Expression of G-CSF and GM-CSF receptors on CD34 positive cells in aplastic anemia and myelodysplastic syndrome patients and its significance / 中国实验血液学杂志

This study was aimed to detect the ratio of CD34+ cells in bone marrow mononuclear cells (BMMNCs) and the expression rate of G(M)-CSFR on CD34+ cells in bone marrow of the patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). The ratio of CD34+ cells in BMMNCs and the expression rate of G(M)-CSFR on cells of 27 AA patients, 45 MDS patients and 20 controls were detected by flow cytometry (FCM). The results showed that the ratio of CD34+ cells in BMMNCs of AA patients reduced and was significantly different from controls (p<0.05), the ratio of CD34+ cells in MDS patients elevated and was significantly different from controls (p<0.05). Compared with controls and MDS-RA patients, the ratio of CD34+ cells in MDS-RAEB patients significantly elevated (p<0.05), but there was no significant difference between MDS-RA patients and controls (p>0.05). The ratio of CD34+ cells in MDS-RA patients was significantly higher than that in AA patients (p<0.05). There was no significant difference in expression rate of G-CSFR on CD34+ cells between AA patients and controls, MDS patients and controls, AA patients and MDS patients, MDS-RA patients and MDS-RAEB patients (p>0.05). The expression rate of GM-CSFR in MDS patients was significantly higher than that in AA patients and controls (p<0.05), but there was no significant difference between AA patients and controls, MDS-RA patients and MDS-RAEB patients (p>0.05). In AA patients, the ratio of CD34+ cells in BMMNCs was less than 0.1% accounts for 6/8 SAA patients, compared with 2/19 in CAA (p<0.05). There was no correlation between the expression rate of either G-CSFR or GM-CSFR and neutrophil count at diagnosis (r=0.058 and r=0.044). In MDS patients, there was no correlation between bone marrow CD34+ cells ratio and peripheral neutrophil count at diagnosis (r=-0.335). And there was no correlation between the expression of either G-CSFR or GM-CSFR and neutrophil count on diagnosis (r=0.064 and r=0.051). It is concluded the detection of CD34+ cells and their surface expression rate of G(M)-CSFR in AA and MDS is useful in diagnosis and differential diagnosis of these two diseases.

Application of denaturing high performance liquid chromatography to mutation detection of the c-kit gene in mastocytosis / 中国实验血液学杂志

The aim of study was to detect mutation of tyrosine domain of c-kit gene in mastocytosis using denaturing high performance liquid chromatography technique, and to investigate the significance of this gene mutation in diagnosis and therapy of mastocytosis. Genomic DNA was obtained from bone marrow or peripheral blood leukocytes using the phenol/chloroform method from 7 mastocytosis patients. PCR was performed with AmpliTaq Gold DNA polymerase and 100 ng genomic DNA to amplify the entire coding sequence and exon-intron boundaries of c-kit exon 17 approximately exon 19. Denaturing high-performance liquid chromatography (DHPLC) analysis was performed on a WAVE DNA Fragment Analysis System. Each PCR product was mixed with an equal quantity of amplified human placental DNA (served as normal control) and was denatured at 95 degrees C for 5 minutes, followed by slowly cooling down to room temperature by 1.5 degrees C per minute to allow heteroduplexes formation. All the conditions for the DHPLC analysis, including melting temperature and buffer gradients were determined using the Transgenomic software Navigator. Samples with extra peaks or with different peak form on DHPLC were directly sequenced using the BigDye Terminator Cycle Sequencing Reaction kit and ABI 3100 Genetics Analyser. The results showed that DHPLC revealed an aberrant peak in one patient in exon 17 and the D816V mutation was identified by direct sequencing. The other two patients had an extra peak for exon 18/19 and direct sequencing revealed a conservative sequence change (L862L) within exon 18. It is concluded that denaturing high performance liquid chromatography is a high efficiency and reliable technique for mutation detection of c-kit gene and the detection results would be helpful for the selection of therapy in mastocytosis.