Antigen expression on residual blast cells in acute megakaryoblastic leukemia (AMKL, classified as AML-M7 by FAB criteria) may change after treatment, potentially affecting both immunophenotypic characterization and minimal/measurable residual disease (MRD) monitoring. This study aimed to characterize post-therapy immunophenotypic alterations in AMKL and to determine whether specific patterns of antigenic change exist between samples obtained at initial presentation (IP group) and those obtained at MRD-positive status after therapy (MRD group). This retrospective descriptive study included 110 patients diagnosed with AMKL at Hebei Yanda Lu Daopei Hospital between January 1, 2009 and December 31, 2024 (male:female = 57:53; 103 pediatric and 7 adult cases). Immunophenotypes at initial diagnosis and after treatment were analyzed by flow cytometry. The chi-square test was used to compare antigen expression between the IP and MRD groups. Flow cytometric immunophenotypes differed by at least three antigens (including CD33, CD61, and CD42b) between initial presentation and post-therapy samples. Compared with the IP group, the MRD group showed a significantly higher frequency of loss of megakaryocytic markers, including CD61 (11/109, 10.1% vs. 30/109, 27.5%; p < 0.05) and CD42b (6/106, 5.7% vs. 22/101, 21.8%; p < 0.05). Partial loss of CD13 expression was also more frequent in the MRD group (18/99, 18.2% vs. 2/83, 2.4%; p < 0.05). No significant differences were observed in the expression of progenitor-associated markers (CD34, CD117), myeloid markers (CD33, CD11b), or other antigens (HLA-DR, CD7, CD56, CD42a) between the two groups (p > 0.05). Lineage-specific markers MPO and CD22, the monocytic marker CD14, and lymphoid markers CD10 and CD5 were negative in both groups. In contrast, aberrant expression of cCD3 (2/89, 2.2%) and CD19 (3/85, 3.5%) was observed in a small subset of IP cases. Overall, 100 of 110 patients (90.9%) showed changes in at least one antigen after therapy. By lineage category, alterations were most frequent in megakaryocytic markers (CD61, CD42b, CD41a, CD42a; 64/110, 58.2%), followed by myeloid antigens (HLA-DR, CD33, CD13, CD11b; 54/108, 50.0%), progenitor-associated antigens (CD34, CD117; 53/110, 48.2%), and lymphoid antigens (CD7, CD56; 24/107, 22.4%). In addition, CD110 was consistently expressed in all 26 AMKL cases tested, whereas only 18% (9/50) of non-AMKL AML cases were CD110-positive (p < 0.05). Significant immunophenotypic differences, particularly involving CD61, CD42b, and CD13, exist between IP and MRD samples in AMKL. Antigenic shifts affecting megakaryocytic, myeloid, progenitor-associated, and lymphoid markers are common after chemotherapy. For MRD assessment, the use of more specific megakaryocytic markers such as CD110, together with comprehensive multiparameter flow cytometry panels, may improve detection accuracy.

Objective: In minimal residual disease (MRD) analysis after allogeneic hematopoietic stem cell transplantation (allo-HSCT), abnormal immunophenotyping is commonly considered as evidence of a secondary recurrence or complications, leading to overtreatment. We aimed to confirm whether such phenotypic abnormality might originate from donors using multicolor flow cytometry (MFC). Materials and Methods: The MRD of bone marrow specimens of 3395 patients who had received allo-HSCT were analyzed using the conventional two-tube, eight-color MFC panel. The frequencies of abnormal immunophenotypes were also evaluated in three groups of patients without malignancies. Results: The frequency of new abnormal polymorphisms was 0.088% (3/3395) among patients who received allo-HSCT. The abnormal cells seen in three patients in complete remission were Fcγ receptor IIIB (FcγRIIIB) gene deletion (CD16- neutrophils), CD2-CD159a-CD159c+ natural killer (NK) cells, and monoclonal B lymphocytosis (MBL), respectively. In addition, abnormal T-cells (CD4+CD8+) were detected in one donor before allo-HSCT. Identical abnormalities were found in the peripheral blood of the corresponding donors of the three patients via MFC. Among the individuals without malignancies, the incidence of FcγRIIIB deletion was 0.2% (11/5256), that of NK cells with the absence of CD2 and single-positive CD159c was 0.05% (1/2000), that of monoclonal CD4/CD8 double-positive T-cells was 0.05% (1/2000), and that of MBL was 1.3% (14/1100). The frequency of NK cells with the absence of CD2 was 1.3% (1/79) and with CD8dim was 14% (11/79) in NK cell lymphoma. The following abnormalities could be identified by the two-tube, eight-color MFC panel: cκ/cλ/CD19/CD5/CD20/ CD38/CD45/CD56 (adding CD10 and CD34 as the ninth and tenth colors) and CD16+CD56/CD5/CD3/CD7/CD4/CD8/CD2/CD45 (adding CD117 as the ninth color). Conclusion: Abnormalities in recipients of allo-HSCT detected by MRD analysis may originate from their donors. Screening of donor specimens with a suitable two-tube, eight- to ten-color MFC panel may be a promising method for minimizing misdiagnoses.

International Journal of Laboratory HematologyVolume 45, Issue 1 p. e10-e14 LETTER TO THE EDITOR Full spectral flow cytometry analysis of the bone marrow immune cells in patients with myelodysplastic syndrome Man Chen, Man Chen Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorMinjing Fu, Minjing Fu Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorWei Zhao, Wei Zhao Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorAixian Wang, Aixian Wang Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorXueying Wu, Xueying Wu Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorMeiwei Gong, Meiwei Gong Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorJunyi Zhen, Junyi Zhen Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorGuanlan Yue, Guanlan Yue Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorHui Wang, Corresponding Author Hui Wang ldpwanghui@163.com orcid.org/0000-0002-3470-4159 Hebei Yanda Lu Daopei Hospital, Langfang, China Correspondence Hui Wang, Hebei Yanda Lu Daopei Hospital, Hebei, Langfang 065201, China. Email: ldpwanghui@163.comSearch for more papers by this author Man Chen, Man Chen Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorMinjing Fu, Minjing Fu Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorWei Zhao, Wei Zhao Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorAixian Wang, Aixian Wang Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorXueying Wu, Xueying Wu Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorMeiwei Gong, Meiwei Gong Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorJunyi Zhen, Junyi Zhen Hebei Yanda Lu Daopei Hospital, Langfang, ChinaSearch for more papers by this authorGuanlan Yue, Guanlan Yue Beijing Lu Daopei Hospital, Beijing, ChinaSearch for more papers by this authorHui Wang, Corresponding Author Hui Wang ldpwanghui@163.com orcid.org/0000-0002-3470-4159 Hebei Yanda Lu Daopei Hospital, Langfang, China Correspondence Hui Wang, Hebei Yanda Lu Daopei Hospital, Hebei, Langfang 065201, China. Email: ldpwanghui@163.comSearch for more papers by this author First published: 11 August 2022 https://doi.org/10.1111/ijlh.13945Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Open Research DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author, Hui Wang, upon reasonable request. Volume45, Issue1February 2023Pages e10-e14 RelatedInformation

Minimal residual disease (MRD) detection is an important prognostic parameter in patients with refractory or relapsed B-cell acute lymphoblastic leukemia (R/R B-ALL). CD79a has been reported to exhibit a high degree of linage-specificity for B-cell differentiation, with a specificity of 88% and a sensitivity of 100%. In this study, we investigated the efficiency and prognostic role of cytoplasmic CD79a (cCD79a) antibody-gated multicolor flow cytometry (MFC) in MRD detection in patients with B-ALL who received CD19-targeted chimeric antigen receptor (CAR) T-cell therapy bridging to allogeneic hematopoietic stem cell transplantation (allo-HSCT). The retrospective analysis was carried on to 59 patients who accepted allo-HSCT after CD19-CAR-T infusion from June 2016 to May 2017. The MFC MRD statuses before and after allo-HSCT were both strongly correlated with the transplantation prognosis, the MFC panel with cCD79a gating can effectively monitor MRD after CD19 CAR T-cell therapy and predict the prognosis after allo-HSCT. Trial registration: ClinicalTrials#: ChiCTR-IIh-16008711.gov: NCT03173417. Registered 30 May 2017 - retrospectively registered, https://www.clinicaltrials.gov/.

Acute myeloid leukemia (AML) is the most common heterogeneous hematopoietic malignancy in adults, accounting for 80% of acute leukemia cases.1 Chromosomal translocation is the most explored in hematological malignancies, which results in rearrangement of proto-oncogenes or key transcription sites often forming new fusion proteins. The AML1-ETO and PML-RARA, respectively, result from the chromosomal translocation t(8;21)(q22;q22) and t(15;17)(q24;q21), and they are the common chromosomal translocations in AML; however, simultaneous occurrence of both translocations in one patient is rare. Here, we first report a case of concurrent AML1-ETO and PML-RARA using flow cytometry sorting (FACSorting) flowed by morphology, immunophenotyping, fluorescence in situ hybridization (FISH), and molecular technique. A 17-year-old boy was admitted to our hospital with fever, vertigo, and scattered bleeding petechiae on the skin and mucous membranes over the whole body in April 2018. The patient had been diagnosed with AML by another hospital. Routine blood examination revealed white blood cells count of 14.68 × 109/L, hemoglobin concentration of 79.5 g/L, and platelet count of 34.1 × 109/L. The karyotype 2 showed 45,X,-Y,add(1)(p36.1),der(8)ins(8;21)(q22;q22q22)inv(8)(p23q22),del(11)(p13p14-15),t(15;17)(q24;q21),der(21)ins(8;21)(q22;q22q22) (Figure 1A). Among the 500 interphase nuclei analyzed, nuc ish (ETO, AML1)×3(ETO con AML1×1)[474]/(ETO×2, AML1×3)(ETO con AML1×1)[25]/(ETO, AML1)×2[1] (Figure 1B,C). Combined with the chromosome results of Figure 1A, it is suggested that insertion translocation of chromosome 8 and chromosome 21 was occurred followed by inversion (Figure 1B,C). Combined with the chromosome results of Figure 1A, it is suggested that insertion translocation of chromosome 8 and chromosome 21 was occurred followed by inversion (Figure 1B,C). Bone marrow smears observed 90% immature myeloid cells with two kinds of morphological characteristics. The majority AML patients possessed the features of PML-RARA (Red arrow), with irregular nuclei, visible distortion or folding, many azurophilic granules, and visible internal and external plasma (Figure 1D). The minority AML patients had the morphological features with AML1-ETO fusion gene (Green arrow), containing many azurophilic granules, and obvious superficial staining areas in the nuclear folds (Figure 1D). PML-RARA and AML1-ETO fusion genes were detected by RT-qPCR of 15.3% and 228.33%, respectively. WT1 and ASXL2 gene mutations were found by next generation sequencing. Flow cytometric analysis revealed two distinct aberrant cell populations: (a) CD117- immature myeloid cells and (b) CD117+ myeloid blasts (Figure 1E). These results together confirmed the previous AML diagnosis, and the co-expression of AML1-ETO and PML-RARA fusion genes. Moreover, the patient was treated with 10 mg arsenious acid for 2 days, 10 mg NIT for 3 days, 4 mg omacetaxine for 3 days, 100 mg cytarabine for 7 days, 3.25 g refined realgar for 7 days, and 20 mg tretinoin for 28 days. One month later, reexamination revealed 50% of bone marrow involvement; PML-RARA and AML1-ETO fusion genes were detected at 221.9% and 27.3%, respectively, indicating that chemotherapy was ineffective. Transplant was indicated for the patient; however, the patient died prior to transplant due to severe infection. After FACSorting, the CD117+/CD123− and CD117−/CD123+ malignant cells were sent to detect morphology, FISH, and PCR. Hybridization showed yellow signals for both PML-RARA and AML1-ETO fusion genes in malignant myeloblasts, as well as in leukemic promyelocytes (Figure 2A-D). In aberrant promyelocytes, PML-RARA and AML1-ETO fusion transcripts were detected at 150.00% and 37.49%, respectively (Figure 2A-D). Additionally, 44.73% PML-RARA and 205.40% AML1-ETO fusion transcripts were detected in malignant myeloblasts. In terms of cell morphology, the leukemic promyelocytes possessed the characteristic morphological features of PML-RARA (Figure 2E). Malignant myeloblasts had the characteristic morphological features of AML with AML1-ETO fusion gene (Figure 2F). The cell characteristic morphological features of PML-RARA and AML1-ETO were consistent with the description in Figure 1D. Up to now, there are few reports of AML with the coexistence of AML1-ETO and PML-RARA fusion genes. Charrin et al3 first reported such a case in 1992, wherein three distinct clones were observed in the bone marrow of a patient with leukemia: t(15;17), t(8;21), t(15;17)/t(8;21). Other cases of coexistence of t(8;21) or t(15;17) in leukemia have been described.4, 5 Although cases of PML-RARA co-expression with AML1-ETO are rare, studies on the proximity of nonrandomly associated translocation genes involved in leukemia subtypes to interphase genomic location suggest that PML and RARA genes are relatively close to each other in hematopoietic cells, as are AML1 and ETO,6 suggesting increased probability of AML1-ETO and PML-RARA fusion gene occurrence. Researchers examined AML cases defined as acute leukemia with malignant myeloblasts accompanied by leukemic promyelocytes and found that both PML-RARA and AML1-ETO fusion genes coexisted in a few cases.7 In this study, the cells in the bone marrow sample were divided into significant difference two cell populations and the expression level of the PML-RARA fusion gene in leukemic promyelocytes was higher than that in malignant myeloblasts, while the reverse was true for the AML1-ETO fusion gene. Therefore, a correlation between differentiation and related fusion gene expression could not be made. A single effect does not directly contribute to the development of AML in patients with AML1-ETO fusion gene, but may require a second-hit factor like the appearance of the PML-RARA fusion gene or other relevant gene mutations.8 The study found that the AML1-S291fs300X mutation was introduced in CD34 stem/progenitor cells and human induced pluripotent stem cells, confirming that the AML1 mutation induced bone marrow differentiation arrest at the myeloblasts stage.9 Among patients with leukemia having both the AML1-ETO fusion and C-KIT gene mutations, most possessed both phenotypic changes, whereas in three patients in complete remission, only the AML1-ETO fusion gene was expressed.8 Therefore, we suspected the C-KIT gene mutation may develop subsequently from the event of the t(8;21), indicating that a stepwise model occurs in leukemogenesis. That is, AML1-ETO represents the first genetic hit to initiate leukemia, while the C-KIT gene mutation may be the second hit for overall leukemia development. Our results indicated that the C-KIT receptor (CD117) was highly expressed in malignant myeloblasts, while the C-KIT gene mutation was negative, ruling out the possibility of a second genetic hit in the C-KIT pathway. Previous reports have implicated ASXL2 gene mutations in AML1-ETO fusion gene pathogenesis,10 but failed to detect WT1 and ASXL2 gene mutations in sorted cells due to the limited sample size. Charrin et al3 asserted that t(8;21) may be the first mutation in early bone marrow stem cells, while t(15;17) is acquired by the patient during the course of the disease. Whether the PML-RARA fusion gene is a secondary genetic hit for the simultaneous occurrence of PML-RARA and AML1-ETO fusion genes in AML needs to be investigated in greater depth. In summary, the coexistence of PML-RARA and AML1-ETO fusion genes is rare in AML. Although further studies with a greater number of patients are needed, this case is the first one applying FACSorting technique combining with morphology, FISH, and PCR techniques to accurately define the leukemogenesis in patients with concurrent AML1-ETO and PML-RARA fusion genes in AML. The authors declare that they have no competing interest. Hui Wang, corresponding author, designed the research and revised paper. Man Chen, first author, analyzed data and wrote the paper. Minjing Fu, Qing Du, and Meiwei Gong involved in test samples and gather clinical data. Junyi Zhen, Ping Wu, Tong Wang, and Hongxing Liu reported results. Xueying Wu and Aixian Wang drew the figure. All procedures were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from the patient for publication of this case. The data that support the findings of this study are available from the corresponding author upon reasonable request.

目的:探讨流式细胞术在检测急性白血病(AL)除脑脊液以外的髓外浸润中的作用.方法:2008年10月到2014年12月在河北燕达陆道培医院进行诊疗的AL患者,出现除脑脊液以外的髓外包块或者浆液渗出的59例患者,取标本做流式细胞术检测.结果:59例髓外标本均发现肿瘤细胞浸润,与组织学检测一致.流式检测肿瘤细胞占有核细胞中位百分比41.93％(0.38％～97.87％).其中51例(86％)患者同时做了骨髓免疫分型检测:29例(57％)伴有骨髓病变,肿瘤细胞占有核细胞比例中位数10.91％(0.02％～85.19％).移植后病例的髓外浸润常不伴有骨髓病变(P<0.001),急性淋巴细胞白血病(ALL)孤立性髓外浸润概率比急性髓系白血病(AML)高(P<0.05).AML中,单核亚型(M4、M5)、CD56阳性病例易于出现髓外浸润(分别为P<0.01,P<0.001).结论:使用流式细胞术可以有效检测急性白血病除脑脊液以外的髓外浸润.

Abstract Background: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is the most prevalent adult leukemia, and its incidence continues to rise year after year. Rapid and precise diagnosis is an essential element in effective case management, however, the clinical diagnosis, treatment, and prognosis of CLL/SLL are not fully elucidated. Case presentation: we report the case of a 66-year-old man with atypical CLL/SLL. The white blood cell (WBC) count (842.0 × 109/L), platelet count (30.6 × 109/L), and abnormal lymphocytes were increased in peripheral blood. Flow cytometry showed 98.34% of nucleated cells were malignant monoclonal mature B cell. Peripheral blood smear found the leukocytes and lymphocytes with abnormal morphology were increased. Fluorescence in situ hybridization showed CCND1 (11q23)/IGH (14q32) and abnormal chromosome 12 were invisible, 91%-93% of interphase nuclei presented D13S319 and TP53, 17p13.1 loss. Histopathology analysis of bone marrow observed the proliferation centers with immunoblasts. Immunohistochemistry showed that bone marrow was positive for PAX-5, CD20, CD23, and CD5, negative for CD3, cyclinD1, and sox11, and partial positive for Ki67. The patient was diagnosed as CLL/SLL based on above clinical and laboratory findings. The patient was managed with oral 50 mg Vinetoc, fluid replacement, hydration and alkalinization, and the symptoms were significantly relieved. Conclusions: This report further expands the knowledge of clinical diagnosis and treatment of atypical CLL/SLL.

Objective:This study aims to analyze the counts (per kilogram of body weight) or percentages of transplanted lymphocyte subgroups in children with non-infectious pulmonary complications (NIPC) and air-leak syndrome (ALS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and explore its significance in the progression of lung complications after transplantation.Methods:The patients with NIPC and ALS after allo-HSCT from January 2013 to December 2019 in Hebei Yanda Ludaopei Hospital were retrospectively studied and the influencing factors in the progress of NIPC after HSCT were statistically analyzed.Results:Of the 2026 children who received HSCT treatment, 59 patients (34 males and 25 females) developed NIPC, the probability was 2.9% (59/2 026), and the probability of combined ALS was 1.4% (28/206). The differences in the comparison between NIPC progressed to ALS group (ALS group) and failed to progress to ALS group (non-ALS group) in the patient′s age( P=0.028), disease condition before transplantation( P=0.022), NIPC onset time( P=0.004) were significant. The P values of the percentage of NKT-like cells in the bone marrow ( P=0.008) or peripheral stem cells ( P=0.003) accounted for the lymphocytes. CD4+CD25+dim cells in bone marrow ( P=0.029) or peripheral stem cells ( P=0.036) accounted for the CD4+lymphocytes and the ratio of CD4/CD8 in bone marrow( P=0.004) or peripheral stem cells ( P=0.020) were less than 0.05, which meant the differences in patients′ refusion cells were significant. In the binary logistic regression model, the percentage of bone marrow NKT-like cells to lymphocytes, the ratio of bone marrow CD4+/CD8+and the percentage of peripheral stem NK cells to lymphocytes were important risk factors for the progression of NIPC to ALS. The rest factors were excluded from the model (AUC=0.918, P<0.05). Conclusion:During allo-HSCT transplantation, a high proportion of NKT-like cell and NK cell levels, and a high CD4+/CD8+ratio in the infusion of donors with high immune tolerance have an important correlation with the progression of the NIPC.

Objective To prepare a monoclonal antibody (mAb) against human CD33 by immunizing mice with recombinant vector and analyze its characteristics and clinical application. Methods The eukaryotic expression vector pcDNA3.1(+)/CD33 was constructed and used to immunize mice. The mouse monoclonal antibody against human CD33 was then harvested using the hybridoma technique. Its properties were evaluated and the clinical performance was validated. Results One hybridoma cell line capable of secreting mouse anti-human CD33 monoclonal antibody was successfully obtained, which was named HI33a for clone identification with a subclass of IgG2a, κ. Flow Cytometry analysis revealed that the antibody could stain myeloid cell lines but not lymphoid cell lines, and it could inhibit the binding of similar imported antibodies with HL-60 cells competitively. Western blotting verified that it could bind a Mr 67 000 membrane protein extracted from HL-60 cells, which was a strong indication of the characteristics of CD33 protein molecule. Labeled with PE fluorescein, CD33-PE was tested as an antibody reagent in comparison with other similar imported products. Its overall performance including the accuracy, linearity, and precision all met the industrial standard. Further clinical evaluation of 558 bone marrow samples showed that the results were highly consistent with those by the imported reagents used as controls. Conclusion A hybridoma cell line stably secreting anti-human CD33 mAb was prepared.

T cell therapy represents a new class of immunotherapies garnering considerable attention. T cell receptor beta chain constant region 1 (TRBC1) is partially expressed in subsets of normal T cells. However, the immunotherapy of T lymphocyte tumors is rarely validated in clinical trials. Here, we aim to explore whether TRBC1 is a promising target for the immunotherapy of T lymphocyte tumors. This study examined TRBC1 expression in 25 healthy bone marrow samples, 39 patients with T-lineage acute lymphocytic leukemia (T-ALL), 4 patients with mature T cell neoplasms, and 5 patients suspected with mature T cell neoplasms with evidence of T cell neoplasia. Moreover, the expression of TRBC1 was evaluated by flow cytometry and through PCR detection of TCR gene rearrangements. The expression of monophasic TRBC1 was identified in all 25 normal bone marrows (23.83% ± 2.74% positive rate). The expression of TRBC1 was positive in 5 patients (12.8%) among the 39 T-ALL patients. TRBC1 was partially expressed in 1 patient (25%) with T cell non-Hodgkin's lymphoma (T-NHL) and in 1 patient (20%) suspected to have T-NHL. Healthy donors showed a pattern of partial expression and patients with T-lymphocyte tumors showed a polytypic TRBC1 expression pattern. Thus, TRBC1 may be a diagnostic and therapeutic marker for T lymphocyte tumors.

Introduction Multicolor flow cytometry (MFC) has been frequently adopted as a method for minimal residual disease (MRD) detection, and is also a promising technique to detect post-transplant lymphoproliferative disorder. Some abnormal donor origin cells might be found when detecting MRD following an allogeneic hematopoietic stem cell transplantation (allo-HSCT). To minimize the effects from donor cells, using MFC prior to allo-HSCT to screen donor peripheral blood (PB) or bone marrow (BM) might be feasible. Methods We performed 3395 allo-HSCTs between January 2013 and December 2019 at Lu Daopei Hospital in Langfang, China. MRD was detected in recipients' BMs according to a conventional two-tube 8 or 9-color MFC panel. Abnormal cells were observed in BMs from three patients in complete remission (CR) one to four months post allo-HSCT. Abnormal neutrophils lacking CD16 expression were found in a patient with secondary acute myeloid leukemia (AML) that developed from a myelodysplastic/ myeloproliferative neoplasm (MDS/MPN). After ruling out MDS and paroxysmal nocturnal hemoglobinuria (PNH), we hypothesized that an Fcγ receptor IIIB (FcγRIIIB) gene deletion was the most likely reason. Abnormal natural killer (NK) cells were detected in the BM from an allo-HSCT recipient with T-cell acute lymphoblastic leukemia (ALL), and monoclonal B lymphocytosis (MBL) in allo-HSCT recipient with B-cell ALL. These three patinets' PBs were detected using MFC after the new finding to decide the cell origin. Besides, 4.54%(in WBC) CD4+ and CD8+ double positive T- cells which were monoclonal cells of the TCRVβ repertoire were detected in a PB sample from a donor prior to allo-HSCT. To evaluate the incidence rate The immunophenotypings were studied in the BMs from 79 NK lymphoma patients. Results Identical phenotypes were recognized in PBs obtained from the three respective donors. The fourth donor did not donate her cells for allo-HSCT, yet. The incidence rate of abnormal cells in donor samples was 0.1% (4/3395 cases), but this rate might be underestimated because MFC screening was not a routine procedure for donors. Additionally, only abnormal immunophenotyping related to patient diagnosis might have been found using an MRD panel as this panel only included markers related to diagnosis. Among general population, the incidence rate of suspicious FcγRIIIB deletion was 0.2% (11/5256 cases), the incidence rate of NK cells without CD2+ and homogeneously expressed CD159c was 0.05% (1/2000 cases) and none among the 79 NK lymphoma samples. The rate of MBL was 0.75% (15/2000 cases) and 1.36% in older than 40 years old people and the rate of monoclonal CD4/CD8 DP T-cells was 0.05% (1/2000 cases). All of these abnormal cells or polymorphism could be analyzed using a two tube MFC panel-- ckappa/clambda/(CD34)/CD19/ CD5/CD20/CD38 /CD45/CD56 and CD16/(CD117)/CD3/ CD4/CD5/ CD8/CD56/CD45/CD2. Conclusion Donor original abnormal cells or phenotypic polymorphisms could have an effect on MFC-based MRD or PTLD detection of recipients following allo-HSCT. These patients might be mis-diagnosed as being MRD positive or having PTLD if the technician lacks experience. To avoid mis-diagnosis and minimize the risk of allo-HSCT, it might be promising to utilize a suitable MFC panel to screen donor PB or BM samples prior to transplantation. Disclosures No relevant conflicts of interest to declare.

Background Post-transplant lymphoproliferative disorder (PTLD) is a serious complication that can occur following an allogenic hematopoietic stem cell transplant (allo-HSCT). PTLD occurs in approximately 0.8% to 20% of patients following an allo-HSCT and is associated with Epstein-Barr virus (EBV) infection in about 60% to 80% of patients. EBV positive (EBV+) PTLD generally arises early, several months following transplantation. The routine methods to diagnose EBV+ PTLD are clinical symptoms, EBV copy number, imageological examination, and pathological diagnosis, of which pathological diagnosis is the gold standard. Yet obtaining a biopsy is not possible with some patients and is not applicable for post-therapy monitoring. Therefore, there is an urgent need to find a simple, highly efficient, feasible, sensitive, and specific diagnostic and monitoring tool. Flow cytometry (FCM) has been established as a highly cost-effective method to diagnose lymphoma over the past several decades, and particularly for screening monoclonal B and/or plasma cells (MC B/P). Several recent publications as well as our own published and unpublished data have found that MC B/P are present in the peripheral blood of most PTLD patients. To this end, FCM detection of MC B/P in PB is a promising screening and monitoring method for EBV(+) PTLD. Objective To investigate the effectiveness of detecting MC B/P in PB by FCM for EBV+ PTLD screening and monitoring. Methods A total of 1470 patients received allo-HSCT at the Hebei Yanda Ludaopei Hospital, China from January 2018 through December 2019. We conducted a retrospective study of 481 patients with fever and lymphadenopathy following allo-HSCT. Patient PB was extracted for FCM MC B/P analysis. Plasma EBV viral loads were detected by PCR. The median fellow-up time was 6 months (range: 2 days to 21 months). The relationships of PB MC B/P, EBV load and clinical EBV-associated PTLD symptoms were investigated. Results: MC B/Ps were detected in the PB of 47 patients, of which 29 cases had monoclonal B cells, 14 cases had detectable co-existence of monoclonal B cells and monoclonal plasma cells, and 4 cases had detectable monoclonal plasma cells. The median time to PTLD onset following allo-HSCT was 70 days (range: 33 days to 491 days). The median percentage of monoclonal B cells was 0.43% (range: 0.1% to 23.41%. The median percentage of monoclonal plasma cells was 0.25%). Forty of 47 patients (85.1%) were finally diagnosed with PTLD using clinical comprehensive examination of symptoms. The incidence of clinical EBV+ PTLD was 2.9% (44 of 1470 cases). By FCM, MC B/Ps were observed in the PB of 91% of the patients (40 of 44 cases). No MC B/Ps were found in the PB of 4 patients. MC B/P detection in PB by FCM screening could effectively predict the incidence of EBV+ PTLD with a sensitivity of 90.91% and specificity of 98.40%. The positive predictive value was 85.17% and the negative predictive value was 99.08%. There was no significant correlation between EBV viral copy number and percentage of MC B/P. The median fellow-up time was 6 months (range: 2 days to 21 months) for all patients. The therapeutic response rate was 87.5% (35 of 40 patients) for MC B/P positive EBV+ PTLD patients. In those patients with a therapeutic response, clinical symptoms improved, lymph nodes significantly shrank, EBV viral loads decreased, MC B/Ps decreased or vanished, and CD20+ cell proportion decreased or vanished. Among the 7 patients who had MC B/Ps in PB but were not diagnosed with PTLD , their clinical symptoms recovered after treatment with immunosuppressive therapy. Conclusion By detecting PB MC B/Ps using FCM, we can successfully screen for EBV+ PTLD and monitor patient therapeutic responses in most cases. FCM to detect MC B/Ps in PB is a new, promising method for the diagnosis and monitoring of EBV+ PTLD. Compared to a biopsy, this appears to be a more simple, easier and more applicable tool to diagnose and monitor EBV+ PTLD. Disclosures No relevant conflicts of interest to declare.

Introduction Flow cytometry(FC) plays an important role in the diagnosis of hematologic diseases and the study of cell maturation. Spectral multicolor flow cytometry(SMFC) has shown an advantage over traditional FC that more fluorescent markers could be detected simultaneously, more antigen combinations could be made, and the expression of cells could be scrutinized. However, published studies focused on lymphocyte subsets and the differentiation between hematogones and B-acute lymphoblastic leukaemia/lymphoma(ALL/LBL) minimal residual disease(MRD) detection, and there are few studies on myeloid development and expression. Besides, more powerful and cutting-edge software are needed for complicated combinations from SMFC because traditional dot plots are unable to meet the demand of analysis. Here we design a one-tube 24-color panel combining with the multidimensional data analysis software to study the expression and maturation of normal and malignant myeloid cells including minus subgroups. We hope to improve the sensitivity of MRD by FC and explore more information about myeloid diseases, finally promote the development of artificial intelligence(AI) in clinical FC diagnosis. Methods: the one-tube 24-color panel was designed according to our experience and Euroflow recommendation. it is composed of backbones including CD45 and myeloblast markers CD34, CD117 and HLA-DR, adding myeloid markers CD33, CD13, CD371, CD15, CD64, CD11c,CD14, CD36 and CD11b, routine leukaemia associated immunophenotyping(LAIP) or different from normal(DFN) markers CD4, CD19, CD7, CD2, CD56,CD96,CD123, CD38, CD200, CD71 and CD9. The control database consisted of 20 normal bone marrow(BM) specimens, including 8 healthy donors and 12 patients with other diseases that were in complete remission(CR) after treatment. To verify the effectiveness of the panel, 4 BM samples from acute myeloblastic leukaemia(AML) patients with MRD positive or relapsed status were selected, with malignant myeloblasts of 0.23% (sample A1), 4.3%, 30.31% (and 6.47% abnormal mast cells, sample A3), 0.29% (16.49% basophils) (sample A4), respectively. The data was acquired by a 3 laser 38-color Cytek spectral FC, and analyzed by Kaluza and Flowjo software. The results were compared with those of conventional 3 - laser 8 - color Canto FC. Results when analyzing the common antibodies acquired by two kinds of FCs and software, the similarities and good correlations were shown about the percentage of myeloid subsets and expression pattern of antigens, no matter in normal or abnormal specimen. However, SMFC can analyze subsets of myeloid cells more clearly and detailedly, especially in minus subgroups like mast cells or basophils. Abnormal expression of tumor cells could be clearly observed in both FCs, and more abnormalities could be found with 24 color analysis, especially mast cell abnormalities. The t-SNE plug-in in Flowjo was used to conduct dimension-reduction analysis on 20 samples (see figure), with hundreds of dot plots being merged into one multi-dimensional picture. The normal samples were similar, but the abnormal samples were different from the normal ones. However, the t-SNE pictures were same only for the specimens that malignant myeloblasts were the only abnormality. The other two samples with abnormal mast cells or basophils showed different t-SNE figures. In the process of automatic clustering, CD117 and CD34 positive myeloblasts, basophils and mast cells were classified as three clusters according to the proportion of cells, surface antigen expression, and of fluorescence intensity of antigen expression. A more obvious aggregation was formed by malignant cells than normal cells, and was positively related to tumor burden. Since mast cells and basophils were low percentages in normal specimens, the abnormal clusters were more obvious when the proportion of these two types of cells increased. Conclusion Combined with cutting-edge software(BD FlowJO), SMFC can offer more cellular information that is unmatched by traditional FC. This study is only the preliminary attempt of our laboratory. With detailed data from more samples and longer-termed clinical trials, it would be a promising method to explore and study the myeloid maturation, improve the sensitivity of MRD, and promote the application of AI. Disclosures No relevant conflicts of interest to declare.

正>CD16是流式细胞术检测发育阶段粒细胞常用标志,因其编码基因和抗体的复杂性,一些相关疾病呈现相似又不同的表型,需要加以区分。CD13表达强度随着粒细胞分化成熟而变化,所以CD16/CD13是临床监测粒细胞发育情况的常用抗体组合。本文介绍正常对照以及几种容易误诊的粒细胞相关疾病的CD16及CD16/CD13表达模式,以研究其在鉴别不同疾病中的作用。

嵌合抗原受体(chimeric antigen receptors,CAR)细胞疗法已广泛用于白血病、淋巴瘤的治疗,CD19和CD22靶向CAR-T已在复发、难治性急性B淋巴细胞白血病(RR-B-ALL)等血液系统疾病的治疗上取得了显著疗效,而在T细胞肿瘤治疗上进展缓慢.介绍了目前国内外利用CAR细胞技术与CRISPR/Cas9基因编码技术,设计了T-ALL相关的CAR细胞免疫疗法并进行了CAR细胞免疫疗法在T-ALL治疗上的初步探索.

Flow cytometry (FCM) uses flow cytometer to detect the expression of immunological markers on cells based on antigen-antibody binding and laser activating fluorescence. FCM plays a very important role in clinical diagnosis especially in acute leukemia diagnosis and subclassification. However, due to the difficulty in acquiring the sample and the existence of normal lymphocytes in the background, its application and improvement should be further strengthened in the diagnosis of mature lymphoid neoplasm. This paper focuses on the similarity and differences between FCM and pathology immunohistology, immune makers in lymphoma detection, immunophenotypic features of common mature lymphoid neoplasm, common issues and key points in FCM detection, to study the diagnostic application of flow cytometry in mature lymphoid neoplasm. This will help clinicians understand this technic, and make a correct diagnosis by FCM combining with pathology. Key words: Flow cytometry; Mature lymphoid; Neoplasm; Diagnosis

Background: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is the most prevalent adult leukemia, and its incidence continues to rise year after year. Rapid and precise diagnosis is an essential element in effective case management, however, the clinical diagnosis, treatment, and prognosis of CLL/SLL are not fully elucidated. Case presentation: we report the case of a 66-year-old man with atypical CLL/SLL. The white blood cell (WBC) count (842.0 × 10 9 /L), platelet count (30.6 × 10 9 /L), and abnormal lymphocytes were increased in peripheral blood. Flow cytometry showed 98.34% of nucleated cells were malignant monoclonal mature B cell. Peripheral blood smear found the leukocytes and lymphocytes with abnormal morphology were increased. Fluorescence in situ hybridization showed CCND1 (11q23)/IGH (14q32) and abnormal chromosome 12 were negative, 91%~93% of interphase nuclei presented D13S319 and TP53, 17p13.1 loss. Histopathology analysis of bone marrow observed the proliferation centers with immunoblasts. Immunohistochemistry showed that bone marrow was positive for PAX-5, CD20, CD23, and CD5, negative for CD3, cyclinD1, and sox11, and partial positive for Ki67. The patient was diagnosed as CLL/SLL based on above clinical and laboratory findings. The patient was managed with oral 50 mg Vinetoc, fluid replacement, hydration and alkalization, and the symptoms were significantly relieved. Conclusions: This report further expands the knowledge of clinical diagnosis and treatment of atypical CLL/SLL.