In vitro and in vivo persistence of reticulocytes from donor red cells

BACKGROUND: Reticulocytes are important in the phenotyping of transfused patients. Reticulocytes can persist in blood units for the shelf life of the unit. STUDY DESIGN AND METHODS: Temperature dependence of reticulocyte persistence was examined in vitro at 4, 24, and 37 degrees C by using thiazole orange staining and flow cytometric analysis. Two-color flow cytometric analysis was used to evaluate the persistence of donor reticulocytes in transfused patients. RESULTS: Flow cytometric analysis using thiazole orange demonstrated that persistence of reticulocytes in units of stored CPDA-1 blood was temperature-dependent. Reticulocytes disappeared over 13 and 6 days at 24 degrees C and 37 degrees C, respectively, but at 4 degrees C the reticulocyte count changed little over 35 days. Two-color flow cytometric analysis of reticulocyte antigens was used to follow donor reticulocytes in 14 transfusion events in nine different patients. Donor reticulocytes persisted through 24 hours in 75 percent of the patients and were detectable at 48 hours in three patients. CONCLUSION: This study demonstrates that reticulocytes persist during refrigerated storage; they are detectable in the circulation of most recipients for the first 24 hours after transfusion and in the circulation of a few recipients after 48 hours. These findings may have relevance for separation techniques based on reticulocyte density in samples drawn shortly after transfusion and for evaluation of reticulocyte counts in patients with hematologic abnormalities.     

Addition of L-carnitine to additive solution-suspended red cells stored at 4 degrees C reduces in vitro hemolysis and improves in vivo viability

BACKGROUND: The role of L-carnitine (LC) as the requisite carrier of long-chain fatty acids into mitochondria is well established. Human red cells (RBCs), which lack mitochondria, possess a substantial amount of LC and its esters. In addition, carnitine palmitoyl transferase, an enzyme that catalyzes the reversible transfer of the acyl moiety from acyl-coenzyme A to LC is found in RBCs. It has recently been shown that LC and carnitine palmitoyl transferase play a major role in modulating the pathway for the turnover of membrane phospholipid fatty acids in intact human RBCs, and that LC improved the membrane stability of RBCs subjected to high shear stress. RBC membrane lesions occur during storage at 4 degrees C; this study investigated whether the addition of LC (5 mM) to a standard RBC preservative solution (AS-3) affected cellular integrity with 42 days' storage. STUDY DESIGN AND METHODS: A paired (n = 10) crossover design was used for RBCs stored in AS-3 with and without LC. Both in vitro RBC properties reflective of metabolic and membrane integrity and in vivo measures of cell viability (24-hour percentage of recovery and circulating lifespan) were measured at the end of the storage. In addition, the turnover of membrane phospholipid and long-chain acylcarnitine fatty acids and the carnitine content of control and LC-stored RBCs were measured. RESULTS: It was shown that LC was irreversibly taken up by RBCs during storage, with a fourfold increase at 42 days. Furthermore, as found by the use of radiolabeled palmitate, the stored RBCs were capable of generating long-chain acylcarnitine. The uptake of LC during storage was associated with less hemolysis and higher RBC ATP levels and by a significantly greater in vivo viability for LC-stored RBCs than for control-stored RBCs: a mean 24-hour percentage of recovery of 83.9 +/? 5.0 vs. 80.1 +/? 6.0 percent and a mean lifespan of 96 +/? 11 vs. 86 +/? 14 days, respectively (p < 0.05). CONCLUSION: A beneficial effect of the addition of LC to RBCs stored at 4 degrees C was evident. This effect may be related to both biophysical and metabolic actions on the cell membrane.     

In vitro and in vivo evaluation of LEUKOSEP HRC-600-C leukoreduction filtration system for red cells

BACKGROUND: Documentation of the benefits of leukoreduction has led to the increased use of this technique and the need for development of efficient and effective techniques for its accomplishment. This study investigated the in vitro properties and in vivo autologous radiolabeled recovery of leukoreduced red cells (RBCs) produced through a leukoreduction filtration system for RBCs (LEUKOSEP HRC-600-C, Hemerus Medical). STUDY DESIGN AND METHODS: Normal subjects donated 36 units of RBCs that were leukoreduced on Days 0, 3, or 5 through a "hands-off" technique. Biochemical studies were performed before and after filtration and at the end of 42 days of storage. Units leukoreduced on Days 0 or 5 were held until Day 42 and used for autologous radiolabeled return to determine recovery with 51Cr single-label radiolabeling techniques. RESULTS: Leukoreduction filtration was accomplished in 16.3 +/- 2 minutes on Day 0 at room temperature or 27 to 30 minutes on Days 3 or 5 after refrigeration. Leukoreduction efficiency was 4.6 +/- 0.6 log with a median residual white blood cell (WBC) content of fewer than 3.3 x 10(4) WBCs per unit. RBC recovery was 90 +/- 2 percent. Hemolysis was 0.34 +/- 0.16 percent at the end of 42 days of storage. The in vivo recovery of radiolabeled RBCs 24 hours after autologous return was 80.6 +/- 4.5 percent for RBC units leukoreduced on Days 0 and 5 combined. CONCLUSION: The LEUKOSEP HRC-600-C WBC reduction filtration system produced leukoreduced RBCs efficiently and effectively with acceptable poststorage biochemical measures and posttransfusion recovery after 42 days of storage.     

In vitro storage and in vivo survival studies of red cells from persons with the In(Lu) gene

Red cells (RBCs) of individuals with the In(Lu) gene are characterized by suppression of the Lutheran, P1, i, and other blood group antigens, acanthocytosis, and abnormal electrolyte metabolism. To determine the clinical significance of these abnormalities, the survival of autologous RBCs was determined by 51Cr in two siblings with the dominant Lu(a-b-) [In(Lu)] phenotype. Both subjects studied had normal hemoglobin, hematocrit, reticulocyte count, haptoglobin, and ferritin values. RBC indices were mildly hypochromic. Examination of the peripheral smear showed mild acanthocytosis in one individual. Analysis of RBC distribution on discontinuous density gradients showed a shift to lighter fractions than normal control RBCs. Storage of these Lu (a-b-) RBCs at 4 degrees C showed significant hemolysis within a few days; this was confirmed by increased autohemolysis, which was reduced by glucose and ATP. RBC cation content (sodium and potassium) was higher than that in control cells, which indicated increased cell hydration, which explains the lighter density and mild hypochromia of the Lu(a-b-) RBCs. 51Cr survival of autologous Lu(a-b-) RBCs was normal in both subjects studied. The data indicate that the morphologic and cation abnormalities of RBCs of persons with the In(Lu) gene are clinically insignificant, as these cells have normal in vivo survival. Such RBCs, however, are susceptible to increased hemolysis in vitro under standard blood banking storage conditions. Individuals of the Lu(a-b-) phenotype, associated with In(Lu), may not be suitable candidates for routine blood donation.     

In vivo survival of K:18 red cells in a recipient with anti-K18

Red cell survival, IgG subclass, and mononuclear phagocyte assay studies were performed on a patient with an anti-K + K18 described previously. The 51Cr survival study with Kell negative K:18 red cells showed 76.6 percent survival at 75 minutes with 30.7 percent survival at 24 hours. The anti-K18 was characterized as IgG4 + IgG1. The mononuclear phagocyte assay was 16 percent when the serum was tested with K:18 cells. Among 54,450 ABO compatible donor units tested with this serum, no K:18- unit was found. These studies were undertaken to evaluate the clinical significance of the antibody when red cell support for a chemotherapy course was considered. Our data suggest that transfusion with K:18+ blood would be ineffective and could be used only to provide red cell support in an emergency should compatible units be unavailable.     

Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association

We have investigated whether sequence 67 to 74 shared by beta chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRB1*0401, DRB1*0404, and the closely related, RA nonassociated DRB1*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRB1*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR beta chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.     

In vivo viability of red blood cells stored in CPDA-2

The marginal viability of erythrocytes stored for 35 days as red blood cell concentrates in citrate-phosphate-dextrose-adenine-one (CPDA-1) was attributed to inadequate nutrient support with adenine and glucose. In an effort to improve the viability of red blood cells following extended storage, a new CPD-adenine and 1.4 times more glucose than CPDA-1. The efficacy of CPDA-2 was evaluated in vivo by measurement of 24-hour postinfusion recovery of 51Cr-labeled erythrocytes which had been stored as whole blood or red blood cell concentrates for 5 to 8 weeks. All red blood cell concentrates, and the whole blood units stored for 35 and 42 days, were held at room temperature for 8 hours prior to processing and/or refrigeration. CPDA-2 yielded significantly higher 51Cr survivals than CPDA-1 and exceeded the accepted criterion for anticoagulant preservative efficacy of 70 percent postinfusion survival of red blood cells after storage for a period of 42 days. Preliminary data supports possible usage to 49 days. Plasma glucose and red blood cell ATP concentration were maintained better in CPDA-2 than in CPDA-1. When compared to historical controls for CPD and CPDA-1 the data suggest that red blood cells stored in CPDA-02 will have superior viability throughout the entire storage period. CPDA-2 is a candidate to replace CPDA-1 as the anticoagulant-preservative solution of choice for red blood cell concentrates.     

In vitro and in vivo distribution and binding of phenytoin to rat brain

The distribution and binding of phenytoin (PHT) were studied in rat brain using anatomically intact tissue. The pattern and kinetics of PHT distribution in vivo were examined with quantitative carbon-14 autoradiography. Initially gray matter levels were greater than white matter levels, but after 30 min the opposite condition was found. At a given time point, the levels of PHT among various gray matter structures as a group or among white matter structures as a group were uniform. The ratio of radioactivity in white matter to that in gray matter was 3:1 120 min after injection of radiolabeled PHT. Thin-layer chromatography showed only PHT in the brain 15 min after injection of PHT and both PHT and its hydroxylated metabolite [5-(p-hydroxyphenyl)-5-phenylhydantoin] 120 min after injection. The proportions of PHT and 5-(p-hydroxyphenyl)-5-phenylhydantoin were the same in white as in gray matter. Direct chemical measurements of brain samples obtained after coinjections of tracer amounts of radiolabeled PHT with increasing doses of unlabeled PHT corroborated the autoradiographic findings and revealed no displacement of the tracer by pharmacologic doses of the unlabeled drug. In vitro binding of PHT was investigated with sections obtained from frozen brains and with physiologically intact brain slices. Both high (4-10 nM) and low (1 microM) affinity conditions were examined. In no case was specific binding detected. Binding was greater in gray matter than in white matter in sections, whereas binding was greater in white matter than in gray matter in slices. We conclude that PHT distribution and binding reflect physico-chemical factors, such as lipid content, and physiological factors, such as blood flow and selective partitioning into white matter in living tissue, rather than specific receptors.     

In vivo viability studies of two additive solutions in the postthaw preservation of red cells held for 3 weeks at 4 degrees C

An optimized additive solution was developed for the postthaw preservation of red cells that contained adenine, glucose, disodium phosphate, and citrate buffer. This solution, called AS-17, was compared to AS-3 solution in a clinical trial using 40 subjects (20 in each arm). Fresh-frozen red cells were thawed and deglycerolized after 1 to 18 months and subjected to a second period of storage in either solution for up to 3 weeks at refrigerator temperatures. Both solutions yielded red cells with 24-hour survivals in excess of 75 percent. Cells stored in AS-3 for 21 days had a mean survival of 77 +/− 8 percent and cells stored in AS-17 a mean survival of 79 +/− 11 percent. The AS-17 solution resulted in improved maintenance of pH, p50, and 2,3 DPG compared to that with AS-3, but both solutions appear adequate for 3 weeks of postthaw storage.     

大鼠胰岛细胞的体外和体内MR成像方法

目的探讨MRI对SPIO标记的大鼠胰岛细胞在体外及体内的扫描参数及序列的选用。方法联合应用SPIO和多聚左旋赖氨酸(PLL),通过受体介导的内吞作用标记胰岛细胞。采用GE3.0T Signa Excite磁共振扫描仪,配合3英寸小动物线圈作GRE序列T2*W成像,比较SPIO标记的胰岛细胞与未标记的细胞,再分别采用两种具有不同分辨率的扫描方案进行扫描,比较图像的差异。而后将两种细胞分别移植入大鼠体内,比较在体内的差异。对标记后的大鼠分别用FSET2W序列和GRET2*W序列扫描,比较各序列的敏感性。结果无论是体外还是体内,只有SPIO标记后的胰岛细胞可以被MRI监测到,表现为较明显的低信号点。采用更高分辨率的参数扫描后所得到的胰岛图像更为清晰。GRET2*WI较FSET2WI序列对SPIO的监测更敏感。结论MRI能对SPIO标记的胰岛细胞进行成像,可用于活体,实时监测胰岛细胞移植术后移植物的存活及排异情况。     

Establishment of correlation between in vitro enzyme binding potency and in vivo pharmacological activity: application to liver glycogen phosphorylase a inhibitors

In drug discovery, establishing a correlation between in vitro potency and in vivo activity is critical for the validation of the selected target and for developing confidence in the in vitro screening strategy. The present study developed a competition equilibrium dialysis assay using a 96-well dialysis technique to determine the intrinsic Kd for 13 inhibitors of human liver glycogen phosphorylase a (GPa) in the presence of liver homogenate to mimic the physiological environment. The results provided evidence that binding of an inhibitor to GPa was affected by extra cofactors present in the liver homogenate. A good correlation was demonstrated between the in vitro Kd determined under liver homogenate environment and free liver concentration of an inhibitor at the minimum efficacious dose in diabetic ob/ob mice. This study revealed important elements (such as endogenous cofactors missing from the in vitro assay and free concentration at the target tissue) that contributed to a better understanding of the linkage between in vitro and in vivo activity. The approach developed here may be applied to many drugs in pharmacology studies in which the correlation between in vitro and in vivo activities for the target tissue (such as solid tumors, brain, and liver) is critical.     

Prolonged storage of red cells at 4 degrees C

One of the events associated with red cell storage at 4 degrees C is the development of an increasing proportion of echinocytes. Vesicles also may bud off the spicules, presumably leading to a decreased surface-to-volume ratio and decreased deformability. Pursuing the hypothesis that increasing the surface tension of the cells by increasing their volume might reduce the tendency toward echinocytosis and extend refrigerated storage time, packed red cells were resuspended in a solution hypotonic (210 mOsm) with respect to solutes that do not penetrate the cell. Since a reduced ionic concentration results in increased membrane permeability for cations, normal ionic concentration was maintained by the addition of NH4C1, which readily penetrates red cells and therefore contributes no osmotic support. Adenine, glucose, mannitol, citrate, and phosphate also were included. Unexpectedly, the predominant effect of red cell storage in this solution was a remarkable elevation of adenosine triphosphate (ATP). At 4, 8, and 10 weeks, (ATP) levels averaged 165, 135, and 110 percent of initial values, respectively. At 16 weeks, ATP still averaged 50 percent of initial values. Twenty-four-hour in vivo survival of red cells measured at 12 to 18 weeks ranged between 70 and 80 percent, and hemolysis ranged from 0.3 to 7.1 percent. Both the hypotonicity and the ammonium salt appear to be necessary for the high ATP.     

Microbubble formation: In vitro and in vivo observation

Injection of liquid through a catheter into the circulation is known to produce clouds of signals detected by sonography. Blood forced through a stenotic conduit produced sonographic clouding, and bubbles of 10–100 μm were observed by light microscopy. The microbubbles persisted up to three and a half minutes. Microbubbles were observed in the microcirculation of the rat by placing the catheter tip into the descending aorta of 15 animals, viewing the mesentery at 400X magnification, and recording the results on videotape. Following injection of the rats' own blood, numerous microbubbles lodged promptly at the arteriolar level and obstructed blood flow for up to 200 sec before shrinking sufficiently to pass downstream and allow restitution of flow.     

Spontaneous agglutination of red cells with a positive direct antiglobulin test in various media

Red cells from 475 individuals (donors and patients) with a positive direct antiglobulin test (DAT) (66% were 1/2+ to 1+, 19% were 1 1/2+ to 2+, and 15% were 2 1/2+ or greater) were examined for spontaneous agglutination following incubation in various media. Twenty-three percent of the samples showed spontaneous agglutination in commercial Rh control solutions, 6 percent in 30 percent albumin, 3 percent in 10 percent albumin, 2 percent in 6 percent albumin, and 1 percent in saline. Cells suspended in serum were more prone to spontaneous agglutination. The strength of spontaneous agglutination varied; more than 50 percent of the samples reacted only 1+ or less, approximately 10 percent reacted 2+ or stronger. There was not a complete correlation with spontaneous agglutination and quantity of IgG on the cells, as determined by the antiglobulin test. Of those samples showing spontaneous agglutination, 50 percent were associated with a DAT strength of more than 2+, 27 percent with a DAT of 1 1/2+ to 2+, and 23 percent with a DAT of 1/2+ to 1+. Spontaneous agglutination occurred with the same frequency whether the cells were sensitized with both IgG and C3 or with IgG alone. Surprisingly, 7 percent of the samples sensitized with C3 but no detectable IgG also demonstrated spontaneous agglutination. Marked differences in reaction strength were seen with Rh control solutions from different manufacturers, and the degree of spontaneous agglutination was inconsistent with individual products.     

In vitro and in vivo activity of murine lymphokine-activated killer cells after cryopreservation

The in vitro and in vivo effects of cryopreservation on the cytotoxic activity of murine lymphokine-activated killer (LAK) cells were studied. LAK cells were generated by incubation of spleen lymphocytes of BALB/c mice for 3 days with recombinant interleukin-2 (rIL-2) and subsequent cryopreservation. Cytotoxicity was determined in a 51Cr release assay. After thawing, cytotoxic activity was reduced (40.4% 51Cr release at an effector:target cell ratio of 40:1 as compared to 68.5% 51Cr release before freezing) and could be restored to precryopreserved levels by reincubation with rIL-2 for 2 days after thawing (78.8% 51Cr release). These cells were then tested in BALB/c mice injected with RAW 112 cells, a pre-B-cell lymphoma line. The results demonstrate that the survival rate of mice injected with cryopreserved and restimulated LAK cells (50% survival greater than 180 days after injection) did not differ significantly from that of mice injected with fresh unfrozen LAK cells (60% survival greater than 120 days, 50% survival greater than 180 days). Cryopreserved LAK cells have potential use in adoptive immunotherapy.