The characteristic of hereditary spherocytosis related gene mutation in 37 Chinese hereditary spherocytisis patients / 中华血液学杂志

Objective@#To reveal the genetic characteristics of erythrocyte membrane protein in hereditary spherocytosis (HS) in China.@*Methods@#Next-generation sequencing technology was used to detect mutations in genes of erythrocyte membrane proteins in 51 clinically diagnosed HS patients. The relationship between gene mutations and clinical phenotypes was analyzed.@*Results@#Mutations in erythrocyte membrane protein genes were detected in 37 patients, including 17 with ANK1 mutations (17/37, 45.9%), 14 with SPTB mutations (14/37, 37.8%), and 5 with SLC4A1 mutations (5/37, 13.5%). One patient carried both heterozygous ANK1 mutation and SPTB mutation (1/37, 2.7%). SPTA1 and EPB42 mutation was not fou nd in any patient. Nonsense mutations (36.8%) and missense mutations (31.6%) were most common. Of the 38 mutations detected, 34 were novel mutations and have not been reported elsewhere (89.5%). Sixteen HS patients underwent parental genetic validation, 6 patients (37.5%) inherited gene mutation from parents and 10 (62.5%) were de novo. The peripheral blood cell parameters of HS patients were not related to the mutant genes and gene mutation types. However, it seems that HS patients with mild clinical status are prone to carry SPTB mutations while more patients with severe clinical status have ANK1 mutations.@*Conclusions@#ANK1 and SPTB are the most common mutant genes in Chinese HS patients, mainly with missense mutations and nonsense mutations. There was no significant correlation between the mutation of HS related genes and the severity of HS.

Using target next-generation sequencing assay in diagnosing of 46 patients with suspected congenital anemias / 中华血液学杂志

Objective@#To evaluate the impact of the targeted next-generation sequencing (NGS) assay for difficult congenital anemias.@*Methods@#Blood Disease Hospital Anemia Panel 2014 (BDHAP-2014) including 217 known genes of congenital anemias was developed. NGS and parental verification were performed for patients who were suspected diagnosed with congenital anaemia from August 2014 to July 2017.@*Results@#A total of 46 patients were enrolled in this study, the clinical suspection were 11 cases Fanconi anemia (FA), 8 cases congenital dyserythropoietic anemia (CDA), 6 cases congenital sideroblast anemia (CSA), 12 cases congenital hemolytic anemia (CHA), 1 case dyskeratosis congenital (DC), 4 cases iron-refractory iron deficiency anemia and 4 cases unexplained cytopenia (Uc), respectively. 28 (60.9%) of 46 patients became confirmed cases after targeted NGS, corresponding to 44 mutations of which 33 were new. 26(56.5%) patients with results of the assay matching to clinical suspection, including FA (5/11, 45.5%), CSA (6/6, 100.0%), CDA (3/8, 37.5%) and CHA (12/12, 100.0%). 2 (4.3%) cases not matching to clinical suspection, including dyskeratosis congenital (DC) was made in 1(2.2%) patients with suspected FA and familial hemophagocytic lymphohistiocytosis (FHL) was made in 1(2.2%) patients with suspected unexplained cytopenia (Uc). In 12 CHA patients, the hemolytic type was further clarified by the NGS. The remaining 18 cases were not clearly diagnosed.@*Conclusion@#Targeted NGS assay is of major impact on congenital anemias. The assay should be used routinely in congenital anemias.

The clinical and laboratory characteristics of congenital pyruvate kinase deficiency / 中华内科杂志

Clinical data of 19 patients with congenital pyruvate kinase deficiency were analyzed. Insufficient pyruvate kinase confirmed the diagnosis. Laboratory parameters of hemolysis were summarized. In cases of neonatal hyperbilirubinemia and unexplained hemolytic anemia, pyruvate kinase activity and next generation sequencing test may help the early diagnosis.

Evaluation of efficacy of immunosuppressive therapy plus recombinant human thrombopoietin for children with severe aplastic anemia / 中华儿科杂志

Objective@#To evaluate the therapeutic efficacy and safety of immunosuppressive therapy (IST) combined with recombinant human thrombopoietin (rhTPO) for severe aplastic anemia (SAA) in pediatric patients.@*Method@#A retrospective case-control study was conducted and the clinical data of 45 pediatric patients with de novo SAA admitted to the Anemia Diagnosis and Treatment Center of Chinese Academy of Medical Sciences & Blood Disease Hospital during the period from December 2009 to December 2014 were analyzed. Among them, 15 patients were treated with the regimen of IST together with rhTPO and 30 patients were given IST treatment only. The variation characteristics of the peripheral blood routine as well as the transfusion of blood products was dynamically observed, and the therapeutic efficacy was assessed respectively after 3, 6 and 12 months after the treatment. In the meantime, adverse effects related to rhTPO application were recorded. Thereafter, the statistics of the two groups were compared by non-parametric rank sum test.@*Result@#Among 45 pediatric patients, there were 26 male and 19 female, and the median age was 11 years (6-14). The number of patients received good hematological response(complete remission (CR) plus good partial response (GPR)) in the combinatory group versus vs. the IST group was 6 vs. 3 patients (χ2=3.906, P=0.048) at the 3rd month, 7 vs. 7 patients (χ2=1.568, P=0.210) at the 6th month, and 13 vs. 14 patients (χ2=6.667, P=0.01) at the 12th month respectively. For those achieved good hematological response at the 3rd month, the amount of platelets transfusion and red blood cells transfusion of the combined group were both less than that of the IST group during the period from the 10th to the 12th weeks (platelets transfusion: 1.4 U vs. 2.9 U, t=-3.523, P=0.002; red blood cells transfusion: 0.8 U vs. 2.6 U, t=-2.392, P=0.026). No serious adverse effect related to rhTPO application was observed in the IST combined with rhTPO group.@*Conclusion@#Application of rhTPO can improve the short-term therapeutic efficacy of IST for pediatric SAA, alleviate transfusion dependence, and has a good safety profile.

Correlation of the degree of band 3 protein absence on erythrocyte membrane by eosin-5′-maleimide binding test and clinical phenotype in hereditary spherocytosis / 中华血液学杂志

Objective@#To investigate the relationship between the eosin-5′-maleimide (EMA) binding test and the clinical severity of hereditary spherocytosis (HS).@*Methods@#A total of 258 un-splenectomize HS patients were consecutively enrolled. Correlation of hemoglobin concentration, hemolytic parameters, compensating erythropoiesis and the EMA binding test were evaluated.@*Results@#258 (128 male and 130 female) patients were included in this study, including 91 compensatory hemolysis patients, 53 patients with mild anemia, 78 patients with moderate anemia and 36 patients with severe anemia. The median age at diagnosis was 23 (2-70) years. The median decreased fluorescence intensity of EMA binding test was 29.97% (16.09%-47.34%) and the average intensity was (29.70±6.28) % of 258 HS patients. The decreased EMA binding fluorescence intensity correlated with MCV (r=-0.343, P<0.001) and MCHC (r=0.223, P<0.001). There was no relationship between EMA fluorescence intensity and absolute reticulocyte count (r=0.080, P=0.198) , reticulocyte percentile (r=-0.015, P=0.813) , IBIL levels (r=-0.009, P=0.902) , HGB levels (r=-0.067, P=0.280). Evaluated as a quartile variable, EMA fluorescence intensity was not correlated with anemia severity (C=0.150, P=0.746).@*Conclusion@#EMA binding test does not related to anemia levels and has no major clinical implications for disease severity in HS.

Effects of pre-immunosupressive therapy iron overload on hematologic response of severe aplastic anemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of serum ferritin (SF) and iron overload (IO) pre-immunosupressive treatment (IST) on hematologic response of severe aplastic anemia (SAA/VSAA) patients treated with IST.</p><p><b>METHODS</b>257 SAA/VSAA patients who underwent first-line IST from Feb, 2003 to Dec, 2011 in Anemia Therapeutic Centre, Institute of Hematology and Blood Diseases Hospital were retrospectively analyzed, the status of SF before IST and the IO-affected factors were studied. The effects of IO on hematologic response of SAA/VSAA patients were evaluated as well.</p><p><b>RESULTS</b>The median level of SF of 257 patients was 387 (6-2 004) μg/L. 36 patients (14%) had IO, including 20 SAA and 16 VSAA patients. According to univariate logistical regression analyses, IO was influenced by age>14 years (P=0.010) and blood transfusion (P<0.001). The multivariate logistic regression analysis showed that blood transfusion [P=0.001, OR=0.218 (95% CI 0.092-0.520)] was the only independent prognostic factor. SAA (but not for VSAA) patients with IO had much lower hematologic response rate in 6 month after IST (P=0.037). Absolute reticulocyte count and IO correlated with response at 6 month by univariate logistical regression analysis (P=0.014, 0.037). The multivariate logistic regression analysis showed that IO [P=0.021, OR=4.092 (95% CI 1.235-13.563)], ARC ≥20×10(9)/L [P=0.040, OR=2.743 (95% CI 1.049-7.175)] were independent prognostic factors.</p><p><b>CONCLUSION</b>84.8% patients had high serum ferritin before IST, and 14.0% reached IO. Adult and more blood transfusion caused IO more likely. IO correlated with response at 6 month, and was independent prognostic factor.</p>

Outcomes of very severe aplastic anemia patients with different absolute neutrophil counts after frontline immnunosuppressive therapy / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze early hematopoietic response and long-term survival of very severe aplastic anemia (VSAA) patients with different absolute neutrophil counts (ANC) after frontline immnunosuppressive therapy (IST).</p><p><b>METHODS</b>Clinical data and outcome of 145 VSAA patients treated with rabbit antithymocyte globulin combined with cyclosporine were retrospectively analyzed. Hematopoietic responses to IST and long-term survival were statistically analyzed for VSAA patients in different ANC subgroups.</p><p><b>RESULTS</b>Pre-IST ANC=0.05×10(9)/L acted as the best cutoff level to predict IST response at 3, 6 months. For 145 VSAA patients, early death rate was 13.4% (11/82) vs 1.6% (1/63), respectively, in the ANC≤0.05×10(9)/L group and ANC>0.05×10(9)/L group (P<0.05). Hematopoietic response rates to IST was 22.0% vs 54.0% (P=0.000) at 3 months, 34.1% vs 63.5% (P=0.000) at 6 months; the overall five-year survival rate was only (62.5±5.4) % vs (91.4±3.7) % (P=0.000) and five-year event-free survival rate was (42.3±5.5) % vs (63.1±6.5) % (P=0.003), respectively, in the ANC≤0.05×10(9)/L group and ANC>0.05×10(9)/L group.</p><p><b>CONCLUSION</b>VSAA patients with extremely low ANC (≤0.05×10(9)/L) had high early death rate and with very low response rate to frontline IST and poor survival, so it is urgent to seek for the alternative frontline therapy that will bring faster and better outcome for these patients.</p>

Effects of peri-immunosupressive therapy period infection on hematologic response and survival of severe aplastic anemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of peri-immunosuppressive treatment（IST）infection on outcomes of severe and very severe aplastic anemia（SAA/VSAA）patients.</p><p><b>METHODS</b>Medical record and follow-up data of 105 SAA/VSAA who underwent first-line IST were retrospectively analyzed to find out the characters of infections（1 month before to 3 months after IST）, and its effects on hematologic response and survival.</p><p><b>RESULTS</b>Of 105 patients, a total of 270 febrile episodes were recorded in 97 patients（92.4%）during their peri- IST periods, with the median infections of 2（1-7）episodes in each patient with the median febrile duration of 7（1-47）days. Respiratory system（35.1%）was the primary anatomic site of infection. Bacteria（88.2%）were common causes of total 169 pathogenic bacteria in 96 clear pathogenic bacteria episodes. And patients who got infection 1 month before IST had much lower 6- month hematologic response rate than their counterpart ones（50.8% vs 80.0%, P=0.004）. Multiple febrile episodes （ ≥3 times） and the total febrile duration ≥4 days showed the best sensitivity and specificity according to the ROC curve analysis. The 5-year overall survival of the 105 patients was 76%. The 5- year OS of patients with multiple febrile episodes （ ≥3 times） were much lower than their counterpart ones［（59.6±7.2）% vs（89.5±4.0）%］（P＜0.01）. The 5-year OS of the total febrile duration ≥4 days was much lower than their counterpart ones［（63.4±5.8）% vs 100.0%］（P＜0.01）.</p><p><b>CONCLUSION</b>Infections 1 month before IST were associated with hematologic response. Multiple febrile episodes（≥3 times） and infections with the febrile duration ≥4 days presented inferior hematologic response and survival.</p>

CD4⁺/CD8⁻ T- cell large granular lymphocytic leukemia: one case report and literatures reviews / 中华血液学杂志

<p><b>OBJECTIVE</b>Presenting the clinical features of one patient with CD4⁺/CD8⁻ T-cell large granular lymphocytic leukemia, to improve the understanding of the disease.</p><p><b>METHODS</b>Clinical data of one patient hospitalized for skin rush and leukocytosis were analyzed, and the related literatures were reviewed.</p><p><b>RESULTS</b>The patient was hospitalized for skin rush and leukocytosis. Routine blood test showed remarkable elevated white blood cell counts and mild anemia. Subsequent hematological examination led to a diagnosis of T- cell large granular lymphocytic leukemia with CD4⁺/CD8⁻ immunophenontype.</p><p><b>CONCLUSION</b>CD3⁺/CD4⁺/CD8⁻ T- cell large granular lymphocytic leukemia is a kind of variant subtype, and is relatively rare, it has different clinical features with classic CD3⁺/CD4⁻/CD8⁺/TCRαβ⁺T- cell large granular lymphocytic leukemia, so differentiating diagnosis is of great importance.</p>

The study of genetic instability in patients with Dyskeratosis congenital / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the genetic instability in patients with Dyskeration congenita.</p><p><b>METHODS</b>The spontaneous chromosome instability of lymphocytes from 4 DC patients, 29 FA patients and 24 healthy volunteers was assessed with comet assay. The percent of DNA in comet head (HeadDNA%）, the percent of DNA in comet tail (TailDNA%）, tail moment (TM), olive tail moment (OTM), the comet cell percentage (CCP) were compared between groups. And the results of MMC test, PNH clones and karotype were analysed additionally. The correlation between TM, OTM, CCP and the severity degree of bone marrow failure in DC group were evaluated.</p><p><b>RESULTS</b>①PNH clones and karotype abnormalities were not found in 4 DC patients. ②TM (6.77 ± 0.90), OTM（6.19 ± 0.80) and CCP [(46.00 ± 5.03) %] in DC were significantly higher than those in normal control group [0.61 ± 0.49, 0.66 ± 0.42, (5.91 ± 3.19)%, P<0.05], however, not distinguished from FA patients [7.81 ± 3.58, 6.65 ± 2.21, (56.03 ± 13.47) %, P ≥ 0.05]. The aberrant cell percent at the MMC concentration of 80 μg/L in DC group was significantly lower than that in FA group [(21.00 ± 3.16) % vs (31.97 ± 6.33)%, P=0.003]. ③The correlation between TM, OTM, CCP and the severity of bone marrow failure in DC group were not found (P>0.05).</p><p><b>CONCLUSION</b>DC patients were of significantly increased genetic instability and normal DNA repair, which was different from that in FA patients. And there was no correlation between the degree of genetic instability and the severity of bone marrow failure in DC patients presenting as aplastic anemia.</p>

Impact of recombinant human thrombopoietin (rhTPO) on short-term response of immunosuppressive therapy in patients with newly diagnosed acquired severe aplastic anemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the impact of recombinant human thrombopoietin (rhTPO) on short-term response of immunosuppressive therapy (IST) in patients with newly diagnosed acquired severe aplastic anemia (SAA).</p><p><b>METHODS</b>The clinical data of forty adult acquired SAA patients, who treated with IST combined with rhTPO, were retrospective analyzed and the hematologic recovery were compared with patients by the IST alone during the same period. The factors affecting the short-term response were also analyzed.</p><p><b>RESULTS</b>At 3 months after IST, both the total response rate and CR+GPR rate in rhTPO group were much higher than those in control group (75.0% vs 50.0%, P=0.022; and 17.5% vs 2.5%, P=0.025). At 6 months after IST, there was no difference of total hematologic response rate in rhTPO group and control group (77.5% vs 57.5%, P=0.058), while the CR+GPR rate was still higher in rhTPO group (45.0% vs 22.5%, P=0.033). The median time of platelet transfusion independence was much shorter in rhTPO group [33(0-90) vs 53(0-75) d, P=0.019]. Patients in rhTPO group needed less platelets transfusion support. The median platelet count in rhTPO group was 29(4-95)×10⁹/L at 3 months after IST, which was much higher than that in control group [29(4-95)×10⁹/L, P=0.006]. There was no significant difference regarding overall survival between the two groups (100.0% vs 91.0%, P=0.276).</p><p><b>CONCLUSION</b>rhTPO is effective in promoting platelet recovery and improving the hematopoietic response for SAA patients with IST.</p>

Increasing peripheral blood neutrophils after G-CSF treatment is a predictor of early response to immunosuppressive therapy in severe aplastic anemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To testify whether absolute neutrophil count (ANC) response to preimmunosuppressive-therapy (pre-IST) granulocyte-stimulating factor (G-CSF) treatment could predict early response to IST in severe aplastic anemia (SAA).</p><p><b>METHODS</b>Clinical data and hematologic response of 125 SAA patients treated with antithymocyte globulin (r-ATG) combined with cyclosporine were retrospectively analyzed. Correlation of ANC response to pre-IST G-CSF treatment and early response to IST were statistically analyzed, and receiver operating characteristic (ROC) curve was used to estimate the value of increased ANC (∆ANC) in predicting early IST response.</p><p><b>RESULTS</b>The hematologic response (HR) rate to IST in ANC reponded patients was significantly higher than non-responded group (3-month HR 49.0% vs 28.9%, P=0.023; 6-month HR 61.2% vs 40.8%, P=0.026). With ∆ANC≥0.5×10⁹/L as cutoff level, the best point to predict early IST response was 10 days after G-CSF (d 10). Response of ANC to pre-IST G-CSF treatment at d 10 was among the independent factors of predicting 3-month (P=0.004), but not for 6-month response to IST. The overall 5-year survival rate was 92.8% and 69.5% in ANC responded and non-responed groups, respectively (P=0.025).</p><p><b>CONCLUSION</b>Responding to pre-IST G-CSF treatment reflected the residual bone marrow hematopoiesis, and could act as a convenient and practical predictor to early IST response as well as long-term survival in SAA.</p>

Residual hematopoiesis is an important prognostic factor of immunosuppressive therapy in severe aplastic anemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the prognostic value of residual bone marrow hematopoiesis in severe aplastic anemia (SAA) patients with immunosuppressive therapy (IST).</p><p><b>METHODS</b>Clinical data and hematologic responses of 38 SAA patients treated with IST regimen (antithymocyte globulin combined with cyclosporine) in our hospital were retrospectively analyzed. Correlation of pre-IST baseline reticulocyte (Ret), absolute neutrophils count (ANC), soluble transferrin receptor (sTfR) concentration, corrected TPO value and hematologic response rate were statistically analyzed and receiver operating characteristic (ROC) curve was used to estimate the value of Ret, ANC, sTfR, and corrected TPO in predicting early IST response.</p><p><b>RESULTS</b>Responders to IST had significantly higher pre-IST baseline Ret, ANC, sTfR concentration [19.9(2.7-84.4)×10⁹/L, 0.59(0.12-2.67)×10⁹/L, 0.82(0.22-1.58) mg/L] and lower corrected TPO value [142.9(31.8-1 035.0)] than non-responders [5.1(1.5-23.1)×10⁹/L, 0.20(0.04-1.33)×10⁹/L, 0.45(0.19-0.72)mg/L and 2 335.0(1 308.3-7 771.2)](P<0.05). Optimizing parameter cutoff levels obtained from ROC curve was Ret 6.75×109/L, ANC 0.30×109/L, sTfR 0.76 mg/L and corrected TPO 148.6, respectively. Combining the four parameters to predict 6 month hemotologic response showed that all the 7 patients with high Ret, ANC, sTfR and low corrected TPO, while only 1 among those 9 with low Ret, ANC, sTfR and high corrected TPO.</p><p><b>CONCLUSION</b>Such parameters evaluating residual bone marrow hematopoiesis as Ret, ANC, sTfR, corrected TPO are practical in predicting early IST response in SAA.</p>

Chronic natural killer cell lymphocytosis: eight cases report and literature review / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the characteristics of chronic natural killer cell lymphocytosis (CNKL).</p><p><b>METHODS</b>The clinical data of eight cases defined by the World Health Organization classification was retrospectively analyzed and related literatures were reviewed.</p><p><b>RESULTS</b>Half of the 8 patients were asymptomatic. Among them, the most common abnormalities were leukocytosis with the median of 11.8(4.5-20.0)×10⁹/L and high proportion of lymphocytes [0.78(0.51-0.89)], while anemia and thrombocytopenia only in 1 patient respectively. The absolute CD3⁻CD16⁺ NK cell count with the median of 5.7(2.4-9.6)×10⁹/L increased in peripheral blood. By the end of follow-up, except one case was lost, the other seven patients were in stable condition,including four cases without any medications and three patients receiving chlorambucil or glucocorticoid.</p><p><b>CONCLUSION</b>As an indolent chronic lymphoproliferative disease, CNKL was presented with mild clinical symptoms and increased number of CD3⁻CD16⁺ NK cells in peripheral blood. Patients with symptoms could be treated with immunosuppressive therapy while those asymptomatic could be followed up without intervention.</p>

Identification and characterization of clinical features and gene mutation in a patient with iron refractory iron deficiency anemia (IRIDA) / 中华血液学杂志

<p><b>OBJECTIVE</b>To report the clinical data of a case of iron-refractory iron deficiency anemia (IRIDA), so as to improve the understanding of IRIDA.</p><p><b>METHODS</b>The IRIDA patient's hematological characteristics were summarized and analyzed. The hepcidin levels were tested by ELISA kit. The TMPRSS6 gene was amplified by PCR reaction and its mutation was analyzed by sequencing. The effect of TMPRSS6 gene mutation on TMPRSS6 protein tertiary structure was predicted by Swiss-Model.</p><p><b>RESULTS</b>The patient was characterized by typical microcytic hypochromic anemia, low transferrin saturation, more reduction of intracellular iron than exocellular iron. The plasma hepcidin level was 213.77 μg/L which was significantly higher than that of IDA patients [5.19(3.31-12.02) μg/L]. The patient also carried a homozygous missense mutation of K253E in exon 7 of TMPRSS6.</p><p><b>CONCLUSION</b>In children and younger IDA patients with no reason for iron deficiency but unresponsiveness to routine iron treatment, the diagnosis of IRIDA needs to be considered. Serum hepcidin level and TMPRSS6 gene mutation should be detected.</p>