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In recent years, chimeric antigen receptor (CAR) T-cell therapy has been widely used to treat relapsed/refractory B-cell malignancies. Before CAR-T, the identification of target antigens on tumor cells is needed for choosing a suitable antigen-specific CAR, which is usually performed by multiparameter flow cytometry (FCM) for bone marrow (BM) samples or immunohistochemical (IHC) staining for tissue samples. However, some patients with tumor masses only and without BM involvement or serous cavity effusions (SCE) may not have much time to wait the results from IHC staining owing to rapidly growing tumor cells, therefore, we established a quick antigen identification by FCM using tissue samples. Fresh tumor tissues taken by fine or coarse needle puncture from different body parts were put in saline with 2% fetal calf serum and sent to the FCM lab as quickly as possible, once in lab, samples were processed immediately. Tissue strips were placed into a 60 mm petri dish, gently pressed by a syringe piston and repeatedly flushed with saline to separate cells from tissues; then the liquid with cells in petri dish were transferred into a 100μm cell strainer on top of 50 mL conical tube to exclude non-cellular tissue components, the strainer was washed with saline for 2-3 times. The strained cell suspension in conical tube was centrifuged at 1000rpm for 5 minutes, the supernatant was discarded by pipetting, cells were counted and resuspended with saline at a concentration of 104-105/ml, then the single cell suspension was ready for flow cytometry assay. 7-AAD and antibodies CD19/CD22/CD7/CD45 were added into the first tube to quantify viable cells and identify cells available for analysis, if dead cells were more than 30% of all cells, 7-AAD was needed for all next tubes, otherwise, it could be omitted. The standard antibody panels consist of CD10/CD20/CD34/CD19/CD38/CD45 for precursor B-cells and kappa/lambda/CD10/CD19/CD20/CD38/CD45 for mature B-cells, extra antibodies could be added according to patient's situations. Samples were performed on the 8-color BD FACSCanto II flow cytometer, and analyzed by BD FACS Diva software or Kaluza software from Beckman Coulter. Antigen expression, partial expression and no expression were defined as >80%, 20-80% and From October 2017 through June 2020, a total of 63 patients with relapsed/refractory B-cell malignancies who had tumor masses only and no BM involvement or malignant SCE were hospitalized in our center for CAR-T treatment after failure of multiple-line therapies, including 11 (17.5%) acute lymphoblastic leukemia (ALL) and 52 (82.5%) non-Hodgkin's lymphoma (NHL), the median age was 41 years (range, 1-71) with both adults (n=47, 74.6%) and children younger than 18 years (n=16, 25.4%). Acquired events from tissue samples varied from 1000 to more than 100, 000 each tube (5 cases 100,000), the percentages of tumor cells in all cells available for analysis varied from 0.03% to 99.44% (5 cases 90%). We have CD19-, CD20- and CD22-specific CAR-T cells for B-ALL and B-NHL, the choice of a CAR-T product depends on the antigen expression on tumor cells, patients with partial or no or dim expression (PND expression) of certain antigen are not suitable for the relevant antigen-specific CAR-T therapy. Among this cohort of patients, CD19 expressed in all but 1 ALL case who had accepted CD19 CAR-T; PND expression of CD20 occurred in 19.2% (10/52) of NHL patients since they were repeatedly administrated anti-CD20 antibodies in previous treatments which resulted in antigen diminishment or loss, all B-ALL patients had no or partial CD20 expression; PND expression of CD22 was seen in 18.2% (2/11) of ALL and 13.5% (7/52) of NHL. All flow results came back within hours (< 4-6 hours), much quicker than results from IHC staining which takes days. In conclusion, the correct selection of a target antigen is the basis of effective CAR-T treatment in relapsed/refractory B-cell malignancies especially for patients having accepted antibody or CAR-T therapies, our antigen detection by tissue flow cytometry provided a quick and accurate antigen identification for patients with no BM or SCE samples. Disclosures No relevant conflicts of interest to declare.

CD19 has been as a classic marker for gating B-cells in flow cytometric analysis. However, with the application of CD19 chimeric antigen receptor (CAR) T-cell therapy for treating relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL), CD19 gating is no longer adequate for B-ALL patients post CD19 CAR-T therapy owing to CD19 antigen loss, under this circumstance, cytoplasmic CD79a (cCD79a) emerged to replace CD19 as a gating marker of B-cells. Nevertheless, cCD79a was intracellular staining and time consuming, sometimes with unspecific stain or without enough cells for analysis after permeabilization and extra washing. CD22 is another pan B marker expressed on the surface of B lymphocytes at different stages, could be as alternative choice of B-cell identification after CD19 CAR-T. Here, we evaluated the possibility of CD22 as a B-cell gating marker by comparing the expression of CD22 and cCD79a in CD19 negative relapsed B-ALL patients. We routinely used cCD79a to gate B-cells in patients after CD19 CAR-T therapy, CD22 expression was detected simultaneously. The standard antibody panel consist of CD45, CD19, CD22, CD10, CD20, CD34, CD38, CD58, cCD79a, CD81, HLA-DR and CD123. CD22 antibody conjugated with phycoerythrin (PE) or allophycocyanin (APC) was used, extra antibodies could be added based on patient's immunophenotype at initial diagnosis. Bone marrow specimens were prepared by a standard stain-lyse-wash procedure, no less than 300,000 events were acquired by 8-color BD FACS Canto II flow cytometer, data were analyzed using FACS Diva 8.0.1 software. Antigen normal expression and partial expression were defined as >80% and 20-80% of gated cells displaying interested antigen, respectively. Dim expression was defined as more than one log weaker than the normal counterpart's mean fluorescence intensity (MFI). From April 2018 to June 2020, a total of 40 CD19-negative relapsed B-ALL patients post CD19 CART therapy were included in this study, consisting of 14 adults and 26 children younger than 18 years old, with a median age of 12 years (range, 1-64). The simultaneous expression of cCD79a and CD22 among these patients were analyzed. All 40 patients (100%) had normal cCD79a expression on B-cells. Although CD22 was expressed on all cases as well, normal expression was seen in 33 (82.5%) patients whereas 7 (17.5%) were partial or dim (P/D) expression, the P/D expression rendered CD22 as a gating marker unsuitable. To figure out whether this P/D CD22 expression was related to CD19 CAR-T treatment, we checked back the immunophenotype of these 7 patients and found that, before CD19 CAR-T, 4 (10%) patients with P/D CD22 expression and 3 (7.5%) with normal CD22 expression. When we excluded these 4 patients with existed P/D CD22 expression, 91.7% (33/36) of patients accordantly expressed normal CD22 and cCD79a, which indicated that CD22 could be a B-cell gating marker for 91.7% of patients with normal CD22 expression before CD19 CAR-T therapy. To obtain optimal signal, CD22 antibody conjugated with bright fluorescein such as PE or APC is recommended. It is noted that CD22 also expressed on basophils, therefore, HLA-DR should be included in the antibody panel to exclude this interference since basophils do not express HLA-DR. Sometimes, there might be CD22 stain on plasmacytoid dendritic cells (pDCs) which express HLA-DR too, it is not difficult for us to differentiate B-cell from pDCs based on the facts that CD22 expression on pDCs is weaker than that on B cells, pDCs have bright CD123 expression and are located at a relatively fixed position in CD45/SSC plot. In conclusion, our data on the comparison between CD22 and cCD79a expression in CD19 negative relapsed B-ALL patients revealed that, under most circumstances (with 82.5%-91.7% chance), CD22 gating alone could efficiently identify B cells in patients after CD19 CAR-T therapy, which is also a cost-effective and labor-saving strategy for routine practice compared to cCD79a gating. In case of partial or dim CD22 expression, the cCD79a gating is needed. Disclosures No relevant conflicts of interest to declare.