Large granular lymphocyte leukaemia is characterized by a clonal T-cell receptor rearrangement in both memory and effector CD8(+) lymphocyte populations

Large granular lymphocyte (LGL) leukaemia is a disease with increased numbers of circulating granular lymphocytes and an increased percentage of clonally rearranged CD8(+)CD57(+) cells. To determine whether LGL cells are also found in other lymphocyte subsets, CD8(+) cells from 10 LGL patients were sorted into CD57(+) and CD57(-) fractions and analysed for clonality using a T-cell receptor gamma (TCR gamma) polymerase chain reaction (PCR). In nine patients, a clonal TCR rearrangement was identified in the CD8(+)CD57(+) cells, and in one patient, the TCR rearrangement was oligoclonal in the CD8(+)CD57(+) fraction. In eight out of nine of the clonally rearranged patients, the same band was also present in the CD8(+)CD57(-) fraction. To define the relationship between CD57(-) and CD57(+) LGL populations, CD8(+)CD57(-) and CD8(+)CD57(+) cells were sorted from five patients and cultured in the presence of anti-CD3 plus CD28 antibodies. The CD57(+) cells died of apoptosis before d 7, while the CD57(-) cells proliferated and differentiated into CD57(+) cells. Clonal analysis identified the same band in both cultured subpopulations and in the uncultured CD8(+) cells. Immunophenotypical analysis showed that CD8(+)CD57(-) cells expressed memory cell markers, while the CD8(+)CD57(+) cells exhibited effector characteristics. These results suggest that LGL disease originates in a CD57(-) memory T-cell compartment that continually generates CD57(+) (effector cell) progeny.     

Impact of acute vivax malaria on the immune system and viral load of HIV-positive subjects

Objective To explore the mechanisms of malariotherapy for human immunodeficiency virus (HIV)-infected patients and to identify which stage(s) of HIV infection is suitable for the treatment of malariotherapy. Methods Therapeutic acute vivax malaria was induced and terminated after 10 fever episodes in 12 HIV-1-infected subjects: Group 1 (G1) had 5 patients with CD4 T-cell counts >=500/μl at baseline, Group 2 (G2) had 5 patients with CD4 at 499-200/μl and Group 3 (G3) had 2 patients with CD4<200/μl (not included in statistical analysis). Enzyme-Linked-Immuno-Sorbent Assay (ELISA) was used to measure plasma levels of cytokines and soluble activation markers. Flow cytometry was used to measure levels of lymphocyte subsets and phenotypes and CD4 cell apoptosis. Bayer bDNA assay was used to test plasma levels of HIV-1 RNA (viral load). Samples were taken and tested twice before malaria (baselines), three times during malaria and seven times after termination of malaria (at day 10 and 1, 3, 6, 12, 18 and 24 months). Results Levels of plasma tumor necrosis factor-α (TNF-α), soluble TNF-α receptor-2 (sTNF-RII), neopterin (NPT) and soluble IL-2 receptor (sIL-2R) significantly increased during malaria and sharply reduced to baselines post malaria in all groups. Stronger responses of the aforementioned factors were seen in G2 than in G1 during malaria (P=0.081, 0.001, 0.013, 0.020). CD4 count and percentage; CD4/CD8 ratio and CD25+ and CD4+CD25+ percentages increased but HLA-DR+ percentage decreased either during or post malaria in G2. Most G2 patients experienced sustained increase but most G1 patients underwent natural history decline of CD4 counts and percentages during 2-year follow-up. Percentage of apoptotic CD4 cells decreased post malaria in all groups. G3 patients had weaker immune responses, however, one advanced AIDS patient in this group experienced clinical improvement after malariotherapy. Most of the 12 patients experienced increase of HIV viral load during malaria but the viral load returned to baseline levels 1-3 months after cure of malaria and remained near baseline levels for up to two years. Conclusions Part of the mechanisms of malariotherapy is to induce high levels of cytokine activities and subsequently the changes of T-lymphocyte subsets and phenotypes in HIV-infected patients.These findings suggest that malariotherapy may treat HIV-1-infected patients whose CD4 baselines are in the range of 500-200/μl.     

肝硬变门静脉高压症脾切除对免疫功能的影响

目的:探讨门静脉高压症患者切除脾脏时免疫功能的影响。方法:采集126例门静脉高压症患者的肝素防凝血,正常对照组30例,利用Epics-XL型流式细胞仪分析测定T淋巴细胞亚群。结果:门静脉高压症患者较正常人细胞免疫功能明显降低,CD3,CD4,CD4/CD8的比值均降低,有非常显著意义(P<0.001)。保脾组手术后CD3值较手术前明显升高(P<0.05)。保脾组与切脾组手术后CD3值有明显差别(P<0.02)。保睥和切脾手术前后的体液免疫指标无明显差别。结论:门静脉高压症的保脾手术可维持机体较高的细胞免疫功能,但对体液免疫无意义。     

成人前B急性淋巴母细胞性白血病CD20表达与预后的关系

目的：探讨白细胞分化抗原CD20表达对成人前B急性淋巴细胞性白血病（Pre B-ALL）临床特征及治疗转归的影响。方法：回顾性分析我院2005-01／2008-01成人前B-ALL患者91例临床特征及治疗转归,比较CD20阳性患者及CD20阴性患者第1疗程诱导治疗完全缓解率、持续完全缓解率、髓外浸润率、复发率及2a生存率。结果：91例成人前B-ALL患者中CD20阳性患者43例（47％）,CD20阴性患者48例（53％）;CD20阳性与阴性患者经第1疗程诱导治疗后获完全缓解率分别为69.8％（30／43）,81.3％（39／48）（P〉0.05）,持续完全缓解率分别为11.6％（5／43）,41.7％（20／48）（P〈0.05）;髓外浸润率分别为46.5％（20／43）,22.9％（11／48）（P〈0.05）;复发率分别为65.1％（28／43）,39.6％（19／48）（P〈0.05）;2a总生存率分别为17.3％,47.2％（P〈0.05）。结论：成人Pre B-ALL瘤细胞表达CD20提示预后不良。     

流式细胞免疫表型分析在恶性淋巴瘤诊断中的应用研究

目的:探讨流式细胞免疫表型分析在恶性淋巴瘤诊断中的价值。方法:128例淋巴结肿大病例同时行细胞病理学、组织病理学、免疫组化及流式细胞免疫表型分析,其中38例同时行淋巴结细针穿刺针吸细胞学检查。结果:共诊断42例恶性淋巴瘤,其中霍奇金淋巴瘤4例、非霍奇金淋巴瘤32例,淋巴结细针穿刺针吸细胞学结合流式细胞免疫表型分析与组织病理结合免疫组化检查比较,对非霍奇金淋巴瘤诊断差异无统计学意义。结论:流式细胞免疫表型分析在非霍奇金淋巴瘤诊断中可发挥重要作用,是诊断非霍奇金淋巴瘤的重要辅助手段。     

Childhood near‐tetraploid acute lymphoblastic leukemia: an EGIL study on 36 cases

Objectives: Patients with near‐tetraploid (karyotype: 81 – 103 chromosomes) acute lymphoblastic leukemia (NT‐ALL) constitute about 1% of childhood ALL and data reported on them are limited and controversial. The aim of the study was to enlarge the knowledge on these rarely occurring ALL. Methods: The members of the European Group for Immunophenotyping of Leukemias (EGIL) searched retrospectively their databases for NT‐ALL patients. Results: We collected data of 36 European children from seven European countries with NT‐ALL diagnosed since 1992. All patients reached complete remission (CR) after induction chemotherapy. Their blasts were negative for peroxidase and BCR‐ABL1. Ten children were diagnosed as T‐cell ALL (T‐ALL) EGIL categories (T‐I n = 2, T‐II n = 2, T‐III n = 3, T‐IV n = 3) and four displayed various structural chromosomal abnormalities. Eight of 10 T‐ALL remained in 1st CR; one died in CR from sepsis and one is alive in 2nd CR. Median survival was 88 (7–213) months. B‐cell precursor (BCP) ALL was diagnosed in 26 children. Thirteen were positive for ETV6‐RUNX1 and are alive in 1st CR for 32–147 months. Ten children were ETV6‐RUNX1 negative and remained in 1st CR for 16–163 months. One girl with hypodiploid and NT metaphases and ETV6‐RUNX1‐negative BCP‐ALL and one of two boys with NT‐BCP‐ALL not examined for ETV6‐RUNX1 died of infection after stem cell transplantation in 2nd/3rd CR. Secondary myelodysplastic syndrome developed in two patients with NT‐BCP‐ALL. Conclusions: Our data demonstrate immunophenotypic, cytogenetic, and molecular heterogeneity of NT‐ALL and favorable prognosis of most NT‐ALL across different immunophenotypic and/or genetic ALL subtypes.     

Pitfalls in the immunophenotyping of leukaemia and leukaemic lymphomas: survey of 9 years of quality control in The Netherlands

During the last decade, biannual quality controls were performed in The Netherlands focusing on the immunophenotyping of leukaemic haematological malignancies. All results on 48 specimens obtained by 18–34 laboratories were analysed. The interlaboratory variability and percentages of discordant results from 30 markers were measured by assessing false positive or negative (cut-off 10%) results in comparison with median results of the group. The quality of the immunophenotypic diagnoses obtained from the interpretation of these markers in relation to clinical data was evaluated by scoring them as ‘correct’, ‘minor fault’, ‘major fault’, ‘not based upon the markers used’, and ‘no diagnosis’. CD3, CD8, CD19, CD61 and Smλ had the lowest percentage discordancy (sum of total negative and positive discordant values 5–7.5% of assays); CD13, CD15, cyCD22, CD33 and TdT scored worst with 14–20% cumulative discordancy. The analysis of each diagnosis yielded 78% acceptable immunophenotypic conclusions (correct 54% and minor fault 24%). It appeared that the major faults in immunophenotyping were caused by suboptimal antibody selection and erroneous interpretation of the results obtained, rather than by technical errors. Large differences per diagnostic category were observed, with the best scores for mature B-cell leukaemias, AMLs and common-ALL, and the poorest scores for T-cell malignancies which were correctly diagnosed in only 24–60% of specimens. Mature T-NHL and T-PLL were mistakenly diagnosed as T-ALL by 40% of the centres. Misinterpretation of TdT immunofluorescence or omitting this marker contributed significantly to these wrong diagnoses. A median of 4% of immunophenotypic diagnoses were not based on a correct panel of antibodies, but upon the morphology of the accompanying blood smear, and was often flawed by overinterpretation. In conclusion, both the technical performance of immunophenotyping of haematological malignancies in The Netherlands and the procedure by which a final diagnosis is obtained needs improvement, especially for T-cell malignancies.     

Acute basophilic leukemia: case report

The term "basophilic leukemia" has been in use for 75 years. However, consistent diagnostic criteria are lacking. This is due to the rarity of the disease and to the routine unavailability of special tests that are often required to confirm a diagnosis. We report an unusual case of acute basophilic leukemia in a child who was referred to our Center, arriving with partially treated acute lymphoblastic leukemia. Basophilic differentiation on light microscopy was evident from the coarse basophilic granules in blasts, a progressive maturation of blasts toward basophils, and toluidine positivity on cytochemistry. Blasts showed a myeloid immunophenotype (CD13+, CD33+, CD117+) with a characteristic dual positivity for CD34 and CD25, highly suggestive of basophilic nature of the blasts. Conventional cytogenetic studies revealed translocation t(8;21)(q22;q22). A diagnosis of acute basophilic leukemia with t(8;21) was made. Review of pre-therapy slides showed features consistent with AML-M2 with basophilia. There were no basophilic blasts. With these features, a diagnosis of acute basophilic leukemia secondary to AML-M2 was made. In our patient, basophilic leukemia appears to have evolved from selective clonal proliferation of "basophil-committed blasts" during the course of the disease in a case of AML-M2 with basophilia.     

Waldenstrom巨球蛋白血症的形态学、细胞免疫表型、细胞遗传学以及分子生物学研究

目的分析Waldenstrom巨球蛋白血症（WM）形态学、细胞免疫表型、细胞遗传学以及分子生物学（MICM）异常的特点。方法收集1999至2010年MICM资料完整的初治WM患者41例,男27例、女14例。回顾性分析其临床表现、骨髓形态、细胞免疫表型、细胞遗传学、免疫球蛋白重链基因重排、黑色素瘤优先表达抗原（PRAME）的表达及其与临床预后之间的关系。结果本组中高危患者占58.5%。细胞免疫表型分析：CD19阳性100.0%,CD20阳性97.6%,CD38阳性74.1%,FMC7阳性36.9%,CD5阳性10.0%,CD23阳性31.6%,HLA-DR阳性83.3%,CXCR4阳性85.7%。常规细胞遗传学以及荧光原位杂交发现特异性细胞遗传学异常。PRAME在WM中表达增加,且与骨髓中淋巴细胞数相关, MAGEC1/CT7在WM中不表达。WM使用含利妥昔单抗联合化疗较环磷酰胺+长春地辛+醋酸泼尼松（COP）方案治疗未见生存优势。结论 WM具有独特的MICM特征,通过MICM的综合检测有助于早期诊断WM并对疾病进行监测,利妥昔单抗治疗未见明显优势。     

Diagnostic and prognostic advances in the immunophenotypic and genetic characterization of acute leukaemia

Abstract: Soluble tumour necrosis factor receptors (sTNF-Rs) play a role as modulators of the biological function of tumour necrosis factor-α (TNF-α) in an agonist/antagonist pattern. In various pathologic states the production and release of sTNF-Rs may mediate host response and determine the course and outcome of disease by interacting with TNF-α and competing with cell surface receptors. The determination of sTNF-Rs in body fluids such as plasma or serum is a new tool to gain information about immune processes and provides valuable insight into a variety of pathological conditions. Regarding its immediate clinical use, sTNF-Rs levels show high accuracy in the follow-up and prognosis of various diseases. In HIV infection and sepsis, sTNF-Rs concentrations strongly correlate with the clinical stage and the progression of disease and can be of predictive value. Determination of sTNF-Rs also gives useful information for monitoring cancer and autoimmune diseases. The information provided is often even superior to that obtained with classical disease markers, probably due to the direct involvement of the “TNF system” in the pathogenetic mechanisms in these patients. The available data imply that the measurement of sTNF-Rs, especially of the sTNF-R 75kD type, is a useful adjunct for quantification of the Th1-type immune response, similar to other immune activation markers such as neopterin and β2-microglobulin. Endogenous sTNF-Rs concentrations appear to reflect the activation state of the TNF-α/TNF receptor system.