of antibodies. CONCLUSION: The THERAD blends containing both IgG1 and IgG3 anti-D appeared to be the most functionally active, as did blends containing antibodies to two distinct D epitopes. This in vitro evidence has important implications for the future formulation of an effective monoclonal preparation for the prevention of Rh HDN.     

Secondary anti-D immunization by Del red blood cells

BACKGROUND: Recent molecular studies of the RHD gene have revealed that D(el) individuals retain a grossly intact RHD gene or have a portion of RHD in their genomes. No D(el) phenotype has yet been shown to induce a primary or secondary alloanti-D immunization, however. CASE REPORT: A 67-year-old D- Japanese woman with a history of allosensitization from transfusion of D+ red blood cells (RBCs) was negative for anti-D at admission. After she received RBCs from 19 apparently D- donors, she developed anti-D with an 8-fold titer. The titer of anti-D increased further to 128-fold after transfusions of cross-match-compatible D- negative RBCs from 40 donors over the next 2 years. Two of 59 donors were found to be RHD gene-positive and antigen D- with a D(el) phenotype, that is, RHD(K409K). CONCLUSION: This is the first case in which RBCs having the D(el) phenotype induced a secondary alloanti-D immunization. A D- donor with the RHD(K409K) allele was associated with the development of anti-D. Adverse episodes or evidence of hemolysis was not observed after the transfusion of RHD(K409K) RBCs. Further clinical evidence is needed to reveal whether the D(el) phenotype has a clinically relevant potential for anti-D immunization.     

Quantification of feto-maternal haemorrhage (FMH) by flow cytometry: anti-fetal haemoglobin labelling potentially underestimates massive FMH in comparison to labelling with anti-D

Many centres now routinely use flow cytometry to quantify feto-maternal haemorrhage (FMH). However, which flow cytometric method is the most accurate in quantifying FMH is currently unknown. An audit of clinical results in which FMH had been estimated by both directly conjugated monoclonal anti-D and anti-fetal haemoglobin (HbF) labelling suggested that the anti-HbF labelling method may underestimate massive FMH in comparison to labelling with anti-D. Subsequent to this audit, 46 samples of adult D-negative blood were spiked with varying amounts of D-positive cord blood (0.05-10% fetal cells per sample), and the number of fetal cells present was quantified by both labelling methods. The percentage of fetal cells detected by anti-D was not significantly different to the estimated percentage of fetal cells added to each sample (P = 0.636). However, anti-HbF labelling significantly underestimated the percentage of fetal cells present (P = 0.0001). In comparison to anti-D, the percentage of fetal cells detected by anti-HbF was also significantly lower (P < 0.0001). The difference in fetal cell detection between anti-D and anti-HbF labelling was only apparent in the spiked samples containing > or =1% fetal cells per sample. In samples containing < or =0.6% fetal cells, no significant difference in the detection of fetal cells between anti-D and anti-HbF labelling was observed (P = 0.11). To allow adequate immunoprophylaxis in D-negative mothers with massive FMH, we recommend that anti-D labelling should be used in the routine flow cytometric estimation of FMH.     

Detection of anti-D in D- recipients transfused with D+ red blood cells

BACKGROUND: The D antigen is highly immunogenic, requiring only a small quantity of transfused red blood cells (RBCs) to cause alloimmunization in D- immunocompetent recipients. The relatively low sensitization rate in oncology patients transfused with D+ platelets is well documented. A study of the alloimmunization rate of primarily nononcology D- recipients transfused with D+ RBCs was undertaken. STUDY DESIGN AND METHODS: Transfusion service records were examined to identify D- recipients who were not alloimmunized to the D antigen and who had a follow-up antibody screen performed at least 10 days after the initial D+ RBC transfusion(s). The age and sex of the recipients, date and number of D+ RBC transfusion(s) and their leukoreduction status, all subsequent serologic investigations, and the hospital ward where the units were issued were recorded. RESULTS: There were 98 study-eligible recipients identified who received a total of 445 D+ RBC units. The mean follow-up length was 182 days. Most recipients (87%) had antibody screens performed more than 21 days after the initial D+ RBC transfusion. In total, 24 recipients made 44 new alloantibodies: 22 anti-D (22%), 11 anti-E, 5 anti-C, 2 anti-K, and 1 each of anti-Kp(a), anti-Jk(a), anti-Bg, and anti-Fy(b). The rate of anti-D alloimmunization among recipients of entirely leukoreduced D+ units was 13 percent (1/8). Reexposure to D+ RBCs after the initial bleeding episode did not increase the rate of alloimmunization. CONCLUSIONS: The 22 percent rate of anti-D alloimmunization in patients requiring urgent RBC transfusion was intermediate between the rates previously reported for D- oncology patients transfused with D+ RBCs and that in immunocompetent volunteer recipients.     

Prospective evaluation of a transfusion policy of D+ red blood cells into D- patients.

BACKGROUND: Although D- patients should receive red blood cells (RBCs) from D- donors, the scarcity of D- blood components in certain situations makes the transfusion of D+ RBCs unavoidable. Therefore it is recommended that guidelines be developed in order to standardize transfusion policy in these scenarios. STUDY DESIGN AND METHODS: We have prospectively evaluated a policy for the use of D+ RBCs in 905 D- patients. The amount of D- RBCs saved as well as the incidence of hemolytic reactions and anti-D alloimmunization were assessed. RESULTS: 554 patients received D- RBCs while 351 received a total of 1032 D+ RBCs, all of them within our criteria for the acceptable use of D+ RBCs. This strategy allowed us to save 25.6 percent of D- RBCs (1032 out of 4024 RBCs requested). No hemolytic reactions were reported. The incidence of alloimmunization was 21.4 percent. Most patients who developed anti-D did so within the first 2 or 4 RBCs transfused (64% after the first 2 RBCs transfused and 88% after the first 4). In multivariate analysis the age of less than 77 years was the only predictor for alloimmuization (HR = 2.48 [95% CI = 1.21-3.81]; p = 0.014). CONCLUSION: The use of D+ RBCs in selected D- patients does not induce adverse reactions and allows the saving of a significant number of D- RBCs.     

Anti-G in a pregnant patient

BACKGROUND: Anti-G is a red cell (RBC) antibody of the Rh system. It has been described in pregnant women only in association with anti-D or anti-C; therefore, the ability of this antibody alone to cause hemolytic disease of the newborn is uncertain. One case in which this antibody caused no clinical sequelae is reported. CASE REPORT: The patient was a 35-year-old primigravida with type O, D-, C-, E-, c+ RBCs who was given 4 units of type O, D- allogeneic RBCs and 2 units of autologous RBCs 2 years antepartum. She was found to have anti-D and anti-C by an outside laboratory as part of a routine prenatal work-up. Further evaluation by our laboratory revealed the presence of anti-G and possible anti-C without anti-D. Titers at 22 weeks' gestation were 64 against r'r RBCs and 16 against R2R2 RBCs; these remained unchanged throughout the pregnancy. Amniocentesis performed at Weeks 28 and 32 showed no evidence of hemolytic disease of the newborn. A healthy 3.3-kg infant was delivered at 36 weeks' gestation. Prophylactic Rh immune globulin was administered antepartum and postpartum. The infant's RBCs were type O, D+, c+ C-, E-, and the direct antiglobulin test was positive. An acid eluate prepared from the baby's RBCs revealed anti-G. The total bilirubin was 5.5 mg per dL at birth, and the hematocrit was 66 percent. Total bilirubin peaked on Day 5 at 11.9 mg per dL, and no therapeutic intervention was required. CONCLUSIONS: Anti-G alone caused little if any fetal or neonatal hemolysis in this case. Although further study is needed, invasive fetal monitoring may be unnecessary if anti-G is the sole cause of fetomaternal RBC incompatibility.