Comparison of three commercially available buffy coat pooling sets for the preparation of platelet concentrates.

BACKGROUND: A disposable set for platelet concentrate (PC) preparation by the buffy coat method allows pooling of buffy coats, centrifugation and cell separation with in-line leucocyte filtration. This study compares three commercially available pooling sets in combination with INTERCEPT pathogen inactivation (PI). MATERIALS AND METHODS: Sets for pooling of buffy coats were from Fresenius Kabi (FRE), Macopharma (MAC) and Terumo BCT (TER). Platelet yield, recovery and concentration were compared before and after PI (n = 20). Platelet quality was assessed by annexin V binding, P-selectin expression and PAC1 binding. RESULTS: The TER pooling set had the highest platelet yield (5·39 ± 0·44 × 1011 ) compared with MAC (4·53 ± 0·77) and FRE (4·56 ± 0·51) prior to PI. This was the result of a significantly higher platelet concentration in the TER storage bag (1·41 ± 0·12 × 106 /µL) compared with MAC (1·18 ± 0·19) and FRE (1·28 ± 0·15). However, the TER platelet content decreased by 15·6% after PI, yielding 4·55 ± 0·47 × 1011 platelets compared with smaller reductions at 9·5% for MAC (4·10 ± 0·69) and 4·4% for FRE (4·36 ± 0·52). None of the individual PC contained >106 leucocytes. The pH in TER PC was lower compared with MAC and FRE caused by a higher lactic acid production rate. Consequently, PAC1 binding after TRAP activation was lowest for TER PC on day 6. P-selectin and annexin V were not different between suppliers. CONCLUSION: This study demonstrates the added value of evaluating the entire component production process when introducing a new consumable. This study helped to inform a decision on what pooling set is ideally suited for routine implementation taking into account PI.

Cognitive deficits and increases in creatine precursors in a brain-specific knockout of the creatine transporter gene Slc6a8.

Creatine transporter (CrT; SLC6A8) deficiency (CTD) is an X-linked disorder characterized by severe cognitive deficits, impairments in language and an absence of brain creatine (Cr). In a previous study, we generated floxed Slc6a8 (Slc6a8 flox ) mice to create ubiquitous Slc6a8 knockout (Slc6a8-/y ) mice. Slc6a8-/y mice lacked whole body Cr and exhibited cognitive deficits. While Slc6a8-/y mice have a similar biochemical phenotype to CTD patients, they also showed a reduction in size and reductions in swim speed that may have contributed to the observed deficits. To address this, we created brain-specific Slc6a8 knockout (bKO) mice by crossing Slc6a8flox mice with Nestin-cre mice. bKO mice had reduced cerebral Cr levels while maintaining normal Cr levels in peripheral tissue. Interestingly, brain concentrations of the Cr synthesis precursor guanidinoacetic acid were increased in bKO mice. bKO mice had longer latencies and path lengths in the Morris water maze, without reductions in swim speed. In accordance with data from Slc6a8 -/y mice, bKO mice showed deficits in novel object recognition as well as contextual and cued fear conditioning. bKO mice were also hyperactive, in contrast with data from the Slc6a8 -/y mice. The results show that the loss of cerebral Cr is responsible for the learning and memory deficits seen in ubiquitous Slc6a8-/y mice.

High platelet content can increase storage lesion rates following Intercept pathogen inactivation primarily in platelet concentrates prepared by apheresis.

BACKGROUND: Pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide. In vitro studies have demonstrated its effects on storage lesion, but little routine quality control data on blood banking outcomes have been reported. MATERIALS AND METHODS: Swirling of distributed products was monitored before and after implementation of Intercept pathogen inactivation. Metabolic parameters pH, glucose and lactic acid were determined in a random cohort of expired pathogen-inactivated products. Storage lesion indicators in apheresis concentrates with premature low swirling were compared to concentrates with normal swirling. RESULTS: During validation for implementing Intercept pathogen inactivation, pH and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pathogen-inactivated pooled buffy coat-derived products. In routine products, glucose exhaustion was more often found in apheresis compared to buffy coat-derived platelet concentrates despite 3-7% more plasma carryover in the former. Annual incidence of premature low swirling increased significantly by 50% following implementation of pathogen inactivation implementation for apheresis but not for pooled buffy coat platelet concentrates. In addition, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5·0 × 1011 ) than unaffected products (3·5 × 1011 ). CONCLUSION: The risk of increased storage lesion rates following Intercept pathogen inactivation is higher for apheresis than for buffy coat-derived platelet concentrates, especially when platelet contents are higher than 5·0 × 1011 .

Persistent aggregates in apheresis platelet concentrates are commonly collected from donors with a history of aggregate donation.

Platelet apheresis sometimes causes persistent aggregates (PA). This study (n = 211) shows that changing the apheresis settings to reach fixed product volumes instead of yields does not influence PA incidence, even though PA products on average contain more platelets than controls. Furthermore, logistic regression was used to model if PA can be predicted on the basis of certain predonation parameters. PA donation history was the only parameter retained, proving a strong determinant of predictability [AUC = 0.735 (SE = 0.022)]. Consequently, donations from a donor with previous PA history are 7.8 times more likely to contain PA than from a donor without preceding history.

Persistent aggregates in apheresis platelet concentrates.

BACKGROUND: Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing product wastage. This study assessed product quality of apheresis concentrates containing persistent aggregates (PA) and aimed to identify the factors that contribute to their formation. METHODS: Donation (n = 180) and platelet indices (n ≥ 10) from apheresis concentrates with PA were compared with aggregate-free products. RESULTS: The proportion of donors with at least one previous PA donation was twofold higher in the PA group (P < 0·0001) indicating a donor dependence. Significantly higher donor whole blood platelet counts (286 ± 50 vs. 266 ± 49 × 10(3) /µl, P < 0·0001) and higher apheresis yields (6·0 ± 1·6 vs. 5·4 ± 1·5 × 10(11) , P < 0·0001) were noted in the PA group. Haematocrit was also slightly higher, but age, gender and body mass were similar. The pH of PA products on day six postdonation was significantly lower (P < 0·001), in line with higher lactic acid concentrations. Flow cytometry showed no differences in GPIbα levels or phosphatidylserine exposure. However, there was slightly more integrin activation as well as increased degranulation measured by P-selectin expression. Cytokine concentrations were also significantly higher in PA concentrates. Aggregation was normal in response to SFLLRN peptide and collagen stimulation, but agglutination at low-dose ristocetin was significantly higher (P = 0.01) in PA products. Finally, PA were disintegrated by plasmin-mediated thrombolysis but not by integrin αIIb ß3 inhibition. CONCLUSION: Products with PA have acceptable quality parameters, but additional functional studies are warranted. Furthermore, PA are more likely to recur in certain donors who have higher platelet counts.

Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion.

BACKGROUND AND OBJECTIVE: Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT). METHODS: Activity and antigen of plasma components were determined following RF-PRT in the presence or absence of dissolved molecular oxygen. RESULTS: Employing ADAMTS13 as a sentinel molecule in plasma, our data show that its activity and antigen are reduced by 23 ± 8% and 29 ± 9% (n = 24), respectively, which corroborates with a mean decrease of 25% observed for other coagulation factors. Western blotting of ADAMTS13 shows decreased molecular integrity, with no obvious indication of additional proteolysis nor is riboflavin able to directly inhibit the enzyme. However, physical removal of dissolved oxygen prior to RF-PRT protects ADAMTS13 as well as FVIII and fibrinogen from damage, indicating a direct role for reactive oxygen species. Redox dye measurements indicate that superoxide anions are specifically generated during RF-PRT. Protein carbonyl content as a marker of disseminated irreversible biomolecular damage was significantly increased (3·1 ± 0·8 vs. 1·6 ± 0·5 nmol/mg protein) following RF-PRT, but not in the absence of dissolved molecular oxygen (1·8 ± 0·4 nmol/mg). CONCLUSIONS: RF-PRT of single plasma units generates reactive oxygen species that adversely affect biomolecular integrity of relevant plasma constituents, a side-effect, which can be bypassed by applying hypoxic conditions during the pathogen inactivation process.

Evidence for specific and non-covalent binding of lipids to natural and recombinant Mycobacterium bovis BCG hsp60 proteins, and to the Escherichia coli homologue GroEL.

Heat-shock proteins (Hsps) from various origins are known to share a conserved structure and are assumed to be key partners in the biogenesis of proteins. Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60, from Mycobacterium bovis BCG zinc-deficient culture filtrate on phenyl-Sepharose followed by Western blotting revealed the existence of four Hsp60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour. Hsp60-2 species were further purified by ion-exchange chromatography and partial amino acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 species showed identity with the amino acid sequence deduced from the hsp60-2 gene, indicating that the various Hsp60-2 forms are encoded by the same gene. In addition, the mycobacterial Hsp60-2 was overexpressed in E. coli using the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose. The elution pattern of the recombinant Hsp60-2, as well as that of Escherichia coli GroEL, was similar to that of the native Hsp60-2 from the culture filtrate of M. bovis BCG and entirely different from that of the mycobacterial antigen 85. Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E. coli GroEL with organic solvents releases various amounts of non-covalently bound lipids. The presence of lipids on Hsp60-2 was confirmed by labelling M. bovis BCG with radioactive palmitate. The radioactivity was specifically associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipoproteins in the Triton X-114 phase. Analysis of the lipids extracted from purified Hsp60-2, recombinant BCG Hsp60-2 and E. coli GroEL by TLC showed the same pattern for all the samples. Acid methanolysis of the lipids followed by GC analysis led to the identification of C(16:0), C(18:0) and C(18:1) as the major fatty acyl constituents, and of methylglycoside in these proteins. Altogether, these data demonstrate that lipids are non-covalently bound to Hsp60-2 and homologous proteins.

Malaria in urban and rural Kinshasa: the entomological input.

Mosquitoes were collected on human bait over a 16-month period (September 1988 to December 1989) in an urban and a rural area of Kinshasa, Zaïre. P. falciparum malaria sporozoite rates were determined by ELISA. In the urban area Culex quinquefasciatus accounts for 96% of the 121 bites/person/night (b/p/n). The only anopheline is Anopheles gambiae, sensu stricto, with an average of 5.1 b/p/n and a sporozoite rate of 1.86%. The entomological inoculation rate (EIR) averages 0.08 infective b/p/n. Malaria transmission is almost interrupted at the end of the dry season. In the rural area mosquito nuisance is small (20 b/p/n), almost entirely due to six species of Anopheles including four vectors of malaria: An.gambiae (13.3 b/p/n), An.funestus (2.4 b/p/n), An.nili (0.4 b/p/n) and An.brunnipes (0.7 b/p/n) with mean sporozoite rates of 7.85%, 6.60%, 6.63% and 0.53% respectively. An.paludis (0.4 b/p/n) and An.hancocki (0.2 b/p/n) were not found infective. Malaria transmission is intense and perennial: the overall EIR varies monthly between 0.60 and 3.29 infective b/p/n. The specific contributions of An.gambiae, An.funestus and An.nili average 1.07, 0.14 and 0.03 infective b/p/n respectively. Malaria transmission peaks during the rainy season in both study areas. The daily mean survival rates for An.gambiae were 0.91 and 0.78 in the rural and urban area, respectively. All An.gambiae examined belonged to the forest cytotype (Coluzzi et al., 1979). Through its effect on the sporozoite rate, the higher vector survival rate in the rural environment appears to be the major determinant of the greater malaria transmission rate in the rural area as compared to urban Kinshasa.

Gas chromatographic determination of alkyl lysophospholipids after solid-phase extraction from cell culture media.

The gas chromatographic determination of 1-O-octadecyl-2-O-methyl-DL-glycero-3-phosphorylcholine (Et-18-OMe), an anti-invasive alkyl lysophospholipid, in cell culture media is described. Sample clean-up was performed by solid-phase extraction on a weak cation-exchange column of the CBA type (carboxylic acid). For quantitation, the structural analogue Et-16-OMe as the internal standard was used after derivatization with trimethylsilyl bromide. The described method was free of interferences in cell culture media. The overall precision for twenty determinations was 14.99%.

Comparison of the isolation of adducts of 2'-deoxycytidine and 2'-deoxyguanosine with phenylglycidyl ether by high-performance liquid chromatography on a reversed-phase column and a polystyrene-divinylbenzene column.

2'-Deoxycitidine (dCyd) and 2'-deoxyguanosine (dGuo) were subjected to reaction with phenylglycidyl ether (PGE) in methanol in order to study the formation of the corresponding 2'-deoxynucleoside adducts. Separation methods were developed on analytical and semi-preparative scales using high-performance liquid chromatography with photodiode-array detection on a reversed-phase column and on a polystyrene-divinylbenzene column. The use of the latter column was prompted by decomposition of the preparatively isolated dGuo-PGE adducts on the reversed-phase column. The use of a polystyrene-divinylbenzene column solved this problem and also revealed the presence of one more peak in both the dCyd- and dGuo-PGE reaction mixtures. The adducts of dCyd and dGuo were isolated on preparative reversed-phase and polystyrene-divinylbenzene columns and characterized by UV, fast atom bombardment mass and 360 MHz 1H NMR spectrometry. The adducts of dCyd were the diastereomers of N-3-(2-hydroxy-3-phenoxypropyl)-2'-deoxycytidine and N4-(2-hydroxy-3-phenoxypropyl)-2'-deoxycytidine whereas those of dGuo were the two diastereomers of N-7-(2-hydroxy-3-phenoxypropyl)-2'-deoxyguanosine and a third peak which appeared to be mainly N2-(2-hydroxy-3-phenoxypropyl)-2'-deoxyguanosine.

Prospects for malaria control in urban and rural Kinshasa.

Malaria is a major cause of paediatric illness and death in Kinshasa, and all 3 million inhabitants are at risk. In view of the increasing chloroquine-resistance of Plasmodium falciparum, the early treatment of fever cases as the sole malaria control measure is no longer acceptable. The prospects for vector control are determined by the effectiveness, the acceptability and the practicability of the various methods in the local conditions of Kinshasa. Pronounced differences in the level of endemicity exist between the various parts of the town. These differences, and the ecological and socio-economic factors that underlie them, must be taken into account when estimating the potential of a control method. The reduction of man-vector contact through personal protection with impregnated bednets is the only realistic goal at this moment, but even a very marked decrease of the inoculation rate will produce little apparent effect in the highly endemic (semi-)rural districts at the periphery of town. In the urbanized center of Kinshasa, where the moderate to low intensity of transmission is more susceptible to a critical reduction, the same method may have an impact on malaria morbidity. Moreover, the big nuisance from non-vector mosquitoes in the urban area is an important motivating factor for the acceptance and the use of bednets. A mass effect, on the other hand, only is to be expected in isolated villages. Field trials are needed to evaluate the short- and long-term effect on malaria transmission and on its' clinical expression, as well as on the build-up of natural immunity, in the epidemiologically distinct areas. However, the final outcome of a large scale implementation of malaria control with impregnated mosquitonets will equally depend on health education, on the availability of bednets at low cost, on the creation of the appropriate structures for the (re)impregnation and distribution of the nets, and finally on the sustainability of the whole effort.