Use of prostaglandin E1 during preparation of platelet concentrates.

A paired study in 10 autologous volunteer donors was undertaken to investigate the efficacy of adding prostaglandin E1 (PGE1) in vitro during routine platelet concentrate (PC) production. After 5 days storage, PCs prepared with PGE1 were compared with control PCs. In vivo platelet recovery, survival and biodistribution were determined following autologous infusion of indium-111 labelled platelets into volunteers, together with the in vitro evaluation of platelet function and biochemistry. PGE1 facilitated easier and faster platelet resuspension following centrifugation. After storage there were few significant in vitro differences between PCs prepared with PGE1 and control PCs. The artifactual leucocyte concentration was significantly lower in the presence of PGE1, suggesting less platelet aggregates had been formed during storage and beta-thromboglobulin release was significantly reduced by PGE1, 14.0 +/- 6.0 micrograms per 10(9) platelets compared with 22.3 +/- 9.8 micrograms per 10(9) platelets in control PCs, (P < 0.01), indicating PGE1 reduced both platelet aggregation and activation probably at the initial preparation stage, known to produce the greatest trauma. Initial in vivo platelet recovery for PCs prepared with PGE1 was similar to that of control PCs, 41.1 +/- 12.5% vs. 44.4 +/- 8.0%, respectively, and there were no differences in organ distribution at 24 h. However, in vivo multiple hit survival was reduced in the presence of PGE1, 5.8 +/- 1.6 days compared with 6.9 +/- 1.4 days in control PCs (P < 0.05). Despite the ability of PGE1 to facilitate platelet resuspension and inhibit platelet aggregation and activation during preparation of the PCs, the reduced in vivo survival time may preclude the use of PGE1 during routine PC preparation.

A comparative study of platelets stored in polyvinyl chloride containers plasticised with butyryl trihexyl citrate or triethylhexyl trimellitate.

Platelet concentrates (PCs), stored for 5 days in PL 2209, a new polyvinyl chloride (PVC) storage container plasticised with butyryl trihexyl citrate, were compared with those stored in PL 1240, a PVC platelet container plasticised with triethylhexyl trimellitate. In part 1 of the study, pooled platelet-rich plasma (PRP) was aliquoted into each type of pack and pH, pCO2, pO2, hypotonic shock response, aggregation responses, lactate, glucose and ATP concentrations, and lactate dehydrogenase and beta-thromboglobulin release were compared at days 1, 3 and 5. In part 2, 12 volunteers gave a unit of blood on two separate occasions and PCs produced by the PRP method were stored in PL 2209 or PL 1240 for 5 days before autologous reinfusion of a 111In-labelled sample. In vitro results demonstrated that PL 2209 was more gas permeable than PL 1240. In part 2 of the study, at day 5, pCO2 was 3.13 +/- 0.62 versus 5.14 +/- 0.69 (p < 0.001), whilst pO2 was not significantly different for PL 2209 versus PL 1240, respectively. pH was better maintained in PL 2209 than in PL 1240 (7.38 +/- 0.13 vs. 7.24 +/- 0.10, respectively, p < 0.01) after storage for 5 days. These results were confirmed by those from part 1. In vivo data were similar for PC stored in the two plastics with a multiple-hit recovery of 40.9 +/- 12.1% for PL 2209 and 37.4 +/- 11.3% for PL 1240, and a multiple-hit survival of 4.89 +/- 1.20 days and 5.28 +/- 2.06 days for PL 2209 and PL 1240, respectively. gamma-Camera imaging of volunteers showed similar biodistribution of radiolabelled platelets stored in each container. These results demonstrate that PL 2209 is a suitable container for storage of PCs for 5 days.

A comparative study in volunteers of apheresis and buffy coat derived platelets.

It is important that, at the time of transfusion, platelets prepared by different techniques are effective in vivo and meet acceptable criteria. A paired study was undertaken in 8 volunteer donors to compare apheresis platelets collected on the COBE Spectra with platelets derived from the buffy coat of whole blood donations after 5 days storage. In vivo recovery, survival and biodistribution were determined following indium-111 labelling of the platelets and autologous infusion into volunteers together with the in vitro evaluation of platelet function and biochemistry. The in vivo and in vitro characteristics of both types of platelet concentrate were not significantly different. Both products were equally viable after 5 d storage and both were of an acceptable quality as judged by currently used platelet products. Mean platelet survival (multiple hit) was 5.4 d for the apheresis platelets and 6.9 d for the buffy coat derived platelets and maximum recovery in circulation was 28.1% and 34.8%, respectively. There was a significantly higher platelet yield, as expected, from apheresis and a 1.5 log lower level of white cell contamination. This would be advantageous for patients in need of prolonged or recurrent transfusions as the number of donor exposures and the risk of alloimmunisation or viral transmission would be reduced.

Effect of agitation on the quality of platelet concentrates.

Platelet concentrates (PCs) were stored for 4 days at 22 degrees C in 400 ml second-generation (PL1240) platelet packs with either constant agitation, manual mixing once every 24 h or without agitation at any time. After 4 days storage, in vivo recovery, survival and biodistribution were determined following indium-111 labelling of platelets and infusion into autologous volunteers. In vitro assays of platelet function and biochemistry were likewise carried out after 4 days storage. The PCs stored without agitation had significantly lower in vivo recoveries, pH and aggregation responses to ionophore A23187 and a combination of collagen and epinephrine and significantly higher beta-thromboglobulin and indium-111 release than the agitated PCs. The manually mixed PCs were not significantly different from the constantly agitated PCs. PCs mixed simply once every 24 h remained viable with active oxidative phosphorylation and a pH above 6.74 in all but 1 case indicating that PCs stored at 22 degrees C for up to 4 days with only intermittent mixing are satisfactory for transfusion. A change from constant agitation would reduce capital costs in mixing equipment and simplify the transport of PCs from the transfusion centre to small hospital blood banks.

Paired in vivo and in vitro comparison of apheresis and "recovered" platelet concentrates stored for 5 days.

Using a paired study, in vivo and in vitro characteristics of apheresis platelets collected on a cell separator and single-donor whole-blood (recovered) platelets via platelet-rich plasma (PRP) were compared after storage for 5 days in similar plastic containers. Autologous platelets from each of 12 volunteers were labeled with 111Indium after storage and reinjected. There was no significant difference in circulating recovery between platelets prepared by the two methods, and only one of five models of survival showed a significant difference. Hypotonic shock recovery was significantly better in apheresis than recovered platelets (57.0% and 32.4%, respectively), whilst aggregation to ADP at 3.2 microM and 32 microM was significantly higher in recovered than in apheresis platelets (17.0% and 45.2% versus 7.8% and 32.9%, respectively). Lactate dehydrogenase (LDH) content was significantly higher in recovered platelets (143.3 versus 77.1 IU/10(11) platelets), but LDH release was similar (15.0% cf. 12.6%). There was no significant difference between the two platelet preparations for platelet concentration, pH, aggregation with the calcium ionophore A23187 or collagen plus epinephrine, or ATP content or release. beta-TG release was lower in apheresis platelets. Neither product was consistently better than the other for the parameters tested, but apheresis platelets have the advantage of lower donor exposure to the patient.

Comparison of platelet concentrates stored in plasma and in three plasmafree media.

Platelet concentrates (PC) were stored for 7 days at 22°C in either CPDA-1 plasma (control) or one of three plasma-free media, namely a Tyrodes solution (BTST) fortified with citrate and with additional phosphate for buffering effect; Plasma-Lyte A solution (PCD) fortified with dextrose and citrate; and Ringers/CPD-50 (10:1) solution (RCPD) buffered with phosphate, all with a pH of 7.4. Parameters monitored included platelet count, pH, hypotonic shock response, aggregation response to single and paired agonists, and release by platelets of endogenous LDH, ATP and 5HT. BTST was shown to be a satisfactory plasma substitute over the entire storage period. PCD gave similar results over 4 days, although the decrease in pH was more marked. In all test systems RCPD was shown to be a poor plasma substitute.

Isozymes of rodent 5'-nucleotidase: evidence for two independent structural loci Umph-1 and Umph-2.

Two distinct isozymes (UMPH-1 and UMPH-2) which hydrolyse pyrimidine 5'-nucleotides have been identified in mouse, rat and Chinese hamster tissues. One of these, UMPH-1 appears to be specific for pyrimidine 5'-nucleotides, whereas the other, UMPH-2 has a broader substrate specificity. UMPH-1 and UMPH-2 show differences in their expression in different tissues. The evidence suggests that UMPH-1 and UMPH-2 are coded by distinct structural gene loci, Umph-1 and Umph-2. These isozymes have not been identified in man, and it is suggested that they may have overlapping electrophoretic mobility.

Biochemical characterization of the genetic variants of human phosphoglycolate phosphatase (PGP).

Phosphoglycolate phosphatase (PGP) exhibits a wide range of activities in normal human red cells. Analysis of blood from 57 individuals of known PGP phenotype revealed no correlation between enzyme activity, electrophoretic phenotype or 2,3-DPG concentration. Neither was there evidence of variation in Km, heat stability, pH optimum or molecular size among the various electrophoretic phenotypes. However, human red cell PGP exhibits high (0.24 mM) and low (0.05 mM) Km values for phosphoglycolate irrespective of phenotype and this requires further analysis.

Genetical and biochemical studies on human phosphoserine phosphatase.

1. Phosphoserine phosphatase (PSP), specific for D- and L-phosphoserine, has been identified in all human tissues. 2. PSP shows no activity towards phosphorylated serine residues in phosphoproteins. 3. In most tissues, PSP consists of a single major isozyme, probably determined by a single autosomal locus. 4. Other isozymes, identified quite frequently in such post-mortem tissues as kidney and liver, appear to be due to post-translational modification, the mode of which has not yet been identified. 5. Two different rare electrophoretic variants have been identified in 1% and 0.25% of 378 post-mortem kidneys (PSP 2-1 and PSP 3-1 respectively). 6. The three banded isozyme pattern of the rare variants suggests PSP is a dimeric enzyme. From gel filtration results the subunit molecular size is 26 000 daltons. 7. Homologous PSP isozymes have been detected in primates with similar electrophoretic mobility to that of the human enzyme.

Secretion of alpha 1-antitrypsin by an established human hepatoma cell line and by human/mouse hybrids.

The human hepatoma cell line SK-HEP-1 has been shown by radioimmunoelectrophoresis to synthesize and secrete a protein which coprecipitates with human alpha 1-antitrypsin. This protein was indistinguishable from serum alpha 1-antitrypsin in terms of electrophoretic mobility, apparent subunit molecular weight (47,000), and binding to concanavalin-A. The protein identified as alpha 1-antitrypsin (alpha 1 AT) was secreted by seven clones derived from SK-HEP-1 and by twelve out of eighteen hybrid clones derived from the fusion of SK-HEP-1 with mouse RAG cells. There was no correlation between the expression of alpha 1 AT and that of human enzymes assigned to sixteen different autosomes. There was an imperfect correlation between the expression of alpha 1 AT and of the two chromosome 9 marker enzymes AK1 and AK3 (two discordant clones).

Metabolic correction of fucosidosis fibroblasts by human alpha-L-fucosidase.

Human alpha-L-fucosidase, purified from placenta, was taken up from the culture medium by skin fibroblasts from patients with fucosidosis (alpha-L-fucosidase deficiency). The rate of uptake was low (uptake coefficient = 6 X 10(-4) ml.mg-1.h-1). Intracellular alpha-L-fucosidase activity was directly proportional to enzyme in the medium up to an activity of at least 40 nmoles/min/ml. No evidence for saturation of specific cell-surface receptors was seen. However, uptake was reduced by 75% by 1 mM mannose-6-phosphate and by 50% by 1mM glucose-6-phosphate, suggesting that uptake may be mediated by a receptor recognising a phosphorylated sugar or an analagous compound. Enzyme taken up by the cells was most active in subcellular fractions enriched with lysosomes and had an isozyme pattern, by isoelectric focusing, identical to that of the original enzyme preparation. Fucosidosis fibroblasts were shown to accumulate low molecular-weight, fucose-containing compounds to a level several times greater than control cells. This stored material was eluted from Sephadex G-25 as an asymmetrical peak with an elution volume of approximately twice the void volume of the column. Addition of placental alpha-L-fucosidase to the culture medium of fucosidosis fibroblasts prevented excessive accumulation of fucose-containing material and accelerated the breakdown of material accumulated prior to enzyme uptake.

Assignment of the gene locus for human alpha-L-fucosidase to chromosome 1 by analysis of somatic cell hybrids.

The alpha-L-fucosidases (EC 3.2.1.51) from human and mouse cells could be separated by isoelectric focusing of neuraminidase-treated cell extracts in acrylamide slab gels. Fourteen hybrid clones derived from the fusion of mouse and human cultured fibroblasts and 37 hybrid clones derived from the fusion of human long-term lymphoid lines with mouse RAG cells were tested for expression of human alpha-L-fucosidase. A strong correlation between the expression of the human enzyme and the presence or absence of human chromosome 1 was found. The presence of human alpha-L-fucosidase in clones scored as positive by isoelectric focusing was confirmed by Ouchterlony double immunodiffusion against IgG from rabbits immunized with purified human alpha-L-fucosidase. It is concluded that the structural gene locus for human alpha-L-fucosidase is located on chromosome 1.

Polymorphism of human alpha fucosidase.

A common polymorphism of human alpha fucosidase has been detected by the technique of isoelectric focusing on thin layer acrylamide gel. Family studies have shown that the three common phenotypes Fu 1, Fu 2, and Fu 2-1 represent homozygosity or heterozygosity for two autosomal codominat alleles, Fu1 and Fu2. Frequencies of the Fu1 and Fu2 alleles in the New York populations studied were .753 and .247 among whites and .926 and .074 among blacks, respectively.