Monoclonal antibody ratios in malignant myeloid diseases: diagnostic and prognostic use in myelodysplastic syndromes

Based on a 6 1/2-year study of bone marrows (BM) from 74 consecutive adult patients with newly diagnosed primary myelodysplastic syndromes (MDS), we have evaluated the role of immunophenotyping (IP) for subclassification purposes with a selected panel of monoclonal antibodies (Mab). Compared to normal BM IP revealed increased numbers of Mab-defined immature myeloid cells (defined by Mab MY7 [CD13] and MY9 [CD33] (P less than 0.05). Furthermore, decreased numbers of mature myeloid cells (defined by Mab NAT-9 II:3F-6F [NAT-9]) (P less than 0.001) were demonstrated in all French-American-British (FAB) subtypes except refractory anaemia with sideroblasts (RA-S). Since expression of CD13 and CD33 antigens on immature myeloid cells is variable, IP based on a single Mab was often found to be non-informative. However, the construction of myeloid antibody ratios (MAR), designed to give higher figures with increasing numbers of immature myeloid cells and/or decreasing numbers of mature myeloid cells in the BM, disclosed increasing mean ratio values with progressing FAB subtypes. More significant however, was that different prognostic subgroups based on the MAR could be identified independently of the FAB classification. We conclude that the use of IP--especially when MAR is included--is useful in prognostic scoring systems in MDS.     

骨髓增生异常综合征免疫表型分析

目的：探讨免疫表型测定在骨髓增生异常综合征（MDS）诊断及分型中的价值。方法：采用一组系列相关单克隆抗体和流式细胞术对19例MDS患者免疫表型进行检测，并对其中的10例进行了细胞遗传学检查。结果：MDS患者骨髓单个核细胞（MNC）CD13，CD33抗原表达率平均分别为36．69％和41．86％，而T淋巴系抗原CD3的表达平均仅为14．49％，且随着低危的难治性贫血（RA）向高危的难治笥贫血伴原始细胞增多（RAEB）或难治性贫血伴原始细胞增多－转变型（RAEB-t)的进展，较早期的髓系抗原CD13，CD33及干（祖）细胞抗原CD34的表达升高，并伴有T淋巴系抗原CD3的表达降低。10例进行了细胞遗传学检查的患者中，5例有染色体核型异常，染色体核型异常的患者与染色体核型正常的患者在抗原表达上存在区别。结论：对MDS患者进行免疫表型检查有助于MDS的诊断分型研究。     

Characterization of CD34+, CD13+, CD33- cells, a rare subset of immature human hematopoietic cells

BACKGROUND AND OBJECTIVES: Hematopoietic progenitor cells that express CD34 are heterogeneous in their lineage affiliation and degree of maturation. Expression of CD13 and CD33 antigens indicates myeloid lineage association, but the precise sequence of expression of these two markers during differentiation is unclear. We noted the presence of CD34+ cells expressing CD13 but lacking CD33, a subset of cells not yet well characterized. In this report we describe the prevalence and the immunophenotype of this cell subset. DESIGN AND METHODS: We studied the immunophenotype of immature myeloid cells in human bone marrow samples from 11 healthy transplantation donors and in 4 cord blood samples. We used four-color flow cytometry and a large panel of monoclonal antibodies directed against lineage and differentiation-associated antigens. Three additional bone marrow samples were analyzed after immunomagnetic sorting of CD34+ cells. We focused our analysis on the subset of cells defined by the expression of CD34 and CD13 and the lack of CD33. RESULTS: We found CD34+, CD13+, CD33- cells in all 11 bone marrow and 4 cord blood samples studied. These cells represented 0.5 0.5% (mean SD) and 0.8 1.2% of mononucleated cells, respectively. CD34+, CD13+, CD33- cells appeared to be more immature than those expressing CD33 because of their light scatter characteristics (smaller size and lower granularity), the expression of markers associated with early hematopoietic cells (CD90, CD133 and CD117), and the absence of lineage-associated markers. INTERPRETATION AND CONCLUSIONS: These findings suggest that the expression of CD13 precedes that of CD33 during myeloid differentiation, and that CD34+, CD13+, CD33- cells are at an early stage of human myeloid cell differentiation.     

Clinical significance of phenotypic features of blasts in patients with myelodysplastic syndrome

Knowledge of the blast phenotype in myelodysplastic syndrome (MDS) would be valuable, as in other malignancies, but remains sparse. This is mainly because MDS blasts are a minor population in clinical samples, making analysis difficult. Thus, for this blast phenotype study, we prepared blast-rich specimens (using a new density centrifugation reagent for harvesting blasts) from blood and marrow samples of 95 patients with various MDS subtypes and 21 patients with acute leukemia transformed from MDS (AL-MDS). Flow cytometry revealed that a high proportion of the enriched blast cells (EBCs) from almost all patients showed an immunophenotype of committed myeloid precursors (CD34(+)CD38(+)HLA-DR(+)CD13(+)CD33(+)), regardless of the disease subtype. The cytochemical reaction for myeloperoxidase was negative in 58% of the cases. Thus, the EBC phenotype is more immature in MDS than in de novo acute myeloid leukemia. MDS EBCs often coexpressed stem cell antigens and late-stage myeloid antigens asynchronously, but rarely expressed T- and B-lymphoid cell-specific antigens. Markers for myeloid cell maturation (CD10 and CD15) were more prevalent on EBCs from low-risk MDS (refractory anemia [RA] and RA with ringed sideroblasts), whereas markers for myeloid cell immaturity (CD7 and CD117) were more prevalent on EBCs from high-risk MDS (chronic myelomonocytic leukemia, RA with excess blasts [RAEB], and RAEB in transformation) and AL-MDS. A shift to a more immature phenotype of EBCs, accompanying disease progression, was also documented by sequential phenotyping of the same patients. Further, CD7 positivity of EBCs was an independent variable for a poor prognosis in MDS. These data represent new, valuable information regarding MDS.     

Evaluation of apoptosis as a prognostic factor in myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a group of disorders characterized by peripheral pancytopenia despite normo- or hypercellular bone marrow. This is thought to be as a result of the apoptosis of haematopoietic bone marrow cells resulting in ineffective haematopoiesis. To clarify the relationship between prognosis and apoptosis and/or cell proliferation in the bone marrow, we studied 51 cases with MDS. Bone marrow biopsies were stained immunohistochemically for MIB-1 (marker for proliferating cells) and CD34 (marker for stem cells). Apoptosis was visualized by detection of DNA fragmentation using TdT incorporation of nucleotides on 3' ends of DNA (TUNEL technique) and expressed as the apoptotic rate. MDS patients included 32 with refractory anaemia (RA), one RA with ringed sideroblasts (RARS) patient, seven RA with excess of blasts (RAEB) patients, eight patients with RAEB in transformation (RAEB-t) and three patients with chronic myelomonocytic leukaemia (CMMoL). In addition, we also studied six cases with acute myeloid leukaemia (AML) arising from MDS (AML-MDS) and ten control subjects. Fatal pancytopenia was the cause of death in 19 out of 51 patients. The apoptotic rate was higher in MDS patients (5.5%) than in control subjects (0.6%) and AML-MDS patients (0.4%). The percentage of MIB-1 positive cells was higher in MDS and AML-MDS than in control. The percentage of CD34-positive cells was higher in AML-MDS, RAEB, RAEB-t and CMMoL patients than control subjects and RA patients. Our findings indicate the activation of both the proliferative and apoptotic rates in MDS. Poor prognosis correlated significantly with higher apoptotic rates, but not with percentages of MIB-1 and CD34-positive cells. Our results suggest that apoptosis might be a useful prognostic factor and inhibition of apoptotic mechanisms may induce leukaemic transformation in MDS.     

High la (HLA-DR) and low CD11b (Mo1) expression may predict early conversion to leukemia in myelodysplastic syndromes

The FAB classification of myelodysplastic syndromes (MDS) has been useful in predicting prognosis; however, additional methods are required to detect patients at high risk for early conversion to acute nonlymphoblastic leukemia (ANLL). Using a panel of monoclonal antibodies to myelomonocytic surface antigens (MMSA) and flow cytometry, we studied bone marrow cells from 26 patients with MDS of all five FAB subtypes. The MMSA studied included la (HLA-DR), CD11b (Mo1), CD14 (Mo2, My4), CD13 (My7), and CD33 (My9). Marrows were considered 'positive' for a given MMSA if the percentage of reactive cells exceeded the upper limit of the normal range. Twenty-four of twenty-six patients (92.3%) were CD13 (My7)+, suggesting that CD13 may serve as a diagnostic marker for MDS. Ten of twelve patients who developed ANLL during a median follow-up of 44 weeks were la(HLA-DR)+. The Kaplan-Meier estimated median time to leukemia (TTL) was 16 weeks for la+ patients and 88 weeks for la– patients (P = 0.004). All six patients who developed ANLL before 16 weeks from diagnosis were la+, while none of the la– patients converted to ANLL before 24 weeks. Nine of thirteen patients with low CD11b (Mo1) expression (<53% reactive cells) developed ANLL, compared with only two of 11 patients with high CD11b expression (>53% reactive cells). Kaplan-Meier estimated TTL was 29 weeks for patients with low CD11b, compared to 160 weeks for patients with high CD11b (P < 0.05). Patients who met both criteria, la+ and low CD11b, represented the poorest prognostic subgroup, with median TTL of 13 weeks compared with 88 weeks for the others (P = 0.017), la and CD11b patterns were not specific for MDS subtype, and their expression did not correlate with blast count. These data suggest that MDS patients whose bone marrow cells demonstrate high la (HLA-DR) and low CD11b (Mo1) expression represent a poor prognostic subgroup with short TTL. These patients may be candidates for early aggressive or investigational treatment.     

NF-kappaB constitutes a potential therapeutic target in high-risk myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a preneoplastic condition that frequently develops into overt acute myeloid leukemia (AML). The P39 MDS/AML cell line manifested constitutive NF-kappaB activation. In this cell line, NF-kappaB inhibition by small interfering RNAs specific for p65 or chemical inhibitors including bortezomib resulted in the down-regulation of apoptosis-inhibitory NF-kappaB target genes and subsequent cell death accompanied by loss of mitochondrial transmembrane potential as well as by the mitochondrial release of the caspase activator cytochrome c and the caspase-independent death effectors endonuclease G and apoptosis-inducing factor (AIF). Bone marrow cells from high-risk MDS patients also exhibited constitutive NF-kappaB activation similar to bone marrow samples from MDS/AML patients. Purified hematopoietic stem cells (CD34+) and immature myeloid cells (CD33+) from high-risk MDS patients demonstrated the nuclear translocation of the p65 NF-kappaB subunit. The frequency of cells with nuclear p65 correlated with blast counts, apoptosis suppression, and disease progression. NF-kappaB activation was confined to those cells that carried MDS-associated cytogenetic alterations. Since NF-kappaB inhibition induced rapid apoptosis of bone marrow cells from high-risk MDS patients, we postulate that NF-kappaB activation is responsible for the progressive suppression of apoptosis affecting differentiating MDS cells and thus contributes to malignant transformation. NF-kappaB inhibition may constitute a novel therapeutic strategy if apoptosis induction of MDS stem cells is the goal.     

Phenotypical characteristics of acute myelocytic leukemia associated with the t(8;21)(q22;q22) chromosomal abnormality: frequent expression of immature B-cell antigen CD19 together with stem cell antigen CD34.

Twenty-three acute myelocytic leukemia (AML) patients with t(8;21) chromosomal abnormality, all classified as M2 (French-American-British [FAB] classification), were investigated. Blastic cells from all patients were positive for the stem cell-associated antigens, CD34 and HLA-DR, and the immature myeloid antigens, CD13 and CD33. The nonblastic leukemic cells expressed the more mature myeloid antigens, CD11b and CD15, with loss of the immature phenotype. The incidence of positivities for the stem cell-associated antigens, CD34 and HLA-DR, in t(8;21) AML cells was significantly higher in comparison with those in other AML showing granulocytic differentiation (M2 or M3). AML cells with t(8;21) also showed some phenotypic abnormalities. Frequent expression of CD19 was found in the blastic population of t(8;21) AML (18 of 23 cases) without other B-cell antigens and Ig gene rearrangements. CD19 expression was confirmed by immunocytochemistry and Northern blotting. The CD19+ blastic cells coexpressed both CD34 and HLA-DR. In addition, CD33+ cells among the blastic fraction in t(8;21) AML cells were fewer in number than in those of M2 or M3 AML without t(8;21). Our findings indicate that leukemic blasts of t(8;21) AML commonly express CD19 while preserving the stem cell-associated antigens, and differentiate into the granulocytic pathway with discordant maturation such as low CD33 expression.     

An unclassifiable case of hypoplastic leukemia in old age treated successfully with vincristine and prednisolone

An 80-year-old male was admitted because of dizziness and palpitation. Laboratory investigation revealed pancytopenia. A bone marrow aspirate showed a markedly hypocellular marrow with 41.6% blast cells. Peroxidase activity was negative and PAS reaction was block positive in the blast cells. Surface markers of these cells were positive for HLA-DR antigen and partially positive for CD13 (MY7). Other markers, such as T, B or myeloid antigens were all negative. These blast cells were classified as L1 according to the FAB system but suggested essentially unclassifiable in cell differentiation. The patient was treated successfully with vincristine and prednisolone and induced into complete remission although repeated marrow examination findings revealed hypocellular. As for the classification of hypoplastic leukemia, lymphoid or primitive "stem cell" leukemia also should be considered as other categories of acute leukemias and be treated according to each case.     

微分化型急性髓系白血病5例临床分析

目的：探讨流式细胞免疫分型在微分化型急性髓系白血病（AML-M0）诊断中的作用。方法：分析5例AML-M0患者的临床资料,观察其骨髓细胞形态学和细胞化学染色的特征以及与流式细胞仪骨髓细胞免疫分型的符合程度。结果：5例患者骨髓细胞学均报告原始及幼稚淋巴细胞增多,POX染色阴性,PAS染色部分阳性,流式细胞免疫分型髓系标志CD13、CD33、CD117至少1项阳性,而淋巴系统及巨核细胞系统抗原阴性。结论：骨髓细胞形态学、细胞化学染色结合骨髓细胞免疫表型和细胞遗传学检查是诊断AML-M0的主要依据,其中原始细胞的免疫分型对诊断AML-M0是非常必要的。     

Analysis of CD34-positive cells in bone marrow from patients with myelodysplastic syndromes and acute myeloid leukemia and in normal individuals: a comparison between FACS analysis and immunohistochemistry

Abstract: In patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), expression of the hematopoietic stem cell marker CD34 has been associated with a poorer prognosis. CD34 is usually analyzed by flow cytometry (FC), but may also be analyzed using immunohistochemistry (IH). The present study was undertaken to compare these 2 methods. Bone marrow from 16 patients with MDS and 12 with AML and from 12 healthy young volunteers was studied. The expression of CD34 was analyzed with FC on fresh bone marrow cells and with IH on sections of paraffin-embedded bone marrow. The correlation between FC and IH was good both for patients with MDS (p<0.0001) and AML (p<0.01). However, in patients with a high number of CD34-positive cells, the FC method seemed to result in a higher percentage of positive cells compared to the IH method. In normal bone marrow, the ratio between the percentage of CD34-positive cells and the percentage of bone marrow blasts was approximately 0.8. In the whole group of MDS patients, this ratio was 1:1, while in patients with refractory anemia (RA) and ring sideroblastic anemia (RAS) it was 1.6. Patients with MDS differed significantly from patients with de novo AML, who showed a ratio of only 0.23 (p<0.01). We conclude that the FC and IH methods for measuring expression of CD34 are well-correlated in MDS and reasonably well correlated in AML. A stem cell phenotype is more commonly expressed on precursor cells from patients with MDS than from patients with AML.     

Shwachman-Diamond syndrome marrow cells show abnormally increased apoptosis mediated through the Fas pathway

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow disorder with varying cytopenias and a strong predilection to myelodysplastic syndrome (MDS) and acute myeloid leukemia. Previously, it was found that the percentage of CD34(+) cells in bone marrow and the in vitro colony formation from CD34(+) cells of patients with SDS were markedly reduced. For these reasons, and because apoptosis is central in the pathogenesis of bone marrow dysfunction in MDS, this study was initiated to delineate the role of apoptosis in the pathogenesis of the marrow failure. Eleven children with SDS were studied. Compared to normal controls, patients' marrow mononuclear cells plated in clonogenic cultures showed a significantly higher tendency to undergo apoptosis. The defect in SDS was found in patients with and without MDS. Patients showed a more prominent decrease in colony formation and increased apoptosis after preincubation with activating anti-Fas antibody. Fas expression on marrow cells from patients was significantly higher than from normal controls. The difference between patients and controls for Fas expression was also significant for the following cell fraction subpopulations: CD34(-)/CD38(-), CD34(-)/CD38(+), and CD34(+). In conclusion, SDS hematopoietic progenitors are intrinsically flawed and have faulty proliferative properties and increased apoptosis. Bone marrow failure in SDS appears mediated by increased apoptosis as the central pathogenetic mechanism. This increased propensity for apoptosis is linked to increased expression of the Fas antigen and to hyperactivation of the Fas signaling pathway.     

Primary myelodysplastic syndromes: diagnostic and prognostic significance of immunohistochemical assessment of bone marrow biopsies.

Material from 63 cases with primary myelodysplastic syndromes (P-MDS) (French-American-British [FAB] types: refractory anemia [RA] = 21; RA with ring sideroblasts [RARS] = 8; RA with excess of blasts (RAEB) = 10; RAEB in transformation (RAEBt) = 6; chronic myelomonocytic leukemia [CMML] = 10 and unclassifiable = 8, ie, bone marrow aspiration was inadequate and stringent FAB criteria were not applicable) was analyzed for bone marrow histologic and immunohistochemical patterns. Standard Giemsa, hematoxylin and eosin (H&E) and reticulin stains were used for morphologic assessment. To identify the cell lineage precisely, chloroacetate esterase staining and an indirect immunoperoxidase technique using mouse monoclonal antibodies CD15, CD68, HLA-DR, and rabbit polyclonal CD3 and UEA-1 (lectin) was developed on formalin-fixed paraffin embedded bone marrow biopsies (BMB). The immunohistochemical assessment permitted accurate identification of dysplastic features such as mononuclear and binuclear megakaryocytes, Pelger-Huet neutrophils, and binuclear erythroblasts. Additional bone marrow histologic and immunohistochemical features observed were heterogeneity of immunohistochemical staining in various cell lineages, megakaryocytic emperipolesis, alteration of bone marrow microarchitecture, intravascular clusters of hematopoietic cells, and the types of benign lymphoid aggregates. The nature of abnormally localized immature precursors (ALIP) was discerned. Three types of clusters of immature cells were found that were difficult to distinguish on Giemsa and H&E morphology, these were erythroid aggregates (n = 18); megakaryocytic aggregates (n = 4), and immature granulocytic and monocytic aggregates (n = 32). The bone marrow histologic and immunohistologic patterns permitted the identification of four groups of clinical relevance: Group 1, cases with predominant erythroid hyperplasia and without ALIP (n = 15); group 2, cases with prominent myeloid hyperplasia and presence of ALIP (n = 32); group 3, cases with hypoplastic MDS (n = 10); and group 4, cases with hyperfibrotic MDS (n = 6). Statistical analysis showed a significant difference in survival and leukemic transformation between groups 1, 2, 3, and 4, with cases in group 2 showing the worst prognosis with early death due to increased propensity to leukemic transformation and cytopenia-related complications (P less than .0001). We conclude that immunohistochemistry is feasible on routinely processed BMB and the information obtained is of diagnostic and prognostic importance in P-MDS. The phenotype of ALIP varies with the morphologic and histologic subtypes of MDS and the term should be reserved for cases in whom the clusters in the intertrabecular region are of myeloid (granulocytic and monocytic) lineage on immunohistochemistry.     

骨髓增生异常综合征患者骨髓CD34+细胞亚群及细胞周期分析

目的 探讨骨髓增生异常综合征(MDS)患者骨髓CD34+细胞亚群及细胞周期分布异常的临床意义.方法 采用流式细胞术检测50例(17例低危、33例高危)MDS患者、8例MDS转化的急性髓系自血病(MDS-AML)及25例对照组原代骨髓CD34+CD38+、CD34+CD38-细胞亚群及G0/G1期、S期和G2/M期细胞比例.结果 高危和MDS-AML组骨髓CD34+细胞比例[(2.29±2.17)%、(18.69±17.47)%]明显高于对照组[(0.36±0.49)%,P＜0.05].低危、高危及MDS-AML组CD34+CD38+细胞相对比例[(86.09±7.79)%、(81.68±11.82)%、(82.88±2.60)%]显著低于对照组[(92.21±3.85)%,P＜0.05],而CD34+CD38-细胞比例[(13.91±7.79)%、(18.32±11.82)%、(17.13±2.60)%]显著高于对照组[(7.79±3.85)%,P＜0.05].MDS组CD34+CD38-细胞比例与国际预后积分系统(IPSS)(r=0.493,P=0.001)、WHO分型预后积分系统(WPSS)积分(r=0.586,P=0.000)均呈正相关.低危、高危及MDS-AML组骨髓单个核细胞(BMMNC)中G0/G1期细胞比例[(94.52±4.32)%,(96.07±3.88)%,(94.65±4.55)%]明显高于对照组[(88.94±7.30)%,P＜0.01],而3组S期[(4.63±3.34)%,(3.45±3.80)%,(5.12±4.55)%]和G2/M期[(0.84±1.52)%,(0.48±0.74)%,(0.22±0.34)%]细胞比例明显低于对照组[(9.06±6.50)%,(2.00±2.93)%,P值均＜0.05],3组S+G2/M期细胞比例明显高于对照组(P＜0.01).CD34+CD38-细胞比例≤12.0%的MDS患者治疗有效率高于CD34+CD38-细胞比例＞12.0%的患者,但差异无统计学意义.结论 MDS患者原代骨髓CD34+细胞亚群分化异常,BMMNC存在G1期阻滞现象,提示CD34+细胞亚群和细胞周期测定可能有助于MDS患者的辅助诊断以及疗效和预后判断.     

Immunophenotypic analysis of myelodysplastic syndromes

BACKGROUND AND OBJECTIVES: In contrast with hematologic malignancies in which the value of immunophenotypic studies is well established, information on the immunophenotypic characteristics of myelodysplastic syndromes (MDS) is scanty. The main goal of the present study was to explore the immunophenotypic differences between patients with MDS and normal individuals, including changes in distribution of cell lineages as well as phenotypic aberrations and blockades in cell maturation pathways. DESIGN AND METHODS: In MDS the proportion of bone marrow CD34+ cells was higher than in normal patients but the most immature progenitors (CD34+CD38-) were less represented. By contrast the proportion of myelomonocytic CD34+ cells was greater than in normal individuals, translating into an increased myeloid/non-myeloid CD34+ hematopoietic progenitor cell ratio. RESULTS: This suggests that in MDS, the majority of CD34+ cells are already committed to the myeloid lineage. Upon analyzing the granulo-monocytic differentiation pathway, MDS patients showed an increased proportion of monocytic cells with a decreased percentage of cells of neutrophil lineage, leading to a lower neutrophil/monocytic cell ratio. Maturational arrests in the monocytic but not in the neutrophil differentiation pathway were observed. In refractory anemia with excess blasts in transformation (RAEB-t) such blockades mainly occurred during the earliest stages of differentiation but in the other MDS subtypes they occurred in later stages. INTERPRETATION AND CONCLUSIONS: Phenotypic aberrations occurred in 90% of patients and a high proportion of cases showed >=2 aberrations. In summary, our results show that, in addition to an abnormal distribution of the bone marrow cell compartment, MDS patients frequently show aberrant phenotypes and maturational arrests. Some of these features may help in cases in which the diagnosis of MDS is questionable.     

再生障碍性贫血和骨髓增生异常综合征患者骨髓CD34+细胞及其粒细胞集落刺激因子受体的研究

目的:检测再生障碍性贫血(AA)和骨髓增生异常综合征(MDS)患者CD34 细胞占骨髓单个核细胞(BMMNC)的比率及其表面粒细胞集落刺激因子受体(G-CSFR)的表达率。方法:用流式细胞术(FCM)检测13例AA、22例MDS及12例非血液病患者CD34 细胞占BMMNC的比率及其表面G-CSFR的表达率。结果:AA组与对照组、AA组与MDS组、MDS-难治性贫血(RA)组与难治性贫血伴原始细胞增多(RAEB)组BMMNC中CD34 细胞的比率比较差异有统计学意义(P<0.05),但G-CSFR的表达率差异无统计学意义(P>0.05)。多数重型AA(SAA)患者(3/4)及少数慢性AA(CAA)患者(1/9)BMMNC中的CD34 细胞少于0.1%。多数G-CSFR表达率低(<14%)的患者(7/9)外周血中性粒细胞减少;而表达率正常(14.0%~28.9%)的患者(1/6)很少见;表达率高(>28.9%)的患者(3/7)也可存在中性粒细胞减少。结论:造血干细胞减少是AA的主要发病机制之一,其表面G-CSFR的表达率不是影响AA的主要因素;MDS患者CD34 细胞比率升高是一个预后不良的指标,G-CSFR的检测可部分解释MDS患者外周血中性粒细胞减少的原因。     

Enzyme histochemical, immuno histochemical and electron microscopic studies of two cases of leukemic malignant histiocytosis.

Two cases of leukemic malignant histiocytosis had similar morphologic and enzyme histochemical findings. Large blasts with low nuclear/cytoplasmic ratios, occasional azurophilic granules, and immature nuclei with nucleoli were seen in peripheral blood and bone marrow smears. Case 1 had occasional erythrophagocytosis, while in Case 2 it was rare. They were peroxidase negative, and very strongly positive by alpha-naphthyl butyrate esterase stain, the latter being inhibited by sodium fluoride. Acid phosphatase stains were also very strongly positive and were inhibited with tartaric acid. They were also stained granularly with PAS. Surface marker analysis revealed myeloid surface antigens, CD11+, CD13+ and HLA-DR+ in Case 1, and CD11+, CD13+, CD33+ and HLA-DR+ in Case 2. Immunoperoxidase stains of bone marrow biopsies revealed that lysozyme was positive in both cases. S-100 protein was strongly positive in Case 1, but weakly so in the skin tumor and negative in the bone marrow of Case 2. Electron microscopy showed both cases to be myeloperoxidase negative and rich in cytoplasmic organelles, such as lysosomes, mitochondria, and endoplasmic reticuli. Nuclei were irregularly shaped and nucleoli were present in virtually all the cells. These findings suggest that the malignant histiocytes in these two cases derive from bone marrow macrophages, and S-100 protein can also be detected in monocyte-macrophage derived histiocytes.     

Proliferation and differentiation of myelodysplastic CD34+ cells: phenotypic subpopulations of marrow CD34+ cells

In a search for a mechanism to explain the impaired growth of progenitor cells in patients with myelodysplastic syndromes (MDS), marrow CD34+ cells were purified up to 94.9% +/- 4.2% for normal individuals and 88.1% +/- 17.6% for MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). Phenotypic subpopulations of these CD34+ cells were analyzed for CD38, HLA-DR, CD33, CD13, CD14, CD41 and CD3 plus CD19, in association with proliferative and differentiative capacities. The 15 studies performed included 12 MDS patients. Coexpression rate of CD13 significantly increased in the MDS CD34+ cell population with a value of 91.4% +/- 11.6% and ranging from 60.3% to 100%, and exceeded 99% in four studies, whereas that of normal CD34+ cells was 49.9% +/- 15.8%, ranging from 28.2% to 70.1% (P < .001). Coexpression rate of CD38, HLA-DR, CD33, CD14, and CD3 plus CD19 in MDS CD34+ cells did not significantly differ from that of normal CD34+ cells. The total number of colonies and clusters grown from 100 normal marrow CD34+ cells was 40.4 +/- 8.6, the range being from 27.2 to 50.3; this varied in MDS marrow CD34+ cells with a value of 34.0 +/- 28.7, the range being 0 to 95.9. The lineage of colonies and clusters promoted by MDS marrow CD34+ cells was predominantly committed to nonerythroid with impaired differentiation in 13 of 15 studies (87%). CD13 is first expressed during hematopoiesis by colony-forming unit granulocyte-macrophage and is absent in erythroid progenitors. Therefore, this study provides direct evidence for the lineage commitment of MDS CD34+ cells to nonerythroid with impaired differentiation and explains the mechanism of nil or low colony expression of MDS progenitor cells to erythroid lineage.     

CD34/QBEND10 immunostaining in bone marrow biopsies: an additional parameter for the diagnosis and classification of myelodysplastic syndromes

CD34/QBEND10 immunostaining has been assessed in 150 bone marrow biopsies (BMB) including 91 myelodysplastic syndromes (MDS), 16 MDS-related AML, 25 reactive BMB, and 18 cases where RA could neither be established nor ruled out. All cases were reviewed and classified according to the clinical and morphological FAB criteria. The percentage of CD34-positive (CD34 +) hematopoietic cells and the number of clusters of CD34+ cells in 10 HPF were determined. In most cases the CD34+ cell count was similar to the blast percentage determined morphologically. In RA, however, not only typical blasts but also less immature hemopoietic cells lying morphologically between blasts and promyelocytes were stained with CD34. The CD34+ cell count and cluster values were significantly higher in RA than in BMB with reactive changes (p<0.0001 for both), in RAEB than in RA (p=0.0006 and p=0.0189, respectively), in RAEBt than in RAEB (p=0.0001 and p=0.0038), and in MDS-AML than in RAEBt (p<0.0001 and p=0.0007). Presence of CD34+ cell clusters in RA correlated with increased risk of progression of the disease. We conclude that CD34 immunostaining in BMB is a useful tool for distinguishing RA from other anemias, assessing blast percentage in MDS cases, classifying them according to FAB, and following their evolution.