ABSTRACT
Objective The effect of different doses of ethylnitrosourea(ENU)and cyclophosphamide(CP)on the loss rate of CD59 on peripheral blood erythrocytes was explored to optimize the detection method of Pig-a gene mutation. Methods According to the weight and loss rate of CD59 on peripheral blood erythrocytes,rats were divided into 4 groups:the control group,CP 40 mg/kg group,ENU 10 mg/kg group and ENU 40 mg/kg group(n=6). The control group was injected i.p. with PBS,other groups were injected i.p. with corresponding solutions. The body weight of rats on days 0,7,14,21, 28, 42 and 56 were recorded. At the same time, blood samples were collected and incubated with antibodies,and the loss rate of RBCCD59-was detected by flow cytometry. Results Compared with the control group, at different time points, the body weight and weight gain of ENU 10 mg/kg group and ENU 40 mg/kg group had no statistically significant difference(P > 0.05),while those in the CP 40 mg/kg group were significantly decreased(P <0.05). The loss rate of RBCCD59-was significantly increased in the CP 40 mg/kg group at 28,42 and 56 days, ENU 10 mg/kg group at 42 and 56 days,and ENU 40 mg/kg group at 7,14,21,28,42 and 56 days,(P < 0.05). The results showed a dose-response relationship. Conclusions Under the conditions of this Pig-a mutation detection method,ENU is superior to CP on raising loss rate of RBCCD59-,ENU 40 mg/kg is better than 10 mg/kg,and 28 days is suitable as the test period.
ABSTRACT
Objective To optimize the detection test of Pig-a gene mutation in peripheral blood of rats by enriching and detecting mutant erythrocytes, using immunomagnetic separation technique in combination with flow cytometry. Methods SD rats were administered with 20,40 and 80 mg/(kg·bw)doses of N-ethyl-N-nitrosourea(ENU) continually for 3 days. The peripheral blood samples of rats were collected on the 7th,14th and 28th days, respectively, after treatment. Immunomagnetic separation columns were used to enrich RETCD59- and RBCCD59- cells, and then flow cytometry was used to count the number of pre-column and post-column peripheral erythrocytes. Results Compared with the control group,the frequencies of RETCD59- and RBCCD59- were significantly increased in each ENU group(P<0.05). With immunomagnetic separation technique, the test of Pig-a gene mutation of a sample could be completed within 3 minutes,and the number of detected RETCD59- or RBCCD59-cells was up to 2×104or 9×104, respectively. Conclusions In this study,immunomagnetic separation in combination with flow cytometry is used to establish and optimize the Pig-a gene mutation test in rat peripheral blood,showing a high-throughput detection and improved accuracy and efficiency of the experiment.
ABSTRACT
BACKGROUND: Aplastic anemia(AA), myelodysplastic syndrome(MDS) and paroxysmal nocturnal hemoglobinuria(PNH) are hematopoietic stem cell disorders. To investigate the pathogenetic links, we performed CD59 analysis and screened PIG-A gene mutation in the patients with AA, MDS, and PNH developed from AA or MDS. METHODS: We analyzed the proportion of the patients with CD59-deficient cells by flow cytometry for CD59 in 42 patients with AA or MDS and eight patients with PNH developed from AA or MDS. The mutations of PIG-A gene were screened with dideoxy fingerprinting(ddF). RESULTS: In normal controls, the proportion of the RBCs normally expressing CD59 was 97.2+/-1.9% and that of the granulocytes was 98.4%+/-1.5%. In patients with AA or MDS, 9.5%(4/42) had CD59 deficiency on RBCs and 10.3%(3/29) on granulocytes. In patients whose CD59 on both RBCs and granulocytes were analyzed, 17.2%(5/29) showed reduced CD59 in at least one cell lineage. Screening test using ddF revealed abnormal band shifts in three patients with PNH developed from AA or MDS. CONCLUSION: We found the presence of PNH clones in the patients with AA or MDS. And it was indirectly confirmed by ddF that PNH arisen from AA or MDS is also associated with the mutations of PIG-A gene as classical PNH. CD59 analysis in AA or MDS will be helpful for the early diagnosis of PNH.
Subject(s)
Humans , Anemia, Aplastic , Cell Lineage , Clone Cells , Early Diagnosis , Flow Cytometry , Granulocytes , Hematopoietic Stem Cells , Hemoglobinuria, Paroxysmal , Mass Screening , Myelodysplastic SyndromesABSTRACT
BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by deficient biosynthesis of the glycosylphosphatidylinositol (GPI) anchor in hemopoietic stem cells. Mutation of phosphatidyl inositol glycan class A (PIG-A) gene, an X-linked gene that participates in the first step of GPI anchor biosynthesis, is responsible for PNH. Characteristics of somatic mutation of PIG-A gene in the Korean patients with PNH and their relationships to clinical characteristics were analyzed. METHODS: Twenty five patients with PNH and a donor of bone marrow transplantation were selected. Ham tests, sucrose hemolysis tests and CD59 expressions of erythrocytes and granulocytes were performed to confirm diagnosis. Dideoxy fingerprinting (ddF) was used to screen mutations, and direct sequencing of DNA was performed to characterize the mutations. RESULTS: The mutations of PIG-A gene were found in twelve cases and ten of them were novel mutations. There were five deletions, six substitutions and a insertion. Therewere six premature terminations, three abnormal splicings, a missense and two nonsense mutations. There were six point mutations and six frameshift mutations. Five cases of hypoplastic PNH showed mutations only in exons, but three in seven cases of hemolytic PNH showed mutations in introns. Two cases with symptoms of venous thrombosis showed mutations in exon 3. CONCLUSION: There were ten novel mutations among twelve mutations in the Korean patients with PNH and characteristics of the mutations were variable without any remarkable hot spot in sites and types. The characteristics of mutation were not correlated with the results of clinical courses of the patients with PNH.