[Clinical and Laboratory Characteristics of Primary Autoimmune Hemolytic Anemia Patients with Negative Results of DAT by Tube Test But Positive Results by Microcolumn Gel Assay].

OBJECTIVE: To investigate the clinical features and laboratory characteristics of primary autoimmune hemolytic anemia (AIHA) patients with negative results of direct antiglobulin test (DAT) by tube test but positive results by microcolumn gel assay, in order to provide references for the diagnosis of these patients. METHODS: 59 patients diagnosed with primary AIHA in our hospital from January 2015 to December 2020 were retrospectively analyzed. According to the results of tube test and microcolumn gel assay, the cases were divided into 3 groups, and the clinical and laboratory characteristics of each group were compared. RESULTS: The cases were grouped as follows: Group I, cases with negative results by both methods of DAT (n=5); Group II, cases with negative results by tube test but positive results by microcolumn gel assay (n=26); Group III, cases with positive results by both methods of DAT (n=28). There was no significant difference in age and sex between Group II and other groups, whereas the positive rate of anti-IgG + anti-C3d of Group II was lower than that in Group III (P=0.015). The main clinical manifestations of Group II were chest tightness, shortness of breath, fatigue, as well as yellow skin and sclera or dark urine, but the incidence rate of these symptoms was not significantly different from other groups. Anemia related indexes in Group II such as red blood cell (RBC) count and hemoglobin (Hb) were lower than the reference intervals, but there was no significant difference compared with other groups. Hemolysis related indexes in Group II such as reticulocyte (Ret) ratio, indirect bilirubin (IBIL), lactate dehydrogenase (LDH) and free-hemoglobin (F-Hb) were higher than the reference intervals, and the latter two items were signficantly higher than those in Group I (P=0.031 and P=0.036). Serum complement C3 and C4 in Group II were higher than those in Group III (P=0.010 and P=0.037). CONCLUSION: Anemia severity of primary AIHA patients who were negative of DAT by tube test but positive by microcolumn gel assay was similar to those with negative or positive results by both DAT methods, but the mechanism and degree of complement system involved in hemolysis might be different. Results above may be helpful for laboratory diagnosis of this kind of patients.

[Clinical Features and Laboratory Data Analysis of Glucose-6- Phosphate Dehydrogenase Deficiency].

OBJECTIVE: To explore clinical features and laboratory data of glucose-6-phosphate dehydrogenase（G6PD）deficiency and to investigate the relationship between them. METHODS: Clinical data of 43 patients with G6PD deficiency was analyzed, the statistical method was applied to investigate the relationship between clinical features and laboratory data. RESULTS: Among 43 patients，neonatal jaundice occurred as the first symptom in 10 cases，while acute hemolytic anemia occurred as the first symptom in 23 cases. The major clinical symptoms of G6PD deficiency included icteric skin and/or sclera，dark urine，fever，gastrointestinal symptoms，fatigue and lethargy. Symptoms of 26 patients were caused by obvious inducement，including fava beans（61.5%），infection（34.6%）and miocardial infarction（3.8%）. All of 43 patients showed decreased G6PD activity，while the level of their indirect serum bilirubin（IBIL）was positively correlated with reticulocyte percentage（Ret%，r=0.5881，P=0.013） and mean corpuscular volume（MCV，r=0.6854，P=0.0024）. Patients with neonatal jaundice as the first symptom，showed higher level of Ret%（P<0.01）and MCV（P<0.001）and low RBC count（P<0.01）and low Hb level（P<0.01）. as compard with patients with acute hemolytic anemia as first symptome. CONCLUSION: Neonatal jaundice and acute hemolytic anemia are common clinical features of G6PD deficiency. Laboratory results of IBIL，Ret% and MCV have auxiliary value to evaluate the severity of hemolysis induced by G6PD deficiency. Patients with neonatal jaundice as their first symptom show more severe hemolysis than those only suffered from acute hemolytic anemia.

[Application of dual-color fluorescence in situ hybridization in acute myeloid leukemia with t (8; 21)].

OBJECTIVE: To investigate the utilities of dual-color fluorescence in situ hybridization (FISH) in diagnosis and monitor of treatment in acute myeloid leukemia (AML) with t (8; 21). METHODS: Seventy patients having FISH results were divided into two groups: untreated and treated group. Comparative analysis was performed between the results of conventional cytogenetic analysis (CCA) and FISH analysis, and in some of them, between FISH and reverse transcriptase polymerase chain reaction (RT-PCR) results. A successive FISH following R-banding was carried out in those with cytogenetic undetermined cases. RESULTS: In untreated group, 30/42 cases of t (8; 21) AML were positive for AML1/ETO in FISH assay. Three cases were positive for AML/ETO by FISH although two of them lacked t (8; 21) by CCA and one negative for AML1/ETO by RT-PCR. Six cases with complex karyotype abnormalities were confirmed to be AML1/ETO positive by the successive R-banding and FISH assay, and the involved genes were clearly visualized in FISH image. In the treated group, there were 28 cases of t (8; 21) AML diagnosed. Three cases without t (8; 21) by CCA were positive by FISH. Two patients were detected relapse earlier by FISH. CONCLUSION: The dual-color FISH technique is a much more sensitive and accurate approach to the diagnosis of t (8; 21) AML and minimal residual disease (MRD) monitoring. It can also provide precise mapping of fusion signals in complex karyotype.

[The application of fluorescence in situ hybridization to diagnosis of acute promyelocytic leukemia].

OBJECTIVES: To explore whether PML/RAR alpha fusion gene presented in patients with typical clinical characteristics of acute promyelocytic leukemia (APL) but normal karyotype or atypical translocation of chromosomes 15 and 17 by conventional cytogenetic analysis (CCA), and to assess the application of fluorescence in situ hybridization (FISH) to diagnosis of APL. METHODS: 193 newly diagnosed APL patients received CCA in our hospital, 32 cases of whom were carried out FISH analysis, and some of the patients received reverse transcriptase polymerase chain reaction (RT-PCR) detection. RESULTS: 132 of 193 (68.4%) cases were identified to have t(15;17) (q22;q12) by CCA. The selected 32 patients were divided into three groups according to CAA results: group 1 included 14 cases with typical t(15;17), group 2 included 13 cases without t(15;17), and group 3 included five cases with complex karyotype involving chromosomes 15 and 17. As expected, all cases in group 1 were detected PML/RAR alpha fusion by FISH. In group 2, all patients presented the same molecular abnormality by FISH in spite of absence of t(15;17), and in group 3, FISH not only detected PML/RAR alpha fusion but also identified the fusion signals located on chromosomes, other than chromosome 15q. CONCLUSION: All the APL with typical clinical characteristics can be detected PML/RAR alpha fusion by FISH or RT-PCR regardless of classical t(15;17). FISH is more sensitive for molecularly diagnosis of APL, and can identify the precise location of the fusion signals in complex karyotype. It is necessary in clinically APL patients with no or atypical chromosomal abnormalities to perform FISH analysis.

[Analysis and identification of variant Ph chromosome translocation in patients with chronic myelogenous leukemia by conventional cytogenetics and fluorescence in situ hybridization].

The objective of this study was to explore the cytogenetic profiles of variant Ph chromosome translocations (VT) in patients with chronic myelogenous leukemia (CML) and to assess the applications of fluorescence in situ hybridization (FISH) technique for analysis of CML patients with variant translocation by using dual color-single fusion signal (DC-SF) and dual color-dual fusion signal (DC-DF) probe. 42 CML patients with VT were studied by conventional cytogenetic analysis (CCA). Among them, nine and eleven cases were analyzed by DC-SF-FISH and DC-DF-FISH, respectively. The results showed that 42 out of 643 (6.5%) CML cases received CCA were found to have VT, which were composed of 18 cases of simple VT, 23 of complex VT and one of masked VT. The VT involved all over the chromosomes but No. 4 and 6. Four patterns of them appeared recurrent because each occurred in at least two cases. VT with additional chromosomal aberrations were shown in 35.7% of patients with VT (15/42). 19 of 20 patients who received FISH detection were positive for bcr/abl fusion. DC-DF-FISH analysis revealed absence of abl/bcr fusion signal in all patients but one (8.8%) with abl/bcr positive cells. However, it was not an implication of gene loss but the translocation led to part of bcr retaining on der (9q34) and other part of bcr translocating to involve another chromosome. It was unable to observe variant signal features by DC-SF-FISH analysis. In conclusion, variant Ph translocations in CML involved almost all chromosomes in a varying frequencies and ways except chromosomes 9 and 22, and some of them showed recurrent aberrations. FISH provides accurate molecular diagnosis for CML with VT, while DC-DF-FISH facilitates the assessment of variant signals.

[Clinical and cytogenetic features of 29 cases of myelodysplastic syndrome associated with del(20q)].

OBJECTIVE: To analyze the clinical and cytogenetic features of myelodysplastic syndrome(MDS) associated with del(20q). METHODS: The cytogenetic profiles, clinical manifestations, laboratory data, and transformation in course of disease were analyzed. RESULTS: (1) Of 29 MDS patients with del(20q), eleven (37.9%) had normal karyotype in addition to del(20q) aberration. Among them, nine patients were categorized into refractory anemia(RA)/RA with ringed sideroblasts(RAS) group and two into RA with excess Hasts(RAEB)/RAEB in transformation(RAEB-T) group. The breakpoint in 20q11 was commonly seen in patients with RA/RAS(63.2%), while del(20q12) was predominant in patients with RAEB/RAEB-T(accounting for 70% in all RAEB/RAEB-T patients). It was observed that RAEB/RAEB-T patients had higher frequencies of extra chromosomal aberrations(50%) and complex karyotype(30%) than did the RA/RAS patients (26.3%, 5.3% respectively); (2) Almost all patients revealed prominent pancytopenia, dyserythropoiesis and dysgranulopoiesis and 58.6% patients showed dysmegakaryopoiesis; positive periodic acid schiff staining of nucleated erythrocytes or reduction of neutrophils were found in 62.5% of patients; 81.8% of patients expressed lymphoid antigens; (3) Two cases transformed to acute myeloid leukemia. CONCLUSION: Del(20q) may be an early and primary cytogenetic event in the development of hematologic malignancies. Pancytopenia and dysplasia of bone marrow cells are prominent in patients with MDS associated with del(20q); lymphoid antigen expression is a common occurrence; more additional chromosomal abnormalities and complex karyotypes appear when the disease becomes worse.

[Study of the childhood acute lymphoblastic leukemia with t(12;21)].

OBJECTIVE: To explore the incidence, clinical characteristics and prognosis of childhood acute lymphoblastic leukemia (ALL) with t(12;21). METHODS: t(12;21)/TEL-AML1 fusion gene was examined in bone marrow or peripheral blood mononuclear cells from 51 newly diagnosed childhood ALL patients by conventional cytogenetic R-banding analysis (CCA), dual colour interphase fluorescence in situ hybridization (I-FISH), and nested reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: t(12; 21)/TEL-AML1 fusion gene was found in 11 cases by FISH or PCR, accounting for 21.6% and 26.9% in childhood ALLs and in non-T lineage ALL cases, respectively. The median age at diagnosis was 6.8 (2.9 to 12) years. All of the t(12;21) patients expressed non-T lineage immunophenotype, and most of them were common-ALL. High myeloid antigen coexpression was not found. In 11 CCA cases, normal karyotype was found in 7, and a dubious t(12;21) in one. TEL allele deletion was found in 8 (72.7%) of t(12;21) positive cases by FISH. By comparison, no statistic difference was found in sex, anemia, hemorrhage, organ enlargement, and initial WBC count between the positive and negative non-T lineage ALLs, but the platelet count and the frequency of IgH gene rearrangement were much lower in positive cases (P = 0.008 and 0.007, respectively). Moreover, no difference was found in overall CR rate, CR rate within 4 weeks, CR duration and relapse rate between the two groups. CONCLUSION: t(12;21) was the most common chromosomal translocation in childhood ALL, but not all of them could be detected by CCA. t(12;21) cases showed non-T cell immunotypes, most of them were CD(10)(+) ALL. TEL allele deletion was common in these cases. There was no significant difference in clinical characteristics and short term outcome between the t(12;21) and the TEL-AML1 negative cases. In our data, Chinese t(12;21) ALL showed older in age, lower BPC, lower IgH rearrangement frequency and more of normal karyotype as compared with the reports abroad.

[Molecular cytogenetic analysis of -7/7q- abnormalities in patients with myeloid malignancies].

OBJECTIVE: To accurately evaluate the incidence of -7/7q- abnormality in acute myeloblastic leukemia (AML) and myelodysplastic syndrome (MDS) patients and investigate the value of fluorescence in situ hybridization (FISH) technique in the detection and identification of -7 and 7q abnormality. METHODS: A FISH assay was performed to analyze 70 AML/MDS patients who had received conventional cytogenetic analysis (CCA). The dual color probes CEP 7 labeled by SpectrumGreen and D7S486 (locus at 7q31) labeled by SpectrumOrange were used. RESULTS: The incidence of -7/7q- in AML and MDS patients was 4.51% (31 out of 687 cases) and 5.71% (28 out of 490 cases), respectively, and was 5.68% and 10.29% in these patients with abnormal karyotype, respectively. The common deletion region of 7q- was 7q21a222 (ten cases) and 7q31-35(ten cases). FISH assay confirmed the -7/7q- aberration in those with clonal -7/7q- abnormalities, but failed in those with random -7/7q- and normal karyotype. In 7q- group, FISH revealed seven of eleven cases with monosomy 7 clone detected in the same specimen, but the numbers of 7q- interphases cells were much greater than those of monosomy 7 cells (average 42.5% vs 8.4%, P=0.025). FISH also provided precise refinement for three chromosomal structural abnormalities associated with 7q seen in CAA, one case with del(7)(q22) being refined as chromosomal translocation, one case with 7q+ being confirmed as dup(7q), and one case with complex translocation involving 7q being also proved to be true. CONCLUSION: FISH is a powerful tool to identify or refine chromosomal structural aberrations involving 7q, and it provides accurate evaluation of -7/7q- in all the patients. -7 and 7q- clone frequently coexist in the same specimen, and the significantly increasing percentage of 7q- cells implies that -7 clone secondary to 7q- clone is a result from loss of 7q-.

[Comparison of detection of trisomy 8 with fluorescence in situ hybridization and conventional karyotype analysis in myelodysplastic syndrome].

The purpose of this study was to compare the detection of trisomy 8 in myelodysplastic syndrome (MDS) patients with interphase fluorescence in situ hybridization (FISH) and cytogenetic karyotype analysis. Using Spectrum Green labeled chromosome 8 centromere probe, interphase FISH was established. The trisomy 8 clones were simultaneously detected in 48 MDS cases with FISH and conventional cytogenetic analysis (CCA). Results showed that the CCA revealed no significant difference of constitutional proportion between MDS-RA and MDS-RAEB with karyotypes of whole +8, partial +8 and one +8. With FISH, detectable rates were 66.1% for whole +8. Partial +8 and sole +8 were significantly higher than one +8 and complex +8, respectively. The percentages of trisomy 8 were similar in MDS-RA and MDS-RAEB. Trisomy 8 was detected in 1 of 15 specimens with normal or abnormal karyotype without trisomy 8 by FISH. There was linear correlation between the percentages of partial +8 detected by FISH and CCA. Two patients received CCA and FISH examination at diagnosis and during treatment, the percentage of trisomy 8 was increased with progress of disease. In conclusion, our results showed that FISH is a sensitive and accurate technique to detect trisomy 8 in MDS patients. It can provide contribute to diagnosis, assessment of curative effect and predicting progress of disease in MDS. Clone size of trisomy 8 does not related to classification of MDS, but sole +8 is seems to see in MDS-RA frequently.