Sensitivity and specificity of standard and rapid HIV-antibody tests evaluated by seroconversion and non-seroconversion low-titre panels.

BACKGROUND AND OBJECTIVES: The aim of this study is to compare the relative sensitivity and specificity of commercial HIV-antibody assays using seroconversion, non-seroconversion panels, and negative blood donor samples. MATERIALS AND METHODS: We evaluated the sensitivity of five standard ELISA HIV-antibody assays: Vironostika HIV Uni-Form II, Abbott recombinant HIV-1/HIV-2 third-generation EIA, Biotest Anti-HIV-1/-2 recombinant, Recombigen HIV-1/ HIV-2 EIA and Wellcozyme HIV 1 + 2 (VK54/55), and three rapid screening tests, Capillus HIV-1/HIV-2, Abott Test Pack HIV-1/HIV-2 third-generation EIA, and Sensy-Test HIV 1/2. All tests were assessed using four panels of plasma samples obtained from individuals who were seroconverting and a low-titre HIV-antibody panel of samples. Specificity of the standard screening tests was determined on 3.500 HIV-antibody-negative blood donor samples. RESULTS: There was no statistically significant difference in sensitivity between the five standard ELISA tests. One of these tests was significantly less specific than the others. The standard ELISA tests detected all the low-titre HIV-antibody-positive samples. Two of the rapid screening tests were significantly less sensitive on the seroconversion panels and all three tests failed to detect at least one of the positive samples in the low-titre panel. CONCLUSIONS: The additional risk of using one or other of the standard ELISA tests under review of not detecting all HIV-positive units of blood is not statistically significant. Using some of the rapid screening tests will, however, add a significant additional risk. A rapid screening test should therefore be adopted only after careful consideration of the effect of a possible lack of sensitivity on the safety of the blood supply.

Influence of platelet membrane sialic acid and platelet-associated IgG on ageing and sequestration of blood platelets in baboons.

Platelets were isolated from blood of baboons and treated with neuraminidase to remove platelet membrane sialic acid, a process which artificially ages the platelets. The platelets were then labelled with 111In and their mean life span, in vivo distribution and sites of sequestration were measured. The effect of removal of sialic acid on the attachment of immunoglobulin to platelets were investigated and related to the sequestration of the platelets by the spleen, liver, and bone marrow. Removal of sialic acid by neuraminidase did not affect the aggregation of platelets by agonists in vitro, nor their sites of sequestration. The removal of 0.51 (median, range 0.01 to 2.10) nmol sialic acid/10(8) platelets shortened their life span by 75 h (median, range 0 to 132) h (n = 19, p < 0.001), and there was an exponential correlation between the shortening of the mean platelet life span and the amount of sialic acid removed. The increase in platelet-associated IgG was 0.112 (median, range 0.007 to 0.309) fg/platelet (n = 25, p < 0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/10(8) platelets was removed (p < 0.001). There was an exponential correlation between the shortening of mean platelet life span after the removal of sialic acid and the increase in platelet-associated IgG. The results suggest that platelet membrane sialic acid influences ageing of circulating platelets, and that the loss of sialic acid may have exposed a senescent cell antigen that binds IgG on the platelet membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vivo effect in humans of pyridoxal-5'-phosphate on platelet function and blood coagulation.

Vitamin B6 has an antithrombotic effect. This, based on the results of in vitro studies, has been attributed to an antiplatelet effect. We assessed the in vivo effect of vitamin B6 by measuring the effect of long-term administration of vitamin B6 on platelet function and blood coagulation. Vitamin B6 (pyridoxine hydrochloride), 100mg twice daily p.o. for fifteen days, was administered to 10 healthy volunteers. The bleeding time was measured before the first dose and 15 days after. A baseline value, the acute effect, chronic effect, and the acute-on-chronic effect of vitamin B6 was estimated by measuring platelet function. The following tests were performed: platelet aggregation induced by collagen, ADP and epinephrine; thromboxane A2 (TxA2)-production and prostacyclin inhibition of ADP-induced aggregation. The effects on the coagulation system were monitored by measuring: the prothrombin time, activated partial thromboplastin time and levels of coagulation factor. Vitamin B6 significantly prolonged the bleeding time from 4.1 +/- 1.1 minutes to 6.8 +/- 1.0 minutes (p = 0.0063). Aggregation of platelets with collagen was slightly but not significantly inhibited. Platelet aggregation induced with the agonists ADP or epinephrine was significantly inhibited by vitamin B6, and the platelets tended to aggregate at a slightly decreased rate. The mean TxA2-production was slightly, but not significantly, decreased. Vitamin B6 had no effect on the sensitivity of platelets to prostacyclin, or on the coagulation system. Our results indicate that the antithrombotic effects of vitamin B6 is limited to inhibition of platelet function; there was no measurable influence on coagulation. The results of this in vivo study are however such that clinical trials are warranted to further assess the efficacy of vitamin B6 as an antiplatelet drug.

The influence of platelet-graft interaction on platelet survival in patients with aortobifemoral Dacron grafts.

In patients with arterial grafts, platelet consumption may be due either to platelet interaction with the graft, and/or concomitant platelet consumption in the rest of the arterial tree. This hypothesis was tested by quantifying the kinetics and platelet-graft interaction of indium-111-labelled platelets with double velour Dacron grafts in 13 patients with arterial insufficiency ascribed to atherosclerosis. Mean platelet lifespan (MPLS), 149 +/- 46 hours, was significantly shorter (P = 0.001) than normal. Labelled platelets were transiently deposited onto the graft surfaces. Peak 111In deposition on the grafts, 1.33 +/- 1.02% of injected labelled platelets, was reached at 70 +/- 33 hours. Thereafter the graft-platelet radioactivity decreased in parallel with platelet radioactivity in the circulation. There was no statistical correlation between MPLS and the factors known to be associated with graft platelet deposition: graft size; peak graft radioactivity; and the time to attain peak graft radioactivity. It is therefore concluded that in patients with arterial disease requiring graft implantation, the observed increased platelet consumption cannot only be ascribed to the interaction of platelets with the graft. Concomitant atherosclerosis is probably the important modifier of platelet consumption. The significant contribution of this factor to the shortening of the MPLS should be taken into account when assessing, by measuring platelet lifespan, the thrombogenicity of grafts.

Blood platelet kinetics in normal subjects modelled by compartmental analysis.

The purpose of this study was to describe the function of platelets throughout their life span by expressing their in vivo distribution and kinetic behaviour in mathematical terms by using multicompartmental analysis. The distribution of indium-111 labelled platelets in five normal subjects was imaged and quantified with a scintillation camera image processing system. Serial blood samples were also obtained. The data were modelled using the SAAM (Simulation Analysis and Modelling) compartmental computer program. Five models were entertained to evaluate the role of platelets that were either functional or injured during collection and their interaction with the liver, spleen and vascular endothelium. Models were evaluated by comparing F values calculated from the least squares estimate obtained from each model. The Dornhorst function was used to describe the sequestration of platelets in the compartmental model. Results indicated that the data could not be satisfactorily simulated when compartments were included that simulated only functional and sequestered platelets (model 1). It was necessary to include compartments that simulated the kinetics of collection-injured platelets in the liver (model 2) and spleen (model 3). The model that simulated the interaction with the vascular endothelium (model 5) showed a visual but not significant improvement in the fitting of the observed data compared to model 3. The mean organ uptake and range indicated in parentheses were calculated at equilibrium. There were 20% (15%-27%) of the injected platelets in the spleen, 10% (8%-11%) in the liver and 70% (64%-75%) in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Reference values for red cell survival times.

The first purpose of this investigation was to investigate in 35 young normal male subjects the use of the Dornhorst function and the weighted-mean method to calculate reference values for mean red cell survival time with and without correction for elution of 51Cr. We compared survival times calculated with the Dornhorst and weighted-mean methods with survival time estimated with linear or exponential models. Two methods to correct for elution of 51Cr from red cells were investigated. For the first method, correction factors were generated using the Dornhorst function fitted to mean survival curves obtained from the normal subjects. In the second method, the new Dornhorst rate constant method, the survival time, corrected for elution of 51Cr, was directly calculated from the experimental survival curve without applying correction factors. Correction for elution using the Dornhorst rate constant method was not successful and resulted in nonphysiologic values. The 95% confidence range of red cell survival time for reference subjects without correction for 51Cr elution was 37-74 days for the weighted-mean method and 37 to 73 days for the Dornhorst method. The 95% confidence range for normal subjects when the survival curves were corrected for elution was 47-179 days for the Dornhorst method and 58-161 days for the weighted-mean method. The poor results obtained with the Dornhorst rate constant method and the large 95% confidence range were due to the rapid and large variation in elution rate of 51Cr from red cells.

Comparison of oxine and tropolone methods for labeling human platelets with indium-111.

The effect of the chelates oxine and tropolone, used to label platelets, on the kinetics of indium-111-(111In) labeled platelets was studied in twelve normal human subjects. Autologous platelets were labeled either in saline with 111In-oxine or in plasma with 111In-tropolone. Mean platelet lifespan was estimated by fitting the disappearance curve of platelets from the circulation to the multiple hit and other mathematical models. The in vivo distribution of platelets was quantitatively imaged with a scintillation camera. The in vivo recovery of 111In-oxine and 111In-tropolone did not differ, and the mean platelet lifespan was also similar (111In-oxine: 230 +/- 29 hr; 111In-tropolone: 226 +/- 13 hr). At equilibrium (90 min after reinjection of labeled platelets) and at the end of platelet lifespan, 111In-oxine and 111In-tropolone radioactivities in the spleen and liver were similar. These results demonstrate that the results of kinetics measured with 111In-oxine or 111In-tropolone do not differ significantly.

Analysis of indium-111 platelet kinetics and imaging in patients with aortic grafts and abdominal aortic aneurysms.

To quantitatively characterize processes of platelet thrombus formation in vivo, the kinetics and incorporation into thrombus of autologous In-111-labeled platelets were compared in six patients with aortic aneurysms and in seven patients with prosthetic aortic grafts. Although platelet survival was comparably shortened in both patient groups (mean, 5.8 days), the maximum radioactivity (percentage of whole body radioactivity) as determined by gamma camera imaging was higher in the aneurysms than in the grafts (3.3% +/- 1.6% vs. 1.6% +/- 1.1%, p = 0.05). Maximum In-111 uptake was also attained more quickly in the aneurysm patients (2.3 +/- 0.8 days vs. 3.5 +/- 1.3 days; p = 0.07). The experimental platelet kinetic and imaging data were subsequently evaluated by compartmental analysis to estimate both normal and disease-related components of platelet destruction. This analysis indicated that deposited platelet radioactivity had a longer residence time on grafts (2.9 +/- 1.7 days vs. 1.4 +/- 0.9 days, p = 0.07) but accumulated at a faster rate in aneurysms (5.0% +/- 3.4% per day vs. 1.4% +/- 0.9% per day, p = 0.02). As determined by imaging, only a proportion of increased platelet destruction was specifically due to the aneurysms (55% +/- 38%) or grafts (17% +/- 11%, p = 0.03). This result indicates additional components of platelet destruction unrelated to graft and aneurysm thrombus formation which, in some graft patients, may reflect a greater severity of vascular disease or other mechanisms causing a preferential shortening of platelet survival. Thus, the analytical approach described may be a useful one for discriminating components of in vivo platelet utilization including platelet removal due to normal hemostatic and senescent mechanisms, localized thrombus formation, and more generalized vascular disease.

The effect of sepsis and short-term exposure to nitrous oxide on the bone marrow and the metabolism of vitamin B12 and folate.

It is recognised that prolonged anaesthesia with nitrous oxide (N2O) induces megaloblastic anaemia by oxidising vitamin B12. To determine whether sepsis aggravates the effect of N2O on haemopoiesis 5 patients with severe sepsis, who required surgery and were exposed to short-term (45-105 minutes) N2O anaesthesia, were studied. None had evidence of pre-operative vitamin B12 or folate deficiency. The effect of the combination of N2O anaesthesia and sepsis on DNA synthesis in bone marrow cells was assessed morphologically, and by the deoxyuridine suppression test. In 3 patients exposed to the longest duration (75-105 minutes) of N2O, addition of folinic acid and vitamin B12 partially improved the utilisation of deoxyuridine in vitro. No patient had evidence of megaloblastic haemopoiesis as judged by bone marrow morphology. It is concluded that prolonged N2O anaesthesia in patients with severe sepsis may adversely affect DNA synthesis. Although this effect did not manifest as overt megaloblastic erythropoiesis, it may be prudent to avoid N2O in such patients.

The sensitivity of tests to detect in vivo platelet activation induced by the removal of arterial endothelium of the baboon (Papio ursinus).

The relative sensitivities for the various in vivo and in vitro tests for platelet activation are unknown. This was studied in a baboon model where limited and more substantial injury to the vascular endothelium was inflicted. The endothelium of a segment of the right carotid artery was removed with a balloon catheter on day 0 (limited de-endothelialisation), and that of the left carotid artery, abdominal aorta and left femoral artery on day 7 (substantial de-endothelialisation). Eight baboons (Papio ursinus) were used. Baseline tests for platelet activation (platelet volume, platelet density, platelet aggregate ratio, and platelet and plasma levels of platelet factor 4 [PF4] and beta-thromboglobulin [beta-TG]) were performed 7 days before de-endothelialisation and repeated on days 1, 9 and 16. The kinetics of indium-111-labelled platelets were measured after substantial de-endothelialisation. Sham operations were done on 3 animals exactly as in the test, except that the balloon injuries were not inflicted. No influence on the results of the platelet function tests was found. The only test capable of detecting limited injury to the endothelium was the measurement of plasma PF4. The mean platelet life-span (MPLS) shortened, mean platelet density decreased, the circulating platelet aggregate ratio decreased, and plasma levels of PF4 and beta-TG increased (P less than 0.05 in all instances) after the substantial endothelial injury. The mean platelet volume, intraplatelet PF4 and beta-TG, and the in vivo distribution and sites of sequestration of labelled platelets were poor tests for in vivo platelet activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Low molecular weight heparin as an anticoagulant for in vitro platelet function studies.

We evaluated the suitability of low molecular weight (LMW) heparin as an anticoagulant for in vitro platelet function tests in 11 normal volunteers. Results with citrated platelets were considered as the standard. Spontaneous platelet aggregation and the aggregation responses to ADP, epinephrine, collagen, ristocetin and thrombin were measured turbidimetrically in an aggregometer. Dose-response and dose-rate curves were constructed for ADP- and epinephrine-induced aggregation. The maximum aggregation response (EDmax) and rate (EDRmax), and the estimated dose of agonist to induce 50% of the maximum response (ED50) and rate (EDR50) were calculated from these curves. The inhibition of ADP-induced aggregation with PGI2 was expressed as per cent inhibition. The release of ATP and TxA2, from platelets aggregated with collagen was measured. No spontaneous aggregation occurred with either anticoagulant. The ED50 and the EDR50 for heparinized platelets were significantly lower for ADP induced aggregation (0.8 +/- 0.3 microM vs 2.1 +/- 1.0 microM [p = 0.001] and 0.4 +/- 0.1 microM vs 0.8 +/- 0.3 microM [p = 0.003]). The EDRmax with ADP was significantly higher (p = 0.004) for heparinized platelets (64.6 +/- 17.0 units/ml vs 50.4 +/- 7.6 units/ml). The heparinized platelets aggregated slightly, but significantly, less in response to ristocetin than the citrated platelets. The response of washed heparinized and citrated platelets to thrombin was not significantly different. The per cent inhibition of ADP aggregation with PGI2, was significantly lower with heparinized platelets. The release of TxA2 and ATP was similar for both anticoagulants. These results indicate that LMW heparin is a satisfactory anticoagulant for platelet aggregation tests.

Platelet survival in patients with beta-thalassemia.

Thromboembolic events associated with significant morbidity and mortality have been observed in patients with beta-thalassemia major (TM). These include arterial as well as venous thrombosis and the development of early arteriosclerosis. To elucidate the possibility that TM patients may develop a hypercoagulable state we carried out a study of platelet kinetics on ten patients with TM and four patients with thalassemia intermedia (TI). Autologous platelets were labeled with indium-111-oxine, and the platelet lifespan (PLS) was determined. A significant shortening of PLS was observed in 13 out of 14 patients examined. The mean PLS (+/- 1 SD) in ten patients (8 TM, 2 TI) who underwent splenectomy was 107 +/- 36 hr (control splenectomized 248 + 51 hr) (P less than .001) and in four nonsplenectomized patients (2 TM, 2 TI) was 102 +/- 64 hr (control 224 + 23 hr) (P less than .01). The short PLS in addition to reported findings of increased circulating platelet aggregates and the decreased response of TM platelets to aggregating agents suggests in vivo platelet activation in thalassemic patients.

Thromboembolic potential of synthetic vascular grafts in baboons.

We have compared in baboons the capacity of two types of synthetic vascular grafts to accumulate thrombus, activate circulating platelets, and generate occlusive platelet microemboli. Grafts were incorporated into femoral arterial-arterial shunts placed unilaterally in 10 baboons; the unoperated contralateral limbs served as controls. The accumulation of indium 111 (111In)-labeled platelets onto the grafts (expanded polytetrafluoroethylene [ePTFE] or knitted Dacron, 4 mm inner diameter) and the appearance of 111In radioactivity in distal microcirculatory beds (calf and foot) were quantified by dynamic scintillation camera imaging. After 1 hour total platelet deposition per graft was higher with Dacron (49.0 +/- 8.0 x 10(9) platelets) than with ePTFE (3.7 +/- 0.6 x 10(9) platelets, p less than 0.01). Platelet counts decreased and beta-thromboglobulin levels increased with Dacron graft placement but were unaffected by ePTFE graft placement (p less than 0.05 and p less than 0.01, respectively). Emboli shed from Dacron grafts were detected as multifocal, irregular, and changing deposits in the calves and feet. Indium 111 platelet activity in the feet distal to the Dacron grafts increased 81.1% +/- 21.4% from baseline values over 1 hour, whereas the activities in the feet distal to the ePTFE grafts were unchanged (p less than 0.05). The increase 111In-platelet radioactivity above the control limb values (excess radioactivity) was higher for the Dacron graft group than for the ePTFE group in both the feet (139.6% +/- 46.9% vs 6.2%, p less than 0.05) and the calves (86.7% +/- 21.7% vs 7.3% +/- 3.6%, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)