Screening for WBC antibodies by lymphocyte indirect immunofluorescence flow cytometry: superior to cytotoxicity and ELISA?

BACKGROUND: Cytotoxic WBC antibodies are found in patients who have refractoriness to platelet transfusion (RPT) or are experiencing febrile transfusion reactions (FTRs) and in sera giving so called nonspecific hemagglutination by IAT (N/S IAT). Sera from such patients were screened for WBC antibodies regardless of the ability to fix complement using a flow cytometric (FC) lymphocyte indirect immunofluorescence test (LIFT) to compare FC-LIFT with a routine lymphocytotoxicity test (LCT) for WBC antibody detection. STUDY DESIGN AND METHODS: Serum from 104 patients with RPT, 87 with FTR, and 147 with N/S IAT were tested in parallel by using FC-LIFT and LCT. Sera giving discrepant results were re-tested with an HLA class I antibody ELISA to assess whether they were HLA-specific. RESULTS: Of the sera tested, 175 were LIFT positive, and 146 were LCT positive. Fifty-five had antibodies that were detectable only by LIFT; 26 were positive only by LCT. Of these 81 discrepant sera, 30 of 63 were positive in HLA ELISA. CONCLUSION: FC-LIFT detects more WBC antibodies than does LCT or ELISA, and it is a superior screening technique. Because some cytotoxic antibodies are detectable only by LCT, comprehensive WBC antibody screening would require the application of both techniques. However, because FC assessments of cytotoxicity have been described, LCTs may become redundant for WBC antibody screening.

Autoimmune thrombocytopenia in Waldenström's macroglobulinemia.

Autoimmune phenomena are well-recognised complications of Waldenström's macroglobulinemia (WM) and IgM monoclonal gammopathy. Peripheral neuropathy and cold agglutinin hemolytic anemia are the most common reported and occur in 5-10% of patients. Autoimmune thrombocytopenia has been rarely reported in WM and its incidence is not known. In this study we report the case of a 67-year-old man who presented with autoimmune thrombocytopenia who was subsequently found to have WM. Laboratory investigation demonstrated that platelet-associated IgM (PAIgM) but not PAIgG was clearly elevated compared to normal controls. In addition the patient's serum reacted strongly with a panel of donor platelets analysed with an indirect platelet immunofluorescence assay utilising an anti-IgM secondary antibody. Glycoprotein specificity could not however be demonstrated by ELISA techniques for platelet glycoproteins IIbIIIa, IaIIa, IbIXa, and IV. We also reviewed the case records of 104 additional cases of WM diagnosed at our institution between 5/93 and 5/99. Three further cases with clinically significant autoimmune thrombocytopenia were identified. The overall incidence of autoimmune thrombocytopenia (4/105, 3.8%) in this cohort of patients was similar to the incidence of peripheral neuropathy (7/105, 6.7%) and cold agglutinins (3/105, 2.9%).

The low-incidence MNS antigens M(v), s(D), and Mit arise from single amino acid substitutions on GPB.

BACKGROUND: GPB carries 'N' at its N:-terminus and S and s, determined by a polymorphism at amino acid position 29 (Met29Thr). The low-incidence antigens M(v), s(D), and Mit are associated with weakened expression of S and/or s, and the purpose of this study was to define their molecular bases. METHODS: The GPB gene (GYPB) was sequenced after RT-PCR of RNA from four samples: two M(v)+, one s(D)+, and one Mit+. The point mutations observed were confirmed by sequencing of genomic DNA from these and other examples of s(D)+ and Mit+ samples. RESULTS: A point mutation of 65C>G observed in the M(v)+ samples predicted a change of Thr3Ser. A mutation of C>G at nucleotide 173 of the GYPB coding sequence, observed in two s(D)+ samples, predicted a change of Pro39Arg. Three Mit+ samples showed a nucleotide substitution of 161G>A, which predicted a change of Arg35His. Altered expression of S or s was confirmed by serologic tests. CONCLUSION: These results confirm that Arg35 is important for full expression of S. Pro39 and, surprisingly, Thr3 are also important for full expression of s. Furthermore, Thr3 must be essential for expression of 'N,' as M(v)+ RBCs lack 'N.'

Positive and negative selection to reduce tumour contamination in peripheral blood stem cell harvests.

Peripheral blood progenitor cells used during high dose treatments for malignancy may be contaminated with tumour cells that could later contribute to recurrence. CD34+ selected harvests still contain tumour cells and an additional negative selection may be capable of reducing this contamination. We have assessed a two-stage technique in which a CD34+ selection is followed by a tumour specific depletion stage using a B cell or breast cancer specific antibody panel. Initial small-scale selections on 11 patients with NHL and breast cancer showed that cell loss was greatest following the CD34+ selection with a median yield of 38.8 per cent (range 17. 2-56.4 per cent). The addition of the depletion stage resulted in a minimal loss of CD34+ cells with a yield for this step of 94.2 per cent (range 77.5-99.3 per cent). Clinical scale selections were performed on seven patients with CLL and a median of 2.8x10(6)/kg CD34+ cells (range 1.5-6.1x10(6)/kg) were collected. Cell recovery was 53.3 per cent following CD34+ selection and 76.9 per cent following the tumour specific depletion stage, resulting in a final product containing a median of 1.0x10(6)/kg CD34+ cells (range 0. 55-2.0x10(6)/kg). All unmanipulated harvests were heavily contaminated with tumour cells (median contamination 10.2 per cent, range 2.0-83.1 per cent) as measured by flow cytometry and a median 4.7 log (range 3-5 log) tumour cell purge was produced following two-stage selection. Six of the patients have received cells manipulated in this way with median engraftment times of neutrophils>0.5x10(9)/l=16 days (range 13-20 days) and platelets>20x10(9)/l=16.5 days (range 11-42 days). At a median follow-up of 25 months, these transplanted patients remain well and in molecular complete remission.

Antigen capture ELISA for platelet antibody detection: choice of conjugate influences assay result.

The performance of an anti-IgG/A/M and two anti-IgG alkaline phosphatase (AP) conjugates in a commercial antigen capture ELISA (ACE) were compared for their ability to detect antibodies to human platelet antigens (HPAs) contained in 11 sera. Three anti-HPA-1a and one anti-HPA-3a were not detected by the anti-IgG/A/M conjugate, but both anti-IgG conjugates detected all HPA antibodies as a consequence of increased sensitivity in detecting specific antibody binding. This increase varied from 10% to 500%, depending on the serum being tested. The increased sensitivity in some instances was also accompanied by an apparent increase in nonspecific binding, as measured by activity against irrelevant glycoproteins that should not have been recognized by the relevant HPA antibodies in the sera in question. Hence, anti-IgG conjugates would appear to be preferable for detecting platelet-reactive antibodies in many clinical situations; however, the choice of anti-IgG conjugate should be made judiciously (and appropriately validated), in order to avoid false positive results arising from increased nonspecific binding, which may otherwise be erroneously attributed to the presence of auto-antibodies in the serum under investigation.

Monitoring the clearance of fetal RhD-positive red cells in FMH following RhD immunoglobulin administration.

Anti-RhD immunoglobulin was administered to RhD-negative women based on estimates of fetal bleed size obtained using a direct immunofluorescence flow cytometric technique employing a FITC-conjugated monoclonal human anti-D (BRAD 3). The effectiveness of the dose administered was assessed by (i) measuring the fraction of RhD-positive fetal cells in the maternal circulation at d0, and between d2 and d10 post RhD Ig administration, (ii) quantifying the amount of anti-D detectable in maternal plasma following RhD Ig injection in the perinatal period and (iii) assessing maternal serum for the presence of immune anti-D in follow-up samples taken 3 months to 3 years after delivery. Fifty-four women were assessed, 29 having fetal bleeds in excess of 4 mL. Follow-up samples were received from 20/29 mothers after RhD Ig administration; 43-99% and 69-99% of fetal cells had been cleared by d2/3 and d5/6, respectively, in 14/20 mothers, whereas less than 50% had been cleared in the remaining mothers. Long-term follow-up samples were obtained from eight of the 29 mothers (four with bleeds >/=20 mL, two with bleeds >95 mL): none had detectable anti-D in the serum 4 months to 3 years after delivery despite the persistence of up to 36% fetal RhD-positive cells in the maternal circulation six days after delivery.

The detection by enzyme-linked immunosorbent assays of non-complement-fixing HLA antibodies in transfusion medicine.

BACKGROUND: Noncomplement-fixing white cell antibodies have been demonstrated by the use of immunofluorescence flow cytometry against intact lymphocytes. However, such antibodies may be either HLA-specific or directed against other white cell antigens. Commercial enzyme-linked immunosorbent assay (ELISA) kits, using solubilized HLA molecules as targets, enable such HLA-specific antibodies to be detected in patients who are refractory to platelet transfusion, patients experiencing febrile transfusion reactions, and patients whose sera give nonspecific hemagglutination in indirect antiglobulin tests. STUDY DESIGN AND METHODS: Sera from all three groups of patients, previously screened for cytotoxic antibodies by using complement-dependent lymphocytotoxicity, were re-investigated with commercial ELISA kits for HLA antibody screening and identification using the manufacturers' recommended test methods. RESULTS: Non-complement fixing HLA antibodies were detected by ELISA in many sera that were lymphocytotoxicity test-negative; that is, 14 (17.5%) of 80 from refractory patients, 8 (23.5%) of 34 from those with febrile reactions, and 11 (22.4%) of 49 from those with nonspecific hemagglutination in the direct antiglobulin test. However, not all cytotoxic white cell antibodies were detectable by ELISA: only 19 (82.6%) of 23, 19 (67.8%) of 28, and 11 (73.6%) of 49, respectively in the three groups. Similarly, only 143 (79.4%) of 181 cytotoxic sera with clear-cut HLA-A or -B locus specificities were detectable by ELISA. CONCLUSION: ELISAs detect some but not all clinically significant HLA antibodies, irrespective of their ability to fix complement in vitro.

Quantitating fetomaternal hemorrhages of D+ red cells using an FITC-conjugated IgG monoclonal anti-D by flow cytometry: a case report.

Several methods for quantitating fetomaternal hemorrhages (FMHs) have been described; these include the Kleihauer-Betke and red cell rosetting tests, and flow cytometry that uses an indirect antiglobulin technique, employing either FITC-conjugated IgG/unlabeled anti-D or streptavidin conjugates with biotinylated anti-D to enumerate D+ red cells in maternal blood. We have used a recently described directly conjugated FITC anti-D for direct flow cytometric (direct FC) quantitation of FMH in a patient who presented with a large fetal bleed (approx. 80 mL) as determined using the Kleihauer method. We compared the efficacy of the direct FC technique to the rosetting and Kleihauer tests in estimating the quantity of Rh immunoglobulin (RhIg) to be administered to the mother to suppress Rh alloimmunization. Both the Kleihauer and the direct FC gave precise estimates of 80 mL for the size of bleed, whereas the rosetting test failed to be as precise. The former tests predicted that a 10,000 iu dose (2,000 microg) of RhIg would be adequate; the lack of alloanti-D in a maternal follow-up sample obtained 9 months after delivery supported this prediction and underlined the reliability of the direct FC method as an alternative to the Kleihauer method for quantitating large FMHs.

Serologic characteristics of H-deficient phenotypes among Chinese in Hong Kong.

BACKGROUND: The occasional presence of H-deficient red cells among both referred and donor blood samples prompted the mass screening of donated blood in Hong Kong for H-deficient phenotypes; 96 percent of the donors tested are Chinese from the southern province of Kwongtung. STUDY DESIGN AND METHODS: Donor blood was screened for H-deficient red cells with the use of Ulex europaeus. Lewis phenotyping was carried out on all H-deficient individuals, and saliva testing was performed on most such individuals. The thermal amplitude and potency of their anti-H and anti-HI in the serum were also estimated. RESULTS: Between 1984 and 1993, 28 H-deficient blood donors were identified; 16 H-deficient patient samples were also identified, and family studies revealed an additional 7 H-deficient subjects. The H-deficient red cells did not react with anti-H lectin, the levels of ABH substances in saliva were normal or near-normal, normal levels of A or B transferase were found in plasma, minute quantities of A or B (in persons who were genetically group A or B) were detected on the red cells, and anti-H or anti-HI was detected in the serum (about 66.7% of which reacted at 37 degrees C). Atypical anti-A or anti-B was demonstrated in 81.8 percent of the cases. CONCLUSION: The H-deficient phenotype among the Hong Kong Chinese seems to represent a homogeneous group. Despite the presence of normal quantities of ABH substance in the saliva, anti-H or anti-HI that was active at 37 degrees C was detected in most cases. The incidence of the H-deficient phenotype was 1 in 15,620.

Cytokine levels in platelet concentrates: quantitation by bioassays and immunoassays.

Some adverse reactions to the transfusion of platelet concentrates (PCs) cannot be attributed to antibodies against blood cells or to subclinical microbial agents. It has been suggested that leucocyte-derived inflammatory cytokines such as interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF) may contribute to a larger number of unexplained non-antibody-mediated adverse reactions. Three types of PCs, containing different levels of leucocytes, are currently produced. Filtration is used on demand to further reduce leucocyte contamination of these components. we have monitored the plasma of PCs prepared by the platelet-rich plasma method (PRP), the buffy-coat method or by apheresis for IL-6, IL-1, transforming growth factor-beta (TGF-beta), TNF and interferon gamma (IFN gamma). Biologically active IL-6 increased in stored PRP-PCs from a mean of 140 pg/ml on day 1 to 2395 pg/ml on day 5/6. Elevated levels of IL-8, as detected by immunoassay, were evident in PRP-PCs during routine storage under blood bank conditions. Small amounts of immunoreactive IL-1 with only minimal biological activity were present in some PRP-PCs by day 5/6. No significant increase in the levels of IL-8, IL-6 or IL-1 were seen in buffy-coat PCs during storage for 5/6 d. For apheresis PCs, an increase in IL-8 content, but not in IL-6 over 6 d was observed. In all three types of PCs, elevated amounts of both bioactive and immunoreactive TGF beta were present, but there was no evidence of any biologically active or immunoreactive TNF alpha. Pre-storage filtration of PRP-PCs for depletion of leucocytes prevented the increase in IL-8 and IL-6 levels of these PCs. Our results show that leucocyte reduction by buffy-coat method reduces cytokine levels to a comparable level to filtered or apheresis PCs, containing low levels of leucocytes, but use of these PCs in minimizing the severity and incidence of reactions in recipients will require clinical evaluation. This is the first comprehensive and comparative study which, on the basis of biological activity of cytokines, directly indicates that the mode of platelet production grossly influences the levels of cytokines.

The GLAM test: a flow cytometric assay for the detection of leukocyte antibodies in autoimmune neutropenia.

The GLAM assay, a combined flow cytometric immunofluorescence test that simultaneously detects antibodies to granulocytes, lymphocytes, and monocytes, was used in the investigation of autoimmune neutropenia. This method avoids the need for a succession of immunofluorescence tests, first against granulocytes and then against lymphocytes, in order to distinguish granulocyte-specific from granulocyte/lymphocyte-reactive antibodies, such as noncomplement- fixing anti-HLA sera. Samples from 18 patients with a suspected diagnosis of autoimmune neutropenia were referred for investigation. Leukocytes were harvested in sufficient quantities from 10 of the patients such that neutrophil and lymphocyte direct antiglobulin tests (DATs) and antibody screening and identification could be undertaken (in one case the results were inconclusive). Only three of these 10 patients had DAT-positive granulocytes, and one of these three also had DAT-positive lymphocytes. One further patient demonstrated DAT-positive lymphocytes in the absence of granulocyte-bound IgG, despite a presumed diagnosis of autoimmune neutropenia, rather than pancytopenia. This was the only patient in this cohort who had demonstrable leukocyte antibodies reacting with lymphocytes but not granulocytes. Of the remaining eight patients (not evaluable for granulocyte or lymphocyte DATs), five had free leukocyte antibodies in the serum; three of these five had both granulocyte- and lymphocyte-reactive antibodies free in the serum and two only had granulocyte-specific antibodies.

Flow cytometric analysis of 68 monoclonal anti-D reagents.

68 monoclonal IgG anti-D were assessed for quantitative and qualitative binding to D variant red cells using a flow cytometric indirect immunofluorescence test. One antibody failed to bind to normal RhD pos. cells, but reacted weakly with D cat VI and D Cat VII cells. Quantitatively, binding varied approximately twenty-fold between different MAbs and different D variants. D cat III cells gave the lowest level of binding when compared with the D pos. controls. Qualitatively, R1VIr, R2VIr, R1VIIr and DFR samples failed to react with various MAbs regardless of the levels of binding of these MAbs to the D pos. controls, thus supporting the idea of missing epitopes in such variants.

Prediction of the severity of haemolytic disease of the newborn. Quantitative IgG anti-D subclass determinations explain the correlation with functional assay results.

Sera containing anti-D, taken from 44 RhD-negative women with RhD-positive infants, were tested in antibody-dependent cellular cytotoxicity (ADCC) and monocyte monolayer assays (MMA) which used similar target and effector cell populations. In addition, the anti-D concentration was measured in the Auto Analyzer and the number of IgG1 and IgG3 anti-D molecules bound to the target red cells was measured by flow cytometry. The results of the functional assays and Auto Analyzer quantitation were examined for correlation with IgG subclass quantitation and all results were compared for their ability to predict the severity of haemolytic disease of the newborn (HDN). ADCC correctly predicted HDN in 39/44 (88.6%) cases, Auto Analyzer quantitation in 35/44 (79.5%) and the MMA in 32/44 (72.7%). For all three assays, the number of correct predictions was highest when the maternal serum contained both IgG1 and IgG3 anti-D. ADCC activity and HDN were correlated with the number of cell-bound IgG1 molecules (r > or = 0.58), but MMA activity was most closely correlated with the number of cell-bound IgG3 molecules (r = 0.68). Hence the superior predictive value of ADCC is due to its ability to reflect the IgG1 component of maternal anti-D, which has a better correlation than IgG3 anti-D with the severity of HDN.