Mechanisms of white cell reduction in red cell concentrates by filtration: the effect of the cellular composition of the red cell concentrates.

The effect of platelets on the removal of white cells (WBCs) from 16 to 24-hour-old red cell (RBC) concentrates by filtration was studied. RBC concentrates with various concentrations of platelets and WBCs were filtered on a cellulose acetate column filter and on three polyester flatbed filters. The microscopic study revealed that lymphocytes and most monocytes were captured in the smaller pores of the fiber network, irrespective of the brand of filter, the type of filter material, or the prefiltration platelet amount in the RBC concentrates. In contrast, efficient granulocyte depletion depended on granulocyte-platelet interaction and on the filter material. In the presence of platelets, granulocytes were captured in the top part of the column filter or in the coarse layers of two of the flatbed filters, where platelets covered the fibers. Platelet depletion of the RBC concentrates prior to filtration diminished the contribution of these parts of the filters to granulocyte capture. A larger part of the column filter or the fine layers of the flatbed filters were now required for granulocyte capture. In one of the flatbed filters, granulocyte-platelet interaction occurred mainly in the fine layers, which ended in blockage of this filter after the filtration of variable volumes (250-600 mL) of standard RBC concentrates. A quantitative estimation of the effect of platelets on the WBC-reduction capacity found that all three flatbed filters had a highly significant decrease (p = 0.001) in WBC-reduction capacity for platelet-depleted or buffy coat-depleted RBC concentrates, as compared with standard RBC concentrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Asymmetric membrane filters for the removal of leukocytes from blood.

As part of a study on the mechanisms of leukocyte filtration, the influence of pore size distribution on filter efficiency was investigated. Conventional leukocyte filters are not suitable for model studies, as these filters are composed of tightly packed synthetic fibers, with a poorly defined porous structure. Therefore, open cellular polyurethane membranes with pore size distributions varying from approximately 15 to 65 microns were prepared. Filtration experiments with stacked packages of these membranes showed that leukocytes are best removed (greater than 99%) by filters with a pore size distribution of 11-19 microns. These pore sizes approach the size of leukocytes (6-12 microns). However, due to fast clogging, blood flow through these filters is rapidly reduced, which results in a low filter capacity. With an asymmetric membrane filter, in which the pore size decreases from about 65 to 15 microns in the direction of blood flow, both moderate removal of leukocytes (greater than 80%) and maintenance of flow (approximately 0.2 mL/s) are obtained. This results in efficient leukocyte removal. From cell analysis of both filtrate and filter, it is concluded that adhesion rather than sieving is the major filtration mechanism. Thus, further optimization of the filter may be achieved by surface modification.

A flow cytometric method for determination of white cell subpopulations in filtered red cells.

A flow cytometric method for the detection of low amounts of lymphocytes, monocytes, and granulocytes in filtered red cells (RBCs) was evaluated. In this procedure, the RBCs in the samples were lysed by ammonium chloride treatment and the white cells (WBCs) were detected by flow cytometry according to their specific light-scattering properties. The identity of the WBC subpopulations was confirmed by immunofluorescence with monoclonal antibodies specific for each cell type. Flow cytometric determination of WBCs in filtered RBCs correlated with numbers obtained by both a hemocytometer (r = 0.76) and a radioimmunoassay (r = 0.79). Total numbers of WBCs in RBCs measured by flow cytometry were 59 +/- 13 percent (n = 7) of those measured by electronic particle counting, 32 +/- 6 percent (n = 25) by hemocytometer, and 48 +/- 11 percent (n = 29) by radioimmunoassay. Lymphocytes added to filtered RBCs in a concentration of 1.37 cells per microL were detected at an average of 0.56 +/- 0.22 cells per microL (n = 3). Results with monoclonal antibodies indicated an altered expression of membrane markers on granulocytes after RBC filtration, as seen with cell activation. The inefficiency of the flow cytometric method to detect the total number of WBCs calculated by other methods may reflect filtration-induced changes in light-scattering properties of the WBCs. Although the method described does not accurately quantitate the total numbers of WBCs present in filtered RBCs, it may provide useful information on qualitative aspects of WBC subpopulations.

A new radioimmunoassay for the detection of small amounts of white cells and platelets in red cell concentrates: implications for blood transfusion.

In the procedure for quality control of red cell concentrates, made white cell (WBC)-poor by filtration, the particle-counting technique was found to be insufficiently sensitive in detecting the remaining WBCs and platelets. Therefore, direct radioimmunoassays were developed using murine monoclonal antibodies specific for platelets, granulocytes, and T lymphocytes. The sensitivity for platelets was 40 x 10(3) per mL, that for granulocytes was 10 x 10(3) per mL (starting from 0.2 mL of red cell filtrate), and that for T lymphocytes was 0.006 x 10(6) per mL (starting from 5 mL) and 0.0015 x 10(6) per mL (starting from 50 mL). These direct assays were used in experiments on filtration with three types of filters: the Cellselect B-1005, B-1014 and the B-1013 (bedside filter). After filtration of 1 unit of blood cell suspension through the B-1005, the number of remaining platelets was found to vary between less than or equal to 0.04 x 10(6) and greater than 15 x 10(6) per mL (n = 16); after filtration through the B-1013 filter, the remaining platelets were greater than 0.04 x 10(6) per mL. Upon filtration of a second unit of blood cell suspension through the B-1013 filter, the number of remaining platelets varied between less than or equal to 0.04 x 10(6) and 5 x 10(6). In both filter types, the number of remaining granulocytes was always less than 0.01 x 10(6) per mL. A study of T-lymphocyte contamination revealed that, upon filtration of 1 unit of blood through the B-1005, T-lymphocyte numbers were less than or equal to 0.0015 x 10(6) to 0.15 x 10(6) per mL (starting from 5 and 50 mL); upon filtration through the B-1013 filter, the number of remaining T lymphocytes varied between less than or equal to 0.006 x 10(6) and 0.2 x 10(6) per mL (starting from 5 mL). After filtration of a second unit of blood cell suspension through the B-1013 filter, the number of remaining T lymphocytes ranged from less than or equal to 0.006 x 10(6) to 0.1 to 0.5 x 10(6) per mL (starting from 5 mL). The direct radioimmunoassay is an improvement over the present electronic particle-counting techniques with regard to both sensitivity and specificity and may therefore be useful in quality control procedures in blood transfusion as well as in the development of new filters.

Monitoring of platelet contamination in filtered red blood cell concentrates. Use of a radioimmunoassay detecting the platelet glycoprotein IIb-IIIa complex.

Monitoring of contaminating platelets, granulocytes, and lymphocytes in leukocyte-poor red blood cell concentrates is usually done by counting in an electronic particle counter. Sensitivity and specificity of this technique are compromised by the contamination of the preparations with other cell types and particles thereof. In this report we studied platelet contamination in filtered red blood cell concentrates by use of a radioimmunoassay for detection of the platelet glycoprotein complex IIb-IIIa. Our results indicate that platelets and/or fragments thereof, not detectable for particle counters, are present in blood cell concentrates. This finding might have important implications for the preparation of pure red blood cell concentrates to avoid unwanted immunization after transfusion.

Electrotypes and formal genetics of red cell glutathione peroxidase (GPX1) in the Djuka of Surinam.

Samples of venous blood from 239 male and 476 female adults including 41 pairs of parents and 123 of their children belonging to a Surinam population called the Djuka or Bush Negroes of West African origin were screened for electrophoretic variants of red cell glutathione peroxidase (GPX1) in Cellogel. The results confirmed an earlier hypothesis that at least a part of the GPX1 variation mainly, if not exclusively, observed in the Africans and people of African origin living elsewhere, is determined by two codominant alleles (called GPX1*1 and GPX1*2), at an autosomal locus. The frequency of GPX1*2 allele in the Djuka was estimated to be .054. A rare variant provisionally designed as GPX1 Djuka (thought to be a heterozygote due to a third allele called GPX1*3 and the GPX1*1) was found in two apparently unrelated individuals. Catalytically, the product of GPX1*2 appears to be about twice more active than that of GPX1*1. For heuristic purposes, it was proposed and discussed that GPX1*2 is a South-Saharan African allele and is amenable for natural selection.

Removal of leukocytes from blood by fibre filtration. A comparison study on the performance of two commercially available filters.

Two different kinds of filters suitable for the almost complete removal of leukocytes from blood-cell concentrates were tested. The maximal retention of filter I was 1.9-3.4 x 10(9) leukocytes per filter, whereas filter II could retain 3.6 - 7.8 x 10(9) leukocytes per filter before the leukocyte concentration in the filtrate passed the level of 500 leukocytes/micrometer. The leukocytes, once absorbed by the fibre material, could be released by washing the filters I, whereas the leukocytes were retained by the material of the filters II. No detectable particles were released after the first 100 ml of filtrate during the washing procedure of either kind of filters. From more than 20% of the filters I, more than 500 pg/ml of endotoxin could be released during the prewashing, whereas none of the filters II was contaminated with endotoxin. The filter II released acetic acid which could be completely removed during the prewashing with 250 ml of saline solution. Operation according to the prescribed conditions of 25 filters of both kinds revealed that the residual leukocyte content in the filtrate was more than 0.25 x 10(9) leukocytes in 8 out of 25 of the filtrates when filters I were used, whereas with all filters II, this content remained lower than 0.1 x 10(9) leukocytes per filtrate. It was concluded that only filter II has sufficient capacity to guarantee the removal of 97% of all leukocytes and 90% of the thrombocytes present in 500 ml of fresh human blood.

Platelet preservation. IV. Preparation and cryopreservation of platelet concentrates from fresh and overnight-stored human blood.

Two methods were used for the preparation of platelet concentrates from fresh and overnight-stored human ACD blood. In a two-step method prostoglandin E1 was added to platelet rich plasma and the mixture was centrifuged to obtain platelet concentrate. The IBM 2991 Blood Cell Processor was used in a one-step method. The concentrates were stored in the frozen state. Concentrates prepared with the two-step method had a higher platelet recovery and a lower leukocyte contamination than did concentrates prepared with the one-step method. Based on serotonin uptake volocity, response to hypotonic stress and available platelet factor 3, no essential differences were observed between platelet concentrates prepared from fresh and overnight-stored blood. Concentrates prepared with the one-step method had a higher serotonin uptake velocity than those prepared with the two-step method. This did not result in a better recovery after cryopreservation.

Large scale preparation of leukocyte concentrates and further purification of lymphocytes and granulocytes with the IBM 2991 cell processor.

A procedure is described for the large-scale production of pure suspensions of human lymphocytes and granulocytes. Platelet-poor buffy-coats, obtained by centrifugation of six units of ABO-compatible ACD blood, were pooled and centrifuged again, resulting in a crude leukocyte concentrate containing approximately 60 per cent of the original amount of leukocytes. Further fractionation of such crude leukocyte concentrates was achieved by scaling up Bøyum's Ficoll-Isopaque density gradient technique with the aid of an IBM 2991 blood cell processor. In this way 2.9 +/- 0.3 X 10(9) mononuclear leukocytes, consisting of 90 +/- 0.7 per cent lymphocytes and 5.3 +/- 0.3 X 10(9) polymorphonuclear leukocytes of 92 +/- 0.7 per cent purity could be isolated in one run. The overall yield was 36 per cent of the original amount of leukocytes.

Experiments on the preparation of blood components with the IBM 2991 blood cell processor.

Experiments were carried out with the IBM 2991 Blood Cell Processor in order to study the sedimentation behaviour of blood cells from human ACD blood during centrifugation. Based on this behaviour procedures were developed for plasmapheresis and leucapheresis using the Blood Cell Processor. Accumulation of platelets was observed to occur at the plasma-cell interface during centrifugation at 1,000 g; this led to the development of a one-step method for the preparation of platelet concentrates.

Serotonin uptake and glycolytic activity of human platelets after prolonged incubation with glucose-poor plasma.

It is confirmed that metabolic energy is a prerequisite for the uptake of serotonin by human blood platelets. Starvation (i.e. incubation at 25 degrees C of platelets resuspended in glucose-poor autologous plasma) generally stops glucose consumption, lactate formation as well as serotonin uptake within 2 h. The addition of glucose restores lactate formation immediately and, independent of total starvation time, to normal or even higher levels. The active uptake of serotonin, however, can be restored only partly after prolonged incubation with glucose, to an extent which seems to be dependent of the preceding starvation time. The results are compatible with the hypothesis that besides an active carbohydrate metabolism another factor, presumably the integrity of the platelet membrane, is obligatory for the active transport of serotonin.