Development of a large volume line scanning, high spectral range and resolution 3D hyperspectral photoluminescence imaging microscope for diamond and other high refractive index materials.

Hyperspectral photoluminescence (PL) imaging is a powerful technique that can be used to understand the spatial distribution of emitting species in many materials. Volumetric hyperspectral imaging of weakly emitting color centers often necessitates considerable data collection times when using commercial systems. We report the development of a line-scanning hyperspectral imaging microscope capable of measuring the luminescence emission spectra for diamond volumes up to 2.20 × 30.00 × 6.30 mm with a high lateral spatial resolution of 1-3 µm. In an single X-λ measurement, spectra covering a 711 nm range, in a band from 400-1100 nm, with a spectral resolution up to 0.25 nm can be acquired. Data sets can be acquired with 723 (X) × 643 (Y) × 1172 (λ) pixels at a rate of 6 minutes/planar image slice, allowing for volumetric hyperspectral imaging with high sampling. This instrument demonstrates the ability to detect emission from several different color centers in diamond both at the surface and internally, providing a non-destructive method to probe their 3D spatial distribution, and is currently not achievable with any other commonly used system or technique.

Rapid gemstone screening and identification using fluorescence spectroscopy.

We demonstrate a gemstone screening device based on fluorescence spectroscopy. The device can be used to rapidly separate colorless and near-colorless (D to J color grades) natural diamonds from laboratory grown diamonds and diamond simulants, detect multi-treated pink diamonds, and identify certain colored gemstones, such as corundum, spinel, beryl, and zoisite. The device's reflection fiber probe enables testing of both loose and mounted gemstones with exposed facet faces that are larger than 0.9 mm. The experimental prototype demonstrates high accuracy for automatic diamond gemstone screening, referring 100% of the laboratory grown diamonds and simulants tested. The pink diamond screening algorithm can detect 100% of pink multi-treated diamonds and laboratory grown diamonds. Finally, the suitability of this device for the fluorescence analysis of corundum, beryl, spinel, zoisite, garnet, and topaz was evaluated.

Emerging Role of T-cell Receptor Constant ß Chain-1 (TRBC1) Expression in the Flow Cytometric Diagnosis of T-cell Malignancies.

T-cell clonality testing is integral to the diagnostic work-up of T-cell malignancies; however, current methods lack specificity and sensitivity, which can make the diagnostic process difficult. The recent discovery of a monoclonal antibody (mAb) specific for human TRBC1 will greatly improve the outlook for T-cell malignancy diagnostics. The anti-TRBC1 mAb can be used in flow cytometry immunophenotyping assays to provide a low-cost, robust, and highly specific test that detects clonality of immunophenotypically distinct T-cell populations. Recent studies demonstrate the clinical utility of this approach in several contexts; use of this antibody in appropriately designed flow cytometry panels improves detection of circulating disease in patients with cutaneous T-cell lymphoma, eliminates the need for molecular clonality testing in the context of large granular lymphocyte leukemia, and provides more conclusive results in the context of many other T-cell disorders. It is worth noting that the increased ability to detect discrete clonal T-cell populations means that identification of T-cell clones of uncertain clinical significance (T-CUS) will become more common. This review discusses this new antibody and describes how it defines clonal T-cells. We present and discuss assay design and summarize findings to date about the use of flow cytometry TRBC1 analysis in the field of diagnostics, including lymph node and fluid sample investigations. We also make suggestions about how to apply the assay results in clinical work-ups, including how to interpret and report findings of T-CUS. Finally, we highlight areas that we think will benefit from further research.

Dapagliflozin slows the progression of the renal and liver fibrosis associated with type 2 diabetes.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic oral agents indicating promising effects on cardiovascular and renal end points. However, the renoprotective effects of SGLT2 inhibitors are not fully understood. Also, metabolic effects of SGLT2 inhibition on other organ systems, such as effects on hepatic steatosis, are not fully understood. This study sought to address these questions by treating 18-wk-old uninephrectomized db/db mice with the selective SGLT2 inhibitor dapagliflozin. Untreated db/db mice developed progressive albuminuria, glomerular mesangial matrix expansion, and fatty liver associated with increased renal expression of TGFß1, PAI-1, type IV collagen and fibronectin, and liver deposition of fibronectin, type I and III collagen, and laminin. Treatment with dapagliflozin (1 mg·kg-1·day-1) via gel diet from 18 to 22 wk of age not only reduced blood glucose (371.14 ± 55.02 mg/dl in treated db/db vs. 573.53 ± 21.73 mg/dl in untreated db/db, P < 0.05) and Hb A1c levels (9.47 ± 0.79% in treated db/db vs. 12.1 ± 0.73% in untreated db/db, P < 0.05) but also ameliorated the increases in albuminuria and markers of glomerulosclerosis and liver injury seen in untreated db/db mice. Furthermore, both renal expressions of NF-kB p65, MCP-1, Nox4, Nox2, and p47phox and urine TBARS levels and liver productions of myeloperoxidase and reactive oxygen species, the markers of tissue inflammation and oxidative stress, were increased in untreated db/db mice, which were reduced by dapagliflozin administration. These results demonstrate that dapagliflozin not only improves hyperglycemia but also slows the progression of diabetes-associated glomerulosclerosis and liver fibrosis by improving hyperglycemia-induced tissue inflammation and oxidative stress.

Immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes. A report from the IMDSFlow working group.

Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117+ erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84-94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86-97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage.

Activated apoptotic cells induce dendritic cell maturation via engagement of Toll-like receptor 4 (TLR4), dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN), and ß2 integrins.

Dendritic cells (DCs) are professional antigen-presenting cells playing a central role in connecting innate and adaptive immunity. Maturation signals are, however, required for DCs to undergo phenotypic and functional changes to acquire a fully competent antigen-presenting capacity. We previously reported that activated apoptotic peripheral lymphocytes (ActApo) provide activation/maturation signals to human monocyte-derived DCs. In this paper, we have characterized the signaling pathways and molecules involved in ActApo-mediated DC maturation. We found that both cellular and supernatant fractions from ActApo are required for DC maturation signaling. ActApoSup-induced CD80 and CD86 expression was significantly blocked in the presence of neutralizing antibodies against tumor necrosis factor-α (TNF-α). Cell-cell contact-dependent signaling involved ß2 integrins, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), and TLR4 because ActApo-induced up-regulation of the maturation markers CD80 and CD86 was significantly inhibited in the presence of neutralizing antibodies against CD18, CD11a, CD11b, and DC-SIGN as well as TLR4. The role of TLR4 was further confirmed by silencing of TLR4 in DCs. In addition, the endogenous adjuvant effect exerted by activated apoptotic splenocytes (ActApoSp) was reduced after immunization with human serum albumin in TLR4(-/-) mice. We detected activation of multiple signaling pathways and transcription factors in DCs upon co-culture with ActApo, including p38, JNK, PI3K-Akt, Src family kinases, NFκB p65, and AP1 transcription factor family members c-Jun and c-Fos, demonstrating the complex interactions occurring between ActApo and DCs. These studies provide important mechanistic insight into the responses of DCs during encounter with cells undergoing immunogenic cell death.

Autism in toddlers: can observation in preschool yield the same information as autism assessment in a specialised clinic?

We wanted to know whether preschool observation of children suspected of suffering from autism can provide the same information about core autism symptoms as the Autism Diagnostic Observation Schedule (ADOS) performed in a clinic. Forty 2-4-year-old children (9 girls, 31 boys), referred for assessment of suspected autism spectrum disorder participated in the study. The symptom areas covered by the ADOS algorithm were scored by an education specialist after free-field observation of each child in the preschool without using the prescribed ADOS materials. The ADOS was then completed in a clinic setting by examiners blind to the preschool results. Excellent agreement across results obtained at the two different types/settings of observations was found. The only significant difference found was with regard to spontaneous initiation of joint attention. The present study does not address the issue of whether or not one of the methods used is superior to the other when it comes to determining the "true" level of "autism problems" in these children. However, it is of interest that free-field preschool observation of children with suspected autism using a structured checklist yields very similar information as that obtained at ADOS assessment performed in a clinic setting.

Flow Cytometric Assessment of Myelodysplastic Syndromes/Neoplasms.

Myelodysplastic syndromes/neoplasms (MDS) are a heterogeneous class of hematopoietic stem cell neoplasms characterized by ineffective hematopoiesis leading to peripheral cytopenias. This group of diseases is typically diagnosed using a combination of clinical, morphologic, and genetic criteria. Many studies have described the value of multiparametric flow cytometry (MFC) in the diagnosis, classification, and prognostication of MDS. This review summarizes the approach to MDS diagnosis and immunophenotypic characterization using MFC and describes the current state while highlighting future opportunities and potential pitfalls.

Erythroid side scatter: A parameter that improves diagnostic accuracy of flow cytometry myelodysplastic syndrome scoring.

BACKGROUND: Flow cytometry immunophenotyping (FCM) is a benchmark test for integrated diagnosis of myelodysplastic syndromes (MDS). Our department's FCM-MDS-score follows international guidelines and additionally includes the maturing erythroid (mEry) side scatter (SSC)/lymphocyte SSC ratio (mErySSCr), often increased in MDS patients. A recent exploratory computational flow analysis study highlighted mErySSC as the top feature for separating MDS from non-MDS. Thus, we sought to systematically evaluate the diagnostic accuracy of mErySSCr in conventional diagnostic FCM as used currently in-house. METHODS: Historical MDS (n = 93), chronic myelomonocytic leukemia (CMML; n = 27) and non-neoplastic cytopenia (n = 57) cohorts were created. Differences between these cohorts and LG-MDS entities were mapped and the mErySSCr cut-off was refined. Prospective bone marrows (n = 213) received for marrow failure work-up were used to determine the sensitivity and specificity of mErySSCr, both as a sole parameter and as a component of the MDS-score. RESULTS: Low-grade (LG)-MDS mErySSCr differed more prominently from controls (p = <0.0001) than high-grade (HG)-MDS (p = 0.024). CMML and controls had a similar mErySSCr. As sole parameter, mErySSCr specificity was 91.1% (n = 112 non-MDS diagnoses) and sensitivity was 36% for LG-MDS (n = 36) and 25% for new HG-MDS diagnoses (n = 16). The specificity of the MDS-score was similar if mErySSCr was omitted (81.3% with and 82.1% without). The MDS-score sensitivity for new HG-MDS diagnoses and CMML (n = 17) was 100%, and was not affected by mErySSCr. The score sensitivity for LG-MDS however, dropped from 86.1% to 72.2% when mErySSCr was excluded. CONCLUSION: mErySSCr increases the diagnostic accuracy of flow-based MDS scoring in our setting, particularly for LG-MDS.

Radial Artery Occlusion after Coronary Angiography with Trans Radial Access A nurse led study employing duplex ultrasonography and the reverse Barbeau test.

AIMS: Trans-radial access (TRA) is the recommended approach for coronary angiography and percutaneous coronary intervention (PCI). Radial artery occlusion (RAO) is the most common complication. We examined the incidence of RAO by means of duplex ultrasonography (DUSG) and the reverse Barbeau test (RBT), after TRA in a clinical setting using conventional compression dressings to achieve haemostasis. All radial artery patency examinations were performed by one dedicated nurse after a brief training course, we assessed the feasibility and quality of this routine in regular clinical practice. METHODS AND RESULTS: In total 97 patients undergoing first time coronary angiograph and in some cases PCI via TRA completed the study. Conventional pressure dressing as means of haemostasis was used. Radial artery patency was examined by DUSG and by RBT, before and at follow-up one month after the procedure. An inter- and intra-observer validation of the ultrasound measurements was performed prior to inclusion. Two cases of RAO (2.1%) were discovered following TRA. All RAO cases were detected by both DUSG and the RBT. Results from the inter-observer validation showed no statistically significant discrepancy between an experienced physician and a newly trained nurse operator (p = 0.403). An intraclass correlation coefficient (ICC) was calculated at 0.89 indicating excellent reproducibility. CONCLUSION: In a high-volume TRA centre, we detected an overall low incidence of RAO using conventional pressure dressing as means of haemostasis. The easy-to-use RBT detected all cases of RAO. Following a short course of training, a nurse from the cardiac catheterisation laboratory was able to perform high quality DUSG examinations of the radial artery to assess patency.

The effect of airborne Palladium nanoparticles on human lung cells, endothelium and blood - A combinatory approach using three in vitro models.

A better understanding of the mechanisms behind adverse health effects caused by airborne fine particles and nanoparticles (NP) is essential to improve risk assessment and identification the most critical particle exposures. While the use of automobile catalytic converters is decreasing the exhausts of harmful gases, concentrations of fine airborne particles and nanoparticles (NPs) from catalytic metals such as Palladium (Pd) are reaching their upper safe level. Here we used a combinatory approach with three in vitro model systems to study the toxicity of Pd particles, to infer their potential effects on human health upon inhalation. The three model systems are 1) a lung system with human lung cells (ALI), 2) an endothelial cell system and 3) a human whole blood loop system. All three model systems were exposed to the exact same type of Pd NPs. The ALI lung cell exposure system showed a clear reduction in cell growth from 24 h onwards and the effect persisted over a longer period of time. In the endothelial cell model, Pd NPs induced apoptosis, but not to the same extent as the most aggressive types of NPs such as TiO2. Similarly, Pd triggered clear coagulation and contact system activation but not as forcefully as the highly thrombogenic TiO2 NPs. In summary, we show that our 3-step in vitro model of the human lung and surrounding vessels can be a useful tool for studying pathological events triggered by airborne fine particles and NPs.

The flow cytometry myeloid progenitor count: A reproducible parameter for diagnosis and prognosis of myelodysplastic syndromes.

BACKGROUND: The bone marrow blast count is central to the diagnosis and monitoring of myelodysplastic syndromes (MDS). It is an independent risk factor for worse prognosis whether based on the morphology blast count or the flow cytometry (FC) myeloid progenitor (MyP) count. It is a principal population in FC MDS analysis also because once defined; it provides significant contributions to the overall FC MDS score. METHODS: We elected to investigate inter-analyst agreement for the most fundamental parameter of the FC MDS diagnostic score: the MyP count. A common gating strategy was agreed and used by seven cytometrists for blind analysis of 34 routine bone marrows sent for MDS work-up. Additionally, we compared the results with a computational approach. RESULTS: Concordance was excellent: Intraclass correlation was 0.993 whether measuring %MyP of total cells or CD45+ cells, and no significant difference was observed between files from different centers or for samples with abnormal MyP phenotypes. Computational and manual results were similar. Applying the common strategy to individual laboratories' control cohorts produced similar MyP reference ranges across centers. CONCLUSION: The FC MyP count offers a reliable diagnostic and prognostic measurement in MDS. The use of manual and computational approaches side by side may allow for optimizing both strategies. Considering its known prognostic power, the MyP count could be considered a useful and reliable addition to existing prognostic scoring systems.

ELN iMDS flow working group validation of the monocyte assay for chronic myelomonocytic leukemia diagnosis by flow cytometry.

BACKGROUND: It was proposed that peripheral blood (PB) monocyte profiles evaluated by flow cytometry, called "monocyte assay," could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay requires a large multicenter validation and the assessment of its feasibility on bone marrow (BM) samples, as some centers may not have access to PB. METHODS: PB and/or BM samples from patients displaying monocytosis were assessed with the "monocyte assay" by 10 ELN iMDS Flow working group centers with harmonized protocols. The corresponding files were reanalyzed in a blind fashion and the cMo percentages obtained by both analyses were compared. Confirmed diagnoses were collected when available. RESULTS: The comparison between cMo percentages from 267 PB files showed a good global significant correlation (r = 0.88) with no bias. Confirmed diagnoses, available for 212 patients, achieved a 94% sensitivity and an 84% specificity. Hence, 95/101 CMML patients displayed cMo ≥94% while cMo <94% was observed in 83/99 patients with reactive monocytosis and in 10/12 patients with myeloproliferative neoplasms (MPN) with monocytosis. The established Receiver Operator Curve again provided a 94% cut-off value of cMo. The 117 BM files reanalysis led to an 87% sensitivity and an 80% specificity, with excellent correlation between the 43 paired samples to PB. CONCLUSIONS: This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples.

Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies in myelodysplastic syndromes by the ELN iMDS flow working group.

BACKGROUND: Myelodysplastic syndromes (MDS) represent a diagnostic challenge. This prospective multicenter study was conducted to evaluate pre-defined flow cytometric markers in the diagnostic work-up of MDS and chronic myelomonocytic leukemia (CMML). METHODS: Thousand six hundred and eighty-two patients with suspected MDS/CMML were analyzed by both cytomorphology according to WHO 2016 criteria and flow cytometry according to ELN recommendations. Flow cytometric readout was categorized 'non-MDS' (i.e. no signs of MDS/CMML and limited signs of MDS/CMML) and 'in agreement with MDS' (i.e., in agreement with MDS/CMML). RESULTS: Flow cytometric readout categorized 60% of patients in agreement with MDS, 28% showed limited signs of MDS and 12% had no signs of MDS. In 81% of cases flow cytometric readouts and cytomorphologic diagnosis correlated. For high-risk MDS, the level of concordance was 92%. A total of 17 immunophenotypic aberrancies were found independently related to MDS/CMML in ≥1 of the subgroups of low-risk MDS, high-risk MDS, CMML. A cut-off of ≥3 of these aberrancies resulted in 80% agreement with cytomorphology (20% cases concordantly negative, 60% positive). Moreover, >3% myeloid progenitor cells were significantly associated with MDS (286/293 such cases, 98%). CONCLUSION: Data from this prospective multicenter study led to recognition of 17 immunophenotypic markers allowing to identify cases 'in agreement with MDS'. Moreover, data emphasizes the clinical utility of immunophenotyping in MDS diagnostics, given the high concordance between cytomorphology and the flow cytometric readout. Results from the current study challenge the application of the cytomorphologically defined cut-off of 5% blasts for flow cytometry and rather suggest a 3% cut-off for the latter.

Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34+ CD19- ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS.

Clinical application of flow cytometry in patients with unexplained cytopenia and suspected myelodysplastic syndrome: A report of the European LeukemiaNet International MDS-Flow Cytometry Working Group.

This article discusses the rationale for inclusion of flow cytometry (FCM) in the diagnostic investigation and evaluation of cytopenias of uncertain origin and suspected myelodysplastic syndromes (MDS) by the European LeukemiaNet international MDS Flow Working Group (ELN iMDS Flow WG). The WHO 2016 classification recognizes that FCM contributes to the diagnosis of MDS and may be useful for prognostication, prediction, and evaluation of response to therapy and follow-up of MDS patients.

Flow cytometric analysis of myelodysplasia: Pre-analytical and technical issues-Recommendations from the European LeukemiaNet.

BACKGROUND: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures. METHODS: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group. RESULTS: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies. CONCLUSIONS: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice.

ESCCA/ISCCA protocol for the analysis of cerebrospinal fluid by multiparametric flow-cytometry in hematological malignancies.

Central nervous system (CNS) involvement is a serious but often underdiagnosed complication of hematological malignancies. Currently, the gold standard to detect CNS involvement is conventional cytology (CC) whose sensitivity though is lower than 50%. Multiparametric flow cytometry (MFC) demonstrated a superior sensitivity over CC, particularly when low levels of CNS infiltrating cells are present. Although prospective studies are few, a positive finding by MFC appears to anticipate an adverse outcome even if CC shows no infiltration. However, the use of MFC to diagnose CNS involvement presents some pitfalls, due to the typical hypocellularity of cerebrospinal fluids (CSF), and low cell vitality. Furthermore, the threshold to be used for defining the MFC positivity is not universally defined. In this paper, a working group of the European Society for Clinical Cell Analysis-ESCCA-and the Italian Society for Clinical Cell Analysis-ISCCA-will discuss the critical aspects of CSF processing, highlighting difficulties in storage and processing of samples, interpretation and reporting of data.

Flow cytometric evaluation of peripheral blood for suspected Sézary syndrome or mycosis fungoides: International guidelines for assay characteristics.

A peripheral blood flow cytometric assay for Sézary syndrome (SS) or circulating mycosis fungoides (MF) cells must be able to reliably identify, characterize, and enumerate T-cells with an immunophenotype that differs from non-neoplastic T-cells. Although it is also important to distinguish SS and MF from other subtypes of T-cell neoplasm, this usually requires information in addition to the immunophenotype, such as clinical and morphologic features. This article outlines the approach recommended by an international group with experience and expertise in this area. The following key points are discussed: (a) At a minimum, a flow cytometric assay for SS and MF should include the following six antibodies: CD3, CD4, CD7, CD8, CD26, and CD45. (b) An analysis template must reliably detect abnormal T-cells, even when they lack staining for CD3 or CD45, or demonstrate a phenotype that is not characteristic of normal T-cells. (c) Gating strategies to identify abnormal T-cells should be based on the identification of subsets with distinctly homogenous immunophenotypic properties that are different from those expected for normal T-cells. (d) The blood concentration of abnormal cells, based on any immunophenotypic abnormalities indicative of MF or SS, should be calculated by either direct enumeration or a dual-platform method, and reported.

Detection of CAR-T19 cells in peripheral blood and cerebrospinal fluid: An assay applicable to routine diagnostic laboratories.

BACKGROUND: Chimeric antigen receptor-modified T-cells targeting CD19 (CAR-T19) are licensed for treating relapsed/refractory diffuse large B-cell lymphoma and B-acute lymphoblastic leukemia. Predicting treatment responses and toxicity (e.g., cytokine release syndrome and neurotoxicity) remains a big challenge. CAR-T19 monitoring could increase our understanding of treatment responses and be of relevance to patient management. A robust method for accurate CAR-T19 detection is therefore extremely desirable. METHODS: An assay that uses fluorochrome-conjugated human recombinant soluble CD19 was tested against two commercially available CAR-T19 therapies and a CAR-T19 cell line developed in-house. Precision, concordance, and analyte stability were tested using peripheral blood obtained from CAR-T19-treated patients and controls. RESULTS: The assay showed good accuracy, and had a limit of blank for whole blood samples of 0.13%. Reproducibility and inter-operator concordance were satisfactory (CVs <15%). The assay distinguished CAR-T19 from reactive T-cells in cerebrospinal fluid (CSF) from patients with suspected immune effector cell-associated neurotoxicity syndrome (ICANS), and was adapted to study memory T-cell compartments in treated patients. CONCLUSION: The assay enabled routine monitoring of CAR-T19 in blood and CSF samples. Despite profound cytopenia in many lymphoma patients, results were obtained regularly from only 4 ml of blood. The assay can be adapted easily to characterize the memory and exhaustion status of CAR-T19 and native T-cells. Importantly, it does not rely on CAR construct specificity; thus, it can be used to detect any CD19-targeted CAR cell. Finally, our validation process can serve as a blueprint for other fluorochrome proteins used to detect CAR cells.