Suntaree Apibal

Effect of genetic alterations of cytarabine-metabolizing enzymes in childhood acute lymphoblastic leukemia

BACKGROUND Single nucleotide polymorphisms (SNPs) of deoxycytidine kinase (dCK) and cytidine deaminase (CDA) are known to alter their enzymatic activities, which affect the metabolism of cytarabine. Currently, treatment of childhood acute lymphoblastic leukemia (ALL) includes cytarabine, especially in high-risk patients. Therefore, we hypothesized that a genetic variation of dCK and CDA genes may influence the risk of cytarabine-related toxicities and early response to treatment. PATIENTS AND METHODS We included children diagnosed with ALL and lymphoblastic lymphoma (LL) stage III and IV. The patients received a modified St Jude Total Therapy Study XV protocol. Cytarabine was used during induction remission (low-dose cytarabine) and reinduction II (high-dose cytarabine) phases. Genotyping of dCK-360C>G and -201C>t and CDA 79A>C and 208G>A was performed. Minimal residual disease (MRD) at the end of the induction phase was measured using flow cytometry. RESULTS Ninety-four children with ALL (n=90) and LL (n=4) were analyzed. The median age at diagnosis was 5.8 years (range, 0.4-15 years). All four SNPs showed predominant wild type alleles. There was no CDA-208A allele in our population. Children with dCK-360G allele were at risk of mucositis after receiving low-dose cytarabine (OR=3.7; 95%CI, 1.2--11.3). neither dCK nor CDA polymorphisms affected the MRD status at the end of the induction phase. CONCLUSION The dCK-360G allele was found to increase the risk of mucositis after exposure to low-dose cytarabine in childhood ALL therapy.

Association Between Polymorphisms of Dihydrofolate Reductase and Gamma Glutamyl Hydrolase Genes and Toxicity of High Dose Methotrexate in Children with Acute Lymphoblastic Leukemia

Methotrexate (MTX) is an important drug for the treatment of childhood acute lymphoblastic leukemia (ALL). However, related toxicity occurs in many organs which may cause interruption of treatment, morbidity, and mortality. Single nucleotide polymorphisms (SNPs) of dihydrofolate reductase (DHFR) and gamma glutamyl hydrolase (GGH) are known to alter their enzymatic activity and thus affect the metabolism of MTX and influence the effectiveness. Therefore, we hypothesized that genetic variations of DHFR and GGH genes may influence the risk of toxicity after high dose MTX. The study population comprised of 105 children with ALL who were treated according to the modified St Jude Total XV protocol. The patients received 2.5 or 5 g/m2 of MTX for 5 doses during the consolidation phase. Genotyping of DHFR 829C>T and GGH -401C>T was performed using a polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). The GGH-401CT and TT genotypes were associated with increased risk of leukopenia and thrombocytopenia after high dose MTX (OR 2.97, 95%CI; 1.24-7.13 and OR 4.02, 95%CI; 1.58-10.26). DHFR 829C>T was not associated with toxicity. In conclusion, the GGH -401CT and TT genotypes were found to increase the risk of severe leukopenia and thrombocytopenia after exposure to high dose MTX for childhood ALL therapy.

Hematology, cytochemistry and ultrastructure of blood cells in fishing cat (Felis viverrina)

Hematological, cytochemical and ultrastructural features of blood cells in fishing cat (Felis viverrina) were evaluated using complete blood cell counts with routine and cytochemical blood stains, and scanning and transmission electron microscopy. No statistically significant difference was found in different genders of this animal. Unique features of blood cells in this animal were identified in hematological, cytochemical and ultrastructural studies. This study contributes to broaden hematological resources in wildlife animals and provides a guideline for identification of blood cells in the fishing cat.

Acute monoblastic leukemia in a FeLV-positive cat

A 1.6-year-old male domestic short hair cat was brought to the Veterinary Medical Teaching Hospital, Kasetsart University, with signs of severe anemia, depression, and general lymph node enlargement. Complete blood count revealed leukocytosis and massive undifferentiated blasts. Testing for antibodies specific to feline leukemia virus (FeLV) was positive, and FeLV nucleic acid was confirmed by nested polymerase chain reaction. Base on cytochemistry and ultrastructure, the cat was diagnosed with acute monoblastic leukemia.

Fetal red cell in thai thalassemia trait patients

Suntaree Apibal, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Bangkok 10400 (Thailand) The presence of fetal red cells (F cells) in the maternal circulation is an indicator of a fetomaternal hemorrhage which may subsequently lead to hemo-lytic disease of the newborn [1]. However, a small amount of F cells were found in some normal adults [4] whereas the increase in hemoglobin F production might be increased in various conditions as thalassemia trait, unstable B chain variant and pregnancy [4]. In Thailand, thalassemia hemoglobin E-trait, ß-trait and α-trait were commonly found [2, 3]. The incidence of E-trait is 13% on average in the central region, 40% in the northeast, 8% in the north and 12% in the south [2]. The incidence of ß-trait is 3–9% and of α-trait is 20–30% [3]. So, the study for the percentage of F cells among these groups of subjects may be able to differentiate the false positive results of fetomaternal hemorrhage from F cells found in some normal persons and subjects with the thalassemia trait. F cells could be demonstrated in the maternal circulation by acid elution techniques. Our study revealed the presence of F cells in each group by the Saquan-sermsri acid elution technique as follows. The air-dried blood smear of not over 24 h was fixed in 80% ethanol for 3 min, then immersed in acid/alcohol/ami-do black solution for 3 min at room temperature (100 mg amido black B CI No. 20470, Merck’s Reagenzien, FRG, in 100 ml of 80% ethanol, pH adjusted to 2 with HC1). The smear was washed with tap water for 15 s and air dried. The stained smear was examined under an oil immersion microscope. The percentage of F cells (X ± SD) in 30 normal persons, 30 with E-trait, 30 with ß-trait and 30 with α-trait were 0.14 ± 0.18, 0.92 ± 0.78, 2.3 ± 1.87, and 0.5 ± 0.45, respectively. So, more investigations to exclude these conditions were needed before diagnosing fetomaternal hemorrhage. Thus, the diagnosis of fetomaternal hemorrhage is only valid in the presence of F cells in the maternal circulation if there are no such hemoglobinopathies in the mother. References Fanaroff AA, Martin RJ, Merleatz IR: Behrman’s Neonatal Perinatal Medicine, Disease of the Fetus and Infant, ed 3. St. Louis, Mosby, 1983. Na-Nakorn S, Minnich V, Chernoff A: Studies on hemoglobin E. II. The incidence of hemoglobin E in Thailand. J Lab Clin Med 1965;47:490. Wasi P, Na-Nakorn S, Pootrakul S, Sookanek M, Disthasong-chan P, Pornpatkul M, Panich V: Alpha and beta thalassemia in Thailand. Ann NY Acad Sci 1969;165:60.

Automated Cytochemistry in Non-Hodgkin’s Lymphoma: A New Method for Determination of Cells from Lymph Node Biopsy

Cell suspension prepared from the lymph node biopsy of patients with non-Hodgkins lymphoma (NHL), metastatic carcinoma (MC) and non-neoplastic lymphadenopathies (NL) were analyzed by the Hemalog D, automated differential counter. The preparation of lymph node cells is described first in this study. The results show that the percentage of large cells (diameter greater than 13.5 micron) stained negatively with peroxidase (LUC, large unstained cells) was remarkably increased in patients with NHL (mean +/- SEM = 18.6 +/- 3.1%) and was particularly increased in the subgroup, diffuse histiocytic type (31.1 +/- 5.3%). Patients with MC had a raised percentage of nonspecific esterase-positive cells (9.2 +/- 1.4%) compared to patients in the NHL and NL groups. Patients in the NL group had low percentages of both LUC (5.3 +/- 0.7%) and nonspecific esterase-positive cells (1.8 +/- 0.2%). Quantitation of cells in the lymph node by using the Hemalog D may assist in the diagnosis of lymph node diseases.