K, Fy(a), and Jk(a) phenotyping of donor RBCs on microplates.

BACKGROUND: In many cases, the search for compatible blood for patients with clinically significant RBC alloantibodies is difficult and time-consuming. To date, it has been considered necessary only to phenotype the blood donors for ABO group and D. There has been long experience with automated routine analysis (ABO, C, c, D, E, and e typing and RBC antibody screening), using robotic dispensers and computerized interpretation of microplate results. The purpose of this study was to explore the possibilities of also phenotyping for K, Fy(a), and Jk(a), as antibodies directed against these antigens (together with Rh antigens) are the most common clinically significant alloantibodies in the Swedish population. STUDY DESIGN AND METHODS: One thousand thirty-one EDTA samples from blood donors were phenotyped for K, Fy(a), and Jk(a) by use of an IAT with PEG on microplates. The findings were compared to those using conventional IAT in tube's and the microcolumn gel test (DiaMed-ID, DiaMed). RESULTS: All typing results with the microplate method were correct. All reactions for K and Fy(a) typing could be interpreted by the computer. The results for Jk(a) were indeterminate in 1.4 percent (14/1031) of the samples. CONCLUSION: The PEG-IAT microplate method gave reliable results that were suitable for routine phenotyping, thus making available a stock of phenotyped blood at reasonable cost, ready for delivery when required.

Clinical evaluation of a solid-phase test for red cell antibody screening of pregnant women.

BACKGROUND: The aims of this study were to evaluate the results of a new solid-phase screening test for detecting atypical red cell (RBC) antibodies in a large number of pregnant women and to compare these results to the clinical outcome of the newborn. STUDY DESIGN AND METHODS: A total of 38,700 infants born in Stockholm were studied retrospectively. Of these infants, 18,500 were born to pregnant women screened with the solid-phase test. Data were collected on all newborns with a positive direct antiglobulin test (DAT) and on infants requiring an exchange transfusion or a blood transfusion. These data were correlated to the screening results for the mothers. RESULTS: Of 409 DAT-positive newborns, a serologic explanation for the positive DAT was found in 349. Three hundred four cases were due to ABO incompatibility between mother and child; 19 of these infants needed an exchange transfusion. Forty-two cases were due to unexpected maternal RBC antibodies; 11 of these infants were given an exchange transfusion. All 11 were identified before birth. Three other infants had DAT-positive tests due to ABO incompatibility and to unexpected maternal RBC antibodies. CONCLUSION: ABO incompatibility is a major indication for exchange transfusion in DAT-positive newborns. There was no evidence that the solid-phase screening test had failed to detect any clinically significant RBC antibodies. Finally, the results of this study do not indicate a need for routine screening of D+ women more than once during each pregnancy.

Evaluation of a solid-phase test for erythrocyte antibody screening of pregnant women, patients and blood donors.

UNLABELLED: We have studied a previously described solid-phase test (SPH) for screening and identification of antibodies directed against erythrocyte antigens, in large sample numbers and throughout routine processing. The screening results of all samples from pregnant women, patients and blood donors (n = 36,701) sent to our blood bank from August 1992 to July 1993 were compiled and evaluated. Pregnant women were screened by the SPH-antiglobulin technique (IAT) and the SPH-enzyme-enhanced technique (ENZ), while the patients and blood donors were screened only by the SPH-IAT. Samples with known alloimmunization were excluded from this study. Positive screening reactions were further investigated by the SPH-IAT and SPH-ENZ techniques, the manual hemagglutination tests low ionic-strength solution IAT and 2-stage papain. Pregnant women: A positive reaction in one or both of the SPH tests was found in 1.2% of the samples. The SPH tests identified 99 (98.0%) and the manual methods 79 (78.2%) of a total of 101 antibodies. Of the identified antibodies, 61 were to antigens in the Rh blood group system, including 34 Rh immune globulin. The remainder had specificity directed against antigens K, Jka, M, S, Le(a) and Le(b). PATIENTS: A positive reaction in the SPH-IAT test occurred in 0.9% of the samples. The antibodies identified had specificity directed against antigens D, C, c and E in the Rh blood group system. The remainder had specificity directed against antigens Fya, K, Jka, M and Le(a). All antibodies identified by SPH-IAT, except 1 anti-M and 2 anti-Le(a), were also identified by manual methods. Blood donors: A positive reaction in the SPH-IAT test was noted in 0.7% of the samples. The identified antibodies had specificity directed against antigens D, C, E, Fya, K and M. All antibodies identified by the SPH-IAT, except 1 anti-D found in a sample from an RhD(+) male, were also identified by the manual methods. This study indicates that the SPH-IAT test is sensitive for detecting anti-M. This was also indicated for the SPH-ENZ test concerning the Jka antibody. An advantage of using solid-phase antibody screening tests for a larger series of samples is the possibility of automated sample identification, dilution, dispensing and interpretation of results. The SPH-IAT and the SPH-ENZ tests permitted us to select test cells, preferably from our own donor pool. We conclude that the SPH-IAT and SPH-ENZ tests are suitable for routine antibody screening of pregnant women and the SPH-IAT test for screening patients and blood donors.

A solid-phase test using antiglobulin and enzyme-enhanced techniques for erythrocyte antibody screening during pregnancy.

We have modified previously described solid-phase tests for erythrocyte antibody screening to develop a method, suitable for antiglobulin- and enzyme-enhanced techniques (SPH-IAT and SPH-ENZ). In this study we compared the SPH tests with an autoanalyzer (AA) technique. The results were more specific with the SPH tests than with the AA. Of 4,234 unselected samples from pregnant women, screen-positive samples were reduced from 96 (2.27%) by the AA, to 56 (1.32%) by the SPH tests. This difference was due to the reduced number of false-positive reactions with the SPH tests, 0.47% compared to 1.44% with the AA. Antibodies detected by the AA and the SPH-IAT and -ENZ were: 9 Rh prophylaxis, 2 anti-c, 1 anti-K and anti-M, and 1 anti-Jka. Antibodies detected only by the SPH tests were 1 anti-K, 1 anti-Le(a) (SPH-IAT and -ENZ), 1 anti-M (SPH-IAT) and 4 anti-Jka (SPH-ENZ). One anti-C, 2 anti-D, 3 Rh prophylaxis and 1 anti-E were detected by the AA and the SPH-ENZ but failed to react by the SPH-IAT. One anti-Le(a) and 8 Rh prophylaxis antibodies were detected by AA only. All clinically important antibodies were detected by the SPH tests. We conclude that the SPH-IAT and SPH-ENZ are screening methods with high specificity that are readily adaptable to larger series of samples from pregnant women and suitable for automated handling throughout the screening and identification process.

Storage of platelets in additive solutions: a new method for storage using sodium chloride solution.

The in vitro effect of 6-day storage of platelets prepared from 6 pooled buffy coat (BC) units and stored in a platelet storage medium containing approximately 40 percent CPD-plasma and 60 percent platelet additive solution (PAS) was evaluated. PAS is composed of sodium and potassium chloride, citrate, phosphate, and mannitol. The total count of platelets per pooled unit included in the in vitro studies (n = 25) was 376 +/- 59 x 10(9) (mean +/- SD). The present study included three steps. 1. Evaluation of platelet storage in one (n = 7) and two (n = 6) 1000-mL polyolefin containers using PAS. During storage in one container, significantly lower values were found for pH, pO2, glucose, ATP, and the ratio of ATP to AMP+ADP+ATP. The values for mean platelet volume, pCO2, lactate, and extracellular adenylate kinase activity were significantly higher. These results indicate that storage in only one polyolefin container is not appropriate for maintaining satisfactory platelet quality. During storage in two polyolefin containers, a remarkably decreased lactate production (0.07 +/- 0.02 mmol/day/10(11) platelets) was noted. 2. PAS was substituted for saline during 6-day storage in two 1000-mL polyolefin containers (n = 12). The composition of the platelet preparations was the same in all other respects. Similar in vitro results were noted with PAS and saline, which indicated that PAS has no specific effect on the storage of platelets different from that of saline.(ABSTRACT TRUNCATED AT 250 WORDS)

A semiautomated method for erythrocyte antigen typing on microtitration plates.

We describe a method for erythrocyte antigen typing on U-bottom microtitration plates. This method permitted phenotyping of erythrocytes by an enzyme-enhanced hemagglutination assay simultaneously with an indirect antihuman globulin assay within the same microtitration plate. A robotic sample processor identified the samples, prepared the red cell suspensions and distributed the suspension along with the diluted antisera onto the microtitration plates. The assay results were read and interpreted using a microtitration plate photometer controlled by software specially designed to interpret agglutination reactions. Sample identities and assay results were automatically connected by the computer system and transmitted to a minicomputer system.

An alloimmunized, thrombocytopenic patient successfully transfused with acid-treated, random-donor platelets.

Alloimmunized, thrombocytopenic patients, refractory to random-donor platelet transfusion, often respond to HLA-identical single-donor platelets. HLA-compatible platelets are expensive, take time to prepare, and donors are sometimes not to be found. We have used random-donor platelets and 'peeled' the HLA-antigen off the platelets, using a modified laboratory method (incubation of platelets with citric acid solution at 0 degrees C). Platelet recovery in two healthy subjects was 72.0% for acid-treated platelets, and 73.5% for untreated control platelets, using 111In-labelled autologous platelets. Survival time (multiple hit) was 6.25 and 7.95 d, respectively. Random-donor platelets that were strongly positive in the crossmatch with serum from a patient became negative after treatment with the acid solution. Furthermore, transfusion of these platelets gave a post-transfusion, platelet-count increment comparable with transfusion of HLA-compatible single-donor platelets.

Minimal hemolytic effect of veno-venous bypass during liver transplantation.

During liver transplantation, extracorporeal veno-venous bypass (VVBP) from the lower to the upper part of the body is used to prevent the negative circulatory effects otherwise seen on clamping the inferior caval vein during the actual change of livers. Extracorporeal circulation can sometimes be complicated by hemolysis. Therefore, we wanted to determine whether the technique used during liver transplantation might also cause hemolysis. This was measured as plasma hemoglobin using a spectrophotometric method including tetramethylbenzidine. Of 11 patients tested, 5 showed an increase in plasma hemoglobin during the use of VVBP; in no case, however, was this clinically significant. Three patients showed short peaks of hemoglobin during bypass. In two patients the rise continued after bypass and they required higher pump speed than the six patients without hemolysis (mean +/- SD 1900 +/- 150 RPM vs 1700 +/- 60 RPM, respectively). Pump flow, time of VVBP, and numbers of transfusions during the transplantation did not differ between the groups. We conclude that VVBP used during liver transplantation may cause hemolysis, but with low frequency and low clinical significance. We further conclude that the spectrophotometric method used is reliable and sensitive enough to determine the degree of hemolysis.

Storage of platelets in a new plastic container. Polyvinyl chloride plasticized with butyryl-n-trihexyl citrate.

The effect of storage of platelets in a new polyvinyl chloride (PVC) plastic material with a butyryl-n-trihexyl citrate (BTHC) plasticizer (PL 2209) was evaluated. The PL 1240 container, i.e. PVC plastic with a different plasticizer, tri-(ethylhexyl)-tri-mellitate, was used as a reference. Measurements of pH, pO2, pCO2, glucose, lactate, adenosine triphosphate, total adenine nucleotide content, lactate dehydrogenase and platelet factor 4 (PF4) were made during 5 days of storage. Similar results were noted comparing PL 2209 and PL 1240. Differences in pO2 and pCO2 indicate greater gas permeability in PL 2209 than in PL 1240. Significantly higher PF4 levels were found in PL 2209, but the difference could not be attributed to the PL 2209 container itself. Paired autologous reinfusion studies (111Indium) of 6 normal donors gave mean recovery values after 5-day storage of 41.1 +/- 7.4% (PL 2209) and 45.5 +/- 7.7% (PL 1240), t1/2 66 +/- 13 and 75 +/- 5 h, survival time (linear model) 6.3 +/- 1.0 and 6.8 +/- 0.7 and survival time (multiple-hit model) 6.0 +/- 0.7 and 6.5 +/- 0.4 days, respectively. Only the difference in survival time (multiple-hit) was significantly higher in PL 1240. The corrected count increments at 12-24 h following transfusion were 13,300 +/- 10,800 (PL 2209) and 13,600 +/- 11,600 (PL 1240) with no statistically significant difference found. These results indicate PL 2209 as an equivalent alternative to PL 1240 for the 5-day storage of platelets.