Evaluation of preanalytical stability of the sample for complete blood count

ObjectiveTo evaluate the effect of time of incubation on complete blood count (CBC) by using HmX analyzer.MethodsA cross sectional study was conducted at Manipal Acunova Limited, Bangalore. Ten blood samples which were stored at room temperature for CBC and differential count by using HmX analyzer were analyzed within one hour of draw and on 24, 48 and 72 hours of draw.ResultsDifferential count measured in an automated instrument changed over time.ConclusionsThe finding of this study shows that some of CBC parameters can be changed with the incubation, therefore it is better to perform on a fresh sample.     

Bone marrow transplantation for feline mucopolysaccharidosis I

Severe mucopolysaccharidosis type I (MPS I) is a fatal neuropathic lysosomal storage disorder with significant skeletal involvement. Treatment involves bone marrow transplantation (BMT), and although effective, is suboptimal, due to treatment sequelae and residual disease. Improved approaches will need to be tested in animal models and compared to BMT. Herein we report on bone marrow transplantation to treat feline mucopolysaccharidosis I (MPS I). Five MPS I stably engrafted kittens, transplanted with unfractionated bone marrow (6.3x10(7)-1.1x10(9) nucleated bone marrow cells per kilogram) were monitored for 13-37 months post-engraftment. The tissue total glycosaminoglycan (GAG) content was reduced to normal levels in liver, spleen, kidney, heart muscle, lung, and thyroid. Aorta GAG content was between normal and affected levels. Treated cats had a significant decrease in the brain GAG levels relative to untreated MPS I cats and a paradoxical decrease relative to normal cats. The alpha-l-iduronidase (IDUA) activity in the livers and spleens of transplanted MPS I cats approached heterozygote levels. In kidney cortex, aorta, heart muscle, and cerebrum, there were decreases in GAG without significant increases in detectable IDUA activity. Treated animals had improved mobility and decreased radiographic signs of disease. However, significant pathology remained, especially in the cervical spine. Corneal clouding appeared improved in some animals. Immunohistochemical and biochemical analysis documented decreased central nervous system ganglioside storage. This large animal MPS I study will serve as a benchmark of future therapies designed to improve on BMT.     

Ambiguous phenotypes and genotypes in 16 children with acute leukemia as characterized by multiparameter analysis

Ambiguous phenotypes and genotypes were observed in 16 children with acute leukemia. Surface marker, cytogenetic, molecular genetic, and DNA flow cytometric analyses as well as standard morphologic and cytochemical studies were used to divide the patients into three groups. The first group comprised five children with acute leukemia whose blast cells were morphologically lymphoid, while immunophenotyping disclosed simultaneous expression of early pre-B cell and myeloid features. Molecular genetic studies showed evidence of heavy-chain immunoglobulin (Ig) gene rearrangements in all patients. Cytogenetic data, available in three of these children, revealed t(4;11). In five of the 16 patients, morphologic and surface marker analyses indicated the coexistence of two separate cell populations, one with myeloid and the other with early pre-B cell features. Further evidence of B cell commitment in these patients was provided by demonstration of Ig heavy-chain gene rearrangements in all five patients. Surprisingly, one of the five patients showed oligoclonal Ig heavy-chain as well as monoclonal gene rearrangement for the beta chain of the T cell receptor (beta-TCR). The last group consisted of four cases with otherwise typical acute lymphoblastic leukemia (ALL), early pre-B cell phenotype, and coexpression of myeloid or T cell-associated antigens, and two children with unequivocal acute myeloid leukemia (AML) and coexpression of T cell antigens. Gene rearrangement of Ig heavy-chain could be demonstrated in five of six patients, additional Ig light-chain gene rearrangement in two children with ALL, and bigenotypic features (Ig heavy-chain and beta-TCR gene rearrangement) in one patient. In none of the 16 patients did flow cytometry disclose clonal abnormalities of leukemic cell DNA content. Based on these findings, we suggest that malignant transformation in the first and second group of patients took place at a stage ontogenetically close to the pluripotent stem cell, whereas ambiguous phenotypes in the third group resulted from aberrant gene expression or insufficient reagent specificity.     

Impaired nicotinamide adenine dinucleotide synthesis in pyruvate kinase- deficient human erythrocytes: a mechanism for decreased total NAD content and a possible secondary cause of hemolysis

Erythrocytes from individuals with pyruvate kinase (PK) deficiency have approximately half the total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD) of normal erythrocytes. In order to elucidate the mechanism(s) for the decrease in total NAD, we examined NAD synthesis in intact erythrocytes. It is demonstrated that NAD synthesis is impaired in PK-deficient erythrocytes to a degree that is dependent on the PK activity and adenosine 5'-triphosphate (ATP) concentration of these cells. After incubation in the presence of fluoride, which simulates the characteristics of PK deficiency by inhibiting enolase, normal erythrocytes had impaired NAD synthesis and decreased ATP concentrations. Fluoride did not inhibit NAD synthesis in a hemolysate system that is not dependent on glycolysis for ATP generation. These data suggest that fluoride does not inhibit the enzymes of NAD synthesis and that impairment of NAD synthesis by fluoride is mediated by decreased ATP formation. Thus, it is concluded that impaired NAD synthesis in PK-deficient erythrocytes is caused by decreased ATP formation due to the PK deficiency. Since the rate of glycolysis is limited by the availability of NAD+, it is suggested that impaired NAD synthesis causes further ATP depletion and thereby may enhance hemolysis in PK-deficient erythrocytes.     

Analysis of p53 mutations in a large series of lymphoid hematologic malignancies of childhood

p53 mutations are found in a wide variety of cancers, including hematologic malignancies. These alterations apparently contribute to development of the malignant phenotype. We analyzed a large series of lymphoid (330 cases) and a smaller series of myeloid (29 cases) malignancies of childhood for p53 mutations by single-strand conformational polymorphism (SSCP) following polymerase chain reaction. Samples with abnormal SSCP were reamplified and analyzed by direct sequencing method. p53 mutations were detected within the known mutational hotspots (exons 5 to 8) in 8 of 330 lymphoid malignancies, and in none of 29 myeloid malignancies, showing that the frequency of p53 mutations in childhood lymphoid malignancies was very low (8 of 330 cases [2%]). Four of these patients had very aggressive, fatal acute lymphocytic leukemia (ALL). None of 13 infants and none of 48 patients with T-lineage leukemia had detectable p53 mutations in their ALL cells. Exceptionally, p53 mutations were comparatively frequent in a small sample of B-cell non-Hodgkin's lymphomas (2 of 8 cases). Mutations were detected in samples from two patients with ALL at relapse; these were not detected in samples at initial diagnosis from the same patients, suggesting that p53 mutations may be associated with progression to a more malignant phenotype. Seven of eight alterations of p53 were missense mutations, and seven of eight samples may be heterozygous for the mutant p53, indicating that p53 protein may act in a dominant negative fashion.     

Platelet storage: changes in cytosolic Ca2+ actin polymerization and shape

Platelets gradually lose their disc shape during storage. The authors studied simultaneous changes in platelet cytosolic Ca2+ (Cai) and the polymerization state of actin as related to the shape. Platelet concentrates were stored under blood bank conditions for up to 10 days. Aliquots were removed and analyzed as follows: platelet Cai and increments in Cai induced by adenosine diphosphate (ADP) were determined by fluorescence of fura-2-loaded cells; loss of disc shape was determined by differences in light scattering intensity induced by stirring; and the ratio of globular and total actin (G/T) of platelets in plasma was determined by a modification of the DNase inhibition assay. Globular actin was found to be 86 +/- 3% of total actin in freshly drawn platelets suspended in plasma. The following changes occurred during storage: G/T in platelet concentrates increased from 63 +/- 5 (day 0) to 74 +/- 2% in the first 24 hours then fell to 33 +/- 6% by day 10. The percent discoid platelets also increased from day 0 to day 1 then fell in the ensuing days. There was an initial drop in Cai from day 0 to day 1, after which Cai increased on days 3 and 6. Globular actin polymerization during storage closely correlated with the change in percent discs (r = 0.95). During 6 days of storage Cai was highly correlated with shape change (r = 0.97) and to a lesser extent (r = 0.87) with the ratio of globular actin. The authors conclude that actin polymerization, shape, and Ca2+ change in a related fashion during storage.