Biochemical and structural changes in RBCs stored with different plasticizers: the role of hexanol.

BACKGROUND: PVC containers are plasticized with di(2-ethyl)hexylphthalate (DEHP) or a related phthalate. The toxicity of DEHP has been questioned. It has been proposed to use butyryltrihexylcitrate (BTHC) as the plasticizer. The purpose of this study was to determine if hexanol, a component of BTHC, plays a role in the preservation of RBCs stored in BTHC-plasticized PVC bags. STUDY DESIGN AND METHODS: WBC-reduced RBCs of ABO- and D-matched blood groups were prepared in 1-L polyolefin (PO) bags (PL732). Six 60-g aliquots were transferred to transfer packs made of PL146 (DEHP-plasticized) and PL2209 (BTHC-plasticized) and four PO (PL732) packs. To the PL146 and PL2209 packs, 30 mL of AS-1 was added. To three of the PO packs, 30 mL of AS-1 with sufficient DEHP, BTHC, or hexanol to achieve a final concentration of 3 mM was added, and to the final PO pack, 30 mL of AS-1 only was added (control). The units were stored for 6 weeks at 1 to 6 degrees C. RBC ATP, hemolysis, morphology, membrane lipids, deformability, and fluidity were measured. RESULTS: ATP levels were not significantly different in any of the systems after 6 weeks. Compared to the PO bags, hemolysis was lowest in the PL146 containers and was also significantly lower (p < 0.006) in the PO bags with added DEHP, BTHC, or hexanol. The accumulation of vesicles was significantly less in the units stored in the PL146 and PL2209 than in the PO plastic with or without added plasticizers or hexanol (p < or = 0.004). There was no significant difference in the formation of vesicles in any of the PO units (p > 0.05). There was no demonstrable change in the membrane fluidity of the RBCs during storage in any of the systems. The decrease in deformability was the same, and the losses of cholesterol and phospholipid during storage were similar in all the studies. CONCLUSIONS: The hexanol component of the BHTC plasticizer in a concentration of 144.6 microg per mL concentration suppresses hemolysis and vesiculation of RBCs during storage. The hexanol and DEHP that are slowly leached during storage have a greater effect in suppressing hemolysis and vesicle formation than when added extraneously to AS-1 in PO containers.

Glutathione protects chemokine-scavenging and antioxidative defense functions in human RBCs.

Oxidant stress, in vivo or in vitro, is known to induce oxidative changes in human red blood cells (RBCs). Our objective was to examine the effect of augmenting RBC glutathione (GSH) synthesis on 1) degenerative protein loss and 2) RBC chemokine- and free radical-scavenging functions in the oxidatively stressed human RBCs by using banked RBCs as a model. Packed RBCs were stored up to 84 days at 1-6 degrees C in Adsol or in the experimental additive solution (Adsol fortified with glutamine, glycine, and N-acetyl-L-cysteine). Supplementing the conventional additive with GSH precursor amino acids improved RBC GSH synthesis and maintenance. The rise in RBC gamma-glutamylcysteine ligase activity was directly proportional to the GSH content and inversely proportional to extracellular homocysteine concentration, methemoglobin formation, and losses of the RBC proteins band 3, band 4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffy antigen (P < 0.01). Reduced loss of Duffy antigen correlated well with a decrease in chemokine RANTES (regulated upon activation, normal T-cell expressed, and secreted) concentration. We conclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSH synthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, in which RBCs are subjected to chronic/acute oxidant stresses.

Glutathione loading prevents free radical injury in red blood cells after storage.

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1-6 degrees C for 0, 42 and 84 days in a conventional additive solution (Adsol) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4-6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (14C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (approximately 50%), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.

Protein and lipid oxidation of banked human erythrocytes: role of glutathione.

In banked human erythrocytes (RBCs), biochemical and functional changes are accompanied with vesiculation and reduced in vivo survival. We hypothesized that some of these changes might have resulted from oxidative modification of membrane lipids, proteins, or both as a result of atrophy of the antioxidant defense system(s). In banked RBCs, we observed a time-dependent increase in protein clustering, especially band 3; carbonyl modification of band 4.1; and malondialdehyde, a lipid peroxidation product. Examination of the antioxidative defense system showed a time-dependent decline in glutathione (GSH) concentration and glutathione-peroxidase (GSH-PX) activity, with a concomitant increase in extracellular GSH, cysteine, and homocysteine, and unchanged catalase activity. When subjected to acute oxidant stress by exposure to ferric/ascorbic acid or tert-butylhydroperoxide (tert-BHT), catalase activity showed a steeper decline compared with GSH-PX. The results demonstrate that GSH and GSH-PX appear to provide the primary antioxidant defense in stored RBCs, and their decline, concurrent with an increase in oxidative modifications of membrane lipids and proteins, may destabilize the membrane skeleton, thereby compromising RBC survival.

The effect of hypotonicity, glutamine, and glycine on red cell preservation.

BACKGROUND: Red cells (RBCs) stored in hypo-osmolar additive solutions with the same concentrations of adenine, dextrose, mannitol, and sodium chloride and varied amounts of ammonium, phosphate, glycerol, and glutamine were better preserved than RBCs in the standard additive solution (Adsol). Cell swelling occurred in all the experimental additives. This observation prompted the evaluation of glutamine and glycine alone, as well as a combination of glutamine and glycine, all of which have been described as producing swelling of rat liver cells. STUDY DESIGN AND METHODS: Aliquots of RBCs were stored at 4 degrees C in Adsol or experimental additive solutions (EASs) all containing adenine, 2 mM; dextrose, 110 mM; mannitol, 55 mM; and sodium chloride, 50 mM. EAS 42 had, in addition, glutamine, 10 mM; glycine 5 mM, and phosphate, 20 mM. EAS 43 had glutamine, 10 mM; glycine, 10 mM; and phosphate 20 mM. EAS 44 had glutamine, 10 mM; EAS 45 had glutamine, 10 mM, and phosphate, 20 mM, and EAS 46 had only glycine, 10 mM. At intervals, measurements were made of mean corpuscular volume, mean corpuscular hemoglobin concentration, morphology, ATP, hemolysis, supernatant potassium, ammonia, pH, and microvesicles shed. RESULTS: The initial mean corpuscular volumes were larger in all EASs than in Adsol, but the greatest difference was between EASs 44 and 46 (108 fL) and Adsol (86 fL) (p < 0.001). The morphology scores were significantly better in all the EASs (p < 0.04). The ATPs were significantly greater in all the EASs (p < 0.001), and highest in those with phosphate. potassium leakage and hemolysis were less in the EASs (p < 0.001). The ammonia levels higher in all the EASs than in Adsol, with the exception of EAS 46. During storage, the extracorpuscular and intracorpuscular pH levels were essentially identical. The shedding of microvesicles was greatly reduced in all the EASs. CONCLUSION: Cell swelling induced in RBCs after collection appears to improve preservation. Ammonia and phosphate enhance RBC ATP maintenance. Glycine decrease the formation of ammonia by RBCs stored in a hypotonic medium.

Effect of a glycerol-containing hypotonic medium on erythrocyte phospholipid asymmetry and aminophospholipid transport during storage.

Previous studies from our laboratory have shown that under blood bank storage conditions red blood cell (RBC) ATP and lipid content were better maintained in a glycerol-containing hypotonic experimental additive solution (EAS 25) than in the conventional storage medium Adsol. The objective of this study was to determine the mechanism of the protective effect of EAS 25, by measuring transmembrane phospholipid asymmetry and the membrane integrity of stored RBCs. Split units of packed RBCs were stored in either EAS 25 or Adsol. RBCs were analyzed after 0, 42, and 84 days and vesicles shed from stored RBCs were analyzed after 84 days of storage. Phospholipid asymmetry was measured by phospholipase A2 digestion (RBCs) and activation of the prothrombinase complex (RBCs, vesicles). RBC membrane exhibited a significantly greater (P < 0.01) amount of phosphatidylethanolamine externalized after storage in Adsol than in EAS 25 (44.3% +/- 11.7 vs. 25.3% +/- 5.7, respectively). Prothrombin converting activities in RBCs were significantly lower than in shed vesicles (P < 0.001) suggesting the presence of phosphatidylserine in the outer monolayer of vesicle, but not in RBC membranes. The rates of inwardly-directed aminophospholipid transport in RBCs decreased by 50% and glutathione levels decreased by approximately 50% in both media. RBC cholesterol and phospholipid content of stored RBCs remained significantly greater (P < 0.01) in EAS 25 than in Adsol. The results indicate that despite comparable reduction in the rate of aminophospholipid transport and reduced GSH concentrations, RBC phospholipid asymmetry was better maintained during storage in EAS 25 than in Adsol. The data suggest that glycerol in the hypotonic EAS helps preserve RBC lipid organization and membrane integrity during storage.

Glutamine- and phosphate-containing hypotonic storage media better maintain erythrocyte membrane physical properties.

We have shown that red blood cell (RBC) adenosine-5'-triphosphate (ATP) is better maintained and that there is less hemolysis and K+ leakage in hypotonic experimental additive solutions (EASs) containing glutamine and glutamine plus phosphate (Pi) than in the conventional additive solution Adsol during blood bank storage. The objective of this study was to determine if the beneficial effect produced in these media correlates with better preservation of RBC membrane properties including lipid content, phospholipid organization, aminophospholipid transport (flippase), and prothrombin converting activity. Aliquots of packed RBCs were stored in EASs containing adenine, glucose, sodium chloride, and mannitol, with 10 mmol/L glutamine (EAS 44) or with 10 mmol/L glutamine and 20 mmol/L Pi(EAS 45), or in Adsol. RBC membranes were studied after 0, 28, 42, and 84 days of storage, and vesicle membranes were studied after 84 days. RBC cholesterol and phospholipid content remained significantly greater (P < .01) in EASs than in Adsol. The degree of membrane vesiculation was more than 50% lower in EASs than in Adsol (P < .01). After 42 days of storage, the accessibility of phosphatidylethanolamine to phospholipases was approximately 1.5 times greater for Adsol and EAS 44 samples than for EAS 45 samples (43.5% v 28%). The rates of phosphatidylserine transport were 43% to 70% lower for stored cells but were not dependent on storage media. The amounts of bands 3 and 4.1 in the microvesicle membranes were not statistically different in any of the preparations. These results suggest that storage of RBCs in glutamine and Pi-medium better maintains ATP, lipid content, and phospholipid asymmetry and results in decreased vesiculation.

Separation of mixed red cell populations by using microbead columns.

The methods available for separating mixed populations of red blood cells (RBCs) are not completely satisfactory. The purpose of this study was to evaluate the utility of microbead columns for the separation of mixtures of RBCs of different blood groups. Suspensions of RBCs positive for nine different blood group antigens were mixed with RBCs lacking the antigen so that 1%, 5%, 10%, 15%, or 25% at the antigen-positive RBCs were represented. After agglutination by the appropriate antiserum, the antigen-positive RBCs were separated from the antigen-negative RBCs by using microbead columns. The average recovery of the antigen-negative RBCs in the effluent of the columns of the 135 mixtures of RBCs tested was 83% +/- 5% (mean +/- SD). The absence of contaminating antigen-positive RBCs was established serologically and by flow cytometry. The procedure was effective in removing as little as 1% of antigen-positive cells from a mixture. Microbead columns offer a simple and efficient method for separating mixtures of RBCs for biochemical, clinical, and serologic studies.

Improved red blood cell preservation correlates with decreased loss of bands 3, 4.1, acetylcholinestrase, and lipids in microvesicles.

In earlier studies we have shown that a final concentration of 0.69% glycerol in blood mixed with an experimental additive solution, EAS 25, improves the in vitro quality and in vivo survival of red blood cells (RBCs). The objective of this study was to determine if the better preservation of RBCs in EAS 25 is correlated with the improved maintenance of membrane lipids and proteins and decreased vesiculation. Split units of RBCs were stored in Adsol or EAS 25 (mmol/L: adenine 2/2, dextrose 122/110, mannitol 42/55, glycerol 0/150, NaCl 154/50). After 12 weeks storage, RBC and microvesicle membranes were analyzed for cholesterol, phospholipid, diphenyl hexatriene fluorescence anisotropy, and acetylcholinesterase (AchE) activity. Bands 3 and 4.1 were identified in the microvesicle membranes by immunoblotting. The RBC membrane cholesterol, phospholipids, and AchE remained higher in EAS 25 than in Adsol (P < .001). Vesicle membrane lipids and AchE in EAS 25 were significantly less than in Adsol (P < .001). The fluidity of stored cells in both the solutions was greater than the prestorage samples. Immunoblotting analyses showed that bands 3 and 4.1 were greatly reduced in the microvesicle membranes shed by the RBCs stored in EAS 25 compared with those formed in Adsol.

Studies in red blood cell preservation 10. 51Cr recovery of red cells after liquid storage in a glycerol-containing additive solution.

The purpose of the present study was to compare the 24-hour recovery of red blood cells stored for 9 weeks in a hypoosmolar additive solution containing 150 mM glycerol to cells stored in Adsol. Seven units of packed red cells were split into 2 aliquots. To one sample, 100 ml of the experimental additive solution (EAS 25) was added, and to the other, 50 ml of Adsol. At the end of the storage period the cells were labeled with 51Cr. A double chromium technique was used to make it possible to perform comparative autologous studies in the same donor. The 24-hour 51Cr recovery value for EAS 25 was 73.0 +/- (SD) 4.2% and for Adsol 60.9 +/- 7.1. At 9 weeks the adenosine triphosphate levels were not significantly better compared to Adsol but the other in vitro measurements were better. New approaches to the study of red cell preservation are suggested.

The protein composition of red cell eluates.

BACKGROUND: The protein composition of red cell (RBC) eluates has not been extensively studied. The purpose of this study was to determine the IgG content and protein composition of RBC eluates prepared by the acid and xylene elution methods. STUDY DESIGN AND METHODS: Six samples of group O R1R1 RBCs sensitized with anti-D in vitro, six nonsensitized samples of the same group O R1R1 RBCs, and six samples from patients with warm autoimmune hemolytic anemia (WAIHA) were studied. The eluate protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoglobulin concentrations were estimated by an immunoblot technique using horseradish peroxidase-conjugated anti-IgG and 3,3' diaminobenzidine. RESULTS: The protein concentrations of the xylene eluates were significantly greater than those of the acid eluates (37.3 +/- 10.7 and 3.0 +/- 0.4 [SD], respectively; p < 0.005). In all samples, the proportion of IgG was less than 0.13 percent of the total protein content. The acid eluates of sensitized RBCs contained more IgG than the xylene eluates. The antibody titers of eluates from WAIHA RBCs were significantly lower than eluates of in vitro sensitized RBCs (p < 0.005). The estimated molecular weights of the Coomassie blue-stainable protein bands from xylene eluates were 97, 78, 63, 45, 31, 23, and 16 kDa, and those of bands from acid eluates were 97, 78, and 55 kDa. No periodic acid-Schiff reagent-stainable bands were detected. CONCLUSION: These data indicate that IgG represented only a small fraction of the proteins in the eluates and that the protein composition varies with the elution procedure.

Selective inhibition by alcohol and cortisol of natural killer cell activity of lymphocytes from cord blood.

1. The immunosuppressive effects of drugs such as alcohol or hormones such as cortisol may be age-related. To test this hypothesis, the authors investigated the in vitro effects of ethanol (EtOH) and cortisol on Natural Killer (NK) cell activity of lymphocytes from normal cord blood in comparison with that of lymphocytes from normal adult peripheral blood. 2. K562, an erythroleukemia cell line, was used as a target in a 4 hr 51Cr release assay. 3. Ethanol at 0.3% (V/V) and cortisol at 0.05, 0.1 and 0.2 microgram/ml concentrations, added directly to a mixture of effector and target cells significantly suppressed the NK activity of cord blood lymphocytes in a dose dependent fashion, whereas similar concentrations of either EtOH or cortisol did not manifest significant immunoregulatory effects on NK cell activity of normal adult lymphocytes. 4. Pre-treatment of the target with either EtOH or cortisol for 4 hours did not affect cytotoxicity. Inhibition of cytotoxicity was also not due to direct toxicity of effector cells because lymphocytes treated with either EtOH or cortisol showed normal 51Cr release and their viability was comparable to that of untreated control cells. 5. This suggests a selective inhibitory effect of EtOH and cortisol on NK activity of neonatal lymphocytes that may be of clinical significance.

Studies in red blood cell preservation. 8. Liquid storage of red cells in a glycerol-containing additive solution.

The purpose of the present study was to determine whether a hypotonic additive containing a low concentration of glycerol as a membrane permeable solute would improve the liquid storage of red blood cells (RBCs). Packed RBCs were stored either with 200 ml of an experimental additive solution, EAS 25, containing (mM): glycerol 150, adenine 2, glucose 110, mannitol 55, and NaCl 50, or with 100 ml/unit of a conventional additive solution Adsol. The results show that the adenosine triphosphate values, hemolysis, potassium leakage, and the morphology scores of RBCs were significantly better with EAS 25 than with Adsol up to 84 days of storage. The ATP values were significantly different only after the first 42 days of storage. The mean corpuscular volumes (MCVs) of the RBCs were significantly higher throughout in the experimental additive accompanied by decreased microvesiculation as compared to Adsol. The total microvesicle membrane protein shed by 100 ml of RBCs was 47.92 +/- 12.31 mg in Adsol and 18.96 +/- 5.49 mg in EAS 25 (p < 0.001). The larger MCVs of the RBCs in EAS 25 may have a favorable effect on maintaining membrane integrity by decreasing the loss of membrane by microvesiculation.

Studies in red blood cell preservation: 9. The role of glutamine in red cell preservation.

Previous studies have demonstrated that an additive solution containing ammonium chloride (NH4+) and phosphate (Pi) in addition to adenine, glucose and mannitol would support red blood cell (RBC) in vitro characteristics and in vivo 24-hour viability after storage for 9 weeks. The purpose of the present study was to determine if NH4+ generated by the action of glutaminase on glutamine could be substituted for added NH4+ salts. Packed RBCs were stored with equal volumes of adenine, glucose, mannitol, and citrate containing additive solutions with 10 mM glutamine (EAS 31) or with 10 mM glutamine and either 10 (EAS 36) or 20 mM (EAS 37) Pi. One aliquot was stored with Adsol. The mean ATP levels of the RBCs stored in the glutamine plus phosphate EASs were 132 (10 mM Pi) and 144% (20 mM Pi) of the initial levels at 28 days, and at 84 days remained at 48 and 56%, respectively. The ATP levels of the RBC stored in Adsol were 105 and 25% at 28 and 84 days of storage, respectively. Percentage hemolysis and vesiculation was significantly lower (p < 0.01) for RBCs stored in glutamine and glutamine plus phosphate as compared to RBCs stored in Adsol. The levels of NH4+ were 22 to 34% higher in the EASs than in Adsol at the end of 84 days of storage, suggesting that glutamine is broken down by glutaminase to generate NH4+. The mean corpuscular volumes (MCVs) of RBCs in EASs 36 and 37 were substantially higher than in Adsol throughout the course of storage (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Studies in red blood cell preservation. 7. In vivo and in vitro studies with a modified phosphate-ammonium additive solution.

Studies were conducted to examine whether an experimental additive solution (EAS-2) containing, in mM: 20 NH4Cl, 30 Na2HPO4, 2 adenine, 110 dextrose, 55 mannitol, pH 7.15, would be useful to extend the storage shelf life of human RBCs. With 6 pairs of split units, ATP concentrations were better maintained for 12 weeks with EAS-2 than with Adsol (1.8 vs. 1.1 mumol/g Hb, respectively, p = 0.002). Autologous 24-hours 51Cr viability values for split units in the same donors were: on 6 paired units at 8 weeks, EAS-2 87.0 +/- 4.5%, Adsol 72.6 +/- 2.3%, p = 0.004; on 11 paired units at 9 weeks, EAS-2 79.5 +/- 7.1%, Adsol 68.2 +/- 10.1%, p = 0.0003. The data suggest that packed RBCs stored for 9 weeks with EAS-2 will be suitable for transfusion following the removal of supernatant with a single washing step.

Expression of blood group antigens on red cell microvesicles.

The purpose of this study was to determine whether epitopes of the A, B, D, Fya, M, N, S, s, and K blood group antigens are present on microvesicle membranes shed by red cells during storage. Vesicles were isolated from outdated units of blood having and lacking the specified antigens. Diluted antisera were absorbed with fixed quantities of vesicles from red cells with the test antigen and red cells lacking that antigen (controls). The adsorbed and unadsorbed antisera were titrated and scored by using panel cells from persons known to be heterozygous for all the non-AB antigens. The mean titration scores following adsorption with the vesicles from A, B, D, M+N-, M-N+, S+s-, S-s+, and Fy(a+b-) units were appreciably lower than the control scores (0, 0, 3, 2, 2, 0, 4, and 4 vs. 19, 23, 34, 13, 12, 16, 18, and 29, respectively), which indicated the presence of these epitopes on the membrane of shed vesicles. The results following adsorption with K:1,2 vesicles were equivocal.

An enzyme-linked antiglobulin test to detect red cell globulins after glutaraldehyde fixation.

The purpose of this study was to develop an enzyme-linked antiglobulin test (ELAT) for IgG on RBC without the hemolysis caused by the high pH of the alkaline phosphatase reaction. This was achieved by fixing the RBC with 0.05% glutaraldehyde after attachment of the antibodies. Assays using anti-D reference standards demonstrated the sensitivity to be 1-2 ng of antibody as compared to 7.5 ng for the manual indirect antiglobulin test. The coefficients of variation of these assays ranged from 10.4 to 20.1%. The mean background absorbance at 405 nm of 105 normal RBC samples was 0.08 +/- 0.03 SD. There was an increase in sensitivity of the test after the fixed RBC were stored. Dilute glutaraldehyde stabilizes the RBC membrane and the antigen-antibody linkage resulting in a more sensitive ELAT.

The quantification of fetomaternal hemorrhage by an enzyme-linked antibody test with glutaraldehyde fixation.

A modified enzyme-linked antiglobulin test (ELAT) for quantifying D-positive red blood cells in the circulation of D-negative pregnant women is described. The antibody-antigen bond was stabilized and the problem of hemolysis eliminated by the use of 0.05% glutaraldehyde. The r value for the standardization curves for measuring cord RBC in mixtures was 0.98; coefficient of variation 5.8%. Reproducible quantitation of 0.125% D-positive cord RBC mixed with D-negative RBC was demonstrated. In comparative studies of artificial mixtures, Kleihauer-Betke results for more than 1% cord RBC were 50-100% greater than the true values. In several examples of large fetomaternal hemorrhages, the ELAT results matched the clinical data more closely than the Kleihauer-Betke based estimates. 2 ml of fetal RBC in a 1,600-ml red cell mass can be quantified using the modified ELAT.

An enzyme-linked antiglobulin test to quantify nanogram quantities of IgG on polystyrene microspheres.

A method is described in which polystyrene latex beads are used for constructing standard curves to estimate the number of protein molecules on cell surfaces by an enzyme-liked immunoassay test (ELAT). A series of immune globulin (IgG) dilutions in pH 9.8 carbonate buffer were coated on 3-microns microbeads by incubation overnight at 4 degrees C and subjected to ELAT. The r value of the curve derived from four assays was 0.9991. This standard curve applied to previously recorded ELAT data resulted in estimating that normal RBC have 63 +/- 19 (SD) IgG molecules and that the lower level of sensitivity of the antiglobulin test is 155 IgG molecules per RBC. The method should be useful for more precise standardization of procedures for measuring proteins on cell surfaces.

Studies in red blood cell preservation. 5. Determining the limiting concentrations of NH4Cl and Na2HPO4 needed to maintain red blood cell ATP during storage.

The purpose of the present study was to define the lowest concentrations of ammonium (NH4+) and phosphate (Pi) in an experimental additive solution (EAS) that would support suitable red blood cell (RBC) ATP levels and other in vitro characteristics for at least 84 days. It was determined that ATP maintenance was dependent upon both NH4+ and Pi concentrations. RBCs stored for 84 days in additive solutions containing 10 mM NH4+ and 0, 15, 25 and 40 mM Pi had ATP values averaging 1.87, 2.49, 2.70 and 2.65 mumol/g Hb, respectively. The shedding of exocytic hemoglobin-containing vesicles and percent hemolysis were significantly (p less than 0.001) elevated in the preservative containing 40 mM Pi. These data suggest that an EAS containing 10 mM NH4+ and 15 mM Pi would be optimal for storing RBCs up to 84 days. The extended storage would be particularly advantageous for autologous transfusion programs.