流式细胞术联合测定保存血小板表达的磷脂酰丝氨酸和CD62p

人类血小板通过不同的技术进行离体保存后，其特性改变差异较大。建立适用于各种保存血小板特性分析的流式细胞术（FCM）对保存血小板的质量监测和新保存方法研究有重要意义。本研究通过对FCM分析方法主要条件的优化评估，建立了联合测定血小板膜表面包括磷脂酰丝氨酸在内的多参数FCM定量分析方法。在标本制作中，血小板不需要洗涤即可直接进行标记，减少了血小板离心洗涤过程中的激活。除了FCM常用的同型对照外，中还将新鲜富含血小板血浆（FPRP）、凝血酶激活FPRP和液氮处理FPRP作为血小板标准阴性、阳性对照，直接应用于FCM分析，且效果良好。该方法中Gly-Pro-Arg-Pro乙酸盐（GPRP）的选择应用，既防上了血小板聚集和纤维蛋白的形成，同时可稳定血小板，减少制备过程中人为激活，克服了在血小板、Ca^2 和血浆共存条件下FCM定量分析血小板的困难，尤其是为深低温处理血小板包括PS在内的多参数分析提供了良好的方法基础。研究表明该方法标本处理简单，结果分析简便、准确，重复性好。作还通过低温、低渗或激活剂的方法制备了几种膜表面分子标记显不同的血小板应用于FCM分析，不仅证实了所建立的FCM分析方法在各种不同膜表面分子标记的血小板分析中的实用性，又表明了中制备的4种不同膜表面分子标记的血小板在FCM分析保存血小板方法学中的实用价值。     

Detection of residual neuroblastoma cells in bone marrow: comparison of flow cytometry with immunocytochemistry

BACKGROUND: Because the cytomorphologic examination of bone marrow (BM) aspirates appears not sensitive enough to detect residual neuroblastoma cells, two four-color flow cytometric assays using different combinations of CD9, CD81, CD56, CD45, and anti-GD2 were evaluated. METHODS: The sensitivity of the flow cytometric assays was assessed by spiking experiments in normal peripheral blood samples. Twenty-eight BM samples, 12 biopsies, and 3 peripheral blood stem cell (PBSC) preparations from 22 patients with neuroblastoma were analyzed. The results were compared with those of an anti-GD2 immunocytochemical reference assay. RESULTS: Flow cytometric and immunocytochemical analyses showed residual neuroblastoma cells in four BM samples. One PBSC preparation and 20 BM samples were negative for both assays. Four BM and two PBSC samples scored positive for the immunocytochemical assay but were negative for the flow cytometric tests. This was due to the limited number of cells that were flow cytometrically analyzed. A strong correlation between the flow cytometric and immunocytochemical tests was found (chi2 = 6.4, P = 0.011). CONCLUSIONS: When an equal amount of cells is analyzed, the sensitivity of the flow cytometric assays is to be about 10 times lower than that of the immunocytochemical test. However, the flow cytometric assays can be used to screen for residual cells in clinical samples with a sensitivity of one neuroblastoma cell in 10(4) to 10(5) normal mononuclear cells. Flow cytometry is simple, quick, and cost effective compared with immunocytochemistry. In addition, the flow cytometric assays can be used to screen for residual neuroblastoma cells in case of a GD2-negative primary tumor. Therefore we recommend flow cytometry for the detection of residual neuroblastoma cells.     

流式细胞术在成分血残留白细胞检测中的应用

目的 探讨流式细胞术(FCM)绝对计数法在成分血残留白细胞检测中的应用价值。方法 采用FCM绝对计数法检测不同稀释度的浓缩血小板样本中自细胞残留量，并与Nageotte血细胞计数板法比较。结果 两种方法计数微量白细胞具有高度相关性(r=0．9964，P＜0．001)。低浓度白细胞计数时，FCM法变异系数小于Nageotte计数板法。两种方法检测结果相比，Nageotte计数板法计数值偏低。结论 流式细胞术绝对计数法具有快速、客观、精确和重复性好等优点，是一种稳定的检测成分血中微量残存白细胞数的方法。     

Flow cytometric analysis of reticulocytes using an RNA-binding fluorochrome.

The reticulocyte count is an important parameter in the diagnosis of anaemia. The commonly used microscopic counting technique is, however, laborious and hampered by poor precision and low sensitivity. An alternative method has recently been developed, which is based on flow cytometric identification and quantification of reticulocytes after staining with an RNA-binding fluorochrome, thiazol orange. The two methods were compared both in a sample of healthy individuals and in patient specimens. The correlation between the methods was good, but the flow cytometric analysis consistently gave values approximately 1.5 times higher (reticulocytes in per cent of erythrocytes) than the microscopic technique. The coefficient of variation was significantly lower for the flow cytometric technique. Reference values for the reticulocyte count were determined and the effects of storage of specimens were evaluated. It is concluded that flow cytometric analysis of reticulocytes is more sensitive and has a better precision that the standard microscopic procedure.     

Multicenter evaluation of two flow cytometric methods for counting low levels of white blood cells

BACKGROUND: Flow cytometric methods can be used to count residual white blood cells (WBCs) in WBC-reduced blood products, which should contain fewer than 1 x 10(6) WBCs per unit (approximately 3.3 WBCs/ microL). In this study two flow cytometric methods for counting WBCs under routine conditions in nine laboratories were evaluated. STUDY DESIGN AND METHODS: Panels of red blood cells (RBCs), platelets (PLTs), and plasma were prepared containing 33.3, 10.0, 3.3, 1.0, and 0.3 WBCs per microL and counted with flow cytometric methods (either LeucoCOUNT, BD Biosciences, four laboratories; or LeukoSure, Beckman Coulter, five laboratories). Requirements were that at the level of 3.3 WBCs per microL, coefficient of variation was < or =20 percent and accuracy was > or =80 percent. Routine flow cytometric quality control (QC) data of WBC-reduced blood products from two laboratories were analyzed. RESULTS: At the level of 3.3 WBCs per microL, none of the laboratories met the requirements for all three blood products. The LeucoCOUNT method met requirements at more laboratories than the LeukoSure method for RBCs and PLTs, but the opposite was true for plasma. Routine QC data showed that >99 percent of the flow cytometric measurements for WBC-reduced products was below the 95 percent prediction interval at 3.3 WBCs per microL. CONCLUSION: None of the laboratories met the requirements for accuracy and precision for all three blood products. Nevertheless, routine results showed that in >99 percent of the products, WBC counts were below guideline limits. Therefore, both flow cytometric methods are suitable for QC with pass-fail criterion.     

Flow cytometric detection of platelet-reactive antibodies and application in platelet crossmatching

Background: Alloimmunization against HLA or platelet antigens can cause refractoriness to platelet transfusions in multiply transfused patients. Crossmatching of platelet concentrates is effective in overcoming this problem. Study Design and Methods: A flow cytometric assay was used for simultaneous detection of lymphocyte-reactive and platelet-reactive antibodies in a single sample using fluorescein isothiocyanate-labeled anti-IgG. This assay was compared with the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay in selected sera containing HLA and platelet antibodies. In a further study, this assay was compared with lymphocytotoxicity test results from thrombocytopenic patients, for whom platelet concentrates were ordered. The results of both assays were then correlated with the 1-hour corrected count increment, with a corrected count increment greater then 7500 considered as an adequate transfusion response. Results: The results of the MAIPA and flow cytometric assay in detecting platelet-reactive antibodies correlated well (p<0.0001, r = 0.84). The sensitivity and specificity of the flow cytometric assay in detecting platelet-reactive antibodies were 94.7 and 96.3 percent, when the MAIPA assay was taken as a reference. In unselected sera from patients, the sensitivity and specificity of the flow cytometric assays were, respectively, 72.7 and 91.7 percent in detecting lymphocyte- reactive antibodies and 70.6 and 77.7 percent in detecting platelet- reactive antibodies, when the lymphocytotoxicity test was used as a reference. With regard to an adequate transfusion response, the sensitivities and efficiencies were 20.0 and 82.1 percent, 33.3 and 84.3 percent, and 70.0 and 88.6 percent for the lymphocytotoxicity test and the lymphocyte-reactive and platelet-reactive flow cytometric assays, respectively. Conclusion: Flow cytometric crossmatching appears to be an effective method of detecting platelet-reactive antibodies that may affect the success of platelet transfusions. This procedure is well-suited for routine conditions and can be performed within 2 hours.     

Microbiology.

Although flow cytometry can detect microorganisms rapidly in low concentrations in some clinical samples, it has not proved cost-effective for clinical use in this application. Adaptation of fluorescence multiplexing methodology recently developed for flow cytometric bead immunoassays, and the introduction of hybrid cytometers based on microfluidic technology, however, may make it possible to place cost-effective cytometric apparatus in clinical microbiology laboratories in the near future.     

Flow cytometric analysis of DNA ploidy pattern in breast cancer: malignant potential and prognostic implication]

Flow cytometric DNA analysis in solid tumors has developed over the past ten years. In human breast cancer, flow cytometric analysis of DNA ploidy pattern recently reported were reviewed. There are conflicting data regarding the value of DNA ploidy pattern obtained by flow cytometric analysis for determining prognosis for breast cancer patients. Some data seem to indicate that S-phase fraction alone or in combination with DNA ploidy pattern may be more important prognostically than DNA ploidy pattern alone, although the results have not been uniform. These suggests that a consensus of determination for DNA ploidy pattern in breast cancer and further investigations on DNA ploidy pattern are required.     

More than just quality control

Providing quality flow cytometric results requires more than monitoring quality control data. Laboratories should standardize all aspects of testing and evaluate each one critically for opportunities to improve. This article discusses a complete quality management system that includes assay validation and change control, specimen collection and delivery, ordering of flow cytometric testing, sample preparation, verification of specimen integrity, flow cytometry data acquisition, analysis and interpretation, reporting of results, document of standard operating procedures, proficiency testing, training, and documentation of ongoing competency.     

多发性骨髓瘤免疫表型流式细胞术研究新进展

多发性骨髓瘤(multiple myeloma,MM)是一种常见的血液系统恶性肿瘤,以浆细胞恶性增殖、溶骨破坏为特征,表现为M蛋白、骨骼破坏、贫血、肾功能受损和免疫功能异常.通过流式细胞术(flow cytometry,FCM)检测其免疫表型特点,已经作为血液系统疾病的常用检测手段,在辅助诊断、评估预后以及监测微小残留病灶方面的价值,已经被越来越多的学者认同.本文就将免疫表型分析在MM中的应用进展方面作一个综述,包括流式细胞术在MM中的应用和MM中流式免疫表型的特点.     

Anti-D quantification by flow cytometry:a comparison of five methods

BACKGROUND Three flow cytometric methods for anti-D quantification have been published. All use different cell sensitization and antibody detection conditions that may lead to varied results. Therefore, a direct comparison of the three methods is timely. STUDY DESIGN AND METHODS: The published flow cytometric methods and two new in-house modifications were compared. Ten serum samples containing anti-D at levels between 11.6 and 915 IU per mL were selected for analysis, and each was tested a minimum of three times. Anti-D bound to cells was detected with fluorescence-labeled anti-human IgG reagents. RESULTS The interassay CV of the standard curves for each of the five methods was less than 10 percent. The intra-assay CV was consistently <10 percent with four out of the five methods, but, by the fifth method, it was >20 percent in more than one-third of the tests. In 72 percent of the sample and method combinations, the interassay CV was <25 percent. Plotting of the mean anti-D value for each sample as a percentage of the value determined by an automated technique (AutoAnalyzer) revealed wide variability between the methods. CONCLUSION: Anti-D quantification by flow cytometry is influenced by the serum antibody characteristics and the method used. The differences between the flow cytometric and AutoAnalyzer techniques indicate that further validation of the flow cytometric method is required before routine use.     

紫外线照射诱导人外周血淋巴细胞凋亡的研究

目的　探讨紫外线照射充氧自血疗法（ＵＢＩＯ）引起的免疫抑制机制。方法　使用不同剂量中、短波紫外线（ＵＶＢ、ＵＶＣ） 照射诱导人外周血淋巴细胞凋亡,采用电镜、ＤＮＡ 电泳、流式细胞仪ＴＵＮＥＬ凋亡定量检测,了解被照射细胞凋亡情况。结果　ＵＶＢ照射细胞发生典型凋亡。定量检测证实ＵＶＢ２００ Ｊ／ｍ２ 照射淋巴细胞凋亡发生率４８ ．２％ ,５００ Ｊ／ｍ２ 凋亡发生率７８．５ ％ 。ＵＶＣ照射细胞未见凋亡改变。双标记检测发现ＵＶＢ照射诱导的淋巴细胞凋亡几乎全部发生于ＣＤ３ ＋ Ｔ 细胞,随着照射剂量的加大,ＣＤ４＋ ／ＣＤ８ ＋ 比值逐渐减小。表明ＵＶＢ照射可诱导人Ｔ 细胞,特别是ＣＤ４ ＋ Ｔ 细胞发生凋亡,ＵＶＣ照射无凋亡诱导作用。结论　诱导淋巴细胞凋亡可能是ＵＢＩＯ引起免疫抑制机制之一,建议ＵＢＩＯ 采用短波紫外线。     

Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry

BACKGROUND: The laboratory determination of the level of fetal cells in maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper dose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the widely used manual Kleihauer-Betke test have prompted an increased utilization of flow cytometric methods for fetal cell detection in maternal blood samples. STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against fetal hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used in a multiparametric flow cytometric assay developed for the quantitation of fetal red cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton X-100 permeabilization prior to monoclonal antibody incubation was developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of the assay was assessed for linearity and precision and correlated with the Kleihauer-Betke acid elution method. RESULTS: The anti-HbF flow cytometric method showed good correlation with the Kleihauer-Betke method (r2 = 0.86) and superior precision with a CV < 15 percent for blood samples with > 0.1 percent fetal cells. Analysis of 150 blood samples from nonpregnant adults, including individuals with elevated HbF due to hemoglobinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent. CONCLUSIONS: The anti-HbF flow cytometric method for detection of fetal cells offers a simple, reliable, and more precise alternative to the Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the study of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies.     

流式细胞术检测类风湿关节炎滑液对中性粒细胞凋亡的影响

目的观察骨关节炎(OA)和类风湿关节炎(RA)患者滑液对中性粒细胞凋亡的影响。方法通过流式细胞仪及荧光显微镜检查19例RA和11例OA患者对体外培养粒细胞凋亡的影响。结果流式细胞仪及荧光显微镜检测的体外培养粒细胞自发凋亡发生率分别为43.6%和37.4%,加RA滑液组分别为18.3%和13.7%(P<0.01),加OA滑液组则分别为40.3%和31.2%(P>0.05)。结论RA滑液可显著降低粒细胞凋亡发生率,流式细胞仪检测凋亡较荧光显微镜检查更具敏感性。     

Analysis of RNA-protein interactions by flow cytometry

Flow cytometry, in combination with advances in bead coding technologies, is maturing as a powerful high-throughput approach for analyzing molecular interactions. Applications of this technology include antibody assays and single nucleotide polymorphism mapping. This review describes the recent development of a microbead flow cytometric approach to analyze RNA-protein interactions and discusses emerging bead coding strategies that together will allow genome-wide identification of RNA-protein complexes. The microbead flow cytometric approach is flexible and provides new opportunities for functional genomic studies and small-molecule screening.     

In vitro and in vivo persistence of reticulocytes from donor red cells

BACKGROUND: Reticulocytes are important in the phenotyping of transfused patients. Reticulocytes can persist in blood units for the shelf life of the unit. STUDY DESIGN AND METHODS: Temperature dependence of reticulocyte persistence was examined in vitro at 4, 24, and 37 degrees C by using thiazole orange staining and flow cytometric analysis. Two-color flow cytometric analysis was used to evaluate the persistence of donor reticulocytes in transfused patients. RESULTS: Flow cytometric analysis using thiazole orange demonstrated that persistence of reticulocytes in units of stored CPDA-1 blood was temperature-dependent. Reticulocytes disappeared over 13 and 6 days at 24 degrees C and 37 degrees C, respectively, but at 4 degrees C the reticulocyte count changed little over 35 days. Two-color flow cytometric analysis of reticulocyte antigens was used to follow donor reticulocytes in 14 transfusion events in nine different patients. Donor reticulocytes persisted through 24 hours in 75 percent of the patients and were detectable at 48 hours in three patients. CONCLUSION: This study demonstrates that reticulocytes persist during refrigerated storage; they are detectable in the circulation of most recipients for the first 24 hours after transfusion and in the circulation of a few recipients after 48 hours. These findings may have relevance for separation techniques based on reticulocyte density in samples drawn shortly after transfusion and for evaluation of reticulocyte counts in patients with hematologic abnormalities.     

Drug resistance in B-cell chronic lymphocytic leukemia: predictable by in vitro evaluation with a multiparameter flow cytometric cytotoxicity assay

BACKGROUND: Patients with B-cell chronic lymphocytic leukemia (B-CLL) often demonstrate variable responses to similar treatments. It would be highly desirable to develop a personalized therapeutic strategy for selection of appropriate drugs or regimens based on the drug sensitivity profiles of leukemic cells from individuals. METHODS: We applied a multiparameter flow cytometric drug cytotoxicity assay to evaluate drug effects specifically on B-CLL cells from 43 individuals after leukemic cells were incubated in vitro with fludarabine, chlorambucil, cladribine, or prednisolone. RESULTS: We demonstrated that different B-CLL cell populations from 43 individuals showed a marked variability in drug sensitivity. In vitro resistance to fludarabine was greatest in B-CLL cells with deletions of p53, a cytogenetic abnormality that is almost invariably associated with a poor therapeutic response clinically. CONCLUSIONS: In vitro drug sensitivity profiles analyzed by a multiparameter flow cytometric cytotoxicity assay may serve as a tool to facilitate individualized selection of appropriate drugs for treatment in B-CLL. Prospective trials will be needed to validate the clinical utility of this flow cytometric cytotoxicity assay.     

Bacterial infection (BI)-INDEX: an improved and simplified rapid flow cytometric bacterial infection marker

The aim of this study was to develop a rapid and simple flow cytometric bacterial infection marker. In this prospective comparative study, quantitative flow cytometric analysis of CD10, CD35, CD66b, CD282, and MHC Class I molecules on human neutrophils, monocytes, and B-lymphocytes from 141 hospitalized febrile patients with suspected infection and from 50 healthy controls was performed. We developed a flow cytometric marker of local and systemic bacterial infections, designated “bacterial infection (BI)-INDEX”, incorporating the quantitative analysis of CD10, CD35, MHCI, CD66b, and CD282 on neutrophils, monocytes, and B-lymphocytes, which displayed 90% sensitivity and 96% specificity in distinguishing between microbiologically confirmed bacterial (n = 31) and viral infections (n = 27) within a 1-h time-frame. We propose that our novel rapid BI-INDEX test will be useful in assisting physicians to ascertain whether antibiotic treatment is required, thus limiting unnecessary antimicrobial usage.     

Flow cytometric measurement of cell surface and intracellular antigens present in leukemia cells]

Recent advances in flow cytometric technology has enabled us to perform multiparameter analysis of cell surface and intracellular antigens. The clinical application of such analyses requires to establish the procedures of sample preparation, fixation, staining, instrument calibration and data analysis. This article describes the basic elements involved in establishing such procedures and explores the use of multiparameter flow cytometric analysis in the characterization of human leukemia cells. Flow cytometric measurements of cell surface and intracellular antigens of leukemia cells can provide important insights into the biologic features of these cells as well as significant diagnostic information.     

Validation of flow cytometric analysis of platelet function in patients with a suspected platelet function defect

Essentials

• The diagnosis of mild platelet function disorders (PFDs) is challenging.
• Validation of flow cytometric testing in patients with suspected PFDs is required.
• Flow cytometry has added value to light transmission aggregometry (LTA) in diagnosis of PFDs.
• There is fair agreement in diagnosing PFDs between LTA and flow cytometry.

Summary

Background

Light transmission aggregometry (LTA) is the most commonly used test for the diagnosis of platelet function disorders (PFDs), but has moderate sensitivity for mild PFDs. Flow cytometry has been recommended for additional diagnostics of PFDs but is not yet standardized as a diagnostic test. We developed a standardized protocol for flow cytometric analysis of platelet function that measures fibrinogen binding and P‐selectin expression as platelet activation markers in response to agonist stimulation.

Objectives

To determine the additional value of flow cytometric platelet function testing to standard LTA screening in a cross‐sectional cohort of patients with a suspected PFD.

Methods

Platelet function was assessed with flow cytometry and LTA in 107 patients suspected of a PFD in whom von Willebrand disease and coagulation factor deficiencies were excluded. Both tests were compared in terms of agreement and discriminative ability for diagnosing patients with PFDs.

Results

Out of 107 patients, 51 patients had an elevated bleeding score; 62.7% of the patients had abnormal platelet function measured with flow cytometry and 54.2% of the patients were abnormal based on LTA. There was fair agreement between LTA and flow cytometry (κ = 0.32). The discriminative ability of flow cytometric analysis in patients with an elevated bleeding score was good (AUC 0.82, 0.74–0.90), but moderate for LTA (AUC 0.70, 0.60–0.80). Both tests combined had a better discriminative ability (AUC 0.87, 0.80–0.94).

Conclusion

Flow cytometric analysis of platelet function has added value in diagnostics of PFDs in patients with unexplained bleeding tendency.