Dried sera for confirming blood-borne virus infections (HCV, HTLV-I, HIV & HBsAg).

For safe blood transfusion, developing countries face considerable problems including serological screening and confirmation of blood-borne virus infections (HCV, HTLV-I, HIV and HBsAg). Confirmation tests are not only costly but also require sophisticated techniques and expertise. In order to provide this support we have attempted to perform a virus antibody confirmation test on samples dried on blotting paper (BP). Forty-nine sera derived from selected patients and donors from Bombay, and nine donors' sera from Bellarussia were transported on BP. In control experiments, dilutions of antibody-positive sera (HIV, HTLV-I & HCV) and 'blinded' HTLV-I antibody-positive and antibody-negative donors were applied on BP. Eluates from snipped BP were tested initially by screening tests, and the reactives were subjected to confirmatory tests for three types of virus antibody tests (HCV, HTLV-I & HIV) by blotting methods and neutralisation tests for HBsAg. There was considerable reduction of titres in dry sera but all BP-derived dry specimens gave excellent qualitative concordance with their liquid-equivalent sera, and the HTLV-I-positive donor was identified and reconfirmed correctly. Presence of only HCV antibody was confirmed in all the nine selected Bellarussian donors. Blood donors in Bombay had 3% HIV antibody, 6% HBsAg and none had HCV antibody, while selected patients showed substantially higher levels of these markers: HIV-antibody 64%, HBsAg 57% and HCV-antibody 17% confirmed positive. The cause of this high level remains to be established. Dry samples received by post seem to be an economical approach to a first step in providing some levels of independent confirmation of reactives in developing countries.

The Use of Fresh Plasma and a Plasma-free Medium to Enhance the Aggregation Response of Stored Platelets.

Platelet aggregation in response to ADP (10 µM) and collagen (4 µg/ml) in fresh and stored platelet concentrates (PC) and the enhancement of aggregation of the stored platelets after resuspension in fresh plasma and plasma-free medium were measured. The ability of platelets in autologous plasma to respond to the two agonists decreased significantly on days 2 and 5 of storage to 18 and 4% (p < 0.001) respectively of that seen in platelet-rich plasma on day 0 (100%). A 2-fold or greater improvement (p < 0.01) in the aggregation response was achieved when the autologous plasma in stored PC was replaced by fresh allogeneic plasma just before testing. The effect was even greater (three-fold or more, p < 0.001) when the autologous plasma was first replaced by plasma-free medium followed by suitable dilution for the test in fresh allogeneic plasma. These observations indicate another way to rejuvenate stored platelets and enhance their residual capacity to aggregate ex vivo. They could provide a basis for a suitable test for use within a quality assurance programme for stored PC.

The use of mean platelet volume for evaluation of quality of platelet concentrates.

Mean platelet volume (MPV) was determined on whole blood and platelet concentrates (PC) prepared from units of the same blood, as well as on samples of venous blood taken from donors before plateletpheresis and on PC collected by Spectra (COBE Laboratories Ltd) and CS-3000 (Baxter Healthcare Ltd) cell separators. The mean (+/- SD) MPV for PC prepared from blood (7.18 +/- 0.76 fl, n = 12) was significantly lower than that for whole blood (8.32 +/- 0.72 fl, P < 0.02) suggesting significant separation of young, large and dense platelets together with the red cells. In contrast, the mean MPV for PC collected with Spectra and CS-3000 cell separators was 8.48 +/- 0.52 fl (n = 20) and 8.94 +/- 0.60 fl (n = 12), respectively, and was significantly higher (P < 0.01) than that determined in venous blood samples of donors taken before plateletpheresis (7.76 +/- 0.74 fl and 8.12 +/- 0.62 fl respectively). This indicates preferential separation of large platelets, which are by inference, of better quality, into PC.

Stabilization of standard platelet concentrates and minimization of the platelet storage lesion by a prostacyclin analogue.

Platelet concentrates were pretreated with a stable synthetic prostacyclin analogue (Iloprost) at two different concentrations before the second centrifugation step (pelleting step) of preparation. This resulted in loss of platelet sensitivity to aggregating agents. To mimic the situation after transfusion and to assess the reversibility of platelet inhibition, platelets were washed during and after storage and resuspended in fresh-frozen autologous plasma. The Iloprost-treated and washed platelets exhibited an increased sensitivity to the aggregating agents, compared with the control platelets (p less than 0.01). Post-storage recovery of the synergistic aggregation was more than 80% of prestorage aggregation. Beta-thromboglobulin (beta TG) release and thromboxane B2 (TXB2) formation were significantly inhibited in Iloprost-treated platelets (p less than 0.01). After the second centrifugation step, beta TG release was 0.7% +/- 0.3%, compared with 2.7% +/- 0.9% for the controls. TXB2 was 99 +/- 91 pg/ml, compared with 495 +/- 356 pg/ml for the controls. Platelet morphology and ultrastructure were well preserved during 5-day storage. In addition, Iloprost exerted a cytoprotective effect, as evidenced by the significant reduction in lactate dehydrogenase leakage. Post-storage LDH was 378 +/- 159 and 415 +/- 239 U/l respectively by the two Iloprost concentrations, compared with 1180 +/- 937 U/l for the control platelets. The inhibitory and cytoprotective effects of Iloprost were sustained throughout storage, in contrast to the effect of PGE1 (Prostin) which was limited to the early phase of storage.

In vitro evaluation of a high-efficiency leukocyte adherence filter.

In view of the currently available data, prevention of alloimmunization requires filters with higher efficiency to achieve a reduction in the number of leukocytes below 10 million per transfusion. Two versions (Pall PL-100 and PL-50) of the new generation leukocyte-depletion filters were studied. Single donor (SDPC)- and pooled multiple donor (MDPC) platelets were run in parallel. At a flow rate of 10 ml/min, the PL-100 filter was shown to effectively reduce the number of residual leukocytes to far below the critical immunogenic threshold of 10 million in all SDPC units and in 77% of MDPC units. Apheresis platelets appear not only to be better depleted than pooled multiple donor platelets, but also to have a better post-filtration platelet recovery (96% versus 84%). The efficiency of the smaller version of the filter (Pall-50) was higher than that of the Pall-100 filter for both single and pooled multiple donor platelet concentrates (PC). Leukocytes were absent in more than 92% of units in both types of concentrates. The maximal number of detected leukocytes was 2.2 million in a pool of 6 units. The outcome of filtration of 5-day-old pooled platelets was less favorable than filtration of 1- or 2-day-old pooled platelets, indicating that filtration soon after preparation is preferred to filtration after storage. Post-filtration platelet integrity, activation state, function, and morphology were all well preserved in both single and multiple PCs.

Effect of EDTA on platelet count and other platelet parameters in blood and blood components collected with CPDA-1.

We have shown in this study that addition of dried K2EDTA (1.5 mg/ml) to blood samples anticoagulated with CPDA-1 increases significantly the platelet count and mean platelet volume (MPV) of whole blood, red cell concentrate (RCC) and platelet concentrate (PC), but not of platelet-rich plasma (PRP) or of platelet-poor plasma (PPP). Transmission and scanning electron microscopy illustrated that platelet aggregates which are present in some components are dispersed on mixing of the sample with EDTA and that this is accompanied by a change in platelet morphology. Determination of the platelet distribution width (PDW) indicated that the platelet populations in whole blood and RCC seem to be more uniform in size than the populations in PRP, PPP and PC. The determination of MPV and PDW changes after addition of EDTA may provide a new approach to quality control of PC.

Differences between the effects of EDTA and citrate anticoagulants on platelet count and mean platelet volume.

Platelet counts on whole blood samples collected into tripotassium salt of EDTA, trisodium citrate (Na3 citr), citrate phosphate dextrose adenine formula 1 (CPDA-1) and acid citrate dextrose formula A (ACD-A), all showed a statistically significant drop (P less than 0.01) after 1 h standing at room temperature (RT) as compared with the immediate (within 30 min) counts. After 1 h the enumeration became stable in the EDTA samples but the drop continued up to 4-6 h in those samples taken into citrate. The decreases in citrate were significant (18-30%, P less than 0.001). The addition of EDTA (1.5 mg/ml) to the citrated samples after the sixth hour count created a significant rise (6-22%, P less than 0.01) in the counts between the sixth and the seventh hour. Our observations show that platelet counts in citrated blood samples are lower than those in EDTA and highlight the necessity to present citrated samples mixed wtih dried EDTA when characterization or quality control of blood and blood components is required. Analysis of the mean platelet volume (MPV) showed significantly lower values (6-13%, P less than 0.05) in the citrated samples as compared to the same samples in EDTA, and a significant increase (4-6%, P less than 0.01) on the addition of EDTA to the citrated samples after the sixth hour analysis.