International Committee for Standardization in Haematology: Recommended Methods for Red‐Cell Enzyme Analysis*

In this study, possible adverse effects of pyruvate kinase (PK) deficiency on the maturation of erythroid progenitors were investigated. A 4-year-old Japanese girl with severe PK deficiency underwent splenectomy to reduce her need for blood transfusions. The spleen was examined a histologically, and the hematopoietic progenitors in the spleen were assayed to evaluate the extramedullary hematopoiesis of this PK-deficient subject. The number of hematopoietic progenitors including CFU-GM, BFU-E and CFU-GEMM in the spleen of the PK-deficient patient was much higher than those found in control spleens, indicating enhanced extramedullary hematopoiesis. TUNEL assay demonstrated apoptotic cells in the splenic red pulp of the PK-deficient patient. The expression of 7A6 antigen was detected in cells isolated from spleen and in cells cultured in vitro, but only in those cells that were positive for glycophorin A. These results provide evidence that the metabolic disturbances in PK deficiency affect not only the survival of red cells but also the maturation of erythroid progenitors, which results in premature cell death, i.e., apoptosis. Am. J. Hematol. 74:68-72, 2003.

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“…Red cell enzymes were measured by protocols standardized previously by the International Committee for Standardization in Haematology [7].…”

Section: Materials and Methods Red Cell Enzyme Assaymentioning

confidence: 99%

Ineffective erythropoiesis in the spleen of a patient with pyruvate kinase deficiency

et al. 2003

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“…DPG-which affects erythrocyte capacity to release oxygen in tissue and generally increases when HB decreases-and ATP in erythrocytes were measured spectrophotometrically. 18,19 Determination of Osmotic Resistance. One hundred milliliters of packed erythrocytes were carefully added to 13 tubes containing 1 mL each of 0.85%-0% saline in distilled water.…”

Section: Methodsmentioning

confidence: 99%

Low membrane protein sulfhydrils but not G6PD deficiency predict ribavirin-induced hemolysis in hepatitis C

et al. 2004

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Hemolysis is a frequent adverse effect of ribavirin (RBV). It has been suggested that oxidative stress plays a role, but mechanisms and predictive risk factors for severe forms remain unknown. Markers of redox status were determined in erythrocytes of 34 patients with hepatitis C-four of them with glucose-6-phosphate-dehydrogenase (G6PD) deficiencybefore and during treatment with RBV and interferon (IFN) and were compared with 10 healthy control subjects. In addition, erythrocytes were incubated with RBV, and the effects of dipyridamole (DPD), diethylmaleate (DEM), and glutathione ester (GSHE) were studied in vitro. Of the 30 patients without G6PD deficiency who were treated with RBV and IFN-␣, five developed major hemolysis (⌬ hemoglobin > 6 g/dL) and 25 developed minor hemolysis (⌬ hemoglobin < 2.5 g/dL). Patients with major hemolysis had lower median pretreatment values of membrane protein sulfhydrils than patients with minor hemolysis (28.4 vs. 36.7 nmol/mg, P < .001). Erythrocytes of G6PD-deficient patients were not more susceptible to RBV-induced hemolysis. In in vitro incubations of erythrocytes, DEM enhanced the RBV-induced decrease of glutathione, protein sulfhydrils, and osmotic resistance. Supplementation of GSHE and DPD prevented the RBV-induced decrease in osmotic resistance, adenosyl triphosphate (ATP), and 2,3-diphosphoglycerate (DPG), the loss of glutathione and protein sulfhydrils, and the formation of thiobarbituric acid reactive substances (TBARs). In conclusion, the data indicate that low membrane protein sulfhydrils prior to therapy but not G6PD deficiency are predictive of RBV-induced major hemolysis. In vitro, GSHE and DPD reduce the RBV-associated oxidative stress in erythrocytes and prevent the increase in osmotic fragility, suggesting that these compounds might decrease the risk of hemolysis in patients. (HEPATOLOGY 2004;39:1248 -1255 R ibavirin (RBV) is a synthetic nucleoside analogue used in the treatment of chronic hepatitis C in combination with interferon (IFN)-␣. The addition of RBV has increased the probability of sustained response to approximately 40% if combined with conventional IFN 1,2 and to over 50% if combined with pegylated IFN. 3 RBV actively enters cells by way of the es-nucleoside membrane transporter 4 and subsequently undergoes phosphorylation at the expense of adenosyl triphosphate (ATP). 5,6 In contrast to nucleated cells such as hepatocytes, phosphorylation is irreversible in the anucleate erythrocytes, and phosphorylated RBV accumulates because it cannot be exported by the nucleoside carrier. Therefore, the concentration of RBV in erythrocytes is 50-to 70-fold higher than the therapeutic plasma concentration of 9 -15 M. 5,7,8 The high concentration of RBV in erythrocytes is probably responsible for the hemolysis occurring during RBV therapy. A decrease in hemoglobin (HB) is frequently observed in patients who receive RBV,

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“…(1977) and the International Committee for Standardization in Haematology (1967). Final concentrations of the assay mixture components (500 pl) were the same as those of Beutler et al (1977), pyridoxal phosphate always being added in a concentration of 0.02 mM. Using an Aminco Rotochem IIa/36, a centrifugal fast analyzer, decrease of optical density of the system at 340 nm and at 30~ was read every 1 min for 10 min after a lag time of 10 rain.…”

Section: Methodsmentioning

confidence: 99%

Electrophoretic variants of blood proteins in Japanese VII. Cytoplasmic glutamate-oxaloacetate transaminase (GOT1)

Satoh

Takahashi

et al. 1986

Jap J Human Genet

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SummaryA total of 16,835 children, of whom 11,737 are unrelated, from Hiroshima and Nagasaki were examined for erythrocyte cytoplasmic glutamate-oxaloacetate transaminase (GOT1) by starch gel electrophoresis. A variant allele named GOTI*2HR1 which seems to be identical with GOT1*2 was encountered in polymorphic frequency. Five kinds of rare variants, 3NG1, 4NG1, 5NG1, 6HR1 and 7NG1 were encountered in a total of 109 children. Except for 7NGI for which complete family study was unable, family studies confirmed the genetic nature of these rare variants, since for all instances in which both parents could be examined, one of the parents exhibited the same variant as that of their child. Thermostability profiles of these six variants were normal. The enzyme activities of five were decreased, while the value of one was normal compared to that of GOTI 1.

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International Committee for Standardization in Haematology: Recommended Methods for Red‐Cell Enzyme Analysis*

Cited by 512 publications

References 9 publications

Ineffective erythropoiesis in the spleen of a patient with pyruvate kinase deficiency

Ineffective erythropoiesis in the spleen of a patient with pyruvate kinase deficiency

Low membrane protein sulfhydrils but not G6PD deficiency predict ribavirin-induced hemolysis in hepatitis C

Electrophoretic variants of blood proteins in Japanese VII. Cytoplasmic glutamate-oxaloacetate transaminase (GOT1)

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