Flow cytometric identification of myeloid disorders by asynchronous expression of the CD14 and CD66 antigens

Abstract: Using a multiparameter flow cytometry assay enumerating cells positive for CD13, CD14 and CD66 antigens, we determined the asynchronous CD14/CD66 co-expression in unselected bone marrow and peripheral blood samples with suspected malignant blood disorders. CD14/CD66 co-expression >5% were found in 131/691 bone marrow samples. Only 55 of these exhibited an identifiable population in 2-parameter flow cytometry histograms. Of the 55 samples 43 (78%) came from patients with myeloid disorders; e.g. 11 with myelodysplastic syndromes, 15 with chronic myeloproliferative disorders and 17 with acute myeloid leukaemia. Only one of these 17 cases was a de novo case, while 8 were secondary to another malignant haematological disease and 8 were from the period after cytoreductive therapy. Notably, CD14/CD66 co-expression patterns were related to disease categories; e.g. in chronic myelomonocytic leukaemia and acute myeloid leukaemia following a dysplastic phase the co-expression displayed two subsets in peripheral blood, low-avidity CD14 and low-avidity CD66, respectively. The latter disease category also exhibited these 2 subsets in bone marrow. In all other cases, the CD14/CD66 co-expression in bone marrow was heterogeneous. In conclusion, abnormal CD14/CD66 co-expression might be a valuable parameter in defining asynchronous myelopoiesis in malignant myeloid disorders, especially myeloproliferative disorders and secondary acute myeloid leukemias.     

Characterizing CD43 expression in haematogones using multicolor flow cytometric analysis

Haematogones are precursor B cells commonly detected in small numbers in the bone marrow. Morphologically, haematogones can mimic lymphoblasts and are best distinguished using multicolour flow cytometry with antibody combinations. Haematogones show characteristic and reproducible patterns of antigen expression representing the B-cell maturation sequence. CD43 expression, widely seen in haematopoietic elements, has not been well characterized in haematogones. We demonstrate that CD43 is consistently expressed in haematogones in a reproducible pattern similar to that of CD10 when combined with CD20. We propose that in combination with other markers, CD43 can be useful in the identification of haematogones.     

Identification of the CD33-related Siglec receptor, Siglec-5 (CD170), as a useful marker in both normal myelopoiesis and acute myeloid leukaemias

Sialic acid-binding immunoglobulin-like lectin (Siglec)-5 or CD170 is a CD33-related receptor, containing cytoplasmic immune receptor-based tyrosine signalling motifs, that has previously been reported to be myeloid-specific like CD33 and thus may be useful in the characterization of both normal and malignant haemopoiesis. This study showed that Siglec-5 had a distinct expression pattern to CD33 both on normal myeloid cells and in acute myeloid leukaemia (AML). In normal bone marrow and cord blood, myeloid cells predominantly expressed Siglec-5 at the later stages of granulocytic differentiation. Siglec-5 was not expressed at significant levels by CD34+ progenitors either from bone marrow or mobilized peripheral blood. During in vitro myeloid differentiation of cord blood purified CD34+ cells, Siglec-5 was upregulated later than CD33. Siglec-5 expression remained absent or very low on cultured CD34+ cells, unlike CD33, which was present on almost all CD34+ cells by day 4. However, analysis of blasts from 23 patients with AML revealed aberrant expression of Siglec-5 with CD34 in 50% (seven of 14) of patients with CD34+ AML; 61% (14 of 23) of AML cases were positive for Siglec-5 with an increased frequency in the French-American-British subtypes M3-5 (80%) compared with M0-2 (25%). All 13 acute lymphoblastic leukaemic (ALL) samples tested, including a CD33+ ALL, were Siglec-5 negative. These results support the further evaluation of Siglec-5 antibodies in the diagnosis and monitoring of AML.     

Flow cytometric platelet enumeration utilizing monoclonal antibody CD42a

Summary We investigated a flow cytometric method with monoclonal antibody CD42a as a potential reference method for platelet enumeration by blood count analysers. Using peripheral blood samples from 25 healthy individuals we obtained significant (P < 0.0001) correlations between the results obtained by a FACScan method and similar cell counters (TOA Medical Electronics, Kobe, Japan): r = 0.960 (FACScan vs SSF), r = 0.958 (FACScan vs SE-9000A,B), r = 0.949 (FACScan vs K-4500A) and r = 0.954 (FACScan vs K-4500B). This flow cytometric method for counting platelets using the monoclonal antibody CD42a can be used to check the calibration of the platelet count by blood cell analysers.     

Flow cytometric analysis of cell-surface and intracellular antigens in the diagnosis of acute leukemia

To evaluate the usefulness of flow cytometric detection of intracellular antigens (Ags) in establishing proper lineage affiliation and its contribution to the diagnosis of acute leukemia, we studied 100 consecutive patients in whom acute leukemia was diagnosed between January 1997 and July 1998. Immunological classification was assessed using a three-line panel of monoclonal antibodies for phenotypic characterization of leukemic blast cells as proposed at the First Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Leukemia. We found 74 cases of B-cell lineage acute lymphoblastic leukemia (ALL), seven cases of T-cell ALL, and 19 cases of acute myeloid leukemia (AML). In this study cytoplasmic (cy) CD79a, cyCD22, cyCD3, and cyMPO were highly sensitive, specific B, T, and myeloid markers that were expressed in virtually all cases of B and T cell ALL and in all subtypes of AML. Applied in combination with immunophenotyping this knowledge led to improvement in diagnostic precision and refinement of immunological classification, ensuring the selection of the most appropriate therapy for the patients studied. In conclusion, intracellular Ags detection was of utmost importance in establishing correct lineage affiliation in cases lacking expression of B, T, or myeloid surface Ags or disclosing equivocal or ambiguous immunophenotypic features and in identifying biphenotypic acute leukemia. In combination with FAB morphology and immunophenotyping, we were able to reliably classify all patients with acute leukemia in this study.     

Flow cytometric analysis of membrane CD11b, CD11c and CD14 expression in acute myeloid leukaemia: relationships with monocytic subtypes and the concept of relative antigen expression.

Blast cells from 26 cases of acute myeloid leukaemia (AML) were examined, by single and "two-colour" flow cytometry, for relationships between membrane CD11b (monoclonal antibody OKM1), CD11c (KB90) and CD14 (Leu-M3). Increased expression of all three determinants was associated with myelomonocytic leukaemias, with their relative diagnostic value in discriminating monocytic (M4 and M5) from non-monocytic (M1, M2 and M3) subtypes being CD14 greater than CD11c greater than CD11b. However, the results also indicated, because of the heterogenous expression of CD11c in particular, and to a lesser extent CD11b, that the patterns or histograms of fluorescent staining were potentially more informative than an empirical subdivision of blasts into positive and negative subpopulations. In addition, analysis of phenotypic correlations by simultaneous two-colour fluorescence showed that the expression of CD11b and CD11c determinants by leukaemic myeloid blasts was highly correlated, in contrast to the expression of CD14 and CD11c which were relatively independent. Consequently, CD11c+ myeloid blasts almost always coexpressed CD11b whereas CD14+ cases of AML often comprised CD14+ CD11c+ and CD14+ CD11c- subpopulations. It is concluded from these observations that CD11c immunophenotyping is a useful supplementary investigation, particularly in CD14- cases of myelomonocytic leukaemia. However, it is also apparent that the presence of membrane CD11c per se is not lineage-specific and that the level of expression is perhaps a more discriminatory factor.     

Flow cytometric analysis of decay-accelerating factor (CD55) on neutrophils from aplastic anaemia patients

Summary. Using a flow cytometric analysis, CD55 (decay-accelerating factor), CD59 and CD58 have been measured on neutrophils from 12 aplastic anaemia (AA) patients who were long-term survivors after immunosuppressive therapy (IS), 17 healthy individuals, four patients with PNH, and six patients with other haematological disorders.
The neutrophils from normal control patients and the six patients with other haematological disorders showed 98 ±2% (mean ±SD) positive granulocytes for CD55. Corresponding values were low (12%, 26%, 51% and 58%) on the primarily PNH patients. Among the 12 AA patients examined, seven had normal and five low values (59% in two, 70%, 71% and 82%).
Among the five A A patients who showed CD 5 5 neutrophil deficiency, four had showed an incomplete response after the initial IS treatment and the other relapsed following an initial haematological complete response; three cases had a positive Ham's test and two were negative.
Our data suggest that the development of PNH clones is a frequent finding in A A long-term survivors, mainly in those who had shown an incomplete response following IS. Neutrophil CD55 expression analysis by flow cytometry could be useful to detect clonal evolution in these patients.     

流式细胞术分析冻存前后脐血CD34+细胞的分布

目的探讨低温冻存对脐血CD34+细胞的影响.方法采用流式细胞仪分析冻存前后脐血CD34+细胞百分率、CD45+细胞和CD34+细胞的荧光强度变化及死细胞群的分布情况.结果冻存后CD34+细胞占CD45+细胞的百分率[(0.84±0.39)%]明显高于冷冻前[(0.51±0.24)%](P<0.01),冻存前后CD34+细胞绝对数无明显变化[(9.372±6.072) ×106/L和(9.246±6.132)×106/L](P>0.05),冻存前后CD34+细胞百分率呈正直线相关(r=0.564, P<0.01).冻存后CD45+细胞荧光强度减弱(P<0.01),CD34+细胞荧光强度无明显变化(P>0.05);中性粒细胞比例下降,淋巴细胞和单核细胞比例增高.死细胞组分中以中性粒细胞为主,占81.52%;活细胞组分中以淋巴细胞为主,占59.44%.结论冻存后CD34+细胞占CD45+细胞的百分率增高,但低温冻存对CD34+细胞绝对数量影响不大.死细胞主要为较成熟的粒细胞,冻存后CD34+细胞的分析需排除死细胞的干扰.     

Flow cytometric analysis of P-glycoprotein in normal and leukemic cells

Summary Classical multidrug resistance is characterized by overexpression of a membrane protein, P-glycoprotein, which acts like a drug-extruding pump, reducing accumulation of cytotoxic drugs inside malignant cells. We have developed a simple method for detecting an intracellular epitope of P-glycoprotein in normal and leukemic cells by the monoclonal antibody JSB-1 and fluorescence-activated flow cytometry. Permeabilization of blood and bone marrow cells in unprocessed samples is achieved by a commercially available red blood cell lysing solution which excellently preserves the light scatter properties of leukocytes. The method is suitable for analyzing samples in clinical routine. Lower than 1% reactivity was seen in the lymphoid gate of normal peripheral blood and bone marrow samples as compared with over 60% of reacting cells in some leukemic samples. Twelve patients with acute de novo leukemia were studied at presentation, 13 patients at a refractory stage, and 28 in remission. There was a positive correlation between the P-glycoprotein and the CD 34 expression in acute myelogenous leukemia and an association between the P-glycoprotein expression and the blast count in both acute myelogenous and lymphatic leukemias.     

A whole-blood flow cytometric assay for leukocyte CD11b expression using fluorescence signal triggering

A flow cytometric assay for measurements of leukocyte CD11b expression in whole blood has been developed and evaluated. The method is based on triggering of the flow cytometer by a fluorescent pan leukocyte marker, RPE-CD45. This enabled flow cytometric analysis in whole blood, and avoidance of in vitro artefacts related to cell purification and hemolysis. Our methodological evaluation suggested the following routine procedure: sampling with sodium citrate as the anticoagulant, sample incubation at 22 degrees C, and mild sample fixation with 0.5% formaldehyde saline. The latter provided good sample stability during 24 h. Moreover, the assay provided good assay reproducibility, low labelling antibody consumption, and minimal sample manipulation (< 30 min) and acquisition time demands. The assay seems to reflect the CD11b expression of circulating leukocytes, and is also suitable for studies of agonist stimulated CD11b expression in leukocyte subpopulations in vitro. When full CD11b responsiveness to agonist stimulation is desired, samples should be incubated at 37 degrees C, but this also elevated CD11b expression in unstimulated samples. The present whole-blood technique is thus suitable for analyses of CD11b expression for both research and clinical routine laboratory use. The assay can easily be modified for measurements of other leukocyte antigens by use of other specific fluorescent antibodies.     

Flow cytometric analysis of defensins in blood and marrow neutrophils

Polymorphonuclear neutrophils (PMN) are vital in host defense against microbial infections. This study provides a flow cytometric method for the quantitative analysis of microbicidal peptides (defensins) in cells of PMN lineage. Rabbit neutrophil peptides, NP-2 and NP-5, were measured in all PMN and in subpopulations of PMN expressing 1-selectin. PMN lineage counts were made on Wright's-stained blood smears and marrow cytospins. Immunoreactivity for NP-2, and NP-5 was detected by using the alkaline phosphatase anti-alkaline phosphatase technique. The results show that marrow PMN express higher levels of NP-2 and NP-5 than blood PMN, p < 0.001 and that these levels are associated with elevated numbers of myeloid precursors. In both blood and marrow, NP-2 occurs in two PMN subpopulations and the mean fluorescence intensity of NP-2 is consistently higher than that of NP-5. Increased levels of defensins are observed in circulating PMN depicting the most 1-selectin p < 0.05. Immunocytochemical results indicate that PMN defensins reside in cytoplasmic granules and are not constitutively expressed on the cell surface. Furthermore, defensins are not detected in monocytes, eosinophils, lymphocytes and erythrocytes. The flow cytometric method described here provides a novel means of quantitating host natural defenses, allows the characterization of PMN subpopulations and has clinical applications.     

上海地区成人不同年龄组间CD4、CD8 淋巴细胞计数正常值调查

目的　了解上海地区成人CD4、CD8计数及CD4/CD8比值正常参考值范围。方法　选取 6 14例成年人不同年龄组的血标本 ,根据不同性别、不同年龄组分别进行比较 ,用流式细胞仪进行CD4、CD8计数检测 ,得出不同性别及不同年龄组的CD4、CD8计数及CD4/CD8比值 ,并进行分析。结果　CD4正常值在不同性别及年龄组之间差异无显著性 ,其正常平均值为 72 6 .99± 2 5 5 .2 1。CD8及CD4/CD8在不同性别及其年龄组之间差异有显著性。CD8计数平均值为 5 39.98± 134 .0 7。CD4/CD8平均值为 1.49± 0 .5 7。结论　上海地区成人CD4淋巴细胞计数较国外低 10 0 /mm3 左右。对获得性免疫缺陷综合征CD4平均值的诊断临界值建议可采用 40 0 /mm3 。     

Evaluation of a quantitative determination of CD4 and CD8 molecules as an alternative to CD4+ and CD8+ T lymphocyte counts in Africans

In the developed word, monitoring HIV-infected patients is routinely determined by CD4+ T lymphocyte absolute counts. The reference procedure, flow cytometry, is expensive, requires sophisticated instrumentation and operators with specific training. Due to these limitations, CD4 counting is often unavailable in developing countries. The Capcellia assay is an enzyme-linked immunoassay for quantitative determination of CD4 and CD8 molecules. We evaluated this method in West Africa on blood samples collected from 39 HIV-uninfected and 44 HIV-infected adult subjects. CD4 concentration ranges were determined according to the clinical stages of the disease. We then studied the relationship between the two methods in the HIV-infected patients. The Spearman's rank correlation was 0.61 (95% confidence interval: 0.38-0.76, P < 0.0001). Nevertheless, determination of limits of agreement revealed discrepancies between the two methods, especially for CD4 counts > 0.4 x 10(9)/l, which are discussed. We conclude that the Capcellia assay is a convenient means to determine the immunodepression level where flow cytometric instrumentation is unavailable, and can be complementary to CD4 T lymphocyte enumeration.     

Flow cytometric scoring system (FCMSS) assisted diagnosis of myelodysplastic syndromes (MDS) and the biological significance of FCMSS‐based immunophenotypes

In the myelodysplastic syndromes (MDS), the haematopoietic cells show various levels of abnormal maturation and differentiation, which can be detected by flow cytometry. Testing the anomalies of stage‐ or lineage‐specific surface antigens in CD34⁺ blasts can distinguish MDS from non‐clonal cytopenic diseases, and also reflect the pathological characteristics of MDS as a class of clonal diseases for providing new clues to basic research. The present study established a flow cytometric scoring system (FCMSS) based on theproportion and antigenic co‐expression of CD34⁺ blasts. This FCMSS showed good sensitivity and specificity (77·8% and 100%) in the assisted diagnosis of low‐risk MDS without chromosome anomalies, ringed sideroblasts and excess marrow blasts. Moreover, we explored and reported different modes of abnormal expression of CD34⁺ blasts antigens in different disease stages and analyzed the biological significance of the immunotypes for the first time. We found expression of mature myeloid antigens and lymphoid antigens gradually decreased, and early functional antigens gradually increased from low‐risk MDS with normal karytype to low‐risk MDS with abnormal karyotype then to high‐risk MDS. The patients with higher FCM scores were generally accompanied with HLA‐DR15 allele or hypocellular marrow. Evolution of clones and immunological factors might have influence on expression of antigens in CD34⁺ blasts.     

Flow cytometric DNA analysis of malignant lymphomas with primary bone manifestation

Summary Malignant lymphomas with primary skeletal manifestation have received controversial evaluation with regard to histological classification and histogenesis. Recent histological and immunohistological studies on the rare bone lymphomas conducted by our team, have shown that they do not differ from primary nodal lymphomas with regard to the spectrum of histological subtypes. The present flow cytometric DNA analysis of paraffin-embedded material from 17 lymphomas documented in the Bone Tumor Registry of Westfalia yielded the following distribution pattern of DNA ploidy: among 12 non-Hodgkin's lymphomas (NHL) (according to the Kiel classification) there was only 1 case of low grade malignancy; this centroblastic-centrocytic lymphoma showed a unimodal diploid DNA histogram. Of 11 highly malignant NHL, 6 were DNA hyperdiploid. Among the 5 cases of Hodgkin's lymphoma, 4 were DNA diploid, (1 nodular sclerosing, 3 mixed types) and one DNA tetraploid (lymphocytic depletion type). Comparison with data from the literature reveals that even with regard to DNA ploidy, malignant lymphomas primarily manifesting in bone do not differ from those of exclusively nodal manifestation.     

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.     

Flow cytometric measurement of reactive oxygen species production by normal and thalassaemic red blood cells

Reactive oxygen species (ROS) contribute to the pathogenesis of several hereditary disorders of red blood cells (RBCs), including thalassaemia. We report here on a modified flow cytometric method for measuring ROS in normal and thalassaemic RBCs. RBCs were incubated with 0.4 mM 2',7'-dichlorofluorescin diacetate (DCFH-DA), then washed and further incubated either with or without 2 mM H2O2. Flow cytometric analysis showed that RBC fluorescence increased with time; it increased faster and reached higher intensity (by 10-30-fold) in H2O2-stimulated RBCs as compared to unstimulated RBCs. In both cases, the antioxidant N-acetyl-l-cysteine reduced fluorescence, confirming previous reports that DCFH fluorescence is mediated by ROS. While the fluorescence of unstimulated RBCs increased with time, probably because of exposure to atmospheric oxygen, in H2O2-stimulated RBCs fluorescence decreased after 30 min. The latter effect is most likely related to H2O2 decomposition by catalase as both sodium azide, an antimetabolite that inhibits catalase and low temperature increased the fluorescence of stimulated RBCs. Washing had a similar effect, suggesting that maintenance of the oxidised DCF requires a constant supply of ROS. We next studied RBCs of beta-thalassaemic patients. The results demonstrated a significantly higher ROS generation by stimulated and unstimulated thalassaemic RBCs compared to their normal counterparts. These results suggest that flow cytometry can be useful for measuring the ROS status of RBCs in various diseases and for studying chemical agents as antioxidants.     

Flow cytometric analysis of cell proliferation kinetics during duodenal ulcer healing

Cell proliferation in the gastroduodenal mucosa of patients with duodenal ulcers was evaluated using flow cytometry. Forty patients with duodenal ulcers and 12 normal subjects were investigated. Biopsy samples were obtained during endoscopic examination and subjected to DNA analysis by flow cytometry. Thirty patients with duodenal ulcers were healed within 3 months with H₂ blockers (tractable or responsive ulcers), whereas 10 patients did not respond to treatment (intractable ulcers). The percentage of cells at the DNA-synthetic phase, an index of cell proliferation, was constant in the adjacent duodenal mucosa 2cm from ulcer margin and antral mucosa during duodenal ulcer healing. The index at the margin of tractable ulcers was elevated during the active stage (12.9 ± 1.3), peaked during the healing stage (15.4 ± 2.8) and returned to the same level at the scarring stage (10.9 ± 2.0) as normal controls (10.3 ± 1.7). However, the index was not elevated in intractable ulcers (10.3 ± 1.7 in the healing stage) and was smaller than in tractable ulcers. These data indicate that augmented mucosal cell proliferation at the ulcer margin plays an important role in duodenal ulcer healing and intractable ulcers are characterized by an abnormal failure to accelerate DNA synthesis to achieve ulcer repair.     

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.     

CD130 rather than CD126 expression is associated with disease activity in multiple myeloma

We analysed the expression of both components of IL-6R, CD126 the ligand binding protein and CD130 the signal transducing protein, on plasma cells from MGUS and multiple myeloma (MM) cases using flow cytometry. CD126 was detectable in 50% of either MGUS or MM patients without any change of expression during disease progression. In contrast, CD130 expression was up-regulated during tumoural expansion (43% of MM patients at diagnosis versus 88% at relapse). Finally, combining CD126 and CD130 expression we found a significant increase of the percentage of CD126+ CD130+ patients at relapse, underlying the crucial role of IL-6 response in the late stage of MM.