慢性淋巴细胞白血病细胞化学染色的特点分析

  目的 应用细胞化学染色方法诊断慢性淋巴细胞白血病（CLL）。方法 用9种细胞化学染色方法对75例CLL患者骨髓片进行染色分析。结果 CLL的骨髓过氧化物酶（MPO）和苏丹黑染色（SBB）为阴性;中性非特异性酯酶（NSE）和α-丁酸酯酶（NBE）染色为弱阳性,糖原（PAS）染色均为阳性,阳性物呈细颗粒、中粗颗粒、粗颗粒散在分布;特异性酯酶（CE）染色4 %可见阳性,阳性率均低于6 %;酸性磷酸酶（AcP）染色呈弱阳性,13.3 %抗酒石酸酸性磷酸酶（TRAP）染色可见阳性,以（+）为主。结论 CLL CE染色大部分阴性,个别病例可见阳性（6 %）,但阳性率明显低于大颗粒淋巴细胞白血病（LGLL）（46.9 %）;AcP染色呈弱阳性,低于LGLL,非霍奇金淋巴瘤（NHL）和HCL;TRAP染色少部分病例虽可见阳性（13.3 %）,但阳性率及强度明显低于毛细胞白血病（HCL）,NHL和幼淋细胞白血病（PLL）。     

恶性淋巴瘤骨髓累及的细胞化学特点

目的 :用细胞化学染色的方法诊断恶性淋巴瘤骨髓累及并与白血病相鉴别。方法 :细胞化学染色方法。结果 :前躯淋巴母细胞性淋巴瘤 (PL B)、中心小裂细胞性淋巴瘤 (S- FCC)和伴毛细胞的脾淋巴瘤 (SL VC) POX、SBB和 CE均为阴性。PAS:S- FCC和 SL VL 反应弱 ,PL B反应较强。淋巴瘤珠状占 2 2 .2 2 %。AL L 珠状占 70 .5 9%。NAE:S- FCC反应强度以 + +为主 ,SL VL 和 PL B较弱。ACP:S- FCC较 SL VC和 PL B略强 ,S- FCC和 SL VC均可见 + + + +。TRAP:S- FCC阳性率 2 %～ 5 4% ;PL B阳性率 10 %～ 16 % ;SL VC阳性率均为 18%。真性组织细胞性淋巴瘤 (THL) ACP4例均为阳性 ,阳性率为 10 0 % ,1例 TRAP阳性。淋巴瘤与 CL L、HCL 和 AL L 相比较发现 ,淋巴瘤较 CL L 和 AL L TRAP明显增高 ,S- FCC和 SL VC的 ACP和 TRAP与 HCL 相同。结论 :细胞化学染色在诊断淋巴瘤骨髓转移 ,并与 AL L 和 CL L 相鉴别上有一定意义 ,淋巴瘤骨髓累及 PAS阳性率和阳性指数较 AL L 弱。ACP阳性较 AL L 和 CL L 强 ,具有较强的抗酒石酸的功能 ,但与 HCL 难鉴别。     

PAS改良染色法及其在病理诊断中的应用

目的:改良高碘酸-无色品红染色方法(PAS),改善PAS染色结果,为临床病理诊断提供可靠依据。方法:脱蜡至水的病理切片。选择2%高碘酸钠作为氧化剂,严格控制氧化剂20分钟作用时间,使用自行配制的Schiff试剂染色孵育5分钟,苏木素衬染,观察染色结果。并将300例含头颈、肺、肾脏、肝脏及胃标本的染色结果与传统的PAS染色法染色结果相互比较。结果:改良后PAS染色方法的PAS阳性物质（黏液、糖原等）颜色鲜艳,特异性强。结论:影响PAS染色的因素很多,使用2%高碘酸钠氧化液,严格控制染色温度和时间,可有效改善PAS染色结果。     

急性髓系白血病Evi1基因表达的研究

目的 :探讨急性髓系白血病中 Evi1基因表达及意义。方法 :应用 RT- PCR方法检测了 94例急性髓系白血病 (AML ,包括初治 4 9例 ,复发 2 3例 ,缓解 2 2例 )患者及 1 0名正常对照 Evi1基因的表达。结果 :1 0名正常人骨髓单个核细胞中无 Evi1 m RNA的表达。AML 患者 Evi1基因总的阳性表达率为 1 8.1 % (1 7/94 ) ,M5型未见表达 ,其中初治、复发、缓解组的 Evi1基因阳性率分别为2 6 .5 %、1 7.4 %、0 ,初治和复发组差别无显著性 (P=0 .5 5 4 )。M3患者中 Evi1、PML /RARα双阳性组与 PML /RARα单阳性组相比复发率高 (P<0 .0 5 ) ,早期死亡率高。 Evi1阳性的 AML患者多数生存期短。结论 :Evi1基因是一个原癌基因 ,在髓系白血病的发病中起重要作用 ,是髓系白血病预后不良的一个指标。     

急性髓细胞白血病微分化型流式细胞术免疫分型分析

目的探讨流式细胞术（FCM）检测免疫表型在诊断急性髓细胞白血病微分化型（AML—M0）中的意义。方法采用多色FCM分析14例AML—M0病例的各相关抗原表达情况。结果14例AML—M0中,仅1例依据骨髓细胞形态学作出诊断,其余13例均依靠FCM作出明确诊断。在AML—M0中,髓系抗原CD33 14例（100％）表达阳性,CD13和CD117 9例（64％）表达阳性,CD34和HLA—DR12例（86％）表达阳性,一般成熟髓系相关抗原如CD16、CD10、CD14等均阴性,B和T淋巴细胞特异抗原如CD79a和CD3均为阴性,少数原始细胞表达细胞内髓过氧化物酶（MPO）、TdT。常常表达一些淋系但并不特异的抗原如CD7、CD2或CD19,但比淋巴细胞白血病表达的荧光强度弱。结论FCM免疫分型在AML—M0诊断中至关重要。     

髓系／NK前体细胞急性白血病1例的诊断分析

目的 分析1例髓系／NK前体细胞急性白血病（M／NKPAL）的诊断过程,提高对M／NKPAL的认识。方法 对1例M/NKPAL采用细胞涂片染色、细胞化学染色方法和流式细胞术进行细胞形态学和免疫表型分析,并应用细胞遗传学和PCR技术进行染色体核型分析及T细胞受体、白血病融合基因的检测,同时结合相关文献进行分析。结果骨髓原始细胞为90.4％,绝大部分白血病细胞形态学类似急性淋巴细胞白血病L2型,过氧化物酶染色阴性,偶见Auer小体。免疫表型为CD34、HLA-DR、CD33、CD7、CD56、CD38阳性和cyCD3弱表达,而cyMPO、CD3、CD4等阴性。存在染色体核型异常,未检测到克隆性T细胞受体基因重排和白血病相关的融合基因。结论M/NKPAL临床少见,诊断复杂,仅依据形态学难以诊断,应注意与伴有髓系抗原表达的T淋巴细胞白血病、急性髓细胞白血病微分化型、T／髓系混合表型急性白血病和母细胞性NK细胞白血病／淋巴瘤等相鉴别。     

急性髓系白血病Evil基因表达的研究

目的:探讨急性髓系白血病中Evil基因表达及意义.方法:应用RT-PCR方法检测了94例急性髓系白血病(AML,包括初治19例,复发23例,缓解22例)患者及10名正常对照Evil基因的表达.结果:10名正常人骨髓单个核细胞中无Evil mRNA的表达.AML患者Evil基因总的阳性表达率为18.1%(17/94),M5型未见表达,其中初治、复发、缓解组的Evil基因阳性率分别为26.5%、17.4%、0,初治和复发组差别无显著性(P=0.554).M3患者中Evil、PML/RARα双阳性组与PML/RARα单阳性组相比复发率高(P＜0.05),早期死亡率高.Evil阳性的AML患者多数生存期短.结论:Evil基因是一个原癌基因,在髓系白血病的发病中起重要作用,是髓系白血病预后不良的一个指标.     

儿童急性白血病免疫表型及临床意义

 目的　探讨儿童急性白血病的免疫表型特征和临床意义。方法　应用10种单克隆抗体,采用免疫酶标技术ABC-AP染色对76例白血病患者骨髓涂片进行免疫表型测定。结果　各型白血病主要表达该系列特异抗原,形态学与免疫学检查符合率为97.6 %（74／76）,1例形态学误诊被免疫分型纠正,1例形态学诊断不明经免疫分型确诊;淋系可见髓系抗原表达,髓系也可见淋系抗原表达,T-ALL与M3不表达HLA-DR。结论　白血病细胞免疫表型具有高度异质性;FAB与免疫分型同时结合可互相补充,提高诊断的准确率。     

P-gp在急性淋巴细胞白血病中的表达及临床意义

目的 :探讨 P-糖蛋白 (P- gp)在急性淋巴细胞白血病的表达 ,评估 P- gp与急性淋巴细胞白血病预后的关系。方法 :用流式细胞仪 ,采用直接免疫荧光法 ,检测 2 0例急性淋巴细胞白血病患者抗 P- gp单克隆抗体 U IC2及 CD3、CD5 、CD7、CD1 0 、CD1 9、CD1 3 、CD1 4 、CD33 、CD34的表达 ,被检患者外周血或骨髓中幼稚细胞数≥ 5 0 % ,并设非耐药细胞系 K5 6 2为阴性对照。结果 :2 0例 AL L患者中 8例 P- gp表达阳性 ,12例 P- gp表达阴性。 P- gp阳性表达率为 40 %。髓系抗原表达阳性者 P- gp阳性率为 33.3 % ,髓系抗原表达阴性者 P- gp阳性率为 45 .5 % ,P>0 .0 5。 2例急性 T淋巴细胞白血病 CD7表达阳性 ,且 P- gp表达皆阳性。 P- gp阳性组 8例患者中 5例治疗有效 ,P- gp阴性组 12例中 10例治疗有效 ,P- gp表达阳性者中 3例 6月内复发。结论 :急性淋巴细胞白血病 P- gp阳性表达率为 40 % ;P-gp表达与髓系抗原表达无相关性 ,与 CD7表达有关 ;P- gp表达阳性与急性淋巴细胞白血病化疗效果有相关性 ,P-gp表达阳性者易早期复发。     

双向分化肿瘤血管生成拟态分子机制初步观察

目的研究肿瘤细胞分泌Ⅳ型胶原和PAS阳性物质在血管生成拟态(VM)中的作用以及VM对肿瘤细胞表达VEGF的影响。方法收集双向分化恶性肿瘤石蜡包埋样本158例，制作成组织芯片，进行CD31和VEGF重复染色及Ⅳ型胶原染色。通过网格计数法，比较CD31与PAS阳性图案围成的管道面积差别及分布特点，以及有VM的点阵和无VM的点阵VEGF表达的差别。结果VM的基底膜样结构表现为PAS及Ⅳ型胶原阳性；无VM的双向分化肿瘤VEGF表达高于有VM者，其中在恶性黑色素瘤及腺泡型横纹肌肉瘤中，二者VEGF的表达差异有显著性(P<0．05)。结论PAS阳性物质和Ⅳ型胶原参与构建了VM的管壁，VEGF表达的差别表明VM的存在可以提供肿瘤细胞氧气和营养物质。     

The cell growth,morphology and immunocytochemistry of novel cell line established from a bone marrow of the patient with therapy-related myelodysplastic syndrome,entitled PC-MDS

We report on cell growth, morphology, and immunocytochemistry of the first human cell line, PC-MDS, derived from a bone marrow of a patient with therapy-related myelodysplastic syndrome who had no overt leukemia post-MDS phase. This cell population consisted of fast-growing mononuclear cells. Standard cytochemistry methods for detection of MPO, lipids, glycogen and ANAE gave results as follows: MPO and SBB negative while PAS and ANAE positive. Positive cytochemical staining and immunophenotype analyses indicated that PC-MDS cells have some characteristics of the early myeloid precursor cell. As the first t-MDS derived cell line it could be a new tool in evaluation of complex biology of MDS and also serves as a model for diverse in-vitro research.     

The development of acute myelomonocytic leukemia in a patient with acute lymphocytic leukemia

A diagnosis of acute lymphocytic leukemia (ALL) was made from a peripheral blood and bone marrow specimen from a 59-year-old woman. Typical-appearing lymphoblasts were positive for periodic acid-Schiff (PAS) reaction, but negative for peroxidase, Sudan black B (SBB) and non-specific esterase (NSE) stains. Lymphoblasts failed to form non-immune rosettes and had no surface membrane immunoglobulins. However, lymphoblasts exhibited an "Ia-like" membrane antigen and markedly stimulated allogeneic lymphocytes in a mixed lymphocyte reaction (MLR). These cytochemical and immunologic studies were considered characteristic of null-cell subtype of ALL. Thirteen months later, the peripheral blood and bone marrow specimens contained numerous myelomonoblasts characterized by a weak or negative PAS stain and strongly positive peroxidase, SBB, and NSE reactions. Electron micrographs of the bone marrow suggested that the majority of leukemic cells were myelomonocytic and a minority of cells were lymphoblasts. In addition, myelomonoblasts in liquid cultures appeared to differentiate into mature macrophages. These data suggest the development of acute myelomonocyte leukemia in a previous case of ALL.     

Hybrid leukemia and the 5q-abnormality

Most cases of acute leukemia with deletions of chromosome 5q (5q-) are acute myelogenous leukemia. 5q- in acute lymphoid leukemia is rare. We studied a case of acute leukemia with 5q- using morphologic, cytochemical, immune and molecular techniques. Morphologic and cytochemical techniques were consistent with ALL (FAB L-2, PAS+, MPO-, ASD-). TdT was present. Immune studies suggested a T-cell phenotype (CD5+, CD7+); however, there was no rearrangement of the T beta-cell receptor gene. Surprisingly, the leukemia cells also expressed the CD13 myeloid antigen. Dual staining analysis showed co-expression of lymphoid and myeloid antigens on most cells. Based on these data and a review of previous reports we suggest that acute leukemia associated with the 5q- abnormality can occur in an immature stem cell resulting in a hybrid leukemia.     

"Pure" monocytic or histiomonocytic leukemia: a revised concept.

In a series of 130 cases of acute leukemia studied by cytochemical staining techniques, 10 cases cytochemically diagnosed as "pure" monocytic leukemia were seen. Cytochemical staining of bone marrow aspirates from these patients revealed all leukemic cells to be Sudan black negative. No positive reactions were observed for peroxidase or naphthol AS-D chloroacetate esterase. All cases demonstrated strong alpha-naphthyl acetate esterase positivity; and fluoride-inhibited naphthol AS-D acetate esterase positivity was observed in 8 of 9 cases tested. The P.A.S. reaction showed diffuse fine to coarse granules. Oil red O stain was positive in 8 of 9 cases, and the beta-glucuronidase activity was strong in 5 of 9 cases. Light microscopy revealed cells with monocytic or histiocytic morphology. Electron microscopic studies in 2 cases demonstrated features consistent with leukemic monocytic or histiocytic morphology; none was suggestive of granulocytic or lymphocytic leukemia. Five of 6 patients treated with drug regimens including prednisone and vincristine entered a complete remission; the other obtained a partial remission. Two patients achieved complete remission after treatment with Adriamycin, 1 following a relapse. Three patients who received cytosine arabinoside as their only therapy died soon after treatment was commenced. It is suggested that the cytochemical similarity but morphological differences in those patients may be objectively used to group them as cases of histiomonocytic leukemia.     

Characterisation of blast cells during blastic phase of chronic myeloid leukaemia by immunophenotyping--experience in 60 patients

The blast cell population of 60 patients with chronic myeloid leukaemia in blast crisis (CML-BC) were analyzed with a panel of monoclonal antibodies to determine the cell surface antigen phenotypes. In addition, cytochemical stains periodic acid Schiff (PAS), myeloperoxidase (MP), Sudan black B (SBB) and terminal deoxynucleotidyl transferase (TdT) were also utilized for subtyping. Nineteen cases (31.6%) expressed lymphoid phenotypes characteristic of common ALL cells and one case with extramedullary lymph node crisis expressed T-cell surface phenotypes. Thirty cases (50%) expressed solely myelomonocytic surface antigens with significant TdT activity in three. Cytochemical stains contributed to recognize only 57% of these myeloid blasts. Seven cases (11.7%) were with a mixture of heterogenous group of cells expressing phenotypic characteristics for various haemopoietic cells of different lineage--five of them from the cells of non-lymphoid series (myelomono-erythromegakaryocytic series) and the other two with cells from both lymphoid and myeloid series. Additionally, in two cases (3.3%), the precursor cells reacted only with the erythroid monoclonals. Finally, in one case, the blast cells remained unclassified due to nonreactivity with any of the monoclonals used but expressed significant TdT positivity. The response to uniform vincristine and prednisolone (V + P) therapy has shown that lymphoid blast crisis cases were highly responsive in contrast to the cases with non-lymphoid blast crisis (complete remission rate 86 vs 21.4%). The results confirm the evidence of multilineage blast crisis involving either single or mixed haemopoietic differentiation pathway and the utility of having phenotypic characterisation for designing protocols for chemotherapy in the CML patients at the time of blast crisis.     

Ultrastructural and cytochemical characteristics of monocytes in various types of acute leukemias

We have examined ultrastructural and cytochemical characteristics of monocytes in lymphoblastic leukemia (ALL) patients and compared them to monocytes in acute monoblastic leukemia (AMoL) patients and acute myeloid nonmonoblastic leukemias (AML), respectively. Blood samples were prepared in the standard way for ultrastructural and cytochemical (alpha-naphthyl butyrate esterase and myeloperoxidase) analyses. Our results indicate that monocytes in ALL and acute phase AML have the same characteristics as the malignant ones in AMoL.     

Diagnosis of acute basophilic leukemia

De novo acute basophilic leukemia (ABL) is a rare form of acute leukemia. Most frequently, the blast cells are morphologically undifferentiated, and the recognition of the presence of coarse basophilic granules may be the first step in diagnosis of this rare disorder. These granules are metachromatic and MPO negative. Immunophenotyping shows myeloid markers and some more specifically associated antigens such as CD9 or CD25 which are strongly expressed. Lymphoid, erythroid or megakaryocytic markers are not significantly expressed. In the absence of basophilic granules, some cases are classified as AML M0 if they express myeloid markers, or undifferentiated leukemia if no markers are present. If specific immature basophilic or theta granules are present, only an electron microscopic study will enable the diagnosis of a basophilic lineage assignment. Some cases may be misdiagnosed if all these steps are not followed. After all these investigations, two types of ABL may be defined: 1-A pure ABL, monophenotypic with basophilic lineage involvement alone, which should be classified as AML M8 . Genetic studies in these cases are very important for understanding the leukemic process and in a few cases, we can suspect c-MYB oncogene involvement but further investigations are still necessary. 2- More frequently, acute leukemia can be a mixture of blasts from different lineages with an important but variable participation of mature or immature basophilic cells. These cases must be classified as AML/Baso or multiphenotypic acute leukemias and often present Phl -chromosomal abnormality.     

Some aspects of immunological and cytochemical markers in leukemia

Summary The value of immunological and of cytochemical markers for understanding of pathophysiology and for diagnosis in different subtypes of leukemia is discussed.Abbreviations used in this paper AML Acute myeloid leukemia - AMoL acute monocytoid leukemia - ALL acute lymphoid leukemia - ANAE acid -naphtyl acetate esterase - APh acid phosphatase - B-ALL (-CPL,-CLL) B-lymphoid acute (prolymphocytic, chronic) leukemia - C-Ag membrane antigen of common ALL - C-ALL common ALL whose cells react with a specifically absorbed antiserum against ALL of non-T, non-B variety - CLL chronic lymphoid leukemia - CPL chronic prolymphocytic leukemia - E rosettes with sheep erythrocytes - Ia immune response antigen - M-Ag myeloid antigen - POX peroxidase - SmIG surface membrane Ig - T-Ag T-lymphocyte antigen - T-ALL (-CPL,-CLL) T-lymphocyte antigen-positive acute (prolymphocytic chronic) lymphatic leukemia - TdT terminal deoxynucleotidyl transferase - UAL undifferentiated acute leukemia     

Heterogeneity of Leukemic Cells with Basophilic Features: Cytochemical, Ultrastructural and Immunophonotypic Analysis of 8 Cases

We have studied leukemic cells, derived from acute nonlymphocytic leukemia with basophilic features and basophilic crisis of chronic myelogenous leukemia (CML), by cytochemical and ultrastructural examination and analysis of surface markers. Cytochemical results varied from case to case, while the ultrastructural appearances of the granules were different from normal granules. The granules had more delicate granular matrices with or without myelinoid figures, whorled or scroll matrix, multivesicular bodies structures, theta granules, and crystalloid structures. Leukemic cells in all cases had myeloid surface markers with some degree of variability. In addition, they were occasionally positive for lymphoid markers, but not for CD10 and IgE receptors. The present results show that leukemic cells with basophilic features are heterogeneous in their morphology, cytochemistry and surface markers.