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Biomedical subjects

L Corash

Publications and source records attributed to L Corash.

At least 37 records · Page 2Linked to original sources
PubMed1994

Photochemical inactivation of pathogenic bacteria in human platelet concentrates.

Platelet concentrates (PC) may be infrequently contaminated with low levels of bacteria that can cause septicemia and death in patients receiving transfusion therapy. We evaluated the efficacy of a photochemical decontamination (PCD) technique using 8-methoxypsoralen (8-MOP) and long wavelength UV light (UVA) to inactivate bacteria in standard therapeutic PC. Twelve phylogenetically distinct pathogenic bacteria, 5 gram-positive and 7 gram-negative organisms, were seeded into PC to a final challenge dose ranging from 10(5) to 10(7) colony-forming units (CFU)/mL. Contaminated PC were treated with 8-MOP (5 micrograms/mL) and 5 J/cm2 of UVA, a PCD treatment regimen found to adequately preserve in vitro platelet function. Greater than 10(5) CFU/mL of all 5 gram-positive (Staphylococcus aureus, Streptococcus epidermidis, Streptococcus pyogenes, Listeria monocytogenes, and Corynebacterium minutissimum) and 2 of the gram-negative (Escherichia coli and Yersinia enterocolitica) organisms were inactivated. The remaining 5 gram-negative organisms were more resistant, with less than 10(1) to 10(3.7) CFU/mL inactivated under these conditions. The inactivation efficiency for this resistant group of gram-negative organisms was improved when PC were resuspended in a synthetic storage medium with reduced plasma protein concentration (15%) and an increased 8-MOP concentration (23.4 micrograms/mL). Illumination with 3 J/cm2 of UVA in this system inactivated greater than 10(5) CFU/mL of 4 resistant gram-negative organisms (Salmonella choleraesuis, Enterobacter cloacae, Serratia marcescens, and Klebsiella pneumoniae) and 10(4.1) CFU/mL of the most resistant gram-negative organism (Pseudomonas aeruginosa). This level of PCD treatment did not adversely affect in vitro platelet function. These results demonstrate that PCD using 8-MOP (5 to 23.4 micrograms/mL) effectively inactivated high levels of pathogenic bacteria in PC with adequate preservation of in vitro platelet properties.

Adult↗
PubMed1994

Flow cytometric reticulocyte analysis. Multiinstitutional interlaboratory correlation study.

Reticulocyte analysis by flow cytometry offers precision and sensitivity greater than those of conventional morphologic methods and permits derivation of a reticulocyte maturity index. However, interlaboratory variability has not yet been reported. The authors analyzed 310 samples at eight sites using 11 instruments over a 4-month period to examine intermethod and interlaboratory variabilities. Stains included thiazole orange, ethidium bromide, and auramine O. Instruments included models by Coulter, Becton Dickinson, TOA Medical Electronics, and Ortho Diagnostics. The coefficient of variation (CV) among all sites and methods on these samples varied as a function of the reticulocyte percentage, ranging from a mean CV of 69% for samples with < .5% reticulocytes to 24.1% for those with > 2.5% reticulocytes. The best performance was observed with the TOA R-1000 dedicated reticulocyte analyzers, with a mean CV of 18.4% for samples with < .5% reticulocytes and 4.6% for samples with > 2.5% reticulocytes. The reticulocyte maturity index showed comparable intersite precision, with a mean CV of 16% for samples with > 2.5% reticulocytes with multipurpose flow cytometers and a mean CV of 7.3% with the TOA R-1000 instruments. Interclass correlations among all sites ranged from .79 to .99 for the reticulocyte counts and .41 to .88 for the reticulocyte maturity index. The authors conclude that flow cytometric reticulocyte analysis is more precise than manual reticulocyte analysis. With greater automation of this methodology, further interlaboratory standardization of reticulocyte counts and the reticulocyte maturity index can be achieved.

Adolescent↗
PubMed1993

Photochemical inactivation of cell-associated human immunodeficiency virus in platelet concentrates.

Photochemical decontamination (PCD) of platelet concentrates, with adequate preservation of platelet function, has been shown using 8-methoxypsoralen (8-MOP) and long wavelength UV light (UVA). To further evaluate this technique, models for the inactivation of pathogenic human cell-associated viruses and integrated proviral sequences are required. We have assessed the ability of the PCD technique to inactivate cell-associated human immunodeficiency virus 1 (HIV-1) in platelet concentrates. We correlated PCD inhibition of HIV-1 infectivity with 8-MOP-DNA adduct formation in contaminating nucleated cells, and measured the inhibition of polymerase chain reaction (PCR)-mediated amplification of cellular DNA sequences as a surrogate for inactivation of integrated proviral nucleic acid sequences. After PCD treatment (8-MOP 300 micrograms/mL, UVA 17 mW/cm2) for 60 minutes, 0.5 x 10(6) plaque-forming units (PFU)/mL of cell-associated HIV-1 were inactivated and no virus was detectable by infectivity assay. After 60 minutes of PCD, 15 8-MOP-DNA adducts per 1,000 bp were formed, while in the absence of UVA, no adducts were formed. PCR-mediated amplification of a 242-bp cellular DNA sequence (HLA-DQ-alpha) was inhibited when greater than eight psoralen-DNA adducts per 1,000 bp were present. These studies indicate that high titers of cell-associated HIV-1 in platelet concentrates were inactivated by PCD, and the numbers of 8-MOP-DNA adducts in nucleated cells were sufficient to inhibit amplification of DNA segments that encode for as few as 80 amino acids. Based on the frequency of 8-MOP-DNA adducts, for the 10-kb HIV-1 genome, the probability of an integrated genome without at least one 8-MOP adduct after 60 minutes of PCD was 10(-33).

Blood Component Transfusion↗
PubMed1993

Failure of the International Normalized Ratio to generate consistent results within a local medical community.

Use of the International Normalized Ratio (INR) has been recommended as a means of standardizing prothrombin time (PT) results for management of oral anticoagulant therapy. During the evaluation of a new lot of thromboplastin reagent, however, INR values were obtained that were inconsistent with results obtained with the prior lot of reagent from the same manufacturer. A local normalized ratio (LNR) was substituted for the INR for the new reagent, based on a calculated local sensitivity index (LSI). Validation of the LSI was performed at the three hospitals in the medical community by testing an identical panel of aliquots from 64 plasmas obtained from patients who were chronically receiving oral anticoagulants. Each test result was classified according to the INR value as low, low therapeutic, high therapeutic, or high. Local normalized ratio values obtained at one hospital in the community were in reasonable agreement with INR determinations in the other two hospital laboratories. Classification mismatches occurred using the INR, however, in 84 of 280 (30%) of the paired samples. Thus, the inability to generate consistent INR values within a local medical community raises serious concern about the reliability of the INR concept in its current form.

Anticoagulants↗
PubMed1992

Use of 8-methoxypsoralen and long wavelength ultraviolet radiation for decontamination of platelet concentrates.

Transmission of viral diseases through blood products remains a problem in transfusion medicine. We have developed a photochemical decontamination system (PCD) for platelet concentrates (PC) utilizing treatment with long wavelength ultraviolet radiation (UVA, 320-400 nm) and 8-methoxypsorlan (8-MOP). This system is capable of inactivating 25-30 logs/hour of bacteria E. coli or S. aureus, 6 logs/hour of bacteriophage fd, 0.9 log/hour of bacteriophage R17, and 1.1 logs/hour of feline leukemia virus (FeLV) in PC. Immediately following 6 hours of PCD treatment, platelet integrity and function of PCD-treated and control PC were equivalent. After overnight storage, PCD-treated and control PC platelet properties were equal, but there was a slight reduction in TXB-2 production of PCD-treated PC compared to controls. Following PCD treatment, PC were stored for 48 to 96 hours. Platelet counts, morphology scores, extracellular LDH levels, aggregation response, dense body (db) content, and alpha granule (alpha g) content of PCD-treated and control PC were comparable. We assessed the ability of the PCD technique to inactivate intracellular and extracellular virus, quantified the degree of DNA adduct formation in contaminating lymphocytes, and measured the inhibition of polymerase chain reaction (PCR) mediated amplification of intracellular DNA. High titers of cell-free murine cytomegalovirus added to human platelet concentrates (final concentration 10(6)) were inactivated by PCD within 30 minutes. Cat renal fibroblasts infected at high levels with feline rhinotracheitis virus (FeRTV) were seeded into PC followed by PCD treatment with inactivation of 4.8 logs of FeRTV within 10 minutes. Purified human lymphocytes were seeded into PC and treated with PCD in the presence of 3H 8-MOP. Six hours of PCD treatment resulted in the formation of 9.3 to 12.8 8-MOP adducts per 1000 base pairs (bp) of DNA. PCR amplification of a 242 bp segment at the HLA-DQ alpha locus was examined. Inhibition of PCR DNA amplification was dependent on the numbers of 8-MOP adducts formed, and no amplification was present when greater than 12 adducts per 1000 bp were formed. These studies indicate that PCD can effectively inactivate high titers of cell-associated and cell-free virus seeded into standard human PC. The efficiency of DNA adduct formation can be quantitated, and the level of 8-MOP adduct formation in lymphocytes contaminating PC is comparable to the level of adduct formation in cellular DNA reported in the absence of platelets.

Blood Component Transfusion↗
PubMed1992

Histologic studies of splenic megakaryocytes after bone marrow ablation with strontium 90.

We previously showed that purified strontium 90 produced sustained bone marrow ablation in mice, lowering platelet levels to less than 10% of normal 11 days after administration. Platelet levels later rose exclusively from splenic production and were maintained at a stable level (58% of normal) from 20 to 115 days after injection. However, there was no change in the total number or ploidy distribution of splenic megakaryocytes, as immunologically detected by flow cytometry. To further study the characteristics of splenic thrombopoiesis after bone marrow ablation by 90Sr, we measured the frequency, cross-sectional area, and endomitotic figures of histologically recognizable megakaryocytes (as well as bare megakaryocyte nuclei) in mouse spleen sections. During the hematopoietic nadir 9 days after injection of yttrium 90-free 90Sr, the size (area) of megakaryocyte cross-sections (mean +/- SD, 1079.3 +/- 661.6 microns 2; normal, 398.7 +/- 192.8 microns 2) was greater than for any other time studied, but megakaryocyte frequency (corrected for size) did not increase until day 16. Overall, splenic megakaryocytes in marrow-ablated mice 16 or more days after 90Sr injection showed substantial increases (p = 0.001 for both comparisons) in mean area (707.5 +/- 386.2 microns 2) and sectional frequency (mean +/- SEM, 4.52 +/- 0.20 per 1.83 mm2; normal, 0.78 +/- 0.06 per 1.83 mm2). Megakaryocyte bare nuclei and endomitotic figures were also more numerous after 90Sr, injection, suggesting acceleration of megakaryocyte maturation and platelet production. The induction of splenic platelet production after bone marrow ablation is associated with increased size of recognizable megakaryocytes, despite lack of change in overall splenic megakaryocyte ploidy.

Animals↗
PubMed1992

Photochemical inactivation of viruses and bacteriophage in plasma and plasma fractions.

Transfusion-associated transmission of viral diseases remains a problem. A number of methods have been developed to inactivate viral pathogens in plasma and plasma fractions, including: dry heating, wet heating, solvent-detergent treatment, and immunoaffinity purification. While some of these methods successfully inactivate pathogenic viruses, inactivation may be incomplete or result in damage to labile plasma proteins. We have developed a method of photochemical decontamination (PCD) using psoralens and long wavelength ultraviolet light to inactivate pathogenic viruses. In the present study, a spectrum of model viruses have been added to plasma and plasma fractions to examine the efficiency of photochemical decontamination and the effects on labile plasma coagulation factors. Both RNA and DNA viruses have been inactivated under conditions which permit preservation of coagulation protein function. PCD technology appears to offer a promising solution to decontamination of blood products.

Bacteriophages↗
PubMed1991

Multiple in vivo effects of interleukin-3 and interleukin-6 on murine megakaryocytopoiesis.

The in vivo effects of interleukin-3 (IL-3), interleukin-6 (IL-6), and a combination of IL-3 plus IL-6 on murine megakaryocytopoiesis and thrombopoiesis were examined. Human recombinant IL-6 was administered subcutaneously as 14 equal injections of 5,000 units each during a 102-hour period. Murine recombinant IL-3 was given as 8 injections of 80,000 units each during the first 54 hours. Megakaryopoiesis and thrombopoiesis were evaluated 120 hours after initial administration of the cytokines. Platelet levels increased by 20% following IL-3 alone, 35% following IL-6 alone and 61% after administration of both IL-3 and IL-6. Platelet production, as measured by 75Se-selenomethionine incorporation, increased by approximately 120% in animals that had received IL-6 or IL-3 plus IL-6. Megakaryocyte ploidy analysis by two-color flow cytometry showed a shift in the modal ploidy class from 16N to 32N and a significant increase in the frequency of 64N cells only in IL-6 treated animals. Both bone marrow and splenic megakaryocyte colony-forming cells were significantly increased following either IL-3 or IL-6. Bone marrow megakaryocyte size increased 18%, 43%, and 38%, respectively, after administration of IL-3, IL-6, or the combination of IL-3 plus IL-6. Leukocyte counts and hematocrits were unaffected by either cytokine. Additional groups of mice received the same injection schedule as above and the serial effects on peripheral blood cell levels were assessed for 30 days. Platelet levels, which had been elevated by IL-3 or IL-6, fell to control values within 4 days following the last injection. Animals given IL-6 or IL-3 plus IL-6 were subsequently thrombocytopenic relative to controls on days 7 through 9 following cessation of treatment. Temporary 'cycling' of platelet levels was observed for 3 weeks following treatment with IL-6 or the combination of IL-3 plus IL-6. We conclude that IL-6 and to a lesser extent IL-3 stimulate platelet production in vivo and that their combined effects on platelet levels are approximately additive. Following discontinuation of IL-3 or IL-6, the effects are rapidly reversed, presumably by negative feedback mechanisms, resulting in a period of 'rebound thrombocytopenia' in mice that had received IL-6.

Animals↗
PubMed1991

Stimulation of megakaryocytopoiesis in mice by human recombinant interleukin-6.

The in vivo effects of purified human recombinant interleukin-6 (IL-6) on murine megakaryocytopoiesis were examined. IL-6 was administered subcutaneously to Swiss Webster mice, followed by evaluation of bone marrow megakaryocyte ploidy, size and frequency, and median platelet volume 24, 48, and 72 hours after the initiation of IL-6 administration. In addition, bone marrow megakaryocyte morphology was examined using electron microscopy at 72 hours. IL-6 (10,000 U per subcutaneous injection) was administered three times during the first 24 hours, three times during the second 24 hours, and twice during the last 24-hour period. IL-6 bioactivity (10 U/ng) was determined using the IL-6-dependent murine hybridoma cell line B9. Megakaryocyte ploidy distribution, measured by two-color flow cytometry, demonstrated a shift in the modal ploidy class from 16N to 32N and a significant increase in the relative frequency of 64N megakaryocytes 48 and 72 hours (but not 24 hours) after initiation of IL-6 administration (cumulative doses of 60,000 and 80,000 U at 48 and 72 hours, respectively). In addition, ploidy levels were increased in animals that received a cumulative IL-6 dose of only 40,000 U (evaluated after 72 hours). The size of recognizable bone marrow megakaryocytes, determined by the cross-sectional areas of plastic embedded bone marrow megakaryocytes, was increased at the 48-hour (60,000 U IL-6) and 72-hour (80,000 U IL-6) time points. Megakaryocyte frequency, measured by flow cytometry, was unaffected at all time points and doses of IL-6. Median platelet volume, measured by electrical impedance, was not consistently altered by administration of IL-6. Electron microscopic examination of bone marrow megakaryocytes showed an increase in the proportion of megakaryocytes with a wide, peripheral, organelle-deficient zone from 20% +/- 9% (SD) in control animals to 50% +/- 7% (SD) (P less than .02) in animals that received IL-6. No changes were observed in the distribution of the demarcation membranes. IL-6 is a potent stimulator of murine megakaryocytopoiesis, in vivo, and appears to act early in megakaryocyte differentiation.

Animals↗
PubMed1991

Neuraminidase-induced thrombocytopenia in mice: effects on thrombopoiesis.

Previous studies to examine the effects of thrombocytopenia on thrombopoiesis have generally utilized immune-mediated platelet depletion. We have developed a nonimmune model to exclude the possibility that adverse immune-mediated effects have been misinterpreted as the physiological response to stimulation of thrombopoiesis. Thrombopoiesis was examined in mice after induction of thrombocytopenia with a single injection of the nonimmunologic agent neuraminidase (Ndase). Utilizing electron microscopy, we examined platelets and megakaryocytes (MK) obtained 8, 12, 24, 48, 72, 96, and 120 hr after administration of Ndase. Eight to 48 hr after induction of acute, severe thrombocytopenia (mean platelet count less than 50,000/microliters), the medians of the platelet sectional area distributions, as measured morphometrically, were significantly greater than the median platelet sectional area of pooled controls. The maximum median value for platelet sectional area was observed at 24 hr. The largest platelets in these samples contained more profiles of endoplasmic reticulum and Golgi cisternae, and a lower concentration of surface-connected canalicular system, as compared with normal platelets. By 72 hr post-injection of Ndase, virtually all platelets exhibited normal size and organelle complement. Mean platelet volumes, determined by electrical impedance analysis, paralleled the serial changes in platelet sectional areas. MK frequency and ploidy, measured by two-color fluorescence activated flow cytometry, were unchanged 12 and 24 hr following Ndase. At 48 hr, total MK frequency increased significantly (P less than 0.01) from 0.11% to 0.17%, and MK ploidy distribution shifted with a reduction in 16N MK (P less than 0.005) and an increase in 32N MK (P less than 0.01). MK ploidy was maximally altered from normal at 72 hr with increased 32N MK frequency (32.0%, P less than 0.001) and increased 64N MK frequency (2.4%, P less than 0.005). Morphologic and morphometric examination of MK at all time points did not reveal detectable changes from normal in cytoplasmic appearance or size, respectively. Therefore, we have demonstrated marked alterations of morphology and size of platelets, and of MK ploidy, using this nonimmunologic model. These studies further support our previous observations that megakaryocyte ploidy and platelet volume are independently regulated in response to depletion of the circulating platelet mass, and they show that these changes are not dependent upon the mechanism of thrombocytopenia.

Animals↗
PubMed1991

The effect of a salmon diet on blood clotting, platelet aggregation and fatty acids in normal adult men.

This study was designed to measure the effect of dietary n-3 fatty acids (FA) on platelets and blood lipids. Healthy men (n = 9), ages 31 to 65, were fed diets in which salmon was the source of n-3 fatty acids. They were confined in a nutrition suite at this Center for 100 days. Food intake and exercise levels were rigidly controlled. Initially they were placed on a stabilization diet for 20 days, then six men were fed the salmon diet for 40 days. The others remained on the stabilization diet. The two groups switched diets for the last 40 days of the study. Both diets were isocaloric [16% protein, 54% carbohydrate, and 30% fat by energy-% (En%)]. The salmon diet contained 7.5% of calories from n-6 FA and 2% from n-3 FA, primarily eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in a 40:60 ratio, while the stabilization diet contained 7.5% of calories from n-6 FA and less than 0.3% n-3 FA, mainly 18:3n-3. The bleeding time was unaffected by the diets in this study. The prothrombin time was shortened (11.6 sec. vs. 12.6 sec., p less than 0.01) for the subjects consuming the salmon diet as compared to that measured after 20 days of the stabilization diet. Mean platelet volume increased significantly during the period in which the volunteers consumed the salmon diet compared to the baseline diet (p less than 0.01), while the mean platelet levels decreased. Platelet aggregation (PA) was measured in platelet rich plasma before, during, and after the salmon diet using collagen, ADP, arachidonic acid (AA), and thrombin agonists. The PA threshold for ADP was significantly increased for the subjects on the salmon diet (p less than 0.05). No change in the PA threshold was detected for collagen or thrombin. The PA threshold for AA was unchanged also, but the platelets in subjects consuming the salmon diet had a prolonged time to maximum aggregation (p less than 0.01) with this reagent compared to platelets from men on the stabilization diet. Plasma, red cell, and platelet total FA composition was determined by capillary GLC. While the men consumed the salmon diets, there were marked increases (3 to 10-fold) in the EPA and DHA levels in all blood components with concomitant decreases in linoleic acid and AA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗
PubMed1991

The automated modified Russell viper venom time test for the lupus anticoagulant.

Antiphospholipid antibodies, both anticardiolipin antibody and lupus anticoagulant, are associated with a hypercoagulable state, manifested as thrombotic events and pregnancy losses. Multiple coagulation tests are available for the lupus anticoagulant, but few are in wide use. The modified Russell viper venom test (RVVT) is one of several excellent tests for the lupus anticoagulant, with high sensitivity and specificity. We present an automated method for measuring the RVVT and demonstrate its reproducibility.

Automation↗
PubMed1990

Measurement of platelet activation by fluorescence-activated flow cytometry.

Platelet activation is postulated to play a critical role in the pathogenesis of thrombotic and hemorrhagic disorders. Previous assays for detection of activated platelets were cumbersome and provided only nonspecific information with limited sensitivity. The recent introduction of fluorescence-activated flow cytometric techniques for platelet analysis used in combination with monoclonal antibodies for detection of specific platelet-activation antigens has introduced the possibility of improved assays to detect activated platelets. The monoclonal antibody S12, directed against the unique platelet-activation antigen GMP-140, has been used to develop a fluorescence-activated flow cytometric assay. Patient samples for this assay can be easily prepared and maintained until analyzed in batch mode. Peripheral blood obtained from normal subjects exhibited low levels of activated platelets, and the assay had sufficient sensitivity to detect as few as 2% to 3% activated platelets among normal platelets. Patients undergoing cardiopulmonary bypass had transiently increased numbers of circulating activated platelets. Evaluation of standard blood bank platelet concentrates has shown the presence of significant numbers of activated platelets. Other studies have suggested that the degree of platelet activation correlated with poor posttransfusion increments and survival. Thus, this assay may also be useful for quality control of platelet concentrates. Future development of the GMP-140 and other platelet-activation antigen assays should improve detection of disorders characterized by inappropriate platelet activation.

Flow Cytometry↗
PubMed1990

Splenic thrombopoiesis after bone marrow ablation with radiostrontium: a murine model.

Murine platelet production is normally supported by high-ploidy bone marrow megakaryocytes without significant contribution from splenic megakaryocytes with predominantly low-ploidy levels. We produced sustained bone marrow ablation using radiostrontium, and examined the processes by which splenic platelet production is initiated and maintained in the absence of bone marrow function. Bone marrow hematopoiesis, measured by total nucleated cell number and viability, megakaryocyte colony-forming cells, and granulocyte-macrophage colony-forming cells, was rapidly ablated in mice by using yttrium 90-free strontium 90. Platelet count declined from normal (1224 x 10(3)/microliters) to a nadir (98 x 10(3)/microliters) 11 days after 90Sr, and then rose to a stable level (705 x 10(3)/microliters) on days 20 through 115. Peripheral leukocyte concentration decreased rapidly and remained below 25% of normal in contrast to hemoglobin levels, which were minimally lowered. Mean spleen weight rose rapidly after 90Sr to 66% above normal. Splenic megakaryocyte frequency, measured by two-color fluorescence-activated flow cytometry, rose from basal levels (0.09% +/- 0.06%) to 0.15% +/- 0.07% (p less than 0.001), total spleen nucleated cells fell to 71% of normal, and the absolute number of spleen megakaryocytes was unchanged. Total spleen megakaryocyte colony-forming cells were not significantly increased above normal whereas total spleen granulocyte-macrophage colony-forming cells increased abruptly after day 13 to 10 times normal levels. Splenectomy after hematopoietic recovery from 90Sr bone marrow ablation resulted in a rapid decline of platelet levels, followed by death. Although the spleen became the sole site of platelet production, the splenic megakaryocyte ploidy distribution was only minimally changed from normal, and the modal ploidy class remained 2N. In contrast to experimental thrombocytopenia in mice with intact bone marrow, in which megakaryocyte ploidy is increased, thrombocytopenia associated with sustained bone marrow ablation does not result in upward regulation of splenic megakaryocyte ploidy as a compensatory mechanism.

Animals↗