Stored red‐blood‐cells inhibit platelet function under physiologic flow

Background and Objectives The DiaMed Impact R tests platelet function under close to physiological flow conditions. The machine is designed to use whole blood but by adding back compatible red cells, it can be used to study stored platelet concentrates. To date, red cells ≤14 days old have been used. In this study, the effect on the assay of using red cells stored for up to 60 days was examined. Material and Methods This study looked at buffy coat‐derived platelet concentrates on day 2 of storage along with various stored red‐blood‐cells (RBC). To determine whether the age of the RBC is a factor in supporting adhesion and aggregation, platelets were assayed with either RBC stored between 2 and 60 days or with separated ‘young’ and ‘old’ red cell populations obtained using a centrifugation method and confirmed by percoll gradient analysis. Results A statistically significant difference was observed between red‐blood‐cells stored for ≤20 days compared with those which have been stored for 21–60 days in respect of their ability to support platelet adhesion (SC) and aggregation (AS) (P < 0·01). Separating red cells by centrifugation into top (young population) and bottom (old population) showed that the effect of storage was much greater than was any difference between young and old at the individual time‐points e.g. ‘young’ red cells from stored units were poorer at supporting platelet adhesion and aggregation than ‘young’ red cells from fresh units. Conclusion Results suggest that the red cells should be stored for less than 21 days when using this assay. This assay may also allow assessment of red cell functionality.     

单采血小板中红细胞混入量超标原因调查分析

目的:探讨单采血小板中红细胞混入量超标的原因。方法:根据所采集的单采血小板是否红细胞混入量超标(有肉眼可见红细胞),分设正常对照组与超标组,正常对照组为红细胞混入量符合标准的献血者80例;超标组为红细胞混入量超标的献血者23例。分别对2组献血者样本进行全血常规及血红蛋白项目检测。结果:超标组中献血者的Hb、Hct、MCV、MCH及血浆总蛋白结果均低于正常对照组献血者,差异有统计学意义(P0.01);而2组献血者RBC计数、MCHC比较差异无统计学意义(P0.05)。结论:单采血小板红细胞混入量超标与外周血的Hb、Hct、MCV、MCH以及血浆总蛋白等参数的水平降低有关;与献血者多次献血或混合献血造成红细胞未完全成熟有关。     

Distribution of cell-associated prion protein in normal adult blood determined by flow cytometry

Leucocyte subpopulations from normally healthy individuals were identified by recognized combinations of fluorochrome-conjugated antibodies to CD markers and stained by different monoclonal antibodies (MAb) to normal cellular prion protein (PrPC), including the 3F4 MAb. Cell preparations were examined by three-colour flow cytometry. All mononuclear leucocyte subpopulations and platelets expressed PrPC, but polymorphonuclear leucocytes and red blood cells expressed little or no PrPC. The amounts of PrPC expressed by the different cells were calculated by comparison to bead standards. Mononuclear leucocytes expressed 3000-4000 molecules of antibody-reactive PrPC per cell. Resting platelets expressed around 1400 molecules of PrPC per cell, whereas activated platelets expressed around 4800 molecules of PrPC per cell. Extrapolation of these values to the amounts of the various cells in whole blood showed that platelet PrPC accounted for at least 96% of cell-expressed PrPC in blood. The PrPC on mononuclear cells and platelets was sensitive to enzymatic treatment of cells by proteinase k and phosphatidylinositol-specific phospholipase C. Certain anti-PrPC MAbs which showed equivalent intensity of staining to MAb 3F4 on fresh cells showed relative reductions of staining compared to MAb 3F4 on stored cells, indicating possible structural alterations of PrPC under these conditions.     

In vitro Effect on Stored Red Blood Cells and Platelets after a 15-Hour Delayed Refrigeration of Whole Blood prior to Component Preparation in CPD-AD

We extended the time of keeping whole blood at 20-24 degrees C to 15 h (overnight) after phlebotomy for preparing platelet concentrates. We have evaluated the in vitro characteristics of platelets and blood cells prepared from whole blood drawn into CPD-AD, an anticoagulant containing 0.4 mM adenine and 1.5 times more dextrose than CPD. We studied in vitro red cell and platelet function of blood cooled either within 4 h after collection or after a 15-hour delay. In vitro platelet function measured as hypotonic shock reaction, aggregation response to ADP and collagen and 14C-serotonin uptake were not significantly different after preparation and after a 5-day storage period. Units held at room temperature for 15 h after blood collection exhibited a level of 2,3-DPG that was 45% of that exhibited by red cells held for 15 h at 1-6 degrees C. All other in vitro parameters of red cell concentrates measured during 35 days of storage were not significantly different. Based on these in vitro data blood drawn into CPD-AD might be kept up to 15 h at room temperature prior to refrigeration in order to prepare platelet concentrates.     

Examination of a rheological profile for blood using micropore filters

Various techniques have been used to assess the flow properties of blood and blood cells in a range of clinical situations. Filtration through microfilters offers a single technique for measuring the flow properties of all cellular components of blood in one experiment but depends on an assumed ability to recognize cells that make up <10% of leucocytes. The remaining leucocytes, labelled fast leucocytes, were previously presumed to be lymphocytes and granulocytes. This study confirmed the identities of these fast leucocytes as those of lymphocytes and granulocytes in undiluted blood. The transit time for lymphocytes (1.2 s) and granulocytes (1.6 s) is close to that recorded for fast leucocytes (1.7 s). The resistance of each type of blood cell to flow through 5 microm filters was defined in this study as the product of the concentration of that cell in blood and its transit time through a pore in the filter. The total resistance to flow of healthy blood through the filter is 4.46 x 10(7) s/ml and is attributed to plasma (2.7%), red cells (25.9%), fast leucocytes (25.3%) and slow leucocytes (46.1%). In a cohort of 21 men with peripheral arterial disease the total resistance was increased to 7.82 x 10(7) s/ml and attributed to plasma (1.5%), red cells (14.5%), fast leucocytes (21.0%) and slow leucocytes (63.00%). This analysis therefore provides a single test for assessing the flow properties of all the cellular components of blood and plasma.     

Monitoring of Platelet Contamination in Filtered Red Blood Cell Concentrates

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.     

The increased effectiveness of platelet concentrates prepared in acidified plasma

Platelet concentrates prepared in acidified plasma (pH 6.5-6.7) are superiorto concentrates prepared by standard methods, and are 80-90 per cent aseffective as platelet rich plasma (PRP). The use of excess citric acid to acidifyplasma promotes resuspension of the concentrate by eliminating clumping,which is a major factor in the decreased effectiveness of standard concentrates.Analysis of posttransfusion recovery and survival of platelets reveals no evidence of platelet injury in an acid medium.

Acidification of PRP inhibits the aggregation of platelets by adenosine diphosphate (ADP). The presence of endogenous ADP may be an importantfactor in clumping during standard concentrate preparation.

A method of acidification of PRP using citric acid is described which allowspreparation of an effective concentrate from fresh whole blood without subjecting the red cells to acid pH. Reconstitution of the acidified platelet poorplasma and its native red cells increases the citrate molarity by less than 6 percent and results in minimal decrease in pH of the whole blood.

Submitted on May 6, 1965 Accepted on July 25, 1965     

New technologies for the inactivation of infectious pathogens in cellular blood components and the development of platelet substitutes

Despite the increased safety of blood components, achieved through improved donor selection and testing, transfusion recipients remain at risk of transfusion-associated diseases. Transfusion of cellular blood components has been implicated in transmission of viral, bacterial and protozoan diseases. While it is commonly recognized that hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), and retroviruses, such as human immunodeficiency virus (HIV) and the human lymphotrophic viruses (HTLV), can be transmitted through cellular components, other pathogens are emerging as potentially significant transfusion-associated infectious agents. For example, transmission of protozoan infections due to trypanosomes and Babesia have been reported. In addition to viral and protozoan infectious agents, cases of bacterial contamination of platelet and red cell concentrates continue to be reported and may be an under-reported transfusion complication. More importantly, new infectious agents continue to enter the donor population, and there is an inherent time delay before the new pathogens are definitively identified and new tests implemented in order to maintain consistent safety of the blood supply. The paradigm for this problem is the HIV pandemic.During the past decade a number of methods for inactivating infectious pathogens in platelet concentrates have been investigated as a strategy to improve the safety of platelet transfusion therapy. One method of treating platelet concentrates to inactive pathogens has now reached the advanced clinical trial phase in the United States and Europe. Similar efforts with a new class of compounds are underway for red cell concentrates, and two of these are in early phase trials. In addition to studies with allogeneic platelets and red cells, a number of laboratories have described methods for developing platelet substitutes or modified platelets to avoid the use of traditional platelet concentrates as a means to improve safety.     

Effect of adhesive properties of buffy coat on the quality of blood components produced with Top & Top and Top & Bottom bags

BackgroundThe Transfusion Medicine Unit of Reggio Emilia currently collects whole blood using conventional quadruple Fresenius Top & Top bags. In this study, new Fresenius Top & Bottom bags were assessed and compared to the routine method with regards to product quality and operational requirements.ResultsCompared to the traditional system, the whole blood units processed with Top & Bottom bags yielded larger plasma volumes (+5.7%) and a similar amount of concentrated red blood cells, but with a much lower contamination of lymphocytes (−61.5%) and platelets (−86.6%). Consequently, the pooled platelets contained less plasma (−26.3%) and were significantly richer in platelets (+17.9%).DiscussionThis study investigated the effect of centrifugation on the adhesiveness of the buffy coat to the bag used for whole blood collection. We analysed the mechanism by which this undesirable phenomenon affects the quality of packed red blood cells in two types of bags. We also documented the incomparability of measurements on platelet concentrates performed with different principles of cell counting: this vexing problem has important implications for biomedical research and for the establishment of universal product standards. Our results support the conclusion that the Top & Bottom bags produce components of higher quality than our usual system, while having equal operational efficiency. Use of the new bags could result in an important quality improvement in blood components manufacturing.     

Red Cell and Platelet Concentrates from Blood Collected into Half-Strength Citrate Anticoagulant: Improved Maintenance of Red Cell 2,3-Diphosphoglycerate in Half-Citrate Red Cells

This study confirms previous work suggesting equivalent in vitro properties in blood components prepared from donations collected into half-citrate preservative (HCPD) compared to components derived from donations collected into standard citrate-phosphate-dextrose (CPD) preservatives. In addition, red cell products harvested from HCPD donations showed significantly improved maintenance of pH over storage, and this was reflected in improved maintenance of intracellular 2,3-diphosphoglycerate (2,3-DPG). This effect was observed in whole blood and in red cells suspended in a phosphate-containing additive solution (Tuta AAS). Collection into HCPD also improved 2,3-DPG maintenance in red cell concentrates processed following an 18-hour hold at 22 degrees C. These improvements were less pronounced in red cells suspended in a non-phosphate-containing medium (Fenwal Adsol) in which a higher pH was maintained even in units collected in CPD. Platelets harvested from HCPD blood and suspended in plasma showed equivalent quality to platelets from standard donations. Some deterioration of platelet properties was observed when HCPD platelets were stored in a non-citrate synthetic medium. Together with data indicating improved coagulation factor stability, these results suggest that collection into HCPD improves stored blood quality and may also allow logistical benefits in blood component preparation.     

Comparison of in vitro responses to fresh whole blood and reconstituted whole blood after collagen stimulation

Background

In patients who have large bleeds, there is a tendency to transfuse more plasma and platelets than recommended in earlier guidelines, and accordingly many hospitals now provide “transfusion packages” with an intended red cell:platelet:plasma ratio of 1:1:1. The purpose of this study was to investigate in vitro functions of transfusion packs compared with fresh whole blood.

Material and methods

“Reconstituted whole blood” was prepared with the same ratio of red cells, platelets and plasma as used in local transfusion packages. The aggregation and thrombin-antithrombin complex formation responses to collagen stimulation of this reconstituted whole blood were compared with those of fresh whole blood. The storage time of red cells and platelets was varied in a systematic manner, giving nine different compositions of reconstituted whole blood that simulated transfusion packs.

Results

The responses varied significantly between whole blood and reconstituted whole blood -and between the reconstituted whole blood of different compositions. A significant decrease (p<0.005) in collagen-induced platelet count reduction was seen with increasing platelet and red blood cell age. Thrombin-antithrombin complex formation peaked in studies with platelets stored for 5 days. The red cells stored for the longest time induced the greatest thrombin-antithrombin complex formation. Fresh whole blood gives more consistent responses, and the aggregation response to collagen is stronger than in reconstituted whole blood.

Discussion

Our results indicate that in vitro responses of reconstituted whole blood vary substantially according to how long the red cells and platelets are stored for. As the responses obtained by testing whole blood are more consistent and usually stronger, the alternative use fresh whole blood in special conditions should not be excluded without further consideration.     

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.     

Evaluation of haemoglobin, haematocrit, haemolysis, residual protein content and leucocytes in 345 red blood cell concentrates used for the treatment of patients with β-thalassaemia

Background

The aim of this study was to evaluate the quality of red blood cell concentrates obtained from donated whole blood, selected for transfusion therapy of thalassaemic patients, by measuring the following parameters: haemoglobin, haematocrit, percentage haemolysis, residual leucocyte count and residual protein content.

Materials and methods

Overall 345 red cell concentrates were evaluated, of which 205 had been filtered in-line pre-storage and washed and 140 were buffy coat-depleted and used within 2 days of collection. Of the buffy coat-depleted concentrates, 62 were leucodepleted and 78 washed and leucodepleted post-storage all within 2 days of collection. The off-line filters used for the leucodepletion were gamma-irradiated polyester with a pore size of 200 μm. The washing procedure was automated (Haemonetics ACP 215, Braintree, MA, USA). The haematological parameters were evaluated by a blood cell counter (Coulter, Ramsey, IL, USA) and the white blood cell count by cytofluorimetry (FACScan).

Results

Ninety-five percent (194/205) of the red cell concentrates that had been filtered pre-storage and washed, 92% (57/62) of the red cell concentrates that had been leucodepleted post-storage and 94% (73/78) of the those subjected to both treatments had normal values of haemoglobin (>40 g/unit), haematocrit (between 50–70%), percentage haemolysis (<0.8/unit), white cell count (<1×10⁶) and residual protein content (<0.5 g/L). Five percent (11/205) of the red cell concentrates that had been filtered pre-storage and washed, 8% (5/62) of those leucodepleted post-storage after 2 days and 6% (5/78) of those that underwent both procedures had a haemoglobin content <40 g/unit and a haematocrit <50%.

Conclusions

The preparation procedures had been carried out satisfactorily; nevertheless, transfusion therapy with some “low dose” normal units could be less effective and might, therefore, result in greater transfusion requirements in patients receiving such units.     

Phosphatidylserine exposure on stored red blood cells as a parameter for donor-dependent variation in product quality

Background

Exposure of phosphatidylserine on the outside of red blood cells contributes to recognition and removal of old and damaged cells. The fraction of phosphatidylserine-exposing red blood cells varies between donors, and increases in red blood cell concentrates during storage. The susceptibility of red blood cells to stress-induced phosphatidylserine exposure increases with storage. Phosphatidylserine exposure may, therefore, constitute a link between donor variation and the quality of red blood cell concentrates.

Materials and methods

In order to examine the relationship between storage parameters and donor characteristics, the percentage of phosphatidylserine-exposing red blood cells was measured in red blood cell concentrates during storage and in fresh red blood cells from blood bank donors. The percentage of phosphatidylserine-exposing red blood cells was compared with red blood cell susceptibility to osmotic stress-induced phosphatidylserine exposure in vitro, with the regular red blood cell concentrate quality parameters, and with the donor characteristics age, body mass index, haemoglobin level, gender and blood group.

Results

Phosphatidylserine exposure varies between donors, both on red blood cells freshly isolated from the blood, and on red blood cells in red blood cell concentrates. Phosphatidylserine exposure increases with storage time, and is correlated with stress-induced phosphatidylserine exposure. Increased phosphatidylserine exposure during storage was found to be associated with haemolysis and vesicle concentration in red blood cell concentrates. The percentage of phosphatidylserine-exposing red blood cells showed a positive correlation with the plasma haemoglobin concentration of the donor.

Discussion

The fraction of phosphatidylserine-exposing red blood cells is a parameter of red blood cell integrity in red blood cell concentrates and may be an indicator of red blood cell survival after transfusion. Measurement of phosphatidylserine exposure may be useful in the selection of donors and red blood cell concentrates for specific groups of patients.     

Evaluation of a new, integral, whole blood filter (RS2000) system for prestorage leucodepletion of SAG-M red cells

Residual donor leucocytes are responsible for many adverse transfusion reactions. Prestorage leucodepletion may ameliorate these effects and enhance product quality. We studied a bottom and top (BAT) system incorporating an integral filter for whole blood leucodepletion. Our evaluation assessed leucodepletion efficiency as well as in vitro SAG-M red cell quality and storage characteristics.   Sixty-six units of blood were collected; test units into the Optipac^®- p L u S system and controls into the standard triple pack configuration. Test units were held for 4–6 h at room temperature (rt) or 12–18 h at 4°C. The mean leucocyte counts for the SAG-M red cells in the quality and storage trial were 0.6×10⁶ (rt hold), 0.05×10⁶ (4°C hold) and 2500×10⁶ (controls). We observed no significant differences between the groups for Na⁺, ATP, 2,3-DPG, glucose, lactate and pH during the 49 d storage. The control group, however, showed a greater increase in haemolysis and K⁺ with time. Autologous in vivo 24 h red cell recovery, after 42 d storage, was >75%. Adjustment of processing parameters in subsequent studies gave leucodepleted SAG-M red cells with minimal cell loss (9–19%) plus acceptable haemoglobin content (46–76 g/U) and haematocrit (54–62%). This system achieved >3.5 log leucodepletion with all but one unit containing <1×10⁶ leucocytes. The product quality is good and the system suitable for routine use in blood centres.     

The annual cost of blood transfusions in the United Kingdom

This study estimated the annual cost of blood transfusions in the UK during 1994/1995. The analysis was based on published data, information derived from interviews with relevant NHS personnel and a purpose-designed structured questionnaire of blood donors. The cost to the UKs blood transfusion services of providing blood and blood products for transfusion was £165.5 million in 1994/1995. During this period, 2.75 million conventional donations of whole blood and 144 000 apheresis donations of platelets and plasma were collected: 2.58 million units of red blood cells were issued, resulting in ≈ 866 000 red blood cell transfusions; 334 000 units of fresh frozen plasma and 1.16 million units of platelets were issued, resulting in ≈ 17 000 and 188 000 isolated plasma and platelet transfusions, respectively. Hospital resource use attributable to providing blood transfusions during 1994/1995 cost the NHS £52.6 million. In total, blood transfusions cost the NHS £218.2 million during 1994/1995. Of this, red blood cell transfusions accounted for 76% of the annual cost, isolated platelet transfusions 16%, isolated plasma transfusions 1% and other products 7%. Donors incurred direct costs of £3.1 million and indirect costs of £11.2 million were accrued due to lost productivity. Additionally, blood donors gave up 2.5 million hours of their leisure time donating blood.     

A study of leucocyte removal by the RemoweLL cardiotomy reservoir

Background

Cerebral damage is a relatively frequent complication of cardiopulmonary perfusion. Leucocyte activation and lipid microembolisation are among the possible causes. The removal of leucocytes and lipid microparticles from the cardiotomy suction blood could prevent this complication.

Methods

We studied the properties of a cardiotomy reservoir (RemoweLL, EUROSETS), which contains a filtering layer designed to remove some leucocytes and lipid microparticles. The reservoir was loaded with red cell concentrates or whole blood units, some of them containing hyperlipidaemic plasma. The extent of leucocyte and lipid removal was evaluated with reference to the storage age and pre-filtration absolute values of the products.

Results

On average, the cardiotomy reservoir removed 35–39% of total leucocytes, with a slight preference for neutrophil granulocytes. This device also retained 26–30% of platelets. The efficiency was not influenced by the storage age of the filtered product nor by the total cell load, within the explored range (323x10⁶ – 1,345 x10⁶ total leucocytes). Lipid (cholesterol, triglycerides) removal was minimal (about 3–6%).

Discussion

The RemoweLL device removed more than a third of the leucocytes from the processed blood. Lipid removal was minimal but the lipid particles in our model (chylomicrons) have a diameter 100-fold smaller than the particles believed to be responsible for clinical effects. This device seems promising and worthy of further studies to document the saturation point of leucocyte removal. On the other hand, lipid removal should be studied in a model more closely resembling the clinical situation in which the device is expected to be used.     

Glutathione redox cycle enzymes and selenium in severe rheumatoid arthritis: lack of antioxidative response to selenium supplementation in polymorphonuclear leucocytes.

The antioxidant capacity of the glutathione redox cycle and the concentrations of selenium in serum, red blood cells or whole blood, and polymorphonuclear leucocytes was evaluated in nine patients with severe rheumatoid arthritis (RA) and eight healthy controls receiving daily supplementation with 250 micrograms selenomethionine for six months. Serum and whole blood concentrations of selenium and the activity of the selenium dependent enzyme glutathione peroxidase (GSH-Px) were low in the serum, red blood cells, and polymorphonuclear leucocytes of patients with RA before selenium supplementation. During supplementation serum and whole blood concentrations of selenium and the activity of GSH-Px in serum and red blood cells of patients with RA and serum GSH-Px in controls increased. Selenium and GSH-Px in polymorphonuclear leucocytes were unaffected in patients with RA in contrast with the controls where both were augmented. Glutathione reductase activity in the red blood cells and polymorphonuclear leucocytes of patients with RA was low but increased during selenium supplementation. Whole blood concentrations of glutathione were slightly lower in patients with RA than controls and no difference in the content in polymorphonuclear leucocytes was found between the groups. The activity in red blood cells of glucose-6-phosphate dehydrogenase was high in patients with RA, indicating sufficient function of the hexose monophosphate pathway. The reduced antioxidant activity of the glutathione redox cycle in patients with severe RA was mainly due to the low availability of selenium. This was further supported by the response to selenium supplementation in serum and red blood cells. In the polymorphonuclear leucocytes, however, no biochemical effects of selenium supplementation were seen. This lack of antioxidative response could play a pathogenetic part in inflammation in patients with RA.     

Amount and Type of Leukocytes in ''Leukocyte-Free'' Red Cell and Platelet Concentrates

The exact number of leukocytes remaining in 'leukocyte-free' red cell and platelet concentrates cannot be measured by standard methods. We have therefore developed methods to harvest all the leukocytes from blood components. The leukocytes were then counted and identified using monoclonal antibodies. The leukocyte-free red cell concentrates were prepared by combining buffy coat removal and filtration through a Cellselect filter. The mean number of leukocytes per unit was 1.0 X 10(5). Most of the leukocytes were granulocytes and T cells. Only a few B cells or monocytes could be detected. Leukocyte-free platelets were prepared by filtering 4 units of PC through a cotton-wool (Imugard) filter. The mean number of leukocytes per PC unit was 0.4 X 10(5) of which 85-95% were T cells.     

Automated separation of whole blood in top and bottom bags into components using the Compomat G4

BACKGROUND AND OBJECTIVES: Whole blood can be separated by hard spin centrifugation into layers of blood components according to their specific gravity. The aim was to develop a program for an automatic separator to subsequently express the various components into their respective satellite bags in top and bottom systems with the following requirements: a red cell concentrate with a low leukocyte and platelet contamination, a 'cell-free' plasma, and a buffy coat with a volume of about 50 ml with an acceptable loss of red cells. MATERIALS AND METHODS: The Compomat G4 possesses an independently moving upper and lower press, to automatically express plasma or red cells to satellite bags of top and bottom systems. The influence of the extension of the lower press was studied by pooling and dividing two units of whole blood, and separating these units after centrifugation (2,960 g, 10 min) either with a program where the lower press was completely extended (program C), or with a program that left approximately 1 mm between the door and the lower press (program D). RESULTS: The program (program D), where the lower press was not completely extended, yielded a buffy coat with a volume of 52+/-1 ml (mean +/- SD, n = 36), which contained >75% leukocytes and >90% platelets of the original whole blood unit, with a red cell loss in the buffy coat of 21+/-1 ml (10.8+/-0.8% of the original volume). The leukocyte content of the red cell concentrates was 775+/-379x10(6) per unit, whereas the plasma contained 3+/-3x10(6) leukocytes and 4+/-3x10(9) platelets per unit. The pooling experiment indicated that complete extension of the lower press (program C) resulted in a significantly higher leukocyte contamination of the red cell concentrate (788+/-431x10(6 ) vs. 658+/-419x10(6); n = 12; p = 0.03), while there was no difference in the yield of red cells or plasma. The buffy coat produced with program D contained significantly more leukocytes (2,242 +/-396x10(6) vs. 2,065+/-327x10(6), p = 0.005) and more platelets (96+/-14x10(9) vs. 92+/-17x10(9), p = 0.02) per unit than with program C, probably because buffy coat cells sticking to the container wall are not expressed to the red cell concentrate, and thus remain in the buffy coat bag. Therefore, program D met our specifications for blood products. CONCLUSIONS: The Compomat G4 warrants reproducible separation of whole blood in top and bottom bags into red cells, buffy coat and plasma meeting our specifications.