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.

Theoretical constraints on the precision and age range of rehydroxylation dating.

Accurate and precise dating methods are of central importance to archaeology, palaeontology and earth science. This paper investigates the expected precision and age range of rehydroxylation dating, a recently proposed technique for fired clays. An expression for combined measurement uncertainty is presented, which takes into account all significant sources of experimental uncertainty. Numerical simulations are performed for comparison. Combined measurement uncertainties of approximately 5% with respect to the age of the ceramic should be possible given well-designed experiments. In this case, the most significant contribution to combined measurement uncertainty is from effective lifetime temperature. In addition, it is shown that precision should be acceptable for recently fired material (less than 1 year). Mismatch of balance resolution to sample mass results in large variation in combined relative uncertainties, which vary by four orders of magnitude (approx. 1-1160%) across recent experimental studies, rendering some recently reported dates meaningless. It is recommended that this ratio be less than 10(-6) for a combined relative uncertainty of less than 1%. The age limits of the technique are set by the value of the rate constant and individual sample mineralogy. This theoretical framework should help future interlaboratory comparison as well as optimizing instrument design.

GIL: a red cell antigen of very high frequency.

A new high-frequency red cell antigen has been identified and named GIL. GIL differs from all high-frequency antigens included in the International Society of Blood Transfusion classification. There is very little family information and GIL has not been shown to be an inherited character. Five women with anti-GIL have been found. All had been pregnant at least twice. Red blood cells of two of the babies gave positive direct antiglobulin tests, but there were no clinical signs of hemolytic disease. Anti-GIL may have been responsible for a hemolytic transfusion reaction and results of monocyte monolayer assays of two of the anti-GIL suggested a potential to cause destruction of transfused GIL+ RBCs.

Comparison of hemagglutination and solid phase titration methods for determination of critical prenatal antibody titers.

The hemagglutination (HA) tube method is the standard method for determination of antibody titer in prenatal samples. Most facilities use a titer between 8 and 32 as their definition of a critical value when amniocentesis may be considered. This study determined if there is a relationship between the results of HA tube and solid phase (SP) titers performed on the same sample. Forty-six paired samples containing known antibody were titrated by both HA tube and SP methods and the results subjected to data analysis. We conclude that there is a strong correlation between HA tube and SP methods in titer end points; i.e., SP was greater than or equal to HA in every case. In addition, the appropriate critical SP titer is 64, which is at least equivalent to an HA critical titer of 16.

Expression of the erythrocyte antigen Henshaw (He; MNS6): serological and immunochemical studies.

Production of murine monoclonal antibodies to the low prevalence MNS antigen Henshaw (He; MNS6) has enabled more detailed study of this antigen. Using these directly hemagglutinating anti-He, red blood cells (RBCs) from 1695 people of African origin were screened in the USA and England. The prevalence of He+ samples among these donors was 2.1%. In Natal, blood samples from 1218 black donors were screened with rabbit anti-He. The prevalence of He+ donors in this population was 7.0%. Immunoblotting confirmed that the He antigen is carried on an erythrocyte membrane component with a molecular mass that is indistinguishable from glycophorin B. Hemagglutination and immunoblotting demonstrated that ten of 56 He+ samples tested more extensively had a reduced expression of the He antigen. The majority of He+ RBCs were S+; those He+ RBC samples that were S-s+ more frequently had a weakened expression of He.

Autologous and allogeneic red cell survival studies in the presence of autoanti-AnWj.

Chromium survival studies were performed with AnWj-positive allogeneic blood in a patient with autoanti-AnWj. 99mTc-labeled autologous RBCs that had depressed AnWj expression had normal survival (77% [94.7% 51Cr equivalent]) at 24 hours, whereas 51Cr-labeled allogeneic AnWj-positive cells had 76 percent survival at 24 hours and 55 percent survival at 7 days. These studies suggest that the specificity of the autoantibody may have implications for transfusion therapy when the development of such autoantibodies is associated with decreased antigen expression on the patient's cells.

Selection of tests and services to generate revenue for reference laboratories.

The 1980s have brought heightened awareness of the operating costs of immunohematology reference laboratories. Revenues can be increased by raising existing charges, expanding services, and acquiring new customers. Selection of tests and services to further increase revenue is best made following a thorough assessment of community needs and the capabilities and resources of the reference Laboratory.

Anti-JMH identified in serum and in eluate from red cells of a JMH-negative man.

Anti-JMH was identified in the serum of an 80-year-old JMH-negative man. Before transfusion, his direct antiglobulin test was weakly positive with polyspecific reagents, anti-C3 and anti-IgG. An eluate prepared from his red cells contained anti-JMH. Chromium-51-labeled JMH-positive cells which were weakly incompatible in vitro appeared to survive normally. Following transfusion with three JMH-positive units, the patient's hematocrit increased from 20.7 percent to 32.1 percent.