Low leukocyte contamination without filtration by preparation of platelet concentrates in cylindrical bags with the buffy-coat method.

BACKGROUND AND OBJECTIVES: The buffy-coat (BC) method for platelet concentrate (PC) preparation was modified in order to obtain leukodepleted PCs from single BCs without filtration. MATERIALS AND METHODS: BCs were centrifuged in cylindrical BC bags and the optimal centrifugation conditions and optimal hematocrit were determined. RESULTS: With optimal conditions, a tenfold lower leukocyte contamination was obtained compared with the conventionally shaped, wide BC bag (0.3 +/- 0.19 versus 3.0 +/- 1.71 x 10(6) leukocytes per unit; 85-ml BCs). The platelet yield obtained with the cylindrical bag did not differ significantly from the yield obtained with the conventional bag (56 +/- 16.4 versus 61 +/- 15 x 10(9) platelets per PC). Furthermore, when PCs were prepared from 100-ml BCs in cylindrical bags, a leukocyte contamination of 0.2 +/- 0.11 x 10(6) and a platelet content of 61 +/- 13.5 x 10(9) per PC were obtained. CONCLUSION: The use of cylindrical BC bags reduced the leukocyte contamination in PCs to a level required for leuko-depletion without affecting platelet recovery.

Pooled platelet concentrates prepared by the platelet-rich-plasma method and filtered with three different filters and stored for 8 days.

Pooled platelet concentrates (PC) prepared by the platelet-rich plasma (PRP) method were filtered with three different filters and stored for 8 days at room temperature. The effect of filtration on leukocyte contamination, platelet concentration, and the in vitro function, morphology, metabolism and activation of platelets were studied. Eight pools of 20 PRP-PC were used, each pool was split into 4 equal volumes; 3 were filtered over a PL50HF, a PL-10A and a Bio P10 filter, the 4 served as a control. After filtration, leukocyte counts exceeded 3 x 10(5) in none of the pooled PC. Platelet loss induced by filtration was about 17%. During storage, no differences in pH, PCO2, and lactate and glucose concentration were found between the filtered and the unfiltered units, nor were any differences observed between filtered and unfiltered pooled PC in aggregation upon stimulation with collagen and/or ADP, adhesion capacity to collagen in flowing blood, nucleotide content of the platelets and nucleobase concentration in the plasma, expression of activation-dependent antigens, or platelet morphology as observed by light microscopy and by the swirling effect. Selective removal of beta-thromboglobulin (22%) by the PL50HF filter was observed. Pooled PC prepared by the PRP-method can be filtered and stored for 8 days without detrimental effect on platelet function, metabolism or activation.

In vitro evaluation of platelet concentrates, prepared from pooled buffy coats, stored for 8 days after filtration.

BACKGROUND: Posttransfusion complications can be prevented by pretransfusion removal of donor white cells from platelet concentrate. The filtration used for this removal seems to have little effect on platelet function and activation, but more information is needed on its effect on function during subsequent long-term storage of concentrate. STUDY DESIGN AND METHODS: The effect of prestorage filtration of buffy coat-prepared platelet concentrates (PCs) on platelet function, metabolism, and activation was investigated. A pool of three PCs, each made of four buffy coats, was split into three equal volumes; two were filtered over two different filters and the third served as a control. Variables monitored immediately after filtration and during the subsequent 8-day storage period at 22 degrees C included aggregation upon stimulation with collagen and/or ADP, platelet adhesion capacity to collagen and fibrinogen in flowing blood, nucleotide content of and nucleobase release by the platelets, expression of activation-dependent antigens, and beta-thromboglobulin release by the platelets. RESULTS: No differences were observed between the PCs filtered over two different filters and the nonfiltered control PCs immediately after filtration and during storage, except for a selective removal (20%) of beta-thromboglobulin by one filter. CONCLUSION: PCs prepared from a pool of four buffy coats can be filtered and subsequently stored for 8 days (starting +/- 24 hours after whole blood collection) without detriment to platelet function, metabolism, or activation.

Effect of filtration on subsequently stored platelet concentrates.

The effect of filtration on the quality of platelet concentrates (PC) during storage was investigated. Two leukocyte depletion filters (Pall PL50HF and Sepacell PL-10A) were applied to filter PC made from a pool of 4 buffy coats. For each experiment 3 PC were pooled and divided into 3 identical PC to eliminate differences between the PC. Two PC were filtered, and the third PC served as an unfiltered control. A total of 12 experiments was performed. Before filtration, volumes of the PC were 263 +/- 11.7 ml (mean +/- SD). Platelet and leukocyte counts per PC were 241 +/- 25.9 x 10(9) and 7.2 +/- 1.8 x 10(6), respectively. After filtration leukocyte counts did not exceed 5 x 10(4) in any of the PC. In the PC filtered with the Pall PL50HF the mean platelet loss was approximately 14% and with the Sepacell PL-10A, 17%. During a 9-day storage period the pH, PO2, PCO2, bicarbonate, lactate and glucose concentration and LDH release as well as the morphology, examined by the swirling effect and microscopically, were not significantly different in filtered and unfiltered units. Filtration through the 2 investigated leukocyte depletion filters for PC did not adversely affect in vitro viability of the platelets during storage.

Platelets stored for 6 days in a polyolefin container in a synthetic medium with minimal plasma carry-over.

Platelet concentrates (PC) were stored for 6 days in either polyolefin (PO) or polyvinylchloride/di-(2-ethylhexyl)phtalate (PVC/DEHP) bags in 100% plasma or in a synthetic medium with 35 or 10% plasma. For all conditions studied the usual in vitro parameters were well maintained, with a pH above 6.8. In both bag types platelets can be satisfactorily stored for 6 days in a synthetic medium with minimal amounts of residual plasma. For this medium, the PO bag offers a slight advantage with respect to the preservation of platelet ATP content (> 80 versus > 70% in the PVC bags) and aggregation and adhesion capacity. The adhesion capacity increased in the PO bags, while it decreased in the PVC bags.

Effect of pre-incubation at 37 degrees C of platelet concentrates on the post-transfusion platelet adhesion capacity to collagen and fibrinogen.

The effect of warming (37 degrees C) of stored platelet concentrates (PC) on the post-transfusion platelet function as measured by the adhesion capacity in a rectangular perfusion system under flow conditions was analyzed in 22 patients undergoing transfusion for stable thrombocytopenia. Nine patients received a PC stored at 22 degrees C and incubated at 37 degrees C for 1 h before transfusion, 10 patients received a non-warmed PC, 3 patients received both a pre-warmed and a non-warmed PC. In the PC the platelet adhesion capacity to collagen was higher in the pre-warmed PC than in the non-warmed PC (33 +/- 5.9% coverage vs. 22 +/- 4.7% coverage, respectively, in a selected group with the same platelet concentration). The adhesion capacity to collagen of the platelets in the patient's blood, measured 10 min after transfusion, had increased considerably in both patient groups and 4 h later the adhesion capacity in both patient groups was similar to that of the pre-warmed PC before transfusion. We conclude that though pre-warming of stored PC had a beneficial effect on the adhesion capacity to collagen of the platelets in the PC, the clinical significance is questionable because already 10 min after transfusion the adhesion capacity to collagen of stored non-warmed platelets had improved to the level of the pre-warmed platelets and 4 h after transfusion this improvement was still present.

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)

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)

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C.

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.

Electrokinetic properties of endotoxins and their significance for the limulus amoebocyte lysate test.

The electrokinetic properties of the endotoxins of Escherichia coli and Serratia marcescens have been examined. Both endotoxins are negatively charged, with the zetapotential being increased by the presence of cations whose relative influence resembles the Schulze-Hardy rule for colloid stability. Fe3+ and Th4+ ions are capable of reversing the negative charge of the endotoxin particles to positive. These cations were found to have a strong inhibitory effect on the activity of endotoxins in the limulus amoebocyte lysate test but the inhibitory effect did not parallel changes in the zetapotential because the effect occurred at concentrations too low to affect this parameter.

Oxygen affinity of hemoglobin solutions modified by coupling with NFPLP and the effects on tissue oxygenation in the isolated perfused rat liver.

From these liver perfusions with Hb and Hb/HbNFPLP solutions the following conclusions can be drawn: In spite of the chemical modification of the hemoglobin molecule, no rheological differences are seen. All parameters measured were sensitive to hypoxia induced by a decrease in perfusion flow rate. The NFPLP-induced decrease in oxygen affinity was reflected in a higher venous PO2. These in-vivo observations are in agreement with the in-vitro measured oxygen dissociation curves. The difference in PO2 did not result in a change in the other oxygen-sensitive parameters in this model under the chosen conditions. Possible causes for these observations are: the level of hypoxia was too low the oxygen supply in the perfusions with the modified hemoglobin solutions was lower than the oxygen supply in the perfusions with normal hemoglobin. Whether or not this observation is due to an intrinsic property of the modified hemoglobin molecule remains to be established.

Platelet concentrates stored in plasma for 72 hours at 22 degrees C prepared from buffycoats of citrate-phosphate-dextrose blood collected in a quadruple-bag saline-adenine-glucose-mannitol system.

A method is described to prepare platelet concentrates from the buffycoat of citrate-phosphate-dextrose (CPD) blood in a closed four-bag system and to store the platelets in autologous plasma under sterile conditions. After separation of the blood into plasma, buffycoat and leukocyte- and thrombocyte-poor red cell concentrate, saline-adenine-glucose-mannitol (SAGM) was added to the red cells. Next the platelets were concentrated in plasma by a second centrifugation and were transferred to the 600-ml bag that previously contained the SAGM. The platelets were stored for 72 h at 22 degrees C on a platform rotator at 1 cycle per second. The mean volume of the platelet concentrates (n = 12) was 61 ml, with an average platelet content of 72 X 10(9). The mean leukocyte contamination was only 17 X 10(6); erythrocytes were not detected. The aggregation of the platelets with 1 microM of ADP was normal. After 72 h of storage, the mean pH was 7.1 +/- 0.1. The PO2 always remained above 17 mm Hg. No loss of thrombocytes had occurred during storage. The platelets had retained their discoid shape. The aggregation response to ADP had disappeared, however. The in vivo viability of the platelets was determined in 8 healthy volunteers. Autologous platelets, stored for 72 h at 22 degrees C, were labeled with 51 Cr and reinfused [43 (+/- 9)% recovery at 15 min after reinfusion]. The mean survival time in vivo of the platelets was 6.8 (+/- 0.7) days.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin in blood products: correlation between the Limulus assay and the rabbit pyrogen test.

The possibility of replacing the rabbit pyrogen test by the Limulus (LAL) test, as a final release test for plasma products, was investigated. The LAL test measured the endotoxin content quantitatively, using a chromogenic substrate. The samples were boiled and centrifuged to remove inhibiting substances, which represent a major problem when testing plasma protein samples. In order to correlate the LAL test to the rabbit test, parallel assays were performed on 85 batches of two different human albumin preparations. For both products, a positive correlation was observed between the two tests. However, the pass/fail limit of the rabbit test was found at different endotoxin levels, corresponding to about 2 and 20 ng/kg, respectively. This discrepancy could be removed by extracting the endotoxin before administration to rabbits. It is concluded that the endotoxin, detected in plasma products by the LAL test, may be present in a non-pyrogenic state.

Hypotensive effects of plasma protein fraction. The relation between prekallikrein activator, bradykinin generation, and blood pressure in an animal model.

In a rat model, the relationship between the activity of PKA in human PPFs, the changes in arterial BK concentration, and the changes in blood pressure on infusion of PPF was investigated. The rat was chosen as a model because it is reported to be one of the few animals sensitive to human PKA. However, hypotensive reactions after infusion of human PKA-containing PPF were observed only in the presence of a bradykinin-potentiating peptide (BPP9a). A correlation was found between the PKA content of the rapidly infused PPF, the BK generation in the rat, and the fall in arterial blood pressure. In control experiments, infusions of BK provoKed a similar fall in blood pressure at corresponding BK levels. After neutralization of the PKA activity in the PPF with C1-esterase inhibitor, neither a rise in BK level nor a fall in blood pressure was observed on infusion. We conclude therefore that the hypotensive reactions were caused by PKA-mediated generation of BK.

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.

Large-scale purification and cryopreservation of human monocytes.

Human blood monocytes were purified by a new method capable of handling 3 X 10(9) mononuclear leukocytes, which does not involve adherence of the cells and takes about 3 h to perform. The yield of monocytes is 70%, the purity about 90% and the viability 99%. Monocytes purified by this method were cryopreserved at -196 degrees C. The function of the cells was tested before freezing and after thawing. We found that the capacity of cryo-preserved monocytes to move to the source of a chemotactic gradient, to ingest particles, to mount a respiratory burst during phagocytosis, to kill intracellular bacteria, to lyse anti-D sensitized erythrocytes and to help autologous lymphocytes in a proliferative response to mitogens or antigens, was preserved by 70% or more as compared with non-frozen cells. Thus, cryopreservation of human blood monocytes is possible with maintenance of functional capacities.