WBC-reduced platelet concentrates from pooled buffy coats in additive solution: an evaluation of in vitro and in vivo measures.

BACKGROUND: The use of a platelet additive solution (PAS-II, Baxter) may have benefits over plasma for storage of platelets. It was the aim of this study to develop a method to produce WBC-reduced platelet concentrates (PCs) in PAS-II with >240 x 10(9) platelets and <1 x 10(6) WBCs per unit, which can be stored for 5 days at pH >6.8 and that will give sufficient platelet increments after transfusion: a 1-hour CCI of >7.5 and a 20-hour CCI of >2.5. STUDY DESIGN AND METHODS: PCs were made from five pooled buffy coats and 250 g of PAS-II. After centrifugation the PCs were WBC-reduced with a filter (Autostop BC, Pall Biomedical) and stored in a 1000-mL polyolefin container. CCIs were assessed in stable hemato-oncologic patients after 5-day old PCs were transfused. RESULTS: Routinely produced PCs contained a median of 310 x 10(9) platelets (n = 5,363) with 3.5 percent containing <240 x 10(9) platelets, in a median volume of 320 mL (n = 11,834). The median number of WBCs was <0.03 x 10(6) (n = 694). The WBC count exceeded 1 x 10(6) in three PCs, but it was always <5 x 10(6), giving 99-percent confidence that more than 99.5 percent of the units will contain <1 x 10(6) WBCs. The pH remained >6.8 on Day 8, provided the concentration was below 1.1 x 10(9) platelets per mL (n = 32). After 28 transfusions in 28 patients, the 1-hour CCI was 12.6 +/- 4.3 (mean +/- SD, with 2/28 CCIs <7.5) and the 20-hour CCI was 8.9 +/- 5.6 (with 4/28 CCIs <2.5). Limitations of this study include the absence of a control group of patients receiving platelets stored in plasma and of in vivo radiolabeled survival studies, but a comparison of these data with previously published data suggested that the in vivo survival of platelets stored in PAS-II is less than that of platelets stored in plasma. CONCLUSION: The WBC-reduced PCs conformed to specifications. These WBC-reduced PCs could be stored at least 5 days with maintenance of pH, and they gave sufficient increments after transfusion to patients.

Collection of heparinized plasma by plasmapheresis.

BACKGROUND AND OBJECTIVES: Heparinized plasma can be used for exchange transfusions in neonates and is usually collected by drawing whole blood using heparin as anticoagulant. The heparinized red blood cells and buffy coat cannot be used and are therefore discarded. To collect heparinized plasma more efficiently, a method was developed using an apheresis machine. MATERIALS AND METHODS: With an MCS3p apheresis machine (Haemonetics), plasma was collected from volunteer donors as anticoagulant, heparin in saline (30,000 IU/l) was added in a 1:9 ratio. The activated partial thromboplastin time (APTT) of the donors was measured before and immediately after the procedure, and various parameters were determined in the collected plasma. RESULTS: In 2 collection cycles, an average of 456+/-52 ml (mean +/- SD; n = 20) of heparinized plasma was collected, and 504+/-57 ml (n = 2; donors with a high hemoglobin level) when 3 cycles were performed. The leukocyte and platelet contamination in the plasma (n = 22) was 1.11+/-0.92x10(6) and 0. 05+/-0.22x10(9) per unit, respectively, which conformed to national specifications. Sodium levels were normal, but due to dilution of the plasma with heparin solution, potassium and calcium levels were about 20% lower than the serum levels in the donors. The donor APTT values were slightly longer after the procedure than before, but remained all within normal values. CONCLUSION: For the collection of heparinized plasma, apheresis has the advantage that (1) high-quality heparinized plasma can be harvested; (2) no blood components need to be discarded; (3) more plasma can be harvested with each donation, and (4) these procedures can be performed more often than whole blood donations.

Preparation of leukodepleted platelet concentrates from pooled buffy coats: prestorage filtration with Autostop BC.

BACKGROUND AND OBJECTIVES: Our requirements for leukocyte-depleted platelet concentrates (LD-PC) for an adult patient are: platelets >240x10(9), leukocytes <5x10(6), volume of 150-400 ml; and at the end of storage a pH between 6.8 and 7.4 and presence of the swirling effect. Our aim was to develop a standardized, semiautomated method for the production of LD-PC, by pooling of buffy coats (BC), and prestorage leukoreduction by filtration. MATERIALS AND METHODS: Whole blood was collected in Top and Bottom systems, and separated automatically with the Compomattrade mark G3 equipment into a red cell concentrate, a plasma and a BC. Subsequently, a pool of 5 BC was made, and 200 g plasma from one of the donors was added. Then, after soft spin centrifugation, the platelet rich plasma was leukocyte depleted by filtration using the Autostoptrade markBC filter, and stored in a 1,000 ml polyolefin platelet storage bag. RESULTS: BC (n = 60) had a volume of 51+/-2 ml (mean +/- SD) with a hematocrit of 0.44+/-0.03 l/l and contained 80+/-5% of the platelets and 74+/-12% of the leukocytes of the whole blood. Routinely prepared LD-PC (n = 15,037) contained a median of 341x10(9) platelets (range 49-599x10(9)), with only 104/15,037 (0.7%) containing fewer than 240x10(9) platelets; the median volume was 263 ml (range 134-373 ml). In 118/917 (13%) LD-PC leukocytes were observed in the Nageotte hemocytometer, but only twice exceeding 1x10(6) leukocytes per unit, and none exceeding 5x10(6) (median <0. 6x10(6); range <0.6-1.41x10(6)). Storage experiments of the LD-PC (n = 12) revealed adequate oxygenation and maintenance of pH and swirling effect up to 9 days. CONCLUSIONS: This method warrants with 99% confidence that LD-PC contain more than 240x10(9) platelets; with 97.5% confidence that 100% of the LD-PC contain <5x10(6) leukocytes, and with 95% confidence that more than 99% of the LD-PC contain fewer than 1x10(6) leukocytes; these LD-PC can be stored satisfactorily for up to 9 days.

Six filters for the removal of white cells from red cell concentrates, evaluated at 4 degrees C and/or at room temperature.

BACKGROUND: Six filters were tested for their ability to remove white cells from buffy coat-depleted red cell concentrates at various temperatures. STUDY DESIGN AND METHODS: Cellselect FR, BPF4, and Sepacell filters were tested at both room temperature (RT) and 4 degrees C. The Leucoflex filter was tested only at 4 degrees C, while the Cellselect Optima Plus and Imugard filters were tested only at RT. Donor-dependent differences were excluded by pooling and subsequently dividing 9 red cell concentrates; 12 sets of experiments were performed. RESULTS: With all filters, red cell concentrates containing <5 x 10(6) white cells per unit were obtained. The lowest numbers of residual white cells were achieved with the Leucoflex (at 4 degrees C, 0.15 +/- 0.11 x 10(6), the Sepacell (at 4 degrees C, 0.23 +/- 0.14 x 10(6), the Imugard (at RT, 0.24 +/- 0.14 x 10(6), and the BPF4 (at 4 degrees C, 0.25 +/- 0.24 x 10(6); differences not significant). With the Cellselect FR, filtration at 4 degrees C resulted in 0.86 +/- 0.37 x 10(6) white cells per unit, a level not significantly different from that obtained with the BPF4 and Sepacell filters at RT (1.16 +/- 0.43 x 10(6) and 0.80 +/- 0.36 x 10(6) white cells, respectively). Filtration at RT with the Cellselect FR and Cellselect Optima Plus resulted in red cell concentrates with 1.79 +/- 0.69 x 10(6) and 2.29 +/- 0.69 x 10(6) white cells, respectively (p<0.01). CONCLUSION: All filters conformed to the current standards for white cell reduction; the process was less efficient at RT than at 4 degrees C. For routine application, the composition of the red cell concentrate, the temperature, and logistic preferences should be taken into account in the final choice of filter; before implementation, the chosen filter must be validated under routine conditions.

Prevention of Yersinia enterocolitica growth in red-blood-cell concentrates.

In response to concern about Yersinia enterocolitica contamination of blood products, we have studied the effects on Y enterocolitica growth of holding whole blood at 22 degrees C for 20 h and then removing leucocytes. Thirty pools of three bags of blood were inoculated with Y enterocolitica (2 x 10(1)-3 x 10(4) colony-forming units/ml). One bag in each pool was processed to red-blood-cell concentrate after 6 h at 4 degrees C (RBC); the other two were held at 22 degrees C for 20 h before processing to buffy-coat-depleted RBC (BCd-RBC). One of these bags was then depleted of leucocytes by filtration (Ld-RBC). All bags were stored at 4 degrees C for 5 weeks. RBC bags showed Y enterocolitica growth after the shortest storage times, followed by BCd-RBC then Ld-RBC (p less than 0.03-0.001). We recommend that whole blood should be held at 22 degrees C to make use of inherent bactericidal activity; leucocytes should then be removed.

Comparison of five different filters for the removal of leukocytes from red cell concentrates.

The leukocyte depletion capacity and performance of 5 filters designed for filtration of red cell concentrates (RCC) were compared by counting leukocytes, measuring red cell volumes and by histological examination of the filters after use. To eliminate interdonor differences, 5 buffy-coat-poor RCC were pooled (in each of 10 experiments) and subsequently split up into the original bags. The RCC were passed over the Cellselect filter, a column filled with cellulose acetate, and over flat-bed polyester filters: the Cellselect Optima, the Pall RC 50, the Leukostop and the Sepacell R-500. The filtration was shortest with the Pall RC 50 (p less than 0.001 compared to the other 4 filters). Leukocyte removal was most effective with the cellulose acetate filter (p less than 0.01 compared to the other 4 filters) followed by the Cellselect Optima polyester filter (p less than 0.02 compared to the remaining 3 filters). Residual leukocytes did not exceed 50 x 10(6) for any brand of filter. Red cell recovery was similar for all 5 filters with mean values from 86.1 to 89.2%. The leukocyte numbers, counted in Türk's solution or in propidium iodide, gave comparable values in hemocytometers applying light microscopy or fluorescent microscopy, respectively. Histological examination showed that lymphocytes were mainly removed by trapping, whereas granulocytes showed a variable pattern: adhesion in presence of platelets or trapping.

Quality of red cell concentrates in relation to the volume of the buffy coat removed by automated processing in a top and bottom system.

The effect of automated removal of increasing volumes of buffy coat in a 'top and bottom' system on the composition of red cell concentrates (RCC) was investigated. The volume of the buffy coat was adjusted to group 1:50 ml (n = 31), group 2: 70 ml (n = 31) and group 3: 100 ml (n = 31), respectively. The numbers of platelets and leukocytes in the buffy coats were comparable between the groups, whereas the red cell volumes in the buffy coats showed a significant difference (17 +/- 3.6 ml group 1, versus 22 +/- 4.1 ml group 2 and 26 +/- 3.88 ml group 3; p less than 0.001). The volumes, hematocrits and cell counts of the RCC were not significantly different. The plasma volumes were inversely correlated with the volume of buffy coat removed, i.e. 268 +/- 19 ml group 1, versus 257 +/- 15 ml group 2 and 233 +/- 20 ml group 3 (p less than 0.001). We conclude that in the 'top and bottom' system an increase of the volume of the buffy coat from 50 to 100 ml did not improve the quality of the RCC regarding contamination with leukocytes and platelets.

Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods.

The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from platelet-rich plasma (PRP) and from the other as nonpelleted platelets from the buffy coat (BC). The activation of platelets in these PCs was studied immediately after preparation and during storage for up to 9 days at 22 degrees C with gentle agitation. The binding of monoclonal antibodies (MoAbs) against the GP IIb/IIIa complex and against activation-dependent antigens (GMP 140 from the alpha granules and a 53-kDa glycoprotein from the lysosomal granules) was measured. Beta-thromboglobulin (beta-TG) release was also determined. Disc-to-sphere transformation was quantitated by measuring on an aggregometer the difference in light transmission during stirring at different rates and also by light microscopy. Immediately after preparation, platelets derived from PRP had a more spheric morphology (p less than 0.01), had a higher beta-TG release (p less than 0.01), bound more MoAbs against GP IIb/IIIa (p less than 0.01), and expressed more GMP 140 and 53-kDa glycoprotein (p less than 0.01) than did BC-derived platelets. However, these differences had disappeared after 2 days of storage. It was concluded that, immediately after preparation, PRP-derived platelets are more activated than BC-derived platelets. This is most likely a result of the pelleting that follows the second high-speed centrifugation of the PRP.

Comparison of a conventional quadruple-bag system with a 'top-and-bottom' system for blood processing.

'Top-and-bottom' bags have an outlet at the top and at the bottom of the collecting bag, allowing simultaneous expression of plasma and red cells, whereas the buffy coat remains in the collecting bag. The composition of blood components was investigated following manual separation of whole blood in a conventional 4-bag system (A) or automated separation in a 'top-and-bottom' system (B). To overcome inter-donor differences, two units of whole blood were pooled and redistributed into the original bags (A and B) prior to centrifugation. Leukocyte-poor platelet concentrates (LPPC) were manufactured from both types of buffy coat (A and B). The volumes of plasma, red cell concentrates (RCC) and buffy coat were similar in both methods. However, the residual leukocytes and platelets in the RCC from the top-and-bottom system were significantly lower than in the conventional system, 140 +/- 59 x 10(6) (mean +/- SD) versus 762 +/- 228 x 10(6), respectively (p less than 0.01). Both types of LPPC contained less than 10(7) leukocytes and could be stored for 7 days maintaining a pH above 6.5. We conclude that the top-and-bottom system enables automated and standardized preparation of RCC and plasma with a significantly better buffy-coat removal than with manual processing.

[A new multiple blood bag system with top and bottom drainage. Comparison with the conventional multiple bag system]. / Untersuchung eines neuen Mehrfach-Blutbeutel-systems mit oberem und unterem Abgang. Vergleich mit konventionellem Mehrfach-Beutelsystem.

Blood components were investigated following manual preparation in a conventional 4-bag system (A) or automatic preparation in a top and bottom system (B). To overcome inter donor differences two units of whole blood were pooled and subsequently split up into the original bags. Leukocyte-poor platelet concentrates (PC) were manufactured from both types of buffy-coat (A and B). The volumes of the various blood components were similar, whereas the residual leukocytes and platelets in the red cell concentrates from the top and bottom system were significantly lower (p less than 0.01). Both types of PC contained less than 10(7) leukocytes and could be stored for 7 days maintaining a pH above 6.5. We conclude that the top and bottom system enables automatic preparation of red cell concentrates and plasma with a significantly better buffy-coat removal than with manual processing.

Storage of leukocyte-poor red cell concentrates: filtration in a closed system using a sterile connection device.

Storage of leukocyte-poor red cell concentrates (LP-RCC) was investigated after filtration in a closed system that was assembled using a Sterile Connection Device (SCD). The LP-RCC were stored for up to 6 weeks following filtration with either 0.9% saline solution (n = 14) or saline-adenine-glucose-mannitol (SAG M) solution (n = 15) to prime and rinse the cellulose acetate filter. The results were compared with the data of nonfiltered buffy-coat-poor red cell concentrates (BC-poor RCC) stored in SAG M solution (n = 10). All LP-RCC contained less than 10(6) leukocytes whereas the nonfiltered BC-poor RCC contained 675 +/- 286 X 10(6) leukocytes at day 1, decreasing to 83 +/- 49 x 10(6) at day 42. Although glucose consumption, lactic acid production and decrease in pH was similar from day 7 through 28 in both groups of LP-RCC, a significantly steeper decline of ATP values as well as a higher hemolysis and LDH release was observed in the LP-RCC filtered with saline. During storage of the nonfiltered BC-poor RCC in SAG M, significantly higher glucose consumption (p less than 0.01), LDH release (p less than 0.001), rate of hemolysis (p less than 0.001) and a lower pH (p less than 0.001) were found, compared to the filtered units. It is postulated that the leukocytes present in the nonfiltered BC-poor RCC were responsible for these differences. The ATP values in the SAG-M-filtered and nonfiltered BC-poor RCC in SAG M were comparable.(ABSTRACT TRUNCATED AT 250 WORDS)

Storage of whole blood for up to 24 hours at ambient temperature prior to component preparation.

The effect of rapid cooling to 20-24 degrees C of whole blood immediately after collection, using 'cooling units' with butane-1,4-diol and prolonged storage up to 24 h at ambient temperature was investigated in the whole blood and the subsequently prepared plasma, buffy coat and buffy-coat-poor red cell concentrate (BC-poor RCC) in saline-adenine-glucose-mannitol (SAG M) solution. Factor VIII:C content of the plasma (n = 10), after 24 h storage was 80 +/- 3% of the initial value. In routine procedures factor VIII:C content in the plasma (n = 129 pools of 20 donor units plasma) was 0.77 +/- 0.078 IU/ml, after storage of the whole blood for 16-20 h. In whole blood (n = 10), the 2,3-diphosphoglycerate (2,3-DPG) content of the red cells decreased from 4.36 +/- 0.55 to 1.47 +/- 0.6 mumol/ml red cells after 24 h storage at 20-24 degrees C. After storage of the BC-poor RCC (n = 10) at 2-6 degrees C for 1 week, the 2,3-DPG had dropped to 0.76 +/- 0.46 mumol/ml red cells. During the first 24 h of storage of whole blood, the adenine triphosphate (ATP) levels of the red cells remained stable. A mean increase of 20% of the initial value was observed after addition of SAG M solution. In the BC-poor RCC the ATP slowly decreased to 81 +/- 5% after 5 weeks and to 68 +/- 6.6% of the initial value after 6 weeks storage.(ABSTRACT TRUNCATED AT 250 WORDS)

A new cellulose acetate filter to remove leukocytes from buffy-coat-poor red cell concentrates.

Transfusion of leukocyte-free red cell concentrates (RCC) prevents or delays HLA immunization in multitransfused patients. We investigated a new cellulose acetate filter which was recently introduced to remove leukocytes from buffy-coat-poor RCC. It was found that the filtration time was only 10 min with buffy-coat-poor RCC in saline-adenine-glucose-mannitol (SAG M; n = 23), hematocrit being 62 +/- 2% (SD). The red cell loss was 13.5 +/- 2.6% and leukocyte removal was more than 99%. Routine filtration in SAG M (n = 179) showed again that more than 99% of the leukocytes were removed from buffy-coat-poor RCC with an original leukocyte content of 804 +/- (SD)458 x 10(6). The red cell loss (12 +/- 8.6%) was not diminished by increasing the amount of saline (0.9% NaCl) from 100 to 300 ml in an attempt to remove the retained red cells from the filter. We conclude that the new filter is reliable in rapidly removing more than 99% of the leukocytes from buffy-coat-poor RCC in SAG M solution.

Preparation of leukocyte-poor platelet concentrates from buffy coats. III. Effect of leukocyte contamination on storage conditions.

To study the influence of contaminating leukocytes on the storage conditions of platelet concentrates (PC), various amounts of leukocytes were added to identical PC. From 12 blood donations, 12 leukocyte-poor PC were prepared and pooled. Subsequently, the pool was divided into 12 identical PC. The plasma volume of the PC was 58.6 +/- 0.6 ml, the platelet concentration was 1.01 +/- 0.04 x 10(9)/ml (mean +/- SD) and the red cell contamination did not exceed 10(7) per PC. To 4 groups of 3 PC, pooled leukocytes were added from the same 12 blood donations. The leukocyte contamination for each group of 3 PC was 0.14 +/- 0.05, 1.96 +/- 0.09, 5.53 +/- 0.98 and 13.0 +/- 0.93 x 10(6)/ml (mean +/- SD) for groups I-IV, respectively. The PC were stored for 7 days at 22 degrees C in normal polyvinylchloride bags. A significant correlation was found between increasing concentrations of leukocytes in the PC and the drop in pH (r = -0.93), glucose consumption (r = -0.91), lactic acid production (r = 0.93) and release of lactate dehydrogenase (r = 0.92) during storage of the PC. The excretion of beta-thromboglobulin, depletion of platelet adenine nucleotides, decreased ability to incorporate 3H-adenosine into metabolic nucleotides and poor morphology of the platelets were also significantly correlated with an increased number of leukocytes in the PC. These data show that high concentrations of leukocytes in PC have a significant detrimental effect on the viability of platelets during storage at 22 degrees C. We conclude that for good storage conditions of PC, the upper limit of leukocytes per PC should not exceed 10(7).

Preparation of leukocyte-poor platelet concentrates from buffy coats. II. Lack of effect on storage of different plastics.

Six citrate phosphate dextrose (CPD)-saline adenine glucose mannitol (SAG M) quadruple systems were evaluated for the preparation and storage of leukocyte-poor platelet concentrates (PC) from buffy coats. The platelet storage bags examined were manufactured from normal polyvinylchloride (PVC) or special-type plastics. Biotest supplied PVC 76 (n = 14) and PVC 763 (n = 16) NPBI supplied PSV 3277 (n = 15) and DPL-110 (n = 14) and Terumo supplied Teruflex (n = 18) and molded Teruflex (n = 14). The PC were stored for 7 days at 22 degrees C on a horizontally shaking platform. Cell counts, pH, PO2, PCO2, morphology score and swirling patterns were monitored at 5, 72, 120 and 168 h. The plasma volumes averaged 63 ml and ranged from 39 to 81 ml, the overall mean +/- SD platelet concentration was 0.89 +/- 0.33 X 10(9)/ml. None of the PC had a leukoyte count higher than 10 X 10(6) per unit. After storage for 168 h, the pH ranged from 6.56 to 7.40 for all brands. The PO2 remained stable and even rose significantly (p less than 0.05) during storage in the NPBI PSV 3277 and Terumo molded Teruflex bags. The PCO2 decreased equally in all bags. Morphology scores were well maintained in 98% of all PC for up to 120 h, and in 83% at 168 h. A swirling pattern score of 2.5 or greater predicted with a specificity of 100% a good morphology score in the PC.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation of leukocyte-poor platelet concentrates from buffy coats. I. Special inserts for centrifuge cups.

A special insert was developed for centrifuge cups in order to prepare leukocyte-poor platelet concentrates from buffy coats by using quadruple citrate phosphate dextrose-saline adenine glucose mannitol systems from different manufacturers. Each centrifuge cup could contain up to 4 sets of double bags allowing the preparation of 24 platelet concentrates per run. Optimal conditions for centrifugation of the buffy coats in the inserts were found to be 6 min at 380 g (2,150 g min). A platelet count of 69 +/- 19 X 10(9) and a leukocyte contamination of 14 +/- 10.5 X 10(6) per platelet concentrate was thereby obtained in a plasma volume of 63 +/- 10.5 ml (mean +/- SD). The method described allows large scale production of leukocyte-poor platelet concentrates from buffy coats in a closed system.