Repeated short-term warming of red blood cell concentrates has minimal effect on their quality.

BACKGROUND AND OBJECTIVES: Blood components must be stored under controlled temperature conditions, for reasons of component quality and safety. However, there are occasions when components may be exposed to conditions outwith the defined limits. This study aimed to generate prospective data on the effect of red cell exposure to extremes of temperature. MATERIALS AND METHODS: Study 1: red cell concentrates (RCC) in saline, adenine, glucose and mannitol (SAGM), made after ambient overnight hold of whole blood, were exposed to either +22°C or -2°C for up to three periods of 3 h on days 3, 8 and 15 of storage, followed by a 5 h exposure on day 29. Study 2: RCC in SAGM were exposed to 25°C for 24 or 48 h from day 2. In vitro markers of cell quality were measured during storage to 43 days, and compared with control units that had been stored at 2-6°C. RESULTS: Multiple short-term exposures to +22°C or -2°C did not cause any significant changes to pH, haemolysis, supernatant potassium, cellular ATP, 2,3-DPG, or deformability, when compared to control units. Exposure of RCC to 25°C for 24 or 48 h caused a significant fall in pH, ATP, and deformability. CONCLUSION: Red cells may be damaged by prolonged exposure to warm temperatures, but repeated short-term exposure to 22°C or -2°C does not appear to affect the in vitro quality of RCC. It is important to note that no bacterial growth studies were performed during this study.

Red cell concentrate storage and transport temperature.

BACKGROUND AND OBJECTIVES: This study investigated the current U.K. guidelines for storage and transport of red cell concentrates (RCC) in saline, adenine, glucose and mannitol (SAGM). The guidelines stipulate storage at 2-6 °C but allow exposure to between 1-10 °C core temperature in a single occurrence of less than 5 h and a surface temperature of 2-10 °C for no more than 12 h during transportation. METHODS AND MATERIALS: Twenty RCC units in SAGM were selected on the day of blood collection (day 0) and in vitro quality was tested pre- and post-temperature deviation at 10 °C and up to day 42 of storage. Each group of 10 RCC units was incubated for either 12 h or for both 5 and 12 h. RESULTS: Haemolysis was below the 0·8% U.K. limit at day 42 in all units, although there was an unexpected trend towards lower haemolysis in packs incubated for 5 and 12 h rather than just 12 h alone. Supernatant potassium was significantly higher than reference data on day 35 (P < 0·05) with a maximum of 58 mmol L(-1) and day 42 (P < 0·001). All units incubated at 10 °C had comparable levels of adenosine triphosphate and, 2,3-diphosphoglycerate to reference data from previous studies, throughout storage. CONCLUSION: These results suggest that exposure to 10 °C for 12 h or for 5 and 12 h did not adversely affect in vitro red cell quality for the remainder of the components shelf life.

Platelet apoptosis and activation in platelet concentrates stored for up to 12 days in plasma or additive solution.

BACKGROUND: Several studies suggest that apoptosis of platelets occurs during storage of platelet concentrates (PC). We sought to determine whether storage of PC in additive solution alters levels of apoptosis during storage beyond the current shelf life (5-7 days). STUDY DESIGN AND METHODS: Pooled buffy coat PC (n = 7) were prepared in either 100% plasma or 70% Composol and stored at 22 °C for 12 days. A third arm of the study stored PC in 100% plasma at 37 °C, which is thought to induce apoptosis. PC were tested for mitochrondrial membrane potential, annexin V binding, microparticles, caspase-3/7 activity and decoy cell death receptor 2, as well as standard platelet quality tests. RESULTS: Composol units remained ≥pH 6·88, with 36% lower lactate and higher pH vs plasma by day 12 (P < 0·001). Platelet function was better maintained, and activation and apoptotic markers tended to be lower in Composol units towards the end of storage. However, levels of all apoptosis markers assessed were not significantly different in units stored in Composol. Storage at 37 °C saw stronger correlation of apoptotic markers with standard quality tests compared to 22 °C, but loss of correlation of caspase-3/7 activity with other apoptosis markers. CONCLUSION: We conclude that storage of platelets in 70% Composol vs 100% plasma does not increase the rate of platelet apoptosis. Our data agree with other studies suggesting that platelet apoptosis is sequential to high levels of activation, but share a significant degree of overlap.

In vitro function of buffy coat-derived platelet concentrates stored for 9 days in CompoSol, PASII or 100% plasma in three different storage bags.

BACKGROUND AND OBJECTIVES: The aim of the study was to compare the in vitro quality of buffy coat-derived platelet concentrates (PC) during extended storage in plasma or additive solution in three different storage bags. MATERIALS AND METHODS: A pooled and split design was chosen so that identical PCs were produced in either 100% plasma, 70% PASII : 30% plasma or 70% CompoSol : 30% plasma (n = 6 each). This was repeated for three different manufacturers' platelet storage bags (Fresenius, Baxter and Pall). PCs were sampled on days 1, 5, 7 and 9 of storage and tested in vitro using a variety of tests of platelet function. For each bag type, storage in PASII or Composol was compared with plasma (data taken across the entire storage period), and differences occurring with time were analysed for all storage media. RESULTS: The pH of all PCs was > 6.8 at day 9 of storage. In vitro platelet function, as assessed by markers of platelet activation and metabolism, of PCs stored in CompoSol appeared to be similar to that of PCs stored in plasma over 9 days of storage. In contrast, PCs stored in PASII tended to have significantly higher levels of platelet activation (almost a twofold increase in % platelets positive for CD62P by day 5) and lower hypotonic shock response (approximately 40%, by day 7) compared to either PCs stored in 100% plasma or 70% CompoSol. The magnitude of the differences observed between platelet storage media appeared to be dependent on the type of platelet storage bag with the highest degree of platelet activation and lowest hypotonic shock response values being observed in Fresenius bags in combination with PASII. CONCLUSIONS: The maintenance of platelet function in vitro during extended storage of PCs in platelet additive solutions is dependent on the combination of type of additive solution and type of platelet storage bag. For all bag types studied, storage in PASII resulted in poorer platelet function in vitro.

Porphyria cutanea tarda: the etiological importance of mutations in the HFE gene and viral infection is population-dependent.

A number of factors, including increased iron stores and alcohol consumption, are known to be associated with the development of porphyria cutanea tarda (PCT) in susceptible individuals. Recent reports have described a significant association between inheritance of the C282Y and H63D mutations in the HFE gene, associated with genetic hemochromatosis (GH) and PCT. A strong association between hepatitis C virus infection and PCT has also been demonstrated, while case reports record a link between human immunodeficiency virus (HIV) and PCT. We have investigated the frequency of these factors in a racially-mixed population of patients with PCT in Cape Town, South Africa. 57 patients with PCT drawn from three ethnic groups were screened for the presence of the C282Y and H63D mutations linked to GH, and the prevalences were compared with corresponding healthy control populations. The seroprevalence of markers for HCV, hepatitis B (HBV) and HIV infection were examined in 28 of these. In the control populations, we found that both the C282Y and H63D mutations are highly prevalent in South Africans of European origin. In a population of mixed or Asian origin, the C282Y mutation is very rare whereas the H63D mutation is common. Neither mutation was encountered in any African subject. Both mutations are associated with PCT, but the association is dependent on the ethnic origins of the population to which the patient belongs. In contrast to other studies, HCV infection is numerically unimportant in PCT in our patients. HIV infection is increasingly encountered in our patients with PCT, but the strength of the association cannot be determined in view of the high background prevalence of HIV infection in some sectors of the South African population. The contribution of specific risk factors may be heavily dependent on the population from which patients are drawn, and care should be taken in extrapolating from observations in one racial or geographic population to any other.

Identification of the first variegate porphyria mutation in an indigenous black South African and further evidence for heterogeneity in variegate porphyria.

Variegate porphyria is an autosomal dominant disorder of haem metabolism resulting from reduced levels of the penultimate enzyme in the pathway, protoporphyrinogen oxidase. Here we investigate the molecular basis of variegate porphyria in four non-R59W South African families. We report the identification of the first mutation in the protoporphyrinogen oxidase gene in a black South African individual (V290M). In addition, we document three further mutations, a missense mutation (L15F), a deletion followed by a substitution [c769delG;770T > A], and a nonsense mutation (Q375X), in individuals of European or mixed ancestry. Our data provide further evidence of genetic heterogeneity in South Africa.

Homozygous variegate porphyria in South Africa: genotypic analysis in two cases.

Variegate porphyria is an autosomal dominant disorder of heme metabolism which results from decreased activity of the enzyme protoporphyrinogen oxidase. Clinically, the disease manifests postpubertally and is characterized by photocutaneous sensitivity and/or acute neurovisceral crises. However, in homozygous variegate porphyria, onset of the disease usually occurs in infancy with severe skin manifestations. The molecular basis of variegate porphyria in two severely affected probands in two South African families is described. Mutation detection included combined SSCP-heteroduplex analysis followed by direct sequencing. The unrelated probands both had the common R59W mutation while the other lesion was Y348C or R138P (both novel mutations), causing homozygous variegate porphyria.

Identification and characterisation of a deletion (537delAT) in the protoporphyrinogen oxidase gene in a South African variegate porphyria family.

Variegate porphyria is an autosomal dominant disorder of haem metabolism resulting from a partial decrease in protoporphyrinogen oxidase activity. Variegate porphyria is highly prevalent in South Africa, the result of a founder effect now confirmed genetically as a single point mutation (R59W) which has been described in nearly all South African variegate porphyria patients studied. Only two other mutations (H20P, R168C) have been reported in South Africa. We utilised simultaneous, single-stranded conformational polymorphism and heteroduplex analysis, and direct sequencing to identify a further mutation; a 2 bp deletion in exon 6 which results in a premature stop codon 11 codons downstream from the mutation and is the first reported deletion in the protoporphyrinogen oxidase gene in a South African family. The familial segregation of this mutation strongly suggests that it is the disease causing mutation for variegate porphyria in this family. This further evidence for allelic heterogeneity limits the utility of tests for the R59W mutation in the diagnosis of variegate porphyria in South Africa.

The expression of mRNA for fibrinogen in megakaryocytes isolated from patients with T-cell lymphoma.

The expression of fibrinogen mRNA was studied by in situ hybridization in freshly isolated megakaryocytes in 14 newly-diagnosed patients: seven with non-Hodgkin's lymphoma (NHL), three with immune thrombocytopenic purpura (ITP) and four haematologically normal patients prior to coronary artery bypass surgery. Fibrinogen mRNA in megakaryocytes was not detected in ITP, B-cell lymphomas or in healthy donors. However, it was present in all patients with the high-grade T-cell lymphomas, both with and without thrombocytopenia.

Megakaryocytes from patients with coronary atherosclerosis express the inducible nitric oxide synthase.

Endothelial and platelet generation of nitric oxide (NO) plays an important role in the regulation of hemostasis. Alterations in NO biosynthesis are described in atherosclerosis. We have investigated the NO pathway in megakaryocytes and platelets from patients with atherosclerosis and age-matched control subjects. Megakaryocytes and platelets were isolated from patients with severe coronary atherosclerosis (n = 19) and normal coronary arteries (n = 9) as demonstrated by selective angiography. Constitutive (Ca(2+)-dependent) and inducible (Ca(2+)-independent) NO synthase (cNOS and iNOS, respectively) activities were measured by using the citrulline assay and by immunostaining techniques using an anti-peptide antibody to iNOS. Megakaryocytes from patients with atherosclerosis expressed significantly greater amounts of iNOS (1.28 +/- 0.46 pmol citrulline.mg-1.min-1) than cNOS (0.29 +/- 0.40 pmol.mg-1.min-1). In contrast, megakaryocytes from patients with normal coronary arteries expressed significantly more cNOS (1.48 +/- 0.23 pmol.mg-1.min-1) than iNOS (0.49 +/- 0.40 pmol.mg-1.min-1). Platelets isolated from both groups showed no significant difference in cNOS expression, and no iNOS was seen in either group. Immunostaining confirmed the presence of the iNOS in megakaryocytes. These results suggest there is a link between the expression of iNOS in the megakaryocyte and atherosclerosis.

The relationship between human megakaryocyte nuclear DNA content and gene expression.

Increased platelet reactivity has been implicated in various vascular diseases. Since the protein content of platelets is determined mainly by the megakaryocyte, alterations in megakaryocyte mRNA expression may influence platelet protein content and therefore activity. In order to determine whether DNA content (ploidy) of a megakaryocyte influences its mRNA expression, we have developed a method to investigate the relationship between megakaryocyte ploidy and gene expression. By measuring the ploidy and mRNA expression in individual cells, we have shown that in the physiological state there is an increase in mRNA expression for beta-actin, glycoprotein IIb and neuropeptide Y with increase in ploidy and that this increase levels off at high ploidy values. This may have relevance in the understanding of platelet reactivity in pathological events.

Antimicrotubule agents induce polyploidization of human leukaemic cell lines with megakaryocytic features.

In most eukaryotic cells the regular alternation of chromosome reduplication and cell division is controlled by interdependent relationships which prevent progression to the next cell-cycle phase unless the preceding phase has been completed. Megakaryocytes become polyploid by allowing many rounds of DNA replication without completion of intervening mitoses. To assess the role of cell-cycle dependencies in megakaryocytopoiesis we examined human cell lines which express megakaryocytic features for their ability to continue DNA synthesis and undergo polyploidization in the presence of mitotic poisons. Treatment of HEL cells with colcemid blocked cell division but not cellular DNA synthesis. DNA content distributions of cells treated with colcemid for 48 h showed a marked increase in the proportion of polyploid cells (57.6% +/- 9.9%, n = 16), an increase in cellular size and nuclear lobation. Identical effects were observed in HEL cells treated with colchicine, nocodazole or taxol but not with the inactive compound lumicolchicine. Induction of polyploidization by antimicrotubule agents was also observed in the megakaryoblastic cell lines MEG-01, DAMI and UT-7 but not in the T-cell line MOLT-4 or the promyelocytic cell line HL-60. These results suggest that dependency of DNA replication on completion of the previous mitosis is suppressed in the megakaryocytic lineage.

Exploitation of a 650 bp probe for quantification of Pneumocystis carinii.

Signals obtained from serial dilutions of rat-derived Pneumocystis carinii DNA were used to assess the sensitivity of a 650-bp DNA probe which recognises both cystic and non-cystic forms. Enhanced chemiluminescence was selected as a non-radioactive detection method and signals could be semi-quantitated by scanning densitometry. This technique was used to examine the inhibitory effect of pentamidine in vitro indicating that DNA probes might be useful tools in the search for a novel therapeutic agent.

Ultrastructural observations on the attachment of Pneumocystis carinii in vitro.

Pneumocystis carinii trophozoites grow in vivo in close contact with host cells. The attachment of Pneumocystis to the lung cells seems to be a critical step in the parasite's development. Up to now, the contact of Pneumocystis with mammalian tissue culture cells was shown using light and scanning electron microscopy. The methods are not sufficient to observed in detail the parasite-feeder cell area of contact. In this work, the attachment of Pneumocystis trophozoites to feeder cells was examined in serial sections using transmission electron microscopy. When the contact of a trophozoite with a feeder cell took place, the development of filopodia penetrating deeply into invaginations of the feeder cell plasma membrane was observed. Then, the apical tips of filopodia become bulged anchoring the parasite to the feeder cell. The behaviour of Pneumocystis in feeder cell cultures is compared to that of the parasite in other in vitro or in vivo experimental models.

Analysis of the dynamics of Pneumocystis carinii in vitro.

Three culture systems employing mammalian lung cell monolayers were evaluated in terms of their ability to support the replication of primary isolates of rat-derived Pneumocystis carinii. For each system, analysis of variance was used to identify changes in the numbers of P. carinii over time. Significant replication was consistently demonstrated over lung fibroblasts (MRC-5), however, increases perceived in the culture supernatant were clearly influenced by the density of the underlying monolayer.

No detection of characteristic fungal protein elongation factor EF-3 in Pneumocystis carinii.

The taxonomic status of Pneumocystis carinii is uncertain, and P. carinii has been categorized both as a fungus and as a protozoan. Recent comparisons of RNA sequence homologies between P. carinii and several genera of fungi and protozoa suggest that P. carinii has closer affinities with the ascomycetes than with the protozoa. The translatory systems of the fungi, however, require three soluble protein factors for peptide chain elongation rather than the two necessary in other eukaryotic systems; to date the additional protein elongation factor (EF-3) appears to be unique to fungi. Western blot analysis of cell-free extracts of P. carinii, derived from rat, was done using a polyclonal antibody raised in rabbits to Saccharomyces cerevisiae EF-3. Anti-EF-3 cross-reacting material was detected only in lysates of Candida albicans and S. cerevisiae included as fungal controls; no cross reaction was detected in lysates of P. carinii, P. carinii-infected rat lung, or a protozoan control (Trichomonas vaginalis).

Identification of mRNA for PDGF B-chain in human megakaryocytes isolated using a novel immunomagnetic separation method.

A rapid and simple method has been developed for the separation of pure populations of intact human megakaryocytes from whole bone marrow. Megakaryocytes were specifically recognized using monoclonal antibodies coupled to magnetizable articles. Cells labelled with the magnetizable probe were separated from unlabelled cells by introduction of a magnetic field. The technique yields megakaryocyte suspensions with a purity of greater than 98%. Electron microscope examination showed that the ultrastructure of the isolated megakaryocytes was well preserved. Using this method of cell purification, we have investigated expression of the gene for platelet-derived growth factor (PDGF). RNA was isolated from cells harvested from either ribs or posterior iliac crest. The RNA was spotted onto nitrocellulose, and then hybridized using a c-sis riboprobe specific for PDGF B chain mRNA. We demonstrate that mRNA for PDGF B-chain is identifiable in samples of 50,000 cells. We conclude that PDGF is synthesized by the megakaryocyte.

The relationship between megakaryocyte nuclear DNA content and gene expression.

Megakaryocytes are a distinct population of bone marrow cells that have the unique feature of increasing their DNA content without undergoing division. The biological effect of ploidy distribution on gene expression, receptor expression and protein synthesis is still unknown. Using molecular hybridization techniques, we have started a systematic analysis of mRNA expression in megakaryocytes for a number of proteins involved in clot formation. These data will be related to ploidy. Platelets are the unnucleated product of megakaryocytes, having their protein content derived from the precursor cell. Therefore, the understanding of the molecular mechanisms regulating megakaryocyte biology and the consequent type and reactivity of platelets produced is of fundamental importance in both physiological and pathological conditions.

Studies on the activity and activation of rat liver microsomal glutathione transferase, in particular with a substrate analogue series.

A number of potential substrates for the microsomal glutathione transferase have been investigated. Out of 11 epoxides tested, only two, i.e. androstenoxide and benzo(a)pyrene-4,5-oxide, were found to be substrates. Upon treatment of the enzyme with N-ethylmaleimide, its activity toward only certain substrates is increased. It appeared upon inspection of the bimolecular rate constants from the corresponding nonenzymatic reactions that the substrates for which the activity is increased are the more reactive ones. This hypothesis was investigated further using a series of para-substituted 1-chloro-2-nitrobenzene derivatives as substrates. Activation was seen only with the more reactive nitro-, aldehyde-, and acetaldehyde-substituted compounds and not with the amide and chloroanalogues, thus demonstrating the predicted effect with a related series of compounds. Interestingly, kcat values are increased 7-20-fold by N-ethylmaleimide treatment, whereas the corresponding kcat/Km value is increased only for the p-nitro derivative. Effective molarity and rate enhancement values were found to increase with decreasing reactivity of the substrate, attaining maximal values of 10(5) M and 10(8), respectively. It is concluded that the glutathione transferases are quite effective catalysts with their less reactive substrates. Hammett rho values for the kcat values of unactivated and activated enzyme were 0.49 and 2.0, respectively. The latter value is close to those found for cytosolic glutathione transferases, indicating that activation changes the catalytic mechanism so that it more closely resembles that of the soluble enzymes. The rho values for kcat/Km values were 3 and 3.5 for the unactivated and activated enzyme, respectively, values close to those observed for the nonenzymatic bimolecular rate constants and thereby demonstrating that these reactions have similar properties. The high coefficients of correlation between resonance sigma- values and all of these parameters demonstrate a strong dependence on substrate electrophilicity, as expected for nucleophilic aromatic substitution.