Validation and quality control of hematolymphoid neoplasm immunophenotyping by flow cytometry

cytometric immunophenotyping has evolved from two-parameter quantitative measurement of peripheral blood lymphocytes to five-or more parameter qualitative evaluation of bone marrow for hematopathology.Leukemia/lymphoma immunophenotyping represent an important addition to histomorphology in the diagnosis,classification and monitoring of hematolymphoid neoplasms.The complexity of five-or more parameter analyses and the interpretation of the data rely on standardization and validation of the instrument,the reagent and the procedure.In addition,clinical flow cytometry laboratories in U.S.are required to document proficiency testing,sample preparation,method accuracy,specificity,sensitivity and precision.CLSI and the U.S.-Canadian Consensus Conference have provided recommendations,but each laboratory is responsible for validating its own qualitative and quantitative procedures.This paper introduces the procedures for quality control of all levels of the operation in a clinical flow cytometry laboratory in USA.

Flowcytometric detection of immunoglobulinlight chain in hematolymphoid immunophenotyping

During B-cell development and maturation,the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH) and light-chain genes,rearrange to associate one of a number of variable,diverse,and joining gene segments.A single mature B-cell expresses an IgH chain and either a kappa or lambda light chain,which is known as allelic or isotypic exclusion.In normal or reactive conditions,lymphoid cells comprise the mixtures of lymphocytes with either kappa or lambda expression.

Application of CD45/SSC Gating Multiparameter Flow Cytometry in the Classification of Acute Leukemia——An Analysis of 139 Cases

In order to study the significance of flow cytometry immunophenotyping in the diagnosis of acute leukemia, CD45/SSC gating multiparameter flow cytometry (FCM) was utilized to analyze the immunophenotypes of 139 cases of acute leukemia. 139 cases of acute leukemia were enrolled in our hospital from April 1998 to April 2000. Morphological analysis and FCM immunophenotypic tests were conducted on all cases. Our results showed that CD45/SSC gating multiparameter flow cytometry immunophenotyping could reflect the origin of leukemic cells specifically. It is one of the important methods for the diagnosis of ALL, AML, and HAL. CD45/SSC gating multiparameter FCM analysis is a good technique for immunophenotyping. FCM immunophenotypic analysis can help improve the diagnosis and classification of acute leukemia, and extend the use of FCM in clinical practice.

Correlated Flow Cytometric Analysis of H－ras p21 and DNA Ploidy in Acute Myelogenous Leukemia

The flow cytometric immunoassay was used to study the correlation between the H-ras oncogene product p21 and the DNA ploidy in 30 de novo cases of acute myelogenous leukemia (AML). The results showed that 17 cases were negative for p21 expression and 13 positive for p21. The patients with positive p21 had higher percentage of bone marrow and peripheral blasts and lower peripheral leukocyte count. The expression of p21 had no influence on the therapeutic effect. Before treatment,DNA diploidy occurred in 18 cases including 13 p21 negative ones,and DNA aneuploidy was revealed in 12 cases including 8 p21 positive ones. Patients with positive p21 or having aneuploidy in complete remission were at risk for early relapse. Our results suggest that p21 may be involved in the process of leukemogenesis and progression in AML.

Application of Flow Cytometry in Examination of Immunophenotypes in Human Cells

[Objectives]This study was conducted to establish a method for detecting the immunophenotypes of venous blood CIK cells in healthy donors and patients with triple-negative breast cancer or lung cancer by flow cytometry,and to further analyze and discuss the proportion of cellular immunophenotypes such as CD3^(+),CD4^(+),CD8^(+),CD3^(+)CD8^(+),CD3^(+)CD56^(+)in CIK cells.[Methods]Human venous blood was drawn,then anticoagulated with heparin and isolated with lymphocyte isolation solution,and the relevant immunophenotypes were detected by flow cytometry after 21 d of culture.[Results]The expression of CD3^(+)CD8^(+)and CD3^(+)CD56^(+)in the venous blood CIK cells was significantly higher in healthy donors than that in triple-negative breast and lung cancer patients.[Conclusions]CD3^(+)CD8^(+)and CD3^(+)CD56^(+)CIK cells have high anti-tumor activity.

CLINICAL SIGNIFICANCE OF THE DETECTION OF MINIMAL RESIDUAL DISEASE IN CHILDHOOD B-ALL BY FLOW CYTOMETRY

Objective To detect the minimal residual disease in children with B-ALL and to evaluate its clinical significance by flow cytometry. Methods 58 childhood B-ALL cases were enrolled into this study and 33 MRD analyses were performed after remission induction therapy.Four-color combinations of fluorochrome labeled monoclonal antibodies against lymphocyte lineage related phenotypes were used to analyze leukemic cells with flow cytometry.The cells from normal bone marrow were used as controls.The combinations of phenotypes that reflect the antigen expression differences between leukemic and normal bone marrow cells on flow cytometry were considered to be the effective phenotype combinations in the first step screening.The effective phenotype combinations were then used to monitor MRD during the disease course after therapy began. Results 58 cases of childhood B-ALL were screened for MRD effective phenotype combinations.The effective phenotype combinations were identified in 89.7% of B-ALL cases in this study.Four-color phenotype combinations were composed of CD10/CD34/CD19 plus another effective marker such as CD38,CD58,CD66c,CD21.The senstitivity of this method was 0.01%,much higher than that of microscopic inspection.In 8 cases,their bone marrow microscopic inspection results showed no remaining leukemic cells;but with flow cytometry,the percentage of leukemic cells were 5.66%,0.36%,1.43%,0.069%,1.55%,2.7%,0.028% and 0.015%,respectively.In risk stratification,all these MRD positive cases were classified into high risk group for relapse and 1 case showed early relapse within 6 months. Conclusion The application of flow cytometry in MRD measurement can significantly improve the sensitivity of detection of remained leukemic cells in childhood B-ALL,and can provide more accurate information on disease progression as well as the efficacy of therapy,thus facilitate future treatment decisions and follow ups.

A Biphenotypic (Mixed Phenotypic) Acute Leukemia: Report of Two Cases with Immunophenotypic Study

Biphenotypic acute leukemia (BAL) is an uncommon clinical entity. It is a type of acute leukemia with features characteristic of both the myeloid and lymphoid lineages and for this reason is designated as mixed-lineage, hybrid or biphenotypic acute leukemia. As strict diagnostic criteria have only recently been established, the precise incidence among acute leukemia is uncertain, although it is likely to account for approximately less then 5% of all acute leukemia. BAL is now collectively considered as “mixed phenotype acute leukemia” (MPAL). We hereby report two cases of a rare disease, BAL from our institution in the light of morphology, cytochemistry, flow cytometry and review of literature regarding these cases are described.

Individualized leukemia cell-population profiles in common B-cell acute lymphoblastic leukemia patients

Immunophenotype is critical for diagnosing common B-cell acute lymphoblastic leukemia (common ALL) and detecting minimal residual disease. We developed a protocol to explore the immunophenotypic profiles of common ALL based on the expression levels of the antigens associated with B lymphoid development, including IL-7Rα (CD127), cytoplasmic CD79a (cCD79a), CD19, VpreB (CD179a), and sIgM, which are successive and essential for progression of B cells along their developmental pathway. Analysis of the immunophenotypes of 48 common ALL cases showed that the immunophenotypic patterns were highly heterogeneous, with the leukemic cell population differing from case to case. Through the comprehensive analysis of immunophenotypic patterns, the profiles of patient-specific composite leukemia cell populations could provide detailed information helpful for the diagnosis, therapeutic monitoring, and individualized therapies for common ALL.

Deep Immunophenotyping of Human Whole Blood by Standardized Multi‑parametric Flow Cytometry Analyses

Immunophenotyping is proving crucial to understanding the role of the immune system in health and disease.High-through-put flow cytometry has been used extensively to reveal changes in immune cell composition and function at the single-cell level.Here,we describe six optimized 11-color flow cytometry panels for deep immunophenotyping of human whole blood.A total of 51 surface antibodies,which are readily available and validated,were selected to identify the key immune cell populations and evaluate their functional state in a single assay.The gating strategies for effective flow cytometry data analysis are included in the protocol.To ensure data reproducibility,we provide detailed procedures in three parts,including(1)instrument characterization and detector gain optimization,(2)antibody titration and sample staining,and(3)data acquisition and quality checks.This standardized approach has been applied to a variety of donors for a better understanding of the complexity of the human immune system.

Correlative Study on the Expression of p53 and DNA Ploidy in Acute Nonlymphocytic Leukemia

We used the flow cytometric immunoassay to study the correlation between the tumor-suppressor gene product p53- and the DNA ploidy in 30 de novo cases of acute nonlymphocytic leukemia (ANLL).The results showed that 15 cases were negative and the other 15 cases were positive expression for p53. As compared with p53 negative (p53) cases, the patients with positive p53 (p53+) had higher percentage of bone marrow blasts and lower peripheral leukocyte and platelet counts,which had no influence on the complete remission rate. Before treatment, DNA diploidy was seen in 18 cases including 12 p53- cases, and DNA aneuploidy in 12 cases including 9 p53+. After therapy, aneuploidy could be transformed into diploidy.Patients with P53+ or having aneuploidy in complete remission were at risk for early relapse. We believe that p53 may be involved in the process of leukemogenesis and progression of ANLL.

CD34-Negative B-Lymphoblastic Leukemia/Lymphoma Presenting as Cutenous Lesions at Infancy: A Case Report

Acute lymphoblastic leukemia/lymphoma is a highly aggressive neoplasm of precursor lymphoid (blast) cells. There are 2 main subtypes based on lymphoid lineage;B lymphoblastic leukemia/lymphoma (B-ALL/LBL) and T lymphoblastic leukemia/lymphoma (T-ALL/LBL). B-ALL/LBL commonly presents with fever, fatigue, bone or joint pain, bleeding or anorexia (signs of bone marrow infiltration), lymphadenopathy, hepatosplenomegaly, involvement of skin, soft tissue and testes, with a predilection for the central nervous system. Immature cell markers, such as CD34 and TdT, can help to differentiate lymphoblasts from Burkitt lymphoma which, is considered a mature high-grade B cell lymphoma that mimics lymphoblastic lymphoma/leukemia. Unfavorable prognostic factors include: infancy and adult age of diagnosis, high white blood cell count, slow response to initial therapy, central nervous system involvement at the time of diagnosis and Minimal residual disease after therapy. We present a case report of a 4 months old infant seen at a Tertiary Hospital with a rare presentation of CD34 Negative B-lymphoblastic leukemia/lymphoma presenting as cutaneous lesions in infancy.

Laboratory Diagnosis of Acute Leukemia in Kenya: The Gaps and Opportunities

Acute leukemia (AL) is a malignant disease of the bone marrow in which hematopoietic precursors are arrested in an early stage of development. The diagnosis of leukemia and lymphomas, beyond morphology, is limited in low-resource countries including Kenya. Morphological diagnosis includes Cytological and Histological assessment of blood, bone marrow aspirates and tissues on suspected Acute leukemia patients. The World Health Organization (WHO, 2016) international guidelines on Acute leukemia diagnosis recommend that cytogenetic analysis, appropriate molecular genetics, Fluorescent in situ Hybridization (FISH) testing, and flow cytometric immuno-phenotyping should be done in addition to a morphologic assessment of Acute Leukemia. In facilities where resources are relatively available, immunophenotypic and genetic features have resulted not only in providing a more accurate leukemia diagnosis but also in identifying antigens or genes that can then be targeted for therapy. This article will look at the gaps in the diagnosis of Acute leukemia in low-resource settings like Kenya and opportunities available to improve diagnosis.

免疫表型在鉴别急性早幼粒细胞白血病与HLA-DR阴性急性髓系白血病中的应用

目的探讨免疫表型在急性早幼粒细胞白血病(APL)和HLA-DR阴性的急性髓系白血病(AML)之间的鉴别诊断中的价值。方法回顾性研究2014年至2024年间收治的42例APL患者和28例初治及复发的HLA-DR阴性AML患者。通过流式细胞免疫表型分析,比较两组患者CD64、MPO、CD7、CD11c、CD9、CD123等抗原的阳性表达率。利用χ2检验进行统计学分析,并应用ROC曲线评估CD9和CD123的表达模式,以及CD64^(+)MPO^(+)CD7^(-)CD11c^(-)对APL的诊断价值。结果AML组患者的CD64、CD9、MPO阳性率显著低于APL组,而CD11c、CD7阳性率显著高于APL组,差异具有统计学意义(P<0.05)。CD64^(+)MPO^(+)CD7^(-)CD11c^(-)的综合抗原积分表达模式在鉴别诊断APL方面的ROC曲线下面积(AUC^(ROC))为0.859,敏感性为93.8%,特异性为75.0%,CD9/CD123的鉴别诊断APL的AUC^(ROC)为0.919,敏感性为83.3%,特异性为84.0%;而联合应用CD9/CD123和综合抗原积分的诊断模式,AUC^(ROC)为0.955,敏感性为83.3%,特异性为100%。结论CD9/CD123表达模式及CD64^(+)MPO^(+)CD7^(-)CD11c^(-)联合应用可作为鉴别诊断APL与HLA-DR阴性的AML的重要依据.

儿童急性髓系白血病早期流式微小残留病检测与预后相关性的临床研究

目的:探索应用多参数流式细胞术(MFC)监测儿童急性髓系白血病(AML)诱导化疗后骨髓微小残留病(MRD)对预测预后的价值。方法:回顾性分析2015年8月-2022年12月武汉儿童医院血液科97例初诊AML患儿接受诱导化疗第Ⅰ周期和第Ⅱ周期后采用MFC检测骨髓MRD的结果,并分析其与预后的相关性。结果:97例AML患儿诱导治疗Ⅰ后57名患儿MRD阳性(MRD1^(+),58.8%),诱导治疗Ⅱ后仍有19名患儿MRD阳性(MRD2^(+),19.6%)。Kaplan-Meier生存分析结果显示37例(64.9%)MRD1^(+)接受移植治疗患儿预计3年总生存(OS)率明显高于20例(35.1%)未接受移植治疗患儿(84.6%vs 40.0%,P=0.0001)。35例MRD1^(+)MRD2^(-)患儿中,接受移植的25例患儿3年OS率高于10例未移植患儿(87.2%vs 70.0%,P=0.3229)。19例MRD1^(+)MRD2^(+)患儿3年OS率低于35例MRD1^(+)MRD2^(-)患儿(57.4%vs 81.8%,P=0.059)。12例MRD2^(+)移植治疗患儿3年OS率高于MRD2+7例未移植患儿(80.8%vs 14.3%,P=0.0007)。12例MRD1^(+)MRD2^(+)移植患儿和25例MRD1^(+)MRD2-移植患儿3年OS差异无统计学意义(80.8%vs 87.2%,P=0.8868)。7例MRD1^(+)MRD2^(+)未移植患儿3年OS明显低于10例MRD1^(+)MRD2^(-)未移植患儿(14.3%vs 70.7%,P=0.0114)。进一步多因素分析结果显示MRD2^(+)和移植治疗是患儿预后的独立影响因素(P=0.031,0.000),MRD1^(+)对预测97例患儿总体生存无统计学差异(P=0.902)。结论:诱导化疗Ⅱ后MRD阳性是AML患儿预后不良的独立危险因素。MRD2^(+)预示着更差的预后,可筛选出需接受移植的患儿。MRD2^(+)不是患儿移植治疗禁忌症尽早移植明显改善高危患儿的预后。

多参数流式细胞技术分析髓系白血病细胞相关免疫表型

目的 探讨常见的急性髓系白血病细胞相关免疫表型,并阐述其在临床中的应用价值。方法 应用多参数流式细胞技术分析47例急性髓系白血病(AML)患者骨髓标本中白血病细胞相关免疫表型,并对其免疫分型结果与治疗、预后的相关性进行统计分析,进而探讨AML免疫分型在临床中的应用。结果 47例AML患者阳性高表达的髓系抗原有CD33(93.62%)、CD13(89.36%)、MPO(89.36%)、CD117(82.98%)、CD64(38.30%);造血干/祖细胞CD分子阳性表达以CD38(93.62%)、CD34(65.96%)、HLA-DR(51.06%)为主;其他抗原以CD56(48.94%)、CD7(23.40%)、CD19(19.15%)为主。经相同时间治疗后,23例(48.94%)AML患者病情得到完全缓解,患者阳性表达T细胞系抗原CD7的完全缓解率显著低于阴性患者(P<0.05)。6例(26.09%)患者在1 a内复发,没有任何一种抗原表达情况与之有关(P>0.05)。随访结束后,有26例(55.32%)患者死亡,阳性表达NK细胞系抗原CD56和T细胞系抗原CD7患者的死亡率显著性高于阴性表达的患者,阳性表达B细胞系抗原CD19的患者死亡率显著性低于阴性表达的患者(P<0.05),幼稚标志与成熟标志不同步的患者死亡率高于幼稚标志与成熟标志同步的患者(P<0.05)。结论 髓系抗原CD33、CD13、MPO、CD117、CD64是AML诊断中可靠的抗原标志物,AML患者的免疫分型结果可为临床医生确定治疗方案和判断预后提供参考。

21色流式检测人非小细胞肺癌组织中免疫细胞亚群方案的建立

背景与目的肺癌组织的免疫微环境已成为关注的重点,随着多色流式的兴起,流式检测肺癌免疫微环境成为重要的手段,但多为检测细胞亚群占比或主要细胞亚群功能,无法同时对两者进行检测。因此本研究建立了一种可靠的21色流式方案,以检测人非小细胞肺癌(non-small cell lung cancer,NSCLC)肿瘤组织中免疫细胞各亚群。方法选用细胞膜表面抗体细胞分化簇(cluster of differentiation,CD)45、CD3、CD19、CD4、CD8、程序性死亡受体1(programmed cell death 1,PD-1)、CD39、CD103、CD25、CD127、趋化因子受体8(chemokine receptor8,CCR8)、CD56、CD11c、人类白细胞抗原(human leukocyte antigen,HLA)-DR、CD38、CD27、CD69、CD62L、CD45RA、CCR7和核酸染料L/D制定方案。首先对各抗体进行抗体滴定实验、电压优化、减一色染色和单色染色实验,确定各实验条件及检测方案后,采用6例健康成年志愿者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)标本验证方案的可行性;检测分析6例NSCLC患者的肿瘤组织样本。结果采用建立的21色流式方案检测了6例NSCLC患者的肿瘤组织样本,可分析出肺癌组织中各细胞亚群的占比及主要细胞群的免疫表型和分化情况。结论成功建立的21色流式方案适用于PBMCs和NSCLC组织样本检测,为监测肺癌中免疫微环境状态提供了一种有效的新思路。

青少年抑郁症患者淋巴细胞亚群免疫表型研究

目的探究青少年抑郁症患者与正常组的淋巴细胞亚群免疫表型的差异,为抑郁症的诊疗提供更多免疫学思路和实验室依据。方法分别对50例青少年抑郁症患者(患者组)和50例健康人群(对照组)通过流式细胞术进行淋巴细胞亚群检测,对检测结果进行分析比较。结果患者组总T淋巴细胞(CD3^(+))、辅助T细胞(CD3^(+)/CD4^(+))、抑制T细胞(CD3^(+)/CD8^(+))、总B细胞(CD3-/CD19^(+))及淋巴值显著高于对照组,CD4/CD8及NK细胞(CD3-/CD16^(+)/CD56^(+))表达显著低于对照组(均P<0.05)。结论青少年抑郁症患者可能存在免疫系统的失衡,特别是T细胞和B细胞的异常免疫反应。

流式细胞分析在慢性粒-单核细胞白血病诊断中的价值探讨

慢性粒-单核细胞白血病(chronic myelomonocytic leukemia,CMML)是一种罕见的异质性血液系统恶性肿瘤,其重要特征之一是持续性单核细胞增多,这也是与其他疾病鉴别的关键,尤其需要与反应性单核细胞增多症进行鉴别。随着检验技术的不断进步以及对人类单核细胞亚群了解的增加,一些新的鉴别方法广泛应用,包括选取特定表面标志物、单核细胞亚群分型等方式。然而,通过流式细胞术选择特定表面标志物进行慢性粒-单核细胞白血病的诊断存在灵敏度低和特异性差的问题,而且至今尚未发现完全符合条件的表面标志物。对单核细胞亚群通过CD14/CD16分型定量,以单核细胞亚群占比进行慢性粒-单核细胞白血病的诊断与鉴别已经得到大量研究支持。但一些学者仍对这一方法在诊断中的准确性提出质疑,本文就该方面问题作一文献综述。

CD4^(+)Foxp3^(+)调节性T细胞在急性髓系白血病中的变化及其预后意义

目的探究CD4^(+)Foxp3^(+)调节性T细胞(Tregs)在急性髓系白血病(AML)中的变化,并研究其与AML预后的关系。方法采用流式细胞术检测来我院就诊的初诊AML患者和健康对照组外周血Tregs水平;根据细胞核型将AML分为预后较好组、预后中等组和预后较差组,比较各组患者Tregs占CD4^(+)T细胞比例,研究外周血Tregs对AML预后的意义。结果AML患者外周血CD4^(+)Foxp3^(+)Tregs占CD4^(+)T细胞比例高于健康对照组(6.171%±0.292%vs.2.437%±0.144%,P<0.0001);但是各AML亚型中Tregs占CD4^(+)T细胞比例差异无统计学意义(P>0.05);预后较差组AML患者外周血Tregs占CD4^(+)T细胞比例高于预后较好组(7.316%±0.592%vs.5.179%±0.429%,P<0.01)。结论AML患者外周血CD4^(+)Foxp3^(+)Tregs占CD4^(+)T细胞比例提高,对AML患者预后评估有一定的意义。

多发性骨髓瘤患者骨髓单核细胞免疫表型特点及其临床意义

目的:探讨多发性骨髓瘤(MM)患者骨髓单核细胞免疫表型表达的特点及其临床意义。方法:采用流式细胞术检测67例MM患者和30例贫血患者(对照组)骨髓单核细胞免疫表型表达,筛选出与对照组有统计学差异的免疫表型。进一步采用单因素及多因素回归分析影响预后的危险因素,分析单核细胞免疫表型对MM预后的影响,进一步分析CD38+单核细胞与临床特点的相关性。结果:MM组中CD138+单核细胞比例(CD138+M%)、CD27+单核细胞比例(CD27+M%)、CD56+单核细胞比例(CD56+M%)显著高于对照组(P<0.05);而CD38+单核细胞比例(CD38+M%)、HLA-DR+单核细胞比例(HLA-DR+M%)显著低于对照组(P<0.01)。低CD38+M%组中位PFS低于高CD38+M%组;低CD138+M%、低CD27+M%、低CD38+M%、低HLA-DR+M%组中位OS显著缩短。Cox回归分析结果显示,低CD38+M%是预后的独立危险因素。低CD38+M%组受累/非受累游离轻链比值≥100比例及1q21+比例较高CD38+M%组明显升高(P<0.05);低CD38+M%组骨髓瘤细胞CD38-所占比例显著高于高CD38+M%组(P<0.05)。结论:MM患者骨髓CD38+单核细胞的表达与患者预后及临床特点密切相关,或可用来评估预后,指导治疗。