Assessment of common red blood cell pretreatments to yield an accurate serologic antigen phenotype compared with genotype-predicted phenotype.

For patients requiring multiple transfusions and patients with positive direct antiglobulin tests (DATs), an extended red blood cell (RBC) phenotype can provide valuable information and help to determine the risk of forming alloantibodies. In some instances, the phenotype may be used for prophylactic matching. Phenotyping in this patient population is often hindered by the presence of circulating donor cells and/or by a positive DAT. Several methods, such as EDTA glycine acid (EGA) treatment to remove IgG, hypotonic saline wash to separate autologous RBCs, or reticulocyte separation, are often used in these situations to isolate patient RBCs for serologic phenotyping. This study aimed to determine the accuracy of each RBC pretreatment method by comparing serologically determined antigen types with those predicted by RBC genotyping. Forty-eight peripheral blood samples from recently transfused patients were phenotyped for selected antigens in the Rh, Kell, MNS, Duffy, and Kidd systems. Treatment methods for the sample sets were reticulocyte separation (N = 12), EGA (N = 16), and hypotonic saline wash (N = 20). DNA was extracted using standard methods, and genotyping was performed using the HEA BeadChip panel. In addition, 21 samples positive for RBC-bound IgG were EGA-treated up to two times. These samples were analyzed pre- and post-EGA treatment for RBC-bound IgG by tube DAT and by flow cytometry with fluorescein isothiocyanate-labeled anti-human IgG. After reticulocyte separation, 3 of the 12 samples had discordant types with one antigen each: Fyb, N, and K; serologic results were negative compared with genotype-predicted positive phenotype results. The EGA-treated sample set showed one discordant type: Fyb; serologic results were negative compared with genotype-predicted positive phenotype results. Four of the 20 samples had discordant types involving the following antigens: Fyb, N, e, and M; serologic results were negative compared with genotype-predicted positive phenotype results. After EGA treatment of 21 samples, 14 (67%) were negative for RBC-bound IgG by tube DAT, and 7 remained positive. Using flow cytometry, EGA treatment rendered only 4 samples negative, and 17 remained positive. In the antigen testing sample set of 48 samples, 10 of 511 total antigen types tested were discordant. Discordant types were most frequent in the hypotonic saline wash sample set (N = 6). In the flow cytometry sample set, 48 percent of the samples negative by tube DAT after EGA elution had detectable RBC-bound IgG by flow cytometry. These findings suggest that caution should be taken when using phenotype results from all pretreated RBCs and support the use of RBC genotyping to predict RBC antigen expression in samples from recently transfused patients.For patients requiring multiple transfusions and patients with positive direct antiglobulin tests (DATs), an extended red blood cell (RBC) phenotype can provide valuable information and help to determine the risk of forming alloantibodies. In some instances, the phenotype may be used for prophylactic matching. Phenotyping in this patient population is often hindered by the presence of circulating donor cells and/or by a positive DAT. Several methods, such as EDTA glycine acid (EGA) treatment to remove IgG, hypotonic saline wash to separate autologous RBCs, or reticulocyte separation, are often used in these situations to isolate patient RBCs for serologic phenotyping. This study aimed to determine the accuracy of each RBC pretreatment method by comparing serologically determined antigen types with those predicted by RBC genotyping. Forty-eight peripheral blood samples from recently transfused patients were phenotyped for selected antigens in the Rh, Kell, MNS, Duffy, and Kidd systems. Treatment methods for the sample sets were reticulocyte separation (N = 12), EGA (N = 16), and hypotonic saline wash (N = 20). DNA was extracted using standard methods, and genotyping was performed using the HEA BeadChip panel. In addition, 21 samples positive for RBC-bound IgG were EGA-treated up to two times. These samples were analyzed pre- and post-EGA treatment for RBC-bound IgG by tube DAT and by flow cytometry with fluorescein isothiocyanate­labeled anti-human IgG. After reticulocyte separation, 3 of the 12 samples had discordant types with one antigen each: Fyb, N, and K; serologic results were negative compared with genotype-predicted positive phenotype results. The EGA-treated sample set showed one discordant type: Fyb; serologic results were negative compared with genotype-predicted positive phenotype results. Four of the 20 samples had discordant types involving the following antigens: Fyb, N, e, and M; serologic results were negative compared with genotype-predicted positive phenotype results. After EGA treatment of 21 samples, 14 (67%) were negative for RBC-bound IgG by tube DAT, and 7 remained positive. Using flow cytometry, EGA treatment rendered only 4 samples negative, and 17 remained positive. In the antigen testing sample set of 48 samples, 10 of 511 total antigen types tested were discordant. Discordant types were most frequent in the hypotonic saline wash sample set (N = 6). In the flow cytometry sample set, 48 percent of the samples negative by tube DAT after EGA elution had detectable RBC-bound IgG by flow cytometry. These findings suggest that caution should be taken when using phenotype results from all pretreated RBCs and support the use of RBC genotyping to predict RBC antigen expression in samples from recently transfused patients.

Confirmation that the JAHK antigen is associated with the rG haplotype.

Anti-JAHK, an antibody directed toward a low-incidence antigen in the Rh system, was detected during routine antibody identification in a male donor who had no history of transfusion. Examples of anti-JAHK have been found in sera containing multiple antibodies to low-incidence antigens. The first report of anti-JAHK was in 1995 and described the association of the JAHK antigen with the rG haplotype. Our results confirm this association.

Mta: review and case report.

Anti-Mta, which recognizes an antigen in the MNS blood group system, was detected during prenatal testing of a para 6, gravida 1 woman with no history of transfusions. Her husband was apparently Mt(a-). Anti-Mta was first reported in 1962 as a naturally occurring antibody directed against a new antigen in the MNS system. The last report in the literature of detection of anti-Mta was in 1972.

Co-presence of a point mutation and a deletion of exon 3 in the glycophorin C gene and concomitant production of a Gerbich-related antibody.

BACKGROUND: Antigens of the human blood group system Gerbich (Ge) are located on sialoglycoproteins glycophorin C (GPC) and glycophorin D (GPD). CASE REPORT: The Ge+ proposita (RW) produced an alloanti-Ge after receiving 2 units of red cells (RBCs) during surgery. Further studies were carried out to characterize the antibody specificity, RBC GPC and/or GPD (GPC/GPD), and the glycophorin C gene (GYPC) from RW and her compatible siblings. RW's serum contained an alloanti-Ge that did not react with RBCs from RW or four of her siblings or with RBCs with Ge-negative phenotypes. An eluate of RW's antibody reacted weakly with GPC in Western blotting. RW's RBCs were positive with 20 alloanti-Ge2, 1 autoanti-Ge2, 4 alloanti-Ge3, and 1 alloanti-Ge4. Titrations revealed weak expression of these antigens on her RBCs and those of her compatible siblings as compared with controls. In contrast, titrations of mouse and rat monoclonal antibodies specific for GPC/GPD showed no differences. Western blotting of RBC membranes using GPC/GPD specific monoclonal antibodies showed a broad diffuse band corresponding to GPC.Ge in addition to GPC and GPD. Blotting of membranes from trypsin-treated RBCs from these individuals revealed an increase of 1500 in M(r) of membrane-bound tryptic fragment over that in the membranes from typsin-treated RBCs from persons with normal GPC/GPD. In RT-PCR, two products were obtained for RW and her compatible siblings: one had a complete deletion of exon 3 and the other had a base change (A-->T) in nucleotide 173 in exon 3 (confirmed by genomic DNA sequencing of exon 3). This point mutation has resulted in the loss of restriction enzyme Tth111 I-sensitive site in the mutant GYPC. CONCLUSION: The specificity of antibody in RW's serum was serologically anti-Ge2. Two genetic events occurred in exon 3 in GYPC of RW and her compatible siblings. The exon 3 deletion confirmed a Ge:-2,-3,4 haplotype. The abnormal tryptic fragment obtained was due to the (A173-->T) base change in exon 3 that resulted in Asp58-->Val in the deduced amino acid sequence at the membrane boundary.

A 4, 2, and 1 stepping algorithm for quick and accurate estimation of cutaneous sensation threshold.

In quantitative sensory testing, certain methods may lead to incorrect estimates of vibratory (VDT), cool (CDT), or warm (WDT) detection thresholds. We have shown that the specific forced-choice algorithm of testing employed in our Computer-Assisted Sensory Examination (CASE IV) system, when compared with other tests of nerve dysfunction, provides accurate and reproducible estimates of these thresholds. Because this forced-choice algorithm is time consuming and performance might be made worse by drowsiness or boredom, we explored other algorithms that might provide estimates of threshold similar to those obtained with the forced-choice algorithm, but more quickly. In a trial of 25 healthy subjects and 25 patients with neuropathy, the 4, 2, and 1 stepping algorithm with null stimuli, based in part on comparative data from computer simulation and insights from patient decision making, provides an accurate estimate of threshold. On average, the time needed for forced-choice testing was 12.8 +/- 2.9 minutes (mean +/- SD). For 4, 2, and 1 stepping testing, it was 2.7 +/- 2.5 minutes--a large saving of time. Since null stimuli were employed in the 4, 2, and 1 stepping algorithm, it was possible to monitor for spurious responses and repeat the test if they occurred at an excessive rate. The algorithm appears to be sufficiently robust to be recommended for clinical use and for some controlled clinical and epidemiologic trials.

Slow rehydration improves the recovery of dried bacterial populations.

Slow rehydration of bacteria from dried inoculant formulations provided higher viable counts than did rapid rehydration. Estimates were higher when clay and peat powder formulations of Rhizobium meliloti, Rhizobium leguminosarum biovar trifolii, and Pseudomonas putida, with water activities between 0.280 and 0.650, were slowly rehydrated to water activities of approximately 0.992 before continuing the dilution plating sequence. Rhizobium meliloti populations averaged 6.8 x 10(8) cfu/g and 1328 cfu/alfalfa seed greater when slowly rehydrated from bulk powder and preinoculated seeds, respectively. Bulk powder samples were slowly rehydrated to 0.992 water activity by the gradual addition of diluent, followed by a 10-min period for moisture equilibration. Preinoculated seed samples were placed in an environmental chamber at 24 degrees C with relative humidity greater than 80% for 1 h to allow moisture absorption. "Upshock," osmotic cellular stresses that occur during rehydration, was reduced when dried microbial formulations were slowly rehydrated and equilibrated before becoming fully hydrated in the dilution plating sequence. These procedures may also be applicable when estimating total viable bacterial populations from dried soil or other dry formulations.