Unique donor-suitability issues.

On occasion, there arise questions or situations involving blood-donor eligibility determination, which are not adequately addressed by the existing regulations and standards. In such instances, even the most experienced blood collector may be uncertain regarding the best course of action and unable to find adequate guidance in the standard blood banking references, regulations and literature. In order to examine this area in greater depth, the American Association of Blood Banks (AABB) sponsored a short topic session on 'Unique Donor Suitability Issues' at their 2004 annual meeting. The invited speakers were four seasoned physician medical directors, with a combined experience of over 40 years in blood collection at both regional and national levels. They were tasked with identifying and researching problematic areas in donor-suitability determination, and suggesting an overall approach to dealing with such issues. They determined that three of the most problematic areas of eligibility evaluation included donors with: (1) disabilities, (2) disorders of haemostasis, and (3) trans-sexual, homosexual and other unusual gender-related issues. Each of these topics was presented in a 10-min lecture, followed by an open format consisting of audience participation and panel discussion by the speakers. The session was additionally enhanced by a representative of the United States Food and Drug Administration (FDA) who participated as a member of the audience. This review presents the contents of the short topic session in an expanded form.

Typicality range deficit in schizophrenics' recognition of emotion in faces.

Severity of thought disorder in schizophrenics was assessed by a task that involved recognition of joy and shame expression, with the expressions varied for typicality of the emotion category. Accuracy, typicality rating, and reaction time were measured in schizophrenic patients who were high or low on Whitaker's Index of Schizophrenic Thinking (WIST) and in depressive and normal controls. All groups had significant variation of typicality ratings for joy (normal typicality range), but the clinical groups had smaller typicality ranges for shame recognition and higher mean typicality ratings than did normals. High WIST schizophrenics were least accurate on low typical shame expressions. Results imply that, for shame recognition, schizophrenic and depressive groups used common categorization (no typicality variation in category members) rather than resemblance categorization.

Modification of glutathione content in platelet concentrates by the use of acivicin.

BACKGROUND: Intracellular glutathione declines progressively in platelet concentrates stored for transfusion, but little is known about the nature of this process. STUDY DESIGN AND METHODS: Acivicin, an irreversible inhibitor of gamma-glutamyl transpeptidase, was used to determine the fate of glutathione lost from platelets during storage. Glutathione, cysteinylglycine, cysteine, and their disulfides were measured by high-performance liquid chromatography. RESULTS: In control and acivicin-treated platelet concentrates, intracellular glutathione declined progressively during 7 days' storage, with half-disappearance times of 2.12 +/- 0.22 and 2.13 +/- 0.23 days, respectively. No glutathione accumulated accumulated in the medium of control concentrates. In concentrates treated with acivicin, glutathione accumulated in plasma to a level equal to 150 percent of that present in platelets and plasma on Day 1 of storage, which suggested a net synthesis of glutathione during storage. The sum of glutathione, cysteinylglycine, and cysteine in plasma was not different in control and acivicin-treated concentrates; however, plasma from control concentrates contained significantly higher amounts of cysteinylglycine and cysteine. Treatment of concentrates with acivicin had a small favorable effect on plasma lactate dehydrogenase, beta-thromboglobulin, and beta-N-acetylglucosaminidase during storage. CONCLUSION: The loss of glutathione from platelets during their storage as concentrates for transfusion is due to its egress into the suspending medium and catabolism by gamma-glutamyl transpeptidase.

Glutathione disulfide production during arachidonic acid oxygenation in human platelets.

Washed human platelets stimulated with 50 microM sodium arachidonate rapidly accumulated glutathione disulfide to a peak concentration of 0.620 nmole per 10(9) cells, 200% of control (unstimulated) levels. Total glutathione remained unchanged. The rise in glutathione disulfide was transitory, returning to control values within 30 seconds in aggregating platelets. Similar findings were observed in washed platelets aggregated with 5 U/ml thrombin. Platelet aggregation was not necessary for the generation of glutathione disulfide. However, cyclooxygenase activity was necessary for the generation of glutathione disulfide. Aspirin treated platelets aggregated with thrombin demonstrated no thromboxane B2 production and no glutathione disulfide generation. Dose response studies with both agonists demonstrated a direct relationship between the amount of thromboxane B2 produced and the amount of glutathione disulfide generated by stimulated platelets. During the conversion of arachidonic acid to thromboxane B2, unesterified arachidonic acid is oxygenated to prostaglandin G2 which is subsequently reduced to prostaglandin H2. Both reactions are catalyzed by the enzyme prostaglandin H synthase. Our data support the hypothesis that glutathione is an important supplier of reducing equivalents to prostaglandin H synthase during the production of prostaglandin H2 in human platelets.

Alterations in glutathione during storage of human platelet concentrates.

Glutathione and glutathione disulfide decline rapidly and progressively in human platelet concentrates stored for up to 7 days at 22 degrees C. Total glutathione declines progressively throughout the storage period, with an estimated half-disappearance time of 2 days. Glutathione disulfide remains constant during the first 2 days of storage but declines progressively thereafter, with an estimated half-disappearance time of 3.2 days. Based on measurements of glutathione disulfide in freshly collected platelets, the authors postulate that glutathione disulfide is elevated during the production of platelet concentrates or during the first 2 days of storage, accounting for the lag in its decay curve. Since total glutathione decreases more rapidly than glutathione disulfide, the fraction of glutathione present in the oxidized form increases throughout the storage period. Glutathione-dependent mechanisms maintain essential cell proteins in the appropriate redox states and provide avenues for the detoxification of potentially noxious compounds generated by the cell or within its environment. Changes in platelet glutathione and glutathione disulfide during storage may be responsible for storage-dependent alterations in in vitro functions and may also affect subsequent in vivo recovery and survival upon reinfusion.

Sodium arachidonate induced platelet aggregation is independent of secreted adenosine diphosphate.

Human gel-filtered platelets or platelet-rich plasma were stimulated by sodium arachidonate or by ADP in the presence of two compounds known to inhibit ADP mediated aggregation and secretion - ATP and N-ethylmaleimide. Using gel-filtered platelets and the lowest concentration of agonist necessary to elicit maximum aggregation, fifty percent inhibition of ADP-mediated aggregation required 9 microM N-ethylmaleimide or 23 microM ATP. Sodium arachidonate-mediated aggregation was significantly less sensitive; equivalent inhibition required 30 microM N-ethylmaleimide or greater than 500 microM ATP. Concentrations of both inhibitors were determined that would completely inhibit ADP-induced aggregation yet would not completely prevent sodium arachidonate-induced aggregation. Furthermore, this concentration of N-ethylmaleimide could not be overcome by up to 500 microM ADP, demonstrating that the observed arachidonate-induced aggregation was not due to the effects of a small amount of secreted ADP acting at the platelet surface. Therefore, aggregation of human platelets induced by arachidonic acid can occur by a mechanism that is independent of secreted ADP.

Effect of atrial pacing on intracoronary thromboxane production in coronary artery disease.

The effect of atrial pacing on intracoronary thromboxane production was investigated in 35 patients with stable (n = 19) or unstable (n = 16) angina. Arterial and coronary sinus thromboxane B2, the stable metabolite of thromboxane A2, myocardial lactate extraction and thermodilution coronary sinus flow were measured before, during and immediately after atrial pacing until the onset of angina. Pacing did not significantly increase coronary sinus thromboxane B2 (rest, 233 +/- 107 pg/ml; pacing, 249 +/- 154 pg/ml; postpacing, 330 +/- 309 pg/ml) (mean +/- standard deviation) despite a moderate increase in arterial thromboxane B2 (rest, 270 +/- 170 pg/ml; pacing, 387 +/- 364 pg/ml; postpacing, 446 +/- 420 pg/ml) (all changes probability [p] less than 0.05). A positive transmyocardial thromboxane B2 gradient, suggesting intracoronary thromboxane A2 production, occurred in only five patients at rest (gradient = 60 +/- 35 pg/ml). During pacing, a transmyocardial thromboxane B2 gradient was not observed despite myocardial lactate production in 18 patients. A postpacing gradient was observed in eight patients (gradient = 284 +/- 349 pg/ml). These gradients were significantly more frequent in patients who produced lactate during pacing (7 of 18) than in patients without lactate production (1 of 17) (p less than 0.05). In patients with and without a postpacing gradient, coronary vascular resistance decreased with pacing and returned to rest levels immediately after pacing, suggesting that a postpacing thromboxane gradient does not significantly alter coronary tone. These data suggest that: 1) pacing-induced angina is usually not associated with substantial intracoronary thromboxane A2 production; 2) in a minority of patients who develop intracoronary thromboxane A2 production, the amount is small and does not produce significant coronary vasoconstriction.

Direct interaction of mepacrine with erythrocyte and platelet membrane phospholipid.

Mepacrine has been used as an inhibitor of the activation of endogenous phospholipases in many systems. These endogenous phospholipases are important in the modification of the lipid environment of membrane proteins and in the release of locally active oxygenated arachidonic acid metabolites. In both human platelets and erythrocytes, mepacrine blocks the release of fatty acid from phospholipid by endogenous phospholipases. However, mepacrine also interacts directly with membrane phospholipids, primarily phosphatidylethanolamine, to form less polar derivatives. This interaction occurs rapidly and is maximal at concentrations of mepacrine greater than 0.2 mM. Such drug-phospholipid interaction may perturb membrane architecture and function and be responsible for the inhibitory effects of mepacrine on cellular responses observed in many systems. Since the alteration in membrane phospholipid composition occurs under the same conditions as phospholipase inhibition, it is not possible to be certain that the inhibition of cellular responses by mepacrine is due to inhibition of phospholipases rather than to direct perturbation of the membrane. It is also possible that inhibition of phospholipase action by mepacrine is in part a consequence of the change in phospholipid composition. These results indicate that caution should be exercised in the interpretation of results obtained using mepacrine and that the usefulness of this compound for the investigation of the biological importance of phospholipase activation is limited.

A role for prostaglandins and thromboxanes in the exposure of platelet fibrinogen receptors.

Exposure of fibrinogen receptors by a variety of agonists is a prerequisite for platelet aggregation. Because the synthesis of prostaglandins and thromboxane A2 also occurs during platelet aggregation we wondered whether these agents participate in the exposure of platelet fibrinogen receptors. Therefore, we measured the binding of human 125I-fibrinogen to gel-filtered normal human platelets after prostaglandin and thromboxane synthesis had been inhibited by aspirin or indomethacin. The fibrinogen binding assay was performed at 37 degrees C but without stirring to prevent the formation of platelet aggregates. Platelet secretion, measured with [14C]serotonin, did not occur during the procedure. Aspirin or indomethacin inhibited fibrinogen binding stimulated by 10 microM epinephrine by 53%, and inhibited fibrinogen binding stimulated by 1-2 microM ADP by 37.1%. However, ADP at concentrations greater than 2 microM returned fibrinogen binding toward control values. Scatchard analysis demonstrated that aspirin decreased the number but not the affinity of the exposed fibrinogen receptors. To determine whether prostaglandins are capable of directly exposing fibrinogen receptors, prostaglandin H2 was used to stimulate platelets in the fibrinogen binding assay. Prostaglandin H2 exposed approximately 54,000 fibrinogen receptors/platelet and corrected the deficit in receptor exposure induced by aspirin. These studies demonstrate that platelet prostaglandins or thromboxane A2 can play a direct role in the exposure of platelet fibrinogen receptors. In addition, they suggest that the synthesis of prostaglandins and thromboxane A2 by stimulated platelets may be all that is required for optimal secondary platelet aggregation.

Inhibition of platelet phospholipid methylation during platelet secretion.

A pathway for the synthesis of membrane phosphatidylcholine involving the N-methylation of phosphatidylethanolamine has been detected in several types of mammalian cells. Furthermore, it has been implicated in the coupling of agonist binding to cell response. We examined whether human platelets exhibit this synthetic pathway and whether platelet agonists influence its activity. When washed platelets were incubated with 0.15 microM L-[methyl-3H]methionine at 37 degrees C, they incorporated methyl-3H into their phospholipids linearly at the rate of 1 pmole/10(9) platelets/hr. When incubated with 20 microM radiolabeled methionine, they incorporated about 15 pmole/10(9) platelets/hr. The radioactivity was found predominantly in phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine. Thrombin caused an immediate (within 15 sec) and sustained (up to 30 min) decrease in the rate and extent of N-methylation of platelet phospholipids. This was accounted for by a decrease in synthesis of methylated phospholipids rather than an increase in their degradation. This thrombin effect correlated with serotonin release and could be dissociated from platelet aggregation and prostaglandin synthesis. Thrombin also decreased the synthesis of phosphatidylcholine when choline was used as the radiolabeled substrate. Other agonists such as epinephrine, adenosine diphosphate (ADP), or A23187 also decreased phospholipid methylation under conditions in which they stimulated serotonin release. These data demonstrate that platelets are capable of synthesizing phosphatidylcholine from phosphatidylethanolamine by N-methylation and that agonists perturb this pathway as they induce platelet secretion. The precise role of phospholipid methylation in either resting or stimulated platelets remains to be established.

Prevention of thrombosis in patients on hemodialysis by low-dose aspirin.

Since platelet cyclo-oxygenase is much more sensitive to inactivation by aspirin than is the enzyme in the arterial wall and low doses of aspirin may prevent thrombosis by blocking thromboxane synthesis, we conducted a randomized, double-blind trial of aspirin (160 mg per day) vs. placebo in 44 patients on chronic hemodialysis. The study was continued until there were 24 patients with thrombi and both groups had been under observation for a mean of nearly five months. Thrombi occurred in 18 of 25 (72 per cent) of patients given placebo and 16 of 19 (32 per cent) of those given aspirin (P less than 0.01). The incidence of thrombosis was reduced from 0.46 thrombi per patient month in the placebo group to 0.16 thrombi per patient month in the aspirin group (p less than 0.005). A dose of 160 mg of aspirin per day is an effective, nontoxic antithrombotic regimen in patients on hemodialysis.

Sensitivity of fatty acid cyclooxygenase from human aorta to acetylation by aspirin.

The rate of acetylation of fatty acid cyclooxygenase (prostaglandin synthase, EC 1.14.99.1) by [acetyl-3H]-aspirin was measured in microsomes from human aortas and coronary arteries and intact and disrupted human platelets. We also measured the inhibition by aspirin of prostacyclin generation from exogenous arachidonic acid in shredded human aorta. Cyclooxygenase in human aorta and coronary artery microsomes is approximately 1/250th as sensitive to aspirin as enzyme in intact platelets, and 1/60th as sensitive to aspirin as enzyme measured in a platelet microsomal preparation. On the basis of the in vitro data presented, we predict that small oral doses of aspirin are sufficient to inhibit platelet prostaglandin production but are not sufficient to substantially affect aorta or coronary artery prostaglandin production.

Inhibition of platelet prostaglandin synthetase by oral aspirin.

Aspirin inhibits platelet function by permanently acetylating the cyclooxygenase that forms prostaglandins. We determined the sensitivity of platelets to aspirin in normal subjects by measuring [3H-acetyl]aspirin-susceptible cyclooxygenase in washed platelets obtained at various times after aspirin ingestion. A single 325-mg aspirin dose inactivated 89% of platelet cyclooxygenase. The inhibition persisted for 2 days suggesting that oral aspirin also inactivated megakaryocyte cyclooxygenase. Thereafter, active enzyme returned with a time-course reflecting platelet turnover (life-span 8.2+/-2 days). Single doses of 20-650 mg aspirin resulted in 34- greater than 95% inhibition after 24 h. Daily doses of 20-325 mg aspirin for brief periods produced 61- greater than 95% inactivation when measured 24 h after cessation of the drug. Platelet cyclooxygenase is more sensitive to inactivation by aspirin than enzyme in sheep seminal vesicles.

Cotransfer of thymidine kinase and galactokinase genes by chromosome-mediated gene transfer.

The Chinese hamster genes for thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) and galactokinase (ATP:D-galactose 1-phosphotransferase, EC 2.7.1.6) have been cotransferred to mouse cells by chromosome-mediated gene transfer. Hamster metaphase chromosomes were incubated with mouse B82 cells and 22 independent colonies were isolated in a selective medium. All of the 12 colonies analyzed expressed the donor form of thymidine kinase; the hamster form of galactokinase was also expressed in 2 of these colonies, indicating cotransfer with a frequency of about 20%. There was coordinate loss of both transferred genes from each colony when selection was applied for the loss of thymidine kinase alone. Comparison of the regional localization of these two linked genes with the frequency of cotransfer suggests that the transgenome is probably not larger than about 0.25% of the donor genome.

Human gene expression in rodent cells after uptake of isolated metaphase chromosomes.

Permanent transfer of genetic information from chromosomes isolated from human diploid cells to recipient cells has been demonstrated. Human metaphase chromosomes were incubated with mouse A9 fibroblasts deficient in hypoxanthine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7). Colonies of cells containing hypoxanthine phosphoribosyltransferase appeared during growth in a selective medium. The hypoxanthine phosphoribosyltransferase gene product in four independent colonies was identified as human donor species by both gel electrophoresis and isoelectric focusing; hence these colonies did not result from reversion of ta9 parental cells. Other X-linked human genes, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NAD(+) 1-oxidoreductase, EC 1.1.1.49) and phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3), were not expressed in these same colonies. Dissociation of expression of these X-linked genes probably results from chromosomal fragmentation during uptake, but other mechanisms have not been excluded.