Cytostatic drug testing in human leukemias by means of multiparametric flow cytometry

Summary Human bone marrow cells from 20 patients as well as the permanent human B-cell lines RPMI 1788, Raji, Daudi, T-cell lines Molt, CEM, Jurkat and the promyelocytic line HL 60 were assayed by means of a newly developed in vitro flow cytometric cytostatic drug assay. The cells were exposed to cytosine-arabinoside, L-asparaginase, daunorubicin, prednisone or vincristine. Surviving cells were stained after an incubation period of 2 to 7 days with esterase and pH-indicator dye ADB (1,4-diacetoxy-2,3-dicyanobenzene), dead cells with DNA-dye PI (propidium iodide). Dose-response curves were established using percent surviving cells. It was possible to evaluate bone marrow samples from 16 out of 20 patients. Seven samples were leukemic (acute myeloid leukemia (AML) n=6, Non-Hodgkin's Lymphoma (NHL) n=1). Nine samples were from patients either in complete remission or with benign diseases. Daunorubicin and cytosine-arabinoside were cytotoxic in both groups, whereas vincristine was effective mainly in the leukemic group (p<0.05). There was significant heterogeneity in the reactivity of AML-marrow cells from different patients to different drugs. The cell lines exhibited different patterns of sensitivity. Vincristine arrested cells in G2/M-phase, cytosine-arabinoside caused an increase of cells in the S-phase. This work was supported in part by grant (Ne 310/1-1) from the Deutsche Forschungsgemeinschaft     

Analysis of acute myeloid leukemia cells by flow cytometry,introducing a new light-scattering classification

A combined flow-cytometric evaluation of light scattering and the immunophenotype of acute myeloid leukemia (AML) cells from 71 newly diagnosed consecutive patients was conducted. Light-scattering characteristic of AML cells examined by flow cytometry and multiple surface markers were also analyzed using the same samples, to enable a comparison with the French-American-British (FAB) classification. Our AML cases could be classified into three light-scattering classification (LSC) types according to their physical properties on flow cytometry. These were type A, where forward light scattering (FSC) of the leukemic cell population was larger than that of lymphocytes, while side light scattering (SSC) was the same or larger than that of lymphocytes but smaller than that of monocytes; type B, where FSC of the leukemic cell population was larger than that of lymphocytes and SSC spread toward that of monocytes; and type C, where both FSC and SSC of the leukemic cell population spread beyond those of monocytes. Although a clear relationship between the FAB classification and LSC classification by the light-scattering profile of AML was not established, we observed the following findings. The majority of cases were classified as type A (58%), while type B comprised 25% and type C comprised 17%. While CD7 expression on AML cells is considered to be an immature characteristic, CD7 was expressed more frequently among LSC type A cases. Furthermore, all but one of the FAB M1 cases were classified as type A. On the other hand, CD7 was not expressed on type C leukemic cells. The percentage of cases in which more than 60% of leukemic cells possessed another immature surface antigen, CD 34ö, was 13/18 (72%) among FAB M1 cases, much higher than among FAB M2 (35%) or FAB M4 (27%) cases. A negative correlation was observed between mature antigen CD33 and CD34 among the FAB M2 cases. The frequency of CD7 expression was 25% among the total cases, and CD7-positive cases were frequent among FAB M1 and M2, but not among FAB M3 cases. These findings concerning LSC and immunophenotyping indicate that the scattergram pattern analysis may contribute towards more precise immunophenotyping, in that it reflects the maturation stage of each AML case.Abbreviations AML acute myeloid leukemia - LSC light-scattering classification - FAB French-American-British classification - CD cluster of differentiation Partly supported by grants in aid from the Ministry of Education, Science and Culture of Japan (03670325, 04247102, 04454572 and 05670916) and from the Fukuoka Anti-cancer Society     

Flow cytometry study of cell cycle, apoptosis and drug resistance in acute leukemia

The response to therapy of leukemic cells is largely determined by their capacity of proliferation and apoptosis in presence of the administered drugs. We describe here the main markers used in flow cytometry (FCM) and involved in the assessment of cell cycle parameters: single labeling by Propidium Iodide (PI) and double labeling anti-Bromodeoxyuridine (BrdUrd)/PI which, both in vitro and in vivo, gives cell percentages in the different cell cycle phases. The markers of cell cycle progression can be divided into proliferation markers such as PCNA (proliferating cell nuclear antigen) or Ki-67 and cell cycle progression markers. The latter, which are the core of the cell cycle machinery, are molecules recently characterized (Cyclins, CDKs (cell dependent kinases), CDIs (cyclin-dependent kinase inhibitors)) and their cell expression can be analyzed using FCM. FCM is also one of the best means to detect and quantitate appoptotic cells. Several techniques are described: Nuclear labeling using Hoechst 33342: mitochondrial labeling using DiOC6(3): detection of DNA fragmentation using 1) labeling of fixed and permeabilized cells with a DNA marker or 2) labeling of the free 3 DNA ends using incorporation of labeled deoxynucleotides; detection of antigens in apoptotic cells (Bcl-2, Fas, phospholipids...). At last, we analyzed flow cytometry methods to study the cell resistance to Ara-C and anthracyclins. In combination with cell kinetic studies and detection of apoptotic cells, they should increase the efficiancy of the acute leukemia treatment.     

Receptor for platelet-activating factor (PAF) is not detectable by flow cytometry on the surface of myeloid leukemic cells

Flow cytometry was applied to test for platelet-activating-factor receptor (PAF-R) presence on the membranes of acute myeloid leukemia (AML) cells. We have used six human AML cell lines and freshly taken density gradient separated blasts from the bone marrow of ten AML patients covering the majority of French–American–British (FAB) subtypes. Additionally, we have used one histiocytic lymphoma cell line and mature human granulocytes/monocytes as controls. Our results indicate lack of membrane PAF-R on AML of all FAB subtypes tested. This was particularly true for the more mature and differentiated subtypes M₄ and M₅, including monocytic cell elements, and the promyelocytic M₃ AML. In contrast, membrane PAF-R could be easily detected in a histiocytic lymphoma cell line and mature granulocytes/monocytes from peripheral blood used as positive controls. In conclusion, this observation precludes the use of membrane PAF-R as an immunophenotypic marker for AML classification or detection of minimal residual disease.     

流式细胞术分析慢性髓性白血病细胞酪氨酸磷酸化水平

目的：探讨慢性髓性白血病（CML）患者骨髓细胞酪氨酸磷酸化水平（p-Tyr），探讨甲磺酸伊马替尼（商品名：格列卫）治疗对p-Tyr水平的作用和影响。方法：对38例CML患者（其中16例接受格列卫治疗）及5例健康对照者的骨髓细胞进行细胞膜CD45和细胞质内PTyr（PY99）荧光标记，应用流式细胞术（FCM）分析不同病期CML各群细胞p-Tyr水平。结果：①未经甲磺酸伊马替尼治疗的慢性期（CP）、加速期（AP）患者骨髓各群细胞p-Tyr水平高于健康对照者，而CP与AP之间p-Tyr水平差异无统计学意义；②CP患者应用甲磺酸伊马替尼治疗〉12个月者的骨髓细胞p-Tyr水平低于用药〈12个月者；AP期用药≥40个月者的幼稚细胞群p-Tyr水平亦低于用药〈12个月者；③随CML病程延长，幼稚细胞群p-Tyr水平增高，但无明显影响；年龄对p-Tyr水平无明显影响。结论：①应用FCM测定细胞p-Tyr水平可作为快速、敏感的方法检测BCR-ABL（＋）细胞；②CML各期细胞的p-Tyr水平均高于正常对照；③甲磺酸伊马替尼对慢性期白血病细胞p-Tyr水平的降低程度随用药时间延长而增强。     

Simultaneous analysis of cell surface antigens and DNA content by flow cytometry

Summary Cells obtained from the blood or bone marrow of patients with haematological malignancies can both be stained with fluorescent labelled monoclonal antibodies to determine an immunophenotype and permeabilized with 30% ethanol then stained with propidium iodide for simultaneous DNA analysis. In the technique described here, no evidence of selective depletion of the malignant cell population was demonstrated and decreases in the mean fluorescence intensity of the surface markers were insufficient to affect the sensitivity of flow cytometric analysis. The combined method is simple and robust enough to allow incorporation of DNA analysis into routine immunophenotyping of leukaemia and lymphoma cells.     

DNA cell-cycle analysis of cervical cancer by flow cytometry using simultaneous cytokeratin labelling for identification of tumour cells

DNA ploidy and cell-cycle distribution were determined by flow cytometry in fresh tumour tissue of 53 cervical carcinomas. Epithelial cells were labelled by a fluorescein-isothiocyanate-conjugated cytokeratin antibody (CK6, CK18) to study the influence of contaminating stromal and inflammatory cells on results of cell-cycle analysis of tumour cells. Without identification of cytokeratin-positive cells 30/53 (57%) tumours were found to be DNA-aneuploid compared to 43/53 (81%) after gating for cytokeratin. Only 7 of 15 DNA-multiploid tumours could be detected without cytokeratin staining. In addition, cytokeratin-negative cells, which are found in all tumours, can be used as an internal standard for the calculation of ploidy and for quality control (coefficient of variation, linearity) of each individual sample. Cell-cycle analysis revealed significantly higher S-phase and G₂M-phase fractions in cytokeratin-gated compared to ungated samples (13.1% versus 10.0% and 8.0% versus 5.4%;P<0.001). This difference was more pronounced in DNA-diploid than DNA-aneuploid tumours. In conclusion, about 30% of DNA-aneuploid tumours could only be detected after cytokeratin labelling of epithelial cells. Owing to the identification of cytokeratin-positive cells the influence of non-tumoural cell elements on cell-cycle analysis was reduced markedly. Therefore, in cervical cancer, cytokeratin labelling can optimize both the determination of DNA ploidy and cell-cycle analysis.     

12例经流式细胞仪确诊的急性混合型白血病分析

目的:对比经流式细胞仪(FCM)确诊的急性混合型白血病(MAL)与经骨髓细胞形态学组化染色发现具有急性混合型白血病特性的急性白血病(AL)患者的符合率。方法:选取12例MAL患者,经FCM确诊,对比其相应的骨髓细胞学组化染色FAB分型结果,并计算经形态学和免疫组化发现MAL特性的AL患者占经FCM确诊MAL患者的百分率。结果:经FCM确诊的MAL患者,其中急性双表型白血病8例,急性双克隆型白血病3例,发现急性细胞系转变型1例。此12例AL患者经骨髓细胞学组化染色确诊7例为急性淋巴细胞白血病(ALL),3例为急性髓系白血病(AML),另2例经细胞形态学未确诊的AL患者中,1例多次细胞形态学显示呈骨髓坏死,但最终FCM技术也确诊为MAL。结论:经骨髓细胞形态学及组化染色发现具有MAL表现的AL患者仅占经FCM确诊的MAL患者的17%,误诊率极高。应重视流式细胞仪免疫分型在MAL诊断中的应用。     

Comparison of acute myeloid leukemia occurring de novo or preceded by a myelodysplastic stage: Differences in cellular DNA content

The DNA content of bone marrow cells in patients with acute leukemia preceded by a myelodysplastic stage (MDS-AML) was compared to that in patients with de novo AML. We studied granulocytes, lymphocytes, monocytes, and blasts/promyelocytes from Feul-gen-stained bone marrow smears of 11 patients with de novo AML, ten patients with MDS-AML, and 13 apparently healthy controls. The mean amount of DNA per cell (DNA index; Dl) in each cell population was determined using a digital video-based imageanalyzing system (CAS-100). Analysis of variance (F test) showed a significant difference in the DNA content between de novo AML on one hand and MDS-AML and controls on the other as regards to blasts/promyelocytes (P < 0.01), lymphocytes (P < 0.05), and monocytes (P < 0.01), respectively. In three of 11 (27%) patients with de novo AML, a lower than normal limit Dl was found both in immature and mature bone marrow cells. Patients with MDS-AML had those of Dl values similar to normal controls. In consequence, a significantly reduced mean Dl was found in patients with de novo AML in blasts/promyelocytes (P < 0.01), and monocytes (P < 0.05) compared to both normal controls and MDS-AML. Together with data published separately, suggesting differences in granulocyte morphology, clonality, and HLA-DR expression, these data suggest biological differences between the two diseases. © 1993 Wiley-Liss, Inc.     

多参数流式细胞术对恶性胸腔积液的诊断价值

目的应用流式细胞仪检测胸腔积液中的DNA、RNA、增殖细胞核抗原(PCNA),探讨多参数流式细胞术对恶性胸腔积液的诊断价值。方法2003年8月至2004年2月在我院呼吸内科住院治疗的胸腔积液患者47例,其中19例非肿瘤性胸腔积液患者作为对照组,28例经病理学检查确诊的恶性胸腔积液患者为试验组。采用碘化丙啶染色检测DNA,哌若宁染色检测RNA,PCNA-FITC法检测PCNA,阴性对照采用鼠-α-2a。用美国Becton Dickinson公司FacS Calibur流式细胞仪进行检测,计算单项检测和联合检测的敏感性和特异性。结果(1)非肿瘤性胸腔积液中DNA指数、RNA指数、PCNA流式细胞术检测结果分别为1.03±0.06、10.03±0.54及(4.86±0.72)%,而恶性胸腔积液为1.26±0.17、11.65±1.45及(11.97±1.50)%。诊断分界点分别为1.10%、10.75%、4.56%,此时的敏感性分别为89.3%、78.6%、75.0%,特异性为89.5%、98.5%、84.2%;(2)恶性胸腔积液中DNA指数正常而RNA指数异常者6例,表明流式细胞术同时检测患者RNA可以弥补DNA检测的不足;(3)5例患者胸腔积液中细胞学检查未发现肿瘤细胞,但其DNA指数和RNA指数均高于正常值,经多次胸膜活检或肺部肿块穿刺证实为恶性胸腔积液,表明流式细胞术对细胞学检查有补充作用;(4)DNA指数+RNA指数、DNA指数+PCNA的流式细胞术检测结果、RNA指数+PCNA的流式细胞术检测结果及三者联合诊断的敏感性分别为98.2%、89.3%、89.3%及92.9%,特异性为84.2%、89.5%、84.2%及94.2%。三者联合检测具有较低的漏诊率和误诊率,而对照组未发现三者同时高于诊断临界点的患者。结论流式细胞术检测胸腔积液DNA指数、RNA指数、PCNA的流式细胞术检测结果对于恶性胸腔积液的诊断具有一定的价值,特别是对于部分细胞学检测阴性的恶性胸腔积液的诊断可能具有重要的临床意义。DNA、RNA同时检测对于诊断DNA正常而RNA发生异常改变的恶性胸腔积液具有重要价值,可以弥补单项DNA检测的不足。三者联合检测对于恶性胸腔积液的诊断价值大于单项或两项联合检测,具有较低的漏诊率和误诊率。     

急性髓系白血病H-ras表达和点突变─流式细胞术和聚合酶链反应比较研究

目的：了解急性髓系白血病细胞H－rasP21表达与点突变的相关性，以及免疫组化法的临床实用价值。方法：用流式细胞术（FCM）分析67例初治急性髓系白血病（AML）H－rasp21表达；对所有P21阳性和部分P21阴性者作聚合酶链反应（PCR），观察H－ras15位密码子点突变。结果：67例AMLFCMP21阳性者45例（67．2％），P21阴性者22例（32．8％）。P21阳性45例中高表达者22例（48．9％），16例（72．7％）检出H－ras15位密码子点突变，11例（50％）完全缓解（CR）。10例P21阴性和23例P21＋低表达均无H－ras15位密码子点突变，CR率分别为77．2％和87．2％。结论：FCMP21高表达者常有H－ras15位密码子点突变，CR低，H－ras点突变与免疫组化P21＋高表达相关良好，提示AML转归不良。     

Effects of thrombopoietin on megakaryocyte colony formation from leukemic cells at diagnosis and from marrow cells after induction chemotherapy for acute leukemias

 We have studied the effects of recombinant human thrombopoietin (TPO, mpl ligand) on the megakaryocyte colony formation from control human bone marrow cells, human leukemia cells at diagnosis, and human bone marrow cells after induction chemotherapy for acute leukemias. In the control human bone marrow cells from four adults and nine children who had localized malignancy and histologically normal-looking marrow, TPO alone effectively stimulated megakaryocyte colony formation, and interleukin-3 (IL-3) synergized this. In 17 patients (13 adults and four children) with acute myeloid leukemia (AML) at diagnosis, TPO stimulated leukemic colony formation in only one patient with FAB M7 subtype. In 11 children with acute lymphoblastic leukemia (ALL) at diagnosis, TPO did not enhance leukemic colony formation. After 17 courses of induction chemotherapy, nine for AML and eight for ALL, TPO stimulated megakaryocyte colony formation to a level of 51% of that in the control human bone marrow cells. This may suggest that the administration of TPO to patients with M7 subtype warrants caution, whereas it is probably safe to give TPO at any time to patients with ALL. The administration of TPO to patients with acute leukemias after induction chemotherapy could stimulate megakaryocytopoiesis. Received: July 29, 1997 / Accepted: May 6, 1998     

急性白血病免疫分型中系列相关抗体的敏感性和特异性研究

目的：确定急性白血病免疫分型中系列相关抗体的敏感性和特异性。方法：应用St Jude研究医院的急性白血病免疫分型方案对63例急性白血病患者进行了流式细胞术分型。结果：B系列急性淋巴细胞白血病(B-ALL)敏感性最高的单克隆抗体(抗体)是CD19[100％(22／22)]，而特异性最高的是CD79a[96．7％(29／30)]。CD79a敏感性(95．0％)和特异性均很高，在所有B细胞系列相关抗体中确定系列来源的准确性最高[96％(48／50)]。在T-ALL，敏感性最高的抗体是CD7[100％(14／14)]和CyCD3[100％(12／12)]，特异性最高的是CD5[(100％(45／45)]和CyCD3[97．7％(42／43)]；CD7、CyCD3和CD5在BALL均无假阳性；在AML的假阳性分别是[12％(3／25)]、[4．3％(1／23)]和0／24；但CD5的敏感性仅E85．7％(12／14)]；CD3的敏感性[61．5％(8／13)]和特异性[88．6％(39／44)]均明显低于上述抗体。综合来看，CyCD3敏感性和特异性均很高，在所有T细胞系列相关抗体中确定系列来源的准确性最高[98．2％(54／55)]。在AML，MPO的敏感性和特异性最高[100％(23／23)]和[92．9％(26／28)]，而CD13和CD33的敏感性仅在61．5％和76．9％，特异性仅为68．8％和75．0％。结论：St Jude急性白血病免疫分型方案的高敏感性和高特异性抗体在本组B-ALL和T-ALL病例得以重复；但在AML仅有MPO表现出高敏感性和高特异性；CD13和CD33未能得到高敏感性。     

Establishment and validation of a standard protocol for the detection of minimal residual disease in B lineage childhood acute lymphoblastic leukemia by flow cytometry in a multi-center setting;

Minimal residual disease detection, used for clinical management of children with acute lymphoblastic leukemia, can be performed by molecular analysis of antigen-receptor gene rearrangements or by flow cytometric analysis of aberrant immunophenotypes. For flow minimal residual disease to be incorporated into larger national and international trials, a quality assured, standardized method is needed which can be performed in a multi-center setting. We report a four color, flow cytometric protocol established and validated by the UK acute lymphoblastic leukemia Flow minimal residual disease group. Quality assurance testing gave high inter-laboratory agreement with no values differing from a median consensus value by more than one point on a logarithmic scale. Prospective screening of B-ALL patients (n=206) showed the method was applicable to 88.3% of patients. The minimal residual disease in bone marrow aspirates was quantified and compared to molecular data. The combined risk category concordance (minimal residual disease levels above or below 0.01%) was 86% (n=134). Thus, this standardized protocol is highly reproducible between laboratories, sensitive, applicable, and shows good concordance with molecular-based analysis.     

流式细胞仪DNA倍体测定鉴别良恶性胸腔积液的诊断价值

目的通过流式细胞仪（FCM）技术测定胸水脱落细胞细胞核的脱氧核糖核酸（DNA）含量，并将此方法与传统的病理学方法，肿瘤标记物癌胚抗原（CEA）的检测相比较，以鉴别胸水的良恶、性。方法病例选自2003-03～2004-03苏州大学附属第二医院呼吸内科的住院患者，留取新鲜的胸腔积液标本，以肝素抗凝，荧光染料染色后，用流式细胞仪检测细胞核DNA倍体，分析细胞的增殖能力。结果根据临床检查将胸水分为良性和恶性两组，在恶性胸腔积液组中，87．5％的标本可以检测到DNA异倍体，在良性胸腔积液中，90％的标本表现为两倍体。结论流式细胞仪DNA倍体测定使得病理诊断有了可依据的客观指标，是病理学检查的有益补充，与CEA联合测定可以提高恶性胸腔积液的检出率。     

High expression of CEACAM6 and CEACAM8 mRNA in acute lymphoblastic leukemias

CEACAM family members are a set of widely expressed proteins involved in several biological functions, including cell adhesion, migration, signal transduction, and the regulation of gene expression. Abnormal overexpression and downregulation of some CEACAMs have been described in tumor cells. Monoclonal antibodies grouped in the CD66 cluster recognize CEACAM members. Ectopic CD66 expression is commonly detected in B-cell lineage acute lymphoblastic leukemia (ALL). To investigate the CEACAM messenger RNA (RNA) expression in leukemic blasts, we performed a quantitative polymerase chain reaction (RQ-PCR) analysis in purified RNA samples from a consecutive series of acute leukemias (135 patients). Most B-cell lineage ALL expressed CD66 (79.5%), whereas no single case of T-cell lineage ALL disclosed CD66 reactivity (0%). All the BCR-ABL+ ALL cases showed CD66 expression. CD66 was positive even in cases without CD10 expression (72.7%) and/or with MLL rearrangements. Despite the sharp contrast between T-ALL and B-ALL in CD66 reactivity, CEACAM patterns were comparable, and only minor differences for CEACAM1 and CEACAM8 were detected. All the leukemic samples showed overexpression of CEACAM6 and 8 when compared with normal granulocytes. These results were confirmed by dilutional experiments. The leukemic pattern paralleled the normal regenerating bone marrow with lower values for CEACAM1. In line with the results for CD66 reactivity, neoplastic cell lines had a uniform low expression of CEACAM family members. It remains to be investigated whether these CEACAM disturbances provide growth advantages to tumoral cells by inhibiting the anoikis process.     

胃癌组织甲基绿染色DNA/RNA图像分析

目的研究甲基绿＿派洛宁（ＭＧ＿Ｐ）染色法进行胃癌细胞的ＤＮＡ／ＲＮＡ双参数图像分析的可行性。方法选择２６例已用Ｆｅｕｌｇｅｎ染色法检测过ＤＮＡ倍体类型的胃癌石蜡组织，采用同一蜡块的连续切片进行ＭＧ＿Ｐ染色。提纯甲基绿并经反复摸索，找到了ＭＧ＿Ｐ染色的理想条件和最佳染色效果。细胞的染色质呈蓝绿色，核仁和胞浆呈红色，两种颜色对比明显；其他细胞结构不着色。经ＤＮＡ酶处理后染色质不着色，ＲＮＡ酶处理后核仁及胞浆不着色。用图像分析法同时测定胃癌细胞的ＤＮＡ／ＲＮＡ含量，并进行ＤＮＡ倍体分析。结果２６例胃癌患者Ｆｅｕｇｅｎ染色的异倍体率为１５／２６（５７８％），而ＭＧ＿Ｐ染色为１７／２６（６５４％），两者符合率为８８２％。采用退色的方法测定相同组织细胞的ＤＮＡ含量，Ｆｅｕｌｇｅｎ染色与ＭＧ＿Ｐ染色的相关系数的０９９。ＲＮＡ含量在肿瘤细胞中明显高于正常胃粘膜细胞。结论ＭＧ＿Ｐ染色法是可靠的同时测定肿瘤细胞ＤＮＡ／ＲＮＡ含量的方法。