The CD69 early activation molecule is overexpressed in human bone marrow mast cells from adults with indolent systemic mast cell disease

We have analysed the quantitative expression of surface CD69 antigen on human mast cells (MC), from both normal and pathological bone marrow (BM) samples, using flow cytometry. Our major aim was to analyse whether CD69 is constitutively expressed by normal BMMC and to explore the possible differences between CD69 expression by BMMC from normal controls and patients suffering from different pathological conditions. The constitutive expression of surface CD69 was clearly demonstrated in BMMC; however, systemic mast cell disease (SMCD) patients showed significantly higher levels of surface CD69 expression than healthy controls (P < 0.001), chronic lymphocytic leukaemia (P = 0.001), monoclonal gammopathy of unknown significance (P < 0.001), multiple myeloma (P < 0.001) patients, and myelodysplastic syndromes (P = 0.002). Furthermore, almost no overlap between the levels of CD69 expression on BMMC was observed between SMCD cases and the remaining groups of individuals except for the paediatric mastocytosis group (P > 0.05). From the other groups of patients, monoclonal gammopathy of unknown significance (P = 0.04), myelodysplastic syndromes (P = 0.03) and paediatric mastocytosis (P = 0.003) cases showed a significantly higher expression of surface CD69 as compared to normal subjects. In summary, our findings show that the CD69 antigen is overexpressed in SMCD patients.     

Altered apoptosis and cell cycling of mast cells in bone marrow lesions of patients with systemic mastocytosis

Enhanced expression of the apoptosis-preventing protein bcl-xL and the cell cycle-regulating protein p21 was observed in bone marrow infiltrates of systemic mastocytosis. Expression of bcl-2, Ki67, and p53 as well as ISEL apoptosis staining were comparable in patients with mastocytosis and in controls. An altered rate of apoptosis and cell cycling may contribute to accumulation of mast cells in mastocytosis.     

Lymphoid cells and tissue mast cells of bone marrow lesions in systemic mastocytosis: a histological and immunohistological study

Systemic mastocytosis (SM) can be regarded as a tumorous proliferation of tissue mast cells (TMC) involving various organs, particularly the bone marrow. The infiltrates, however, are by no means composed exclusively of TMC, but also contain eosinophils and lymphocytes. The varying composition of the TMC infiltrates and the immunohistological characteristics of the lymphatic cells were the main subjects of investigation in this study. Three different types of bone marrow infiltrates could be identified: (1) A pure mastocytic infiltrate. (2) A mixed mastocytic/lymphocytic infiltrate. (3) A predominantly lymphocytic infiltrate containing loosely-scattered TMC. The mixed mastocytic/lymphocytic infiltrate seems to be a unique finding confined to the bone marrow in cases of SM, and is not detected in this conformation in other tissue sites normally involved in SM (spleen, liver, lymph nodes and skin), nor in cases of malignant mastocytosis. The lymphoid cells could be identified immunohistologically as being an admixture of T lymphocytes and B lymphocytes, while NK cells were virtually absent from the composite nodules. The TMC reacted strongly with antibodies (monoclonal or polyclonal) against vimentin, common leucocyte antigen, lysozyme, alpha 1-antichymotrypsin and alpha 1-antitrypsin, but were negative with a variety of other antibodies tested (UCHL1, MB1, Ki-B3, Leu-7, KL1, desmin, S-100 protein, F VIII-related antigen and chromogranin A).     

系统性红斑狼疮患者骨髓CD34+细胞表面标志及与病情活动的相关性分析

目的研究系统性红斑狼疮(SLE)患者骨髓CD34+细胞表面标志的变化,了解SLE患者造血干细胞是否存在异常。方法应用流式细胞术CD45/SSC设门分析10例SLE患者和10例正常人骨髓CD34+细胞CD90、CD117、CD123、CD164、CD166、CD95(FAS)、FAS-L、人类白细胞抗原(HLA)-DR等表面分子的表达及其与病情活动指标的相关性。结果活动期SLE患者骨髓CD34+细胞比例(1.5±0.4)%,明显低于正常人(2.3±0.8)%,P<0.01;非活动期患者(2.0±0.4)%与正常人相比差异无统计学意义。SLE患者CD34+、CD95+的表达明显高于正常人[(48.3±10.6)%vs(24.3±11.1)%,P<0.05],患者CDl23和CDl66也明显高于正常人[(45±22)%vs(20±4)%,P<0.05];[(31±20)%vs(11±6)%,P<0.05]。其余表面标志的表达与正常人相比差异无统计学意义。CDl23表达率与患者外周血白细胞计数负相关(r=-0.700,P< 0.05),与SLE疾病活动指数(SLEDAI)评分无相关性。CD166表达与SLEDAI(r=0.472,P<0.05),血清C3 (r=-0.712,P<0.01),尿蛋白定量(24h)(r=0.558,P<0.05)显著相关。结论SLE患者骨髓CD34+细胞CD95、CDl23、CDl66的表达率增加,CDl23的表达率与外周血白细胞计数显著负相关:CDl66的表达率与SLEDAI评分、24h尿蛋白呈显著正相关,与血清C3呈显著负相关,CDl66可能是一个新的SLE疾病活动性标志。     

Variation in cell yield and proliferative activity of positive selected human CD34+ bone marrow cells along the circadian time scale

Abstract: Variations in cell yield and proliferative activity of human bone marrow (BM) progenitor cells were determined with flow cytometry along the 24-h (circadian) time scale. Equal volumes of BM were aspirated every 5 h, altogether 5 times in 5 healthy men. An average 6-fold higher yield of positive selected CD34+ cells occurred in each subject when BM was aspirated during the daytime and late afternoon, while a lower yield occurred during the night. Using all CD34+ cell yield data normalized to percentage of mean, a significant time–effect was found by ANOVA (p=0.02) and a significant circadian rhythm was detected by the least-squares fit of a 24 h cosine (p=0.02). The 95% confidence limits of the acrophase (time of highest values) were computed to be at midday between 10:24 and 14:48 h. A highly significant correlation (p=0.001) was found between proliferation of positive selected CD34+ cells and the more mature myeloid precursor cells from the same BM aspirates, suggesting a common temporal pattern along the circadian time scale. However, no correlation was demonstrated between proliferation and cell yield of CD34+ selected cells, suggesting that mechanisms other than variation in proliferation may cause the circadian rhythm in stem cell yield. These circadian variations in stem cell yield and proliferation suggest that proper timing within 24 h may potentially be important regarding outcome from progenitor cell harvesting and treatment with haematopoietic growth factors.     

Analysis of natural killer-associated antigens in peripheral blood and bone marrow of multiple myeloma patients and prognostic implications

The aim of this study was to analyse the expression of NK-associated antigens in both peripheral blood and bone marrow lymphocytes from a large series of newly diagnosed multiple myeloma patients. 112 patients with untreated multiple myeloma (MM) were included in the study. 36 sex- and age-matched healthy volunteers were used as controls for peripheral blood (PB) studies and 14 for the bone marrow (BM) studies. Simultaneous stainings with the CD3/CD56, CD2/CD16 and CD8/CD57 monoclonal antibodies were systematically performed in PB and CD3/CD56 and CD2/CD16 in BM in order to analyse their relationship with the clinical and biological characteristics of the disease and survival. The expression of NK-associated antigens (CD56, CD16 and CD57) assessed within the lymphoid gate, was significantly increased (P < 0.001) in the PB of MM patients both in relative and absolute numbers. In the BM a significant increase in the percentage of CD56⁺ lymphocytes (P < 0.001) was also observed; in contrast, the proportion of CD16⁺ cells did not differ significantly from that of normal BM samples. The number of CD56⁺CD3^? lymphocytes increased significantly within high-risk patients (869 ± 671) as compared to intermediate (388 ± 212) and low-risk patients (274 ± 199) (P = 0.04). Moreover, patients with high values of CD56⁺CD3^? lymphocytes showed a statistically significant association with several adverse prognostic factors including anaemia, hypoalbuminaemia, renal failure, high β2M, DNA diploidy and high S-phase plasma cells. In addition, patients with higher absolute numbers of PB CD56⁺CD3^? lymphocytes displayed a poorer prognosis, whereas patients with higher values of CD57⁺CD8^? cells had a better outcome.     

Varying populations of CD59-negative,partly positive,and normally positive blood cells in different cell lineages in peripheral blood of paroxysmal nocturnal hemoglobinuria patients

CD59-antigen expression on the surface membranes of erythrocytes, granulocytes, monocytes, lymphocytes, and platelets was determined by flow cytometry in 34 healthy controls and 17 patients with paroxysmal nocturnal hemoglobinuria (PNH). In all PNH patients, CD59-negative erythrocytes accounted for > 10% of the total erythrocyte population. Two erythrocyte populations (CD59-negative and normally positlve or CD59-negative and partly positive), three populations (CD59-negative, partly positive, and normally positive), and one population (CD59-negative) were demonstrated in ten, six, and one patients, respectively. However, CD59-negative granulocytes did not account for > 10% of the total granulocytes in two patients, and one of them had only a CD59 normally positive granulocyte population. A particular granulocyte population extended over both CD59-negative and partly positive areas was shown in two patients. Two populations (CD59-negative and normally positive) and one population (CD59-negatlve) were demonstrated in monocytes and lymphocytes. CD59-negative lymphocytes accounted for >50% of the total lymphocytes in only two patients. Three patients had a CD59 normally positive lymphocyte population. Percentages of CD59-positive platelet population in normal controls were widely various. Therefore, it was usually difficult to discriminate between PNH-affected and normal platelets. Thus, the flow cytometric profiles of CD59-antigen expression varied not only between PNH patients but between cell lineages. The present results and our prior study indicate that CD59 flow cytometry using erythrocytes and granulocytes is most suitable for diagnosing PNH. © 1994 Wiley-Liss, Inc.     

Flow cytometric measurement of DNA S-phase in human bone marrow cells: correcting for peripheral blood contamination

Abstract: The relationship between bone marrow (BM) cells with S-phase DNA content and the amount of peripheral blood contamination estimated as percentage lymphocytes+monocytes (L+MO) present in BM samples has been investigated in a total of 136 BM aspirates and biopsy expellates from 35 hematologically healthy individuals. A significant negative correlation was demonstrated between total, erythroid and myeloid BM cells in S-phase and the percentage of L+MO in the aspirates (r = 0.84, 0.57 and 0.49, respectively; p<0.0001). Based on the equation of the slope of the regression line, a correction formula adjusting the measured value of BM cells in S-phase to varying amounts of L+MO percentage has been worked out for the total and erythroid BM cells. In contrast, highly proliferating myelomonocytic cells and CD34⁺ cells did not show any significant correlation between cells in S-phase and percentage L+MO, indicating that peripheral blood contamination of BM aspirates is not a problem regarding kinetic investigations of these cells. In conclusion, the described flow cytometric method of analysing BM aspirates estimates the degree of peripheral blood contamination, as well as make possible a correct estimation of the DNA synthesis of several BM populations. The method is especially applicable when frequent BM sampling is required.     

骨髓粒细胞与红细胞表面CD55 CD59缺失对血液疾病的诊断意义研究

目的探讨骨髓粒细胞、红细胞表面糖基化磷脂酰肌醇(GPI)锚定蛋白CD55、CD59缺失(CD55-、CD59-,也称PNH细胞)在血液系统疾病中的意义。方法采用流式细胞仪检测中山大学附属第一医院血液科2008年9月至2010年11月诊治的正常人、阵发性睡眠性血红蛋白尿、再生障碍性贫血(AA)、骨髓增生异常综合征(MDS)、急性髓细胞白血病(AML)、多发性骨髓瘤(MM)及营养不良性贫血患者外周血及骨髓中红细胞和粒细胞CD55、CD59缺失,并对结果进行分析。结果正常人骨髓粒细胞CD55-高于外周血(P<0.05);PNH患者骨髓红细胞CD55-、CD59-高于外周血(P<0.05);正常人、AA、MDS、AML、MM及营养不良性贫血各组间骨髓粒细胞CD55-表达无显著差异(P>0.05)。结论单一骨髓粒细胞CD55-表达升高特异性差。     

Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats

Background

The presence of paroxysmal nocturnal hemoglobinuria clones in the setting of aplastic anemia or myelodysplastic syndrome has been shown to have prognostic and therapeutic implications. However, the status of paroxysmal nocturnal hemoglobinuria clones in various categories of myelodysplastic syndrome and in other bone marrow disorders is not well-studied.

Design and Methods

By using multiparameter flow cytometry immunophenotypic analysis with antibodies specific for four glycosylphosphatidylinositol-anchored proteins (CD55, CD59, CD16, CD66b) and performing an aerolysin lysis confirmatory test in representative cases, we assessed the paroxysmal nocturnal hemoglobinuria-phenotype granulocytes in 110 patients with myelodysplastic syndrome, 15 with myelodysplastic/myeloproliferative disease, 5 with idiopathic myelofibrosis and 6 with acute myeloid leukemia.

Results

Paroxysmal nocturnal hemoglobinuria-phenotype granulocytes were detected in nine patients with low grade myelodysplastic syndrome who showed clinicopathological features of bone marrow failure, similar to aplastic anemia. All paroxysmal nocturnal hemoglobinuria-positive cases demonstrated loss of the four glycosylphosphatidylinositol-anchored proteins, with CD16⁻CD66b⁻ clones being larger than those of CD55⁻CD59⁻ (p<0.05). Altered glycosylphosphatidylinositol-anchored protein expression secondary to granulocytic hypogranulation, immaturity, and/or immunophenotypic abnormalities was present in a substantial number of cases and diagnostically challenging.

Conclusions

These results show that routine screening for paroxysmal nocturnal hemoglobinuria clones in patients with an intrinsic bone marrow disease who show no clinical evidence of hemolysis has an appreciable yield in patients with low grade myelodysplastic syndromes. The recognition of diagnostic caveats and pitfalls associated with the underlying intrinsic bone marrow disease is essential in interpreting paroxysmal nocturnal hemoglobinuria testing correctly. In our experience, the CD16/CD66b antibody combination is superior to CD55/CD59 in screening for subclinical paroxysmal nocturnal hemoglobinuria because it detects a large clone size and is less subject to analytical interference.     

急性髓系白血病骨髓单个核细胞CD11a的表达及临床意义

目的:探讨淋巴细胞功能相关抗原-1(LFA-1)的α链CD11a在急性髓系白血病(AML)的表达情况及临床意义。方法:采用免疫酶标ABC法检测25例初治AML患者和8例血液系统非恶性肿瘤患者为对照的骨髓单个核细胞CD11a的表达,并追踪观察AML患者的疗效。结果:CD11a在AML患者骨髓单个核细胞的表达率(37.02±13.30)%,明显低于对照组(87.13±5.38)%(P<0.05)。化疗后未缓解组AML患者发病时骨髓单个核细胞CD11a的表达率(47.09±10.55)较完全缓解组(29.11±9.36)%高(P<0.05)。结论:CD11a在AML患者骨髓单个核细胞表达异常,可能与白血病细胞逃脱机体免疫监控及从造血微环境释出有关。检测AML患者骨髓单个核细胞CD11a的表达水平对AML的预后判断有一定意义。     

高脂血症患者瘦素与补体调节蛋白CD55、CD59的关系研究

目的观察高脂血症患者血浆瘦素水平和补体调节蛋白CD55、CD59表达的关系。方法选高脂血症患者58例,年龄、性别、体重相匹配正常人21名作为对照,用ELISA法测定血浆瘦素,用流式细胞术测定外周血白细胞补体调节蛋白CD55及CD59的表达,分析瘦素水平和补体调节蛋白表达之间的关系。结果与正常对照比较,高脂血症患者血浆瘦素较正常对照组明显升高(非正态分布,中位数为8357 ng/L比5348 ng/L,P=0.024);高脂血症患者淋巴细胞CD55平均荧光强度及百分率较正常对照组明显下调[(2.07±0.26)比(2.34±0.44),(35.72±13.53)%比(44.14±15.67)%,P=0.012,0.022];血浆瘦素与TC、LDL、TG、体重指数、腰围及臀围正相关(r=0.285-0.451,P=0.011-0.000),与补体调节蛋白CD55、CD59表达不相关(P>0.05)。结论高脂血症患者血浆瘦素浓度升高,白细胞补体调节蛋白CD55表达下调,但瘦素水平与补体调节蛋白表达水平之间无数量关系,因此,瘦素可能不直接影响补体调节蛋白表达。     

A flow cytometric assay of CD34-positive cell populations in the bone marrow

Summary CD34⁺ BM cells form a heterogenous population of primitive stem cells and more mature progenitors committed to different lineages of differentiation. By combining CD45 expression with SSC, it is possible to separate immature cells from more diferentiated BM cells, and, by three-colour flow cytometry, analyse the antigens expressed in various subsets of cells. In this paper we show that in the normal BM at least four distinct CD34⁺ cell populations can be identified by their different patterns of CD45 expression and SSC. The most immature CD34⁺ cell population (0.1% of the BM cellularity) lacked all signs of lineage commitment and was CD45RA negative and only weakly CD45 positive. With increasing expression of the CD45 antigens, a second CD34⁺ population (0.2% of the BM cellularity) was formed expressing mainly primitive lymphatic antigens. However. 30% of the cells co-expressed B-cell line antigens and myeloid antigens. Cells committed to the myeloid cell line lost B-cell line antigens, gained CD45 antigen expression and SSC and formed two CD34⁺ cell population (0.2% and 0.1% of the BM cellularity. respectively) differing only with respect to the pattern of myeloid antigen expression and SSC characteristics. Similarly, differentiation along the lymphatic pathway implicated down-regulation of myeloid antigens, loss of the stem cell antigen and immature lymphatic antigens and gain of CD45 expression and mature lymphatic antigens.     

Phenotypic analysis of plasma cells in bone marrow using flow cytometry in AL amyloidosis

AL amyloidosis is an intractable disease resulting from a plasma cell dyscrasia which has a wide clinical spectrum. To investigate the phenotype of plasma cells in the bone marrow, a flow cytometric analysis was carried out in 10 patients with this disease (mean age, 57.8 ± 7.9 years) and controls with M-protein (positive controls, n=4) and without it (negative controls, n=8). All patients were shown to have either Aκ- or A$LD-immunoreactive amyloid deposits on the biopsied tissues.

On flow cytometry CD38⁺⁺CD19⁺CD56^-- cells (polyclonal plasma cells) showed no significant difference between patients (0.59 ± 0.37%) and either negative (2.25 ± 2.84%) or positive controls (0.38 ± 0.20%), while CD38⁺⁺CD19-CD56⁺ cells (monoclonal plasma cells) showed a significantly higher level in the patients (1.34 ± 1.54%) than in either negative (0.041 ± 0.004%, p<0.005) or positive controls (0.11 ± 0.09%, p<0.05). With respect to maturation of plasma cells, five of the patients (50%), three of the positive controls (75%) and all of the negative controls showed a dominant proliferation of mature subtype (CD45⁺MPC-1⁺CD49e^- or CD45⁺MPC-1⁺CD49e⁺). Immature (CD45⁺MPC-1^- or CD45-MPC-1^--) and intermediate (CD45^-MPC-1⁺CD49e-) subtypes were dominantly present in the bone marrow in 2 and 3 patients, respectively.

In AL amyloidosis monoclonal plasma cells producing M-protein can be easily and reliably detected in the bone marrow by flow cytometry. This analysis might provide plasma cell phenotypic markers useful for assessing the prognosis and for monitoring the response to treatment.     

Differential expression of CD2 on neoplastic mast cells in patients with systemic mast cell disease with and without an associated clonal haematological disorder

Recently, aberrant coexpression of CD2 and CD25 has been reported to reliably distinguish neoplastic mast cells from normal or so-called reactive mast cells. Such expression is included in the consensus diagnostic criteria for systemic mast cell disease (SMCD). In our study of patients with SMCD, we found CD2 expression to be more prevalent on mast cells from patients without an associated haematological disorder (P = 0.04). Furthermore, no correlation was found between mast cell CD2 expression and other clinicopathological features in these patients.     

AA和MDS患者骨髓CD+34细胞表面GM-CSFR的表达及意义

目的 探讨再生障碍性贫血（AA）和骨髓增生异常综合征（MDS）患者骨髓CD34^＋细胞表面粒细胞-巨噬细胞集落刺激因子受体（GM—CSFR）的表达情况。方法 用流式细胞术（FCM）和单克隆抗体技术检测14例AA、23例MDS和8例非血液病患者骨髓CD34^＋细胞表面的GM—CSFR表达率。结果 MDS组骨髓CD34^＋细胞表面GM-CSFR的表达率明显高于对照组及AA组（P〈O．05）,而AA组与对照组间差异无统计学意义（P〉0．05）。结论 AA患者造血干细胞没有质的缺陷;造血干细胞异常可能是MDS的主要发病机制之一。     

贫血患者外周血CD55-和CD59-细胞检测结果及其意义

目的检测贫血患者外周血中红细胞和中性粒细胞膜糖化磷脂酰肌醇(GPI)连接的补体调节蛋白CD55和CD59表达情况,探讨其临床意义.方法采用荧光素标记的CD55和CD59单克隆抗体,流式细胞术检测35例正常人、5例阵发性睡眠性血红蛋白尿症(PNH)、32例再生障碍性贫血、47例小细胞低色素性贫血、10例巨幼细胞性贫血、12例自身免疫溶血性贫血和15例造血系统肿瘤伴有贫血患者外周血中CD55和CD59-红细胞和中性粒细胞的百分率.结果正常人CD55-、CD59-红细胞和中性粒细胞的百分率均＜5%,PNH患者均＞40%,部分再障患者＞5%(均＜15%).约有50%的小细胞低色素贫血患者CD55-、CD59-红细胞＞5%(均＜15%),但中性粒细胞的结果全部正常.巨幼贫、自身免疫溶血性贫血和造血系统肿瘤伴有贫血患者的阴性红细胞和中性粒细胞比率均正常.结论利用流式细胞术同时检测外周血中CD55和CD59-红细胞和中性粒细胞是目前诊断PNH的最可靠,最敏感的方法,也可作为判断疗效的手段.本法也是临床鉴别诊断PNH与再障和小细胞低色素性贫血的较好方法.     

Amount of mpl on bone marrow haemopoietic precursor cells from healthy volunteers and patients with refractory anaemia

Using a non-isotopic ligand binding assay using multi-colour flow cytometry, we quantitatively examined the amount of mpl in megakaryocyte-platelet lineage cells. Firstly, we quantified the amount of mpl on cell lines. Mpl gene-transfected BaF3 cells expressed a large amount of mpl , whereas original BaF3, K562, HL-60 and NOMO-1 cells showed no mpl . In bone marrow cells from healthy volunteers, mpl was expressed on CD34⁺ cells from the very early stage of differentiation when they had no CD38 antigen. The amount of mpl increased with differentiation to CD34⁺CD41⁺ cells, but decreased with further differentiation to CD34⁻CD41⁺ cells. In CD34⁺CD41⁺ cells the amount of mpl varied according to cell size: abundant in large cells, moderate in medium-size cells and a little in small cells.
In bone marrow cells from patients with refractory anaemia (RA), the amount of mpl was decreased compared with that in bone marrow cells from healthy volunteers. When analysed by the same CD phenotype and same cell size, the amount of mpl was less in RA patients compared with that in healthy volunteers in all phenotypes and sizes tested. The proportion of large CD34⁺CD41⁺ cells was less in RA patients than in normal volunteers.