Absolute Cell Research Articles

How Reliable Is the Absolute Synovial Polymorphonuclear Neutrophil Cell Count in Diagnosing Periprosthetic Joint Infection?

BackgroundDiagnosing periprosthetic joint infection (PJI) is a daunting task for even the most experienced orthopedic surgeons, as there is currently no test available that can provide absolute accuracy. Utilizing an established synovial indicator for detecting PJI without incurring additional costs or resources would be the optimal solution for predicting the presence of infection. Therefore, we hypothesized that synovial absolute neutrophil count (ANC) would improve the diagnostic accuracy of chronic knee and hip PJI. MethodsThe study included 260 patients (134 men and 126 women, mean age of 70 years [range, 26 to 89]) who underwent aspiration during preoperative workup. Of these, 109 patients (41.9%) were diagnosed with chronic PJI (50 knees, 59 hips), and 151 patients (58.1%) were diagnosed as aseptic (94 knees, 57 hips). Data obtained from all patients included age, sex, procedure type (total hip or total knee arthroplasty), operation side, synovial white blood cell count (cells/μL), synovial polymorphonuclear cells percentage, and synovial α-defensin immunoassay value at the admission were retrieved from the electronic medical record. ResultsThe calculated optimal threshold for synovial ANC of 1,415.5 cells/μL was associated with an area under the receiver operating characteristic curve (AUC) of 0.930 for chronic knee PJI diagnosis. The calculated optimal threshold for synovial ANC of 2,247 cells/μL was associated with an AUC of 0.905 for chronic hip PJI diagnosis. ConclusionsThis study has conclusively shown that the synovial ANC serves as a valuable marker in the complicated diagnosis of PJI. This highly effective and efficient approach should be utilized for obtaining further information through standard tests, thereby ruling out the possibility of PJI. Level of evidenceIII.

Tumour area infiltration and cell count in endoscopic biopsies of therapy-naive upper GI tract carcinomas by QuPath analysis: implications for predictive biomarker testing

Guidelines regulate how many (tumour-bearing) tissue particles should be sampled during gastric cancer biopsy to obtain representative results in predictive biomarker testing. Little is known about how well these guidelines are applied, how the number of tissue particles correlates with the actual tumour-infiltrated area and how many absolute tumour cells are captured. The study included endoscopic biopsies of untreated carcinomas of the upper gastrointestinal (GI)-tract during the 2016–2020 review period. Archival (H&E)-stained histological sections were digitised and the tumour areas were manually annotated. The tumour-bearing tissue area and absolute carcinoma cell count per case were determined by image analysis and compared with a reference primary surgical specimen. Biopsies from 253 patients were analysed. The following mean values were determined: (a) tumour tissue particle number: 6.5 (range: 1–25, standard deviation (SD) = 3.33), (b) number of tumour-bearing tissue particles: 4.7 (range: 1–20, SD = 2.80), (c) tumour-infiltrated area: 7.5 mm2 (range: 0.18–59.46 mm2, SD = 6.67 mm2), (d) absolute tumour cell count: 13,492 (range: 193–92,834, SD = 14,185) and (e) tumour cell count in a primary surgical specimen (tumour size: 6.7 cm): 105,200,176. The guideline-recommended tissue particle count of 10 was not achieved in 208 patients (82.2%) and the required tumour-bearing tissue particle count of 5 was not achieved in 133 patients (52.6%). Tissue particle count, tumour-infiltrated area and tumour cell count were only weakly correlated. Most cases featured an infiltrated area ≥ 4.5 mm2 (156, 61.7%). Cases with more tissue particles showed only a moderate increase in infiltrated area and tumour cells compared to cases with fewer particles. Biopsies are often used to determine predictive biomarkers, particularly Her2/neu and PD-L1. Diagnostic standards to ensure representative material have been suggested in guidelines to reduce false-negative predictions. However, the real-world practice seems to substantially deviate from recommended standards. To the best of our knowledge, this is the first systematic study describing the relationships between endoscopic tissue fragment number, actual infiltrated tumour area and carcinoma cell number. The data question the tissue particle number as a quality assessment parameter. We advocate histopathological reports indicating on which basis statements on therapy-relevant biomarkers were made. Digital pathology has the potential to objectively quantify the tissue for documentation, quality assessment and future clinical studies.

Macro- and micro-scale culture environment differentially regulate the effects of crowding on macrophage function.

Macrophages hold vital roles in immune defense, wound healing, and tissue homeostasis, and have the exquisite ability to sense and respond to dynamically changing cues in their microenvironment. Much of our understanding of their behavior has been derived from studies performed using in vitro culture systems, in which the cell environment can be precisely controlled. Recent advances in miniaturized culture platforms also offer the ability to recapitulate some features of the in vivo environment and analyze cellular responses at the single-cell level. Since macrophages are sensitive to their surrounding environments, the specific conditions in both macro- and micro-scale cultures likely contribute to observed responses. In this study, we investigate how the presence of neighboring cells influence macrophage activation following proinflammatory stimulation in both bulk and micro-scale culture. We found that in bulk cultures, higher seeding density negatively regulated the average TNF-α secretion from individual macrophages in response to inflammatory agonists, and this effect was partially caused by the reduced cell-to-media volume ratio. In contrast, studies conducted using microwells to isolate single cells and groups of cells revealed that increasing numbers of cells positively influences their inflammatory activation, suggesting that the absolute cell numbers in the system may be important. In addition, a single inflammatory cell enhanced the inflammatory state of a small group of cells. Overall, this work helps to better understand how variations of macroscopic and microscopic culture environments influence studies in macrophage biology and provides insight into how the presence of neighboring cells and the soluble environment influences macrophage activation.

Adrenal Stereotactic Body Radiation Therapy: Do Dosimetric Factors Predict for Grade 2+ Lymphopenia?

Recent studies suggest improvement in outcomes in multiple oligometastatic tumor sites with immunotherapy (IO) and radiation therapy (RT). However, RT may induce lymphopenia that can be immunosuppressive and dosimetric consensus is lacking on parameters predicting grade ≥2 lymphopenia. In this study, we evaluated lymphopenia post receipt of stereotactic body RT (SBRT) for adrenal metastases to see if we could identify any modifiable treatment planning factors. Patients with adrenal oligometastases who were treated with conventional SBRT or MRI-guided (MRgSBRT), had absolute lymphocyte count (ALC) and white blood cell count (WBC) data available within 6 weeks before and after their radiation treatment, and had contouring data available for review were included in this IRB approved retrospective study. Lymphopenia was graded for ALC as: Grade 1 (1.0-0.8 no/ul), Grade 2 (<0.8 - 0.5 no/ul), Grade 3 (<0.5-0.2 no/ul), Grade 4 (<0.2 no/ul). The vertebral bodies (VB), at the level of the planning treatment volume (PTV) and one above and below, and the spleen were contoured. Dosimetric variables of interest included spleen and VB mean dose (Gy) as well as V2.5-20 Gy in 2.5 Gy increments. Regression analyses were used to identify dosimetric variables associated with absolute and relative difference in ALC post-SBRT. A total of 30 patients were identified with a slight male predominance (N = 17, 57%) and most commonly left adrenal (N = 17) versus right adrenal (N = 10, 33%) or bilateral metastases (N = 3, 10%). Grade 2+ lymphopenia was observed in N = 7 patients pre-SBRT (23%) and N = 17 patients post-SBRT (57%). N = 26 (87%) of the patients had previous chemotherapies and N = 17 (57%) had previous immunotherapy. Multiple primary tumor types were represented with the most common non-small cell lung cancer (N = 14, 47%), followed by melanoma (N = 4, 13%) and small cell lung cancer (N = 3, 10%). Most patients were treated with non-MRgSBRT (N = 19, 63%) vs MRgRT (N = 11, 37%) and all received SBRT in 5 fractions with median dose 50 Gy (Range: 25 - 60 Gy). The median pre-SBRT ALC was 1.13 (Range: 0.39 - 2.96) and median post-SBRT ALC was 0.72 (Range: 0.11 - 4.15). On linear regression analysis, there was no significant association between post SBRT ALC nor lymphopenia grade with splenic or vertebral body dose, treatment laterality, or SBRT dose. In this retrospective series of predominately metastatic lung cancer patients treated with adrenal SBRT, we observed high rates of post SBRT grade 2+ lymphopenia, yet no dosimetric parameter was predictive, not even for left-sided lesions. Future work includes clinical validation of these findings in a larger population, likely with multi-institutional collaboration, and further study of the investigation of the IO/RT sequencing issues in such heavily pre-treated patients.

Grade 4 Lymphopenia Might Associate with Pericardial Irradiation Dose and Worse Prognosis in Patients with Locally Advanced Esophageal Cancer Receiving Concurrent Chemo-Radiotherapy

The immune system may influence prognosis, and lymphopenia is a frequent side effect of concurrent chemo-radiotherapy (CCRT). Radical irradiation for locally advanced esophageal cancer (LA-EC) exposes significant vascular and heart volumes, and we hypothesized that lymphopenia is linked to cardiac and pericardial doses and affects patient prognosis. We identified 190 LA-EC patients who received radical CCRT between 2011 and 2019. Cardiac, pericardial, and lung dosimetric parameters were obtained and multivariate analysis (MVA) was performed to correlate clinical factors and dosimetric parameters with overall survival (OS). Absolute lymphocyte count (ALC), absolute platelet count (PLT), absolute white blood cell count (WBC), absolute neutrophil count (ANC), neutrophil-lymphocyte ratio (NLR = ANC/ALC), and platelet-lymphocyte ratio (PLR = PLT/ALC) were collected before and during CRT. Grade 4 (G4) lymphopenia was defined as Lymphocyte count nadir <0.2 103/mL during CRT and it was used to dichotomize the lymphocyte count nadir. MVA was performed to correlate hematologic toxicity with OS. Logistic stepwise regression was performed to determine the relationship between dosimetric parameters and G4 lymphopenia. Finally, a nomogram of G4 lymphopenia was developed and validated externally. Median follow-up time for all patients was 27.5 months (range 12-118 months). On MVA for OS (n = 190), higher pericardial V30 (PV30) was linked to worse survival (HR = 1.013, 95% CI 1.001-1.026, p = 0.039). The median OS stratified by PV30>55.3% and PV30≤55.3% was 24 months and 54 months, respectively (p = 0.004). G4 lymphopenia was shown to be linked with worse OS in the MVA of hematological toxicity (n = 161) with OS (HR = 2.042, 95% CI 1.335-3.126, p = 0.001). 24 (24%) of the 100 patients in the training set had G4 lymphopenia. Our final model comprised Stage-IVA (p = 0.017), PLR during CRT (p = 0.008), Heart V50 (p = 0.046), and PV30 (p = 0.048). External validation 26 of 90 patients (29%) had grade 4 lymphocytopenia. The ROC curve displays an AUC for internal validation of 0.775 and external validation of 0.843. Higher doses of pericardial radiation might affect LA-EC patients' prognosis by inducing G4 lymphopenia in CCRT process. Further prospective studies are warranted to confirm these findings, especially in the era of immune-checkpoint inhibitor treatment.

Qualification of the differential leukocyte count and immunophenotyping in cryopreserved ex vivo whole blood assay.

We developed a flow cytometry-based assay, termed Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC-ICE), that allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers. We evaluated the performance of the DLC-ICE assay by determining inter-operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. In addition, we assessed inter-operator variability for staining of fixed cells and robustness across different anticoagulants. Accuracy was evaluated by comparing DLC-ICE measurements to real-time cell enumeration using an accredited hematology analyzer. Finally, we developed and tested the performance of a 27-colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB. Overall, we observed <20% variability in absolute counts and frequencies of granulocytes, monocytes and lymphocytes (T, B and NK cells) when fresh WB was collected in different anti-coagulant tubes, processed or stained by independent operators. Absolute cell counts measured across operators and anti-coagulants using the DLC-ICE method exhibited excellent correlation with the reference method, complete blood count (CBC) with differential, measured using a hematology analyzer (r2 > 0.9 for majority of measurements). A comparison of leukocyte immunophenotyping on fresh WB versus DLC-ICE processed blood yielded equivalent and linear results over a wide dynamic range (r2 = 0.94 over 10-104 cells/μL). These results demonstrate low variability across trained operators, high robustness, linearity and accuracy, supporting utility of the DLC-ICE assay for large cohort studies involving multiple clinical research sites.

A-156 White Blood Cell Measurands, Flagging and Scatter Plot Patterns in Chronic Lymphocytic Leukemia

Abstract Background In this study, we use Alinity hq hematology analyzer to investigate the differences between white blood cell measurands of a healthy control population vs Chronic Lymphocytic Leukemia (CLL). A repeatability study was also conducted to determine the precision of the white blood cell parameter results on the Alinity hq. In addition, we used Alinity hq to identify unique flagging and patterns in the optical channels for CLL. Methods One hundred and fifteen peripheral blood samples of CLL were compared to a population of four hundred and fourteen healthy controls on the Alinity hq analyzer. Statistical differences in Alinity hq’s white blood cell measurands compared between the control population and CLL. For the repeatability study, five CLL samples (with sufficient volume) will be tested in 10 replicates for WBC measurands. These samples can be part of the 115 samples above or can be different samples. Comparison of scatterplots were also analyzed between CLL and control samples. Lastly, a peripheral smear was performed on all CLL samples. Results Statistical analysis of the means of the white and red cell parameters showed that in comparison to the means of the control population there was a significant statistical (P &amp;lt; 0.05) and clinical difference with CLL for the following parameters: absolute white blood cells (WBC) X 103/µL (43.6 CLL vs 7.41 control), percent neutrophils (%N) (18.25 CLL vs 53.71 control), percent lymphocytes (%L) (77.72 CLL vs 33.47 control), and percent monocytes (%M) (2.71 CLL vs 8.75 control). The WBC repeatability study results showed good concordance on the Alinity hq CBC + Diff + Retic test mode with the following percent coefficient of variance (%CV) results: absolute WBC X 103/µL (0.64, 5.31), percent neutrophils (%N) (1.32, 5.57), percent lymphocytes (%L) (0.20, 2.49), and percent monocytes (%M) (3.99, 26.39). As expected, the %CV range for monocytes is wide due to lymphocyte/variant lymphocyte population extending into the monocyte cluster. Evaluation of flagging showed 67 of the 115 (58.26%) CLL samples had a blast flag and 88 out of the 115 (76.52%) CLL samples had a variant lymphocyte flag. Review of the raw data from the optical channels shows a dominant and elongated lymphocyte cluster on the IAS, FL1 and PSS channels vs ALL for the CLL sample group compared to the control population. In the IAS and PSS vs ALL scatter plots in the CLL samples the lymphocyte cluster extends into the monocyte population triggering the blast flag. Peripheral smear review confirmed the presence of CLL (i.e., numerous smudge cells, mature-looking lymphocytes with characteristic condensed “soccer ball” pattern chromatin and scant cytoplasm) in the experimental group. Conclusion CLL is a cancer of the blood and bone marrow that predominately affects the quantity, and morphology of lymphocytes. Patients present with an abnormally high relative and variant lymphocyte populations. As such, there are a number of signs found in WBC measurands, flagging, and scatter plots on the Alinity hq that can be used as an early screening tool for CLL.

Zeb1 Regulates the Function of Lympho-Myeloid Primed Progenitors after Transplantation.

Zeb1, a zinc finger E-box binding homeobox epithelial-mesenchymal (EMT) transcription factor, acts as a critical regulator of hematopoietic stem cell (HSC) self-renewal and multi-lineage differentiation. Whether Zeb1 directly regulates the function of multi-potent progenitors primed for hematopoietic lineage commitment remains ill defined. By using an inducible Mx-1 Cre conditional mouse model where Zeb1 was genetically engineered to be deficient in the adult hematopoietic system (hereafter Zeb1-/-), we found that the absolute cell number of immunophenotypically defined lympho-myeloid primed progenitors (LMPPs) from Zeb1-/- mice was reduced. Myeloid- and lymphoid-biased HSCs in Zeb1-/- mice were unchanged, implying that defective LMPP generation from Zeb1-/- mice was not directly caused by an imbalance of lineage-biased HSCs. Functional analysis of LMPP from Zeb1-/- mice, as judged by competitive transplantation, revealed an overall reduction in engraftment to hematopoietic organs over 4 weeks, which correlated with minimal T-cell engraftment, reduced B-cell and monocyte/macrophage engraftment, and unperturbed granulocyte engraftment. Thus, Zeb1 regulates LMPP differentiation potential to select lympho-myeloid lineages in the context of transplantation.

Causal role of immune cells in schizophrenia: Mendelian randomization (MR) study

BackgroundComplex immune-brain interactions that affect neural development, survival and function might have causal and therapeutic implications for psychiatric illnesses. However, previous studies examining the association between immune inflammation and schizophrenia (SCZ) have yielded inconsistent findings.MethodsComprehensive two-sample Mendelian randomization (MR) analysis was performed to determine the causal association between immune cell signatures and SCZ in this study. Based on publicly available genetic data, we explored causal associations between 731 immune cell signatures and SCZ risk. A total of four types of immune signatures (median fluorescence intensities (MFI), relative cell (RC), absolute cell (AC), and morphological parameters (MP)) were included. Comprehensive sensitivity analyses were used to verify the robustness, heterogeneity, and horizontal pleiotropy of the results.ResultsAfter FDR correction, SCZ had no statistically significant effect on immunophenotypes. It was worth mentioning some phenotypes with unadjusted low P-values, including FSC-A on NKT (β = 0.119, 95% CI = 0.044 ~ 0.194, P = 0.002), DN (CD4-CD8-) NKT %T cell (β = 0.131, 95% CI = 0.054 ~ 0.208, P = 9.03 × 10− 4), and SSC-A on lymphocytes (β = 0.136, 95% CI = 0.059 ~ 0.213, P = 5.43 × 10− 4). The causal effect of SCZ IgD on transitional was estimated to 0.127 (95% CI = 0.051 ~ 0.203, P = 1.09 × 10− 3). SCZ also had a causal effect on IgD+ %B cell (β = 0.130, 95% CI = 0.054 ~ 0.207, P = 8.69 × 10− 4), and DP (CD4+CD8+) %T cell (β = 0.131, 95% CI = 0.054 ~ 0.207, P = 8.05 × 10− 4). Furthermore, four immunophenotypes were identified to be significantly associated with SCZ risk: naive CD4+ %T cell (OR = 0.986, 95% CI = 0.979 ~ 0.992, P = 1.37 × 10− 5), HLA DR on CD14− CD16− (OR = 0.738 (95% CI = 0.642 ~ 0.849, P = 2.00 × 10− 5), CD33dim HLA DR+ CD11b− AC (OR = 0.631, 95% CI = 0.529 ~ 0.753, P = 3.40 × 10− 7) and activated & resting Treg % CD4 Treg (OR = 0.937, 95% CI = 0.906 ~ 0.970, P = 1.96 × 10− 4).ConclusionsOur study has demonstrated the close connection between immune cells and SCZ by genetic means, thus providing guidance for future clinical research.

Pronounced decline of absolute cell numbers in the brain of a newborn with congenital syphilis.

Pronounced decline of absolute cell numbers in the brain of a newborn with congenital syphilis.

Pulmonary and cerebral leukostasis as an unusual case in a patient with chronic leukemia in chronic phase: Case report and literature review

Hyperleukocytosis is defined as an absolute white blood cells count greater than 100.000cel/ml, being more common in acute myeloid leukemia (AML) than in chronic myeloid leukemia (CML). CML is the predominance of mature granulocytes, its main manifestation is hyperleukocytosis, that can increase the risk of pulmonary and cerebral leukostasis; although this event is uncommon in patients with CML in chronic phase. We present the case of a 38-year old male who presents with hyperleukocytosis that progress to hypoxemic respiratory failure and multiple intracerebral hemorrhage, treated with cytoreduction and tyrosine kinase inhibitors; without leukapheresis, with appropriate clinical evolution. Leukostasis is a high mortality complication that should be considered in patients with hyperleukocytosis who presents with neurological or respiratory distress, and constitutes a medical emergency in which treatment can’t be delayed.

Increasing the collection flow rate to 2 mL/min is effective and reduces the procedure time in off-line photopheresis.

Extracorporeal photopheresis (ECP) treatment, mostly based on apheresis technology, is used for immunomodulation in various diseases such as cutaneous T-cell lymphoma, graft versus host disease and other (auto)immune disorders. The aim of this study was to collect high cell counts and purity in shorter procedure times using an ECP off-line system with an increased collection flow rate of 2 mL/min to a target volume of 200 mL buffy coat. In this prospective study, data of routinely performed off-line photopheresis treatments were collected and analyzed at the Central Institute for Blood Transfusion & Department of Immunology (ZIB) of the Tirol Kliniken, to assess absolute cell counts and procedure times and to calculate collection efficiencies (CE2). A total of 22 patients participated in this study. The processed blood volume was 4312 mL, the collection time 120 min, overall procedure time 157 min and the absolute cell counts of treated white blood cells (WBC) and mononuclear cells (MNC) were 5.0 and 4.3 × 109 respectively (median values). The calculated CE2 for WBC and MNC was 21.1% and 58.5%, the proportion of treated MNCs of the total number of MNCs present was 55.0%. The data presented in this study show high therapeutically effective cell counts collected with a high MNC purity within a shorter overall collection/procedure time due to an increased collection flow rate.

IL-8 in bone marrow and peripheral blood of patients with B-cell acute lymphoblastic leukemia is associated with high regulatory T cell counts, degree of tumor infiltration and expression of CXCR1 in blasts

IntroductionRegulatory T cells (Treg cells) in a tumor environment and the expression of forkhead box P3 (FOXP3) in tumor cells have been associated with a poor prognosis. There are few studies evaluating Treg cells and FOXP3 in B-cell acute lymphoblastic leukemia (B-cell ALL). This study aimed to evaluate the frequencies of Treg cells in bone marrow (BM) and peripheral blood (PB) of patients with B-cell ALL and to determine their associations with the circulating cytokine profile and the expression of CXCR1 (IL-8 receptor) in Treg cells, as well as to compare FOXP3 expression in blasts of patients with B-cell ALL and normal lymphoid precursors. MethodsSamples of BM and PB from patients with B-cell ALL and healthy controls were studied. Treg cells, cytokines, FOXP3 and CXCR1 were evaluated using flow cytometry and analyzed. ResultsA total of 20 patients with B-cell ALL and 10 healthy controls were included. In B-cell ALL patients, Treg cell frequencies increased significantly, with higher percentages in the PB. Absolute Treg cell counts were associated with absolute blast counts in the BM and PB and with an IL-8 concentration. The IL-8 and IL-6 levels were associated with the CXCR1 expression in PB Treg cells. In addition, a greater expression of FOXP3 was observed in leukemic blasts than in normal lymphoid precursors. ConclusionsThese results suggest that the presence of Treg cells and cytokines in the tumor environment may correspond to mechanisms to evade the immune response. For that reason, it would be important to monitor these parameters in B-cell ALL to establish their effect on the disease prognosis.

Hematological Parameters on Llamas Blood Samples: Reliability Across a Range of Storage Times

Background: Llamas are becoming increasingly popular as livestock and as companion animals and their blood samples may require transportation over long distances and storage for extended periods prior to hematological analyses. Hematological parameters are influenced by a range of factors, including age, sex, diet and environment. Examining how these parameters change over time in stored blood samples can provide insights into the natural variation of these parameters in llamas, which can inform future research on the health and biology of these animals. This study sought to evaluate the stability of hematological markers at 0, 6, 12, 18, 24, 30, 36 and 42 hours of storage in blood samples of llamas (Lama glama) stored at 4°C. Methods: The study was conducted on blood from eight clinically healthy llamas captivated at Sarajevo Zoo-Pionirska dolina, Sarajevo, Bosnia and Herzegovina. The effect of storage was observed on the following hematological parameters: Red Blood Cell count (RBC), hematocrit (HCT), hemoglobin (HGB), Mean Cell Volume (MCV), Mean Cell Hemoglobin (MCH), Mean Cell Hemoglobin Concentration (MCHC), Reticulocyte count (RETIC), White Blood Cell (WBC). White blood cell differentiation, absolute white blood cell counts, platelet count (PLT) and mean platelet volume (MPV) are also included. Result: During 42 hours of storage at 4°C, it was discovered that red blood cells, hemoglobin, white blood cells, mean cell hemoglobin and reticulocyte, as well as the differentiation of white blood cells and measurement of their absolute values, remained stable. However, hematocrit increased at 12 h, while mean cell hemoglobin concentration decreased at 12 h when stored at 4°C.

Cytotoxic NK cells phenotype and activated lymphocytes are the main characteristics of patients with alcohol-associated liver disease

T cells, natural killer (NK) and NKT cells have opposing actions in the development of alcohol-associated liver fibrosis. We aimed to evaluate the phenotype of NK cells, NKT cells and activated T cells in patients with alcohol use disorder (AUD) according to the presence of advanced liver fibrosis (ALF). Totally, 79 patients (51-years, 71% males) were admitted to treatment of AUD. ALF was defined as FIB4-score > 2.67. Immunophenotyping of NK cells (CD3−CD56+CD16+, CD3−CD56+CD16−, CD3−CD56−CD16+), NKT-like (CD3+CD56+), and the activation status of CD4+, CD8+ and regulatory T cells (Tregs) were evaluated according to the HLA-DR expression. Patients had an AUD duration of 18 ± 11 years with a daily alcohol consumption of 155 ± 77 gr/day prior to hospital admission. The values of absolute cells were 2 ± 0.9 cells/L for total lymphocytes, 1054 ± 501 cells/µL for CD4+, 540 ± 335 cells/µL for CD8+, 49.3 ± 24.8 cells/µL for Tregs, 150.3 ± 97.5 cells/µL for NK cells and 69.8 ± 78.3 cells/µL for NKT-like. The percentage of total NK cells (11.3 ± 5.5% vs. 7 ± 4.3%, p < 0.01), CD3−CD56+CD16+ regarding total lymphocytes (9.7 ± 5.1% vs. 5.8 ± 3.9%, p < 0.01), activated CD4+ cells (5.2 ± 3.2% vs. 3.9 ± 3%, p = 0.04) and activated CD8+ cells (15.7 ± 9.1% vs. 12.2 ± 9%, p = 0.05) were significantly higher in patients with ALF. The percentage of CD3−CD56+CD16− regarding NK cells (5.1 ± 3.4% vs. 7.6 ± 6.2%, p = 0.03) was significantly lower in patients with ALF. Activated Tregs (39.9 ± 11.5 vs. 32.4 ± 9.2, p = 0.06) showed a tendency to be higher in patients with ALF. The proportion of activated CD4+ cells (r = 0.40, p < 0.01) and activated CD8+ cells (r = 0.51, p < 0.01) was correlated with the proportion of NKT-like in patients without ALF. Patients with ALF presented an increased NK cytotoxic phenotype and activated T cells concomitant with a decreased NK cytokine-secreting phenotype.

P54 Blood tumour burden does not correlate with skin tumour burden in early-stage mycosis fungoides: analysis of the PROspective Cutaneous Lymphoma International Prognostic Index international registry

Abstract Blood involvement B1, defined as absolute counts 250–1000 IU of an aberrant CD4+CD7– or CD4+CD26– phenotype, may be found in early-stage mycosis fungoides (MF). There is a suggestion that B1 blood involvement is a poor prognostic marker, but there is limited evidence investigating the prognostic significance of B0 or B1 blood involvement in early-stage MF. Loss of CD26 and/or CD7 expression on CD4 cells may occur in MF; however, it is unclear whether loss of either one of these immunophenotypic markers bears more significance on prognosis. For B2 blood involvement (&amp;gt; 1000 IU), the International Society for Cutaneous Lymphomas (ISCL) criteria define Sézary syndrome as CD26 loss of &amp;gt; 30% or CD7 loss of &amp;gt; 40%. Understanding the impact of B1 and comparative loss of CD26 and/or CD7 in early-stage MF may allow patients at higher risk of stage progression to be identified and selected for better therapeutic options. The aims of this study were to analyse B0 vs. B1 blood involvement at diagnosis in patients with early-stage MF (IA–IIA) and evaluate the relative CD4+CD7– and CD4+CD26– absolute cell counts. Prospective data from the PROCLIPI registry were analysed. Ninety-four patients with early-stage MF were identified from our database, 67 (71%) of whom were B0 [stage IA, n = 23 (34%); stage IB, n = 24 (36%); stage IIA, n = 20 (30%)] and 27 (29%) were B1 [stage IA, n = 12 (44%); stage IB, n = 10 (37%); stage IIA, n = 5 (18%)]. Median age at diagnosis in B0 patients was 59 years (range 20–91) vs. 61 years (range 21–81) in B1 patients. Median CD4+CD7– cell count was 16.4% (range 0–41%) in patients with B0 involvement and 28.1% (range12–45%) in B1 patients. Median CD4+CD26– cell count was 16% (range 0–39.1%) in B0 and 28% (range 0–44%) in B1 patients. The Median Modified Severity-Weighted Assessment Tool score in B1 patients was 11 (range 1–73) vs. 20 (range 0.5–100) in B0 patients. There was no significant difference in progression to the advanced stage between B0 and B1 (P = 0.112) patients or overall survival between B0 and B1 patients during median follow-ups of 49 months and 45 months, respectively. These results suggest that blood tumour burden in early-stage MF, measured by the percentage aberrancy in CD4+CD7– and CD4+CD26– cell counts, does not correlate with clinical stage, skin tumour burden or stage progression. This suggests that in B1 involvement, atypical lymphocytes may lose their skin-homing properties. Interestingly, levels of CD4+7– and CD26– are similar in B1 involvement, despite the ISCL criteria defining B2 involvement as a lower relative loss of CD26–. Identifying B1 patients is important as certain therapeutic options such as extracorporeal photopheresis and mogamulizumab may be indicated in patients with early-stage MF, refractory to skin-directed therapies.

Increased CD8+ tissue resident memory T cells, regulatory T cells and activated natural killer cells in systemic sclerosis lungs.

Multiple observations indicate a role for lymphocytes in driving autoimmunity in SSc. While T and NK cells have been studied in SSc whole blood and bronchoalveolar lavage fluid, their role remains unclear, partly because no studies have analysed these cell types in SSc-interstitial lung disease (ILD) lung tissue. This research aimed to identify and analyse the lymphoid subpopulations in SSc-ILD lung explants. Lymphoid populations from 13 SSc-ILD and 6 healthy control (HC) lung explants were analysed using Seurat following single-cell RNA sequencing. Lymphoid clusters were identified by their differential gene expression. Absolute cell numbers and cell proportions in each cluster were compared between cohorts. Additional analyses were performed using pathway analysis, pseudotime and cell ligand-receptor interactions. Activated CD16+ NK cells, CD8+ tissue resident memory T cells and Treg cells were proportionately higher in SSc-ILD compared with HC lungs. Activated CD16+ NK cells in SSc-ILD showed upregulated granzyme B, IFN-γ and CD226. Amphiregulin, highly upregulated by NK cells, was predicted to interact with epidermal growth factor receptor on several bronchial epithelial cell populations. Shifts in CD8+ T cell populations indicated a transition from resting to effector to tissue resident phenotypes in SSc-ILD. SSc-ILD lungs show activated lymphoid populations. Activated cytotoxic NK cells suggest they may kill alveolar epithelial cells, while their expression of amphiregulin suggests they may also induce bronchial epithelial cell hyperplasia. CD8+ T cells in SSc-ILD appear to transition from resting to the tissue resident memory phenotype.

Plasma Cell Leukemia-Clinicopathological Profile from a Tertiary Care Center in Western India.

Poornima ManimaranIntroduction Plasma cell leukemia (PCL) is very uncommon and aggressive neoplasm constituting 2 to 4% of all plasma cell dyscrasias. By definition, clonal plasma cells should make up 20% of peripheral blood or have an absolute plasma cell count of 2 × 10 9 cells/cu.mm. PCL can be primary or secondary. In this study, the clinicohematological features of PCL, and correlation of immunophenotypic profile and conventional therapies with overall survival was analyzed. Materials and Methods This retrospective study involved PCL patients who were diagnosed across a 12-year period, from 2010 to 2021, at a tertiary care center in western India. Clinical, biochemical, peripheral smear, bone marrow aspirate, immunophenotyping, and molecular analysis were performed. Results Total 39 PCL patients were included in the study among which 36 were primary PCL patients. Splenomegaly (10/27), hepatomegaly (6/26), and lymphadenopathy (5/23) were noted. At presentation, all patients had anemia (<11g/dL), thrombocytopenia (33/39), hypercalcemia (>11mg/dl) 10/33 (30.3%) and lytic lesions was noted in 18/26 (69.2%). Immunophenotype of these patients showed CD 38 positivity, CD 138 positivity, CD56 positivity, and CD 117 negativity were 100, 62, 41.6, and 89%, respectively. Overall survival of our patients was 4.1 months and overall survival of patients treated with VTD (bortezomib, thalidomide, dexamethasone) and VCD (bortezomib, cyclophosphamide, dexamethasone) regimen was 3.4 and 4.1 months, respectively, which was not statically significant ( p -value 0.816). CD117 and CD56 markers were also not having any prognostic significance ( p -value 1.000 and 0.873, respectively). Conclusion Because of rarity of the disease, prospective studies are very limited and hence management and outcome of the disease are difficult to analyze. The current treatment protocols have no survival advantage and hence newer therapeutic approach is mandatory to attain better outcome.

Ocrelizumab extended-interval dosing in multiple sclerosis during SARS-CoV-2 pandemic: a real-world experience.

During the COVID-19 pandemic, ocrelizumab administration was frequently postponed because of a lack of safety information and to favour vaccination. The clinical implications of ocrelizumab administration delay in multiple sclerosis (MS) patients were assessed. Relapsing (RMS) and primary progressive (PPMS) MS patients receiving ocrelizumab for at least 6 months at our centre were retrospectively classified, according to the possible occurrence of a delay (≥4 weeks) in treatment administration. Patients were categorized in the extended-interval dosing (EID) group in the presence of at least one delayed infusion; otherwise they were considered as part of the standard interval dosing (SID) cohort. MS history, magnetic resonance imaging examinations and B-cell counts were also retrospectively collected and analysed. A total of 213 RMS and 61 PPMS patients were enrolled; 115 RMS and 29 PPMS patients had been treated according to the SID regimen, whilst 98 RMS and 32 PPMS patients were included in the EID cohort. Average follow-up after delay was 1.28 ± 0.7 years in the EID cohort. In RMS, comparing SID and EID patients, no differences were found considering the occurrence of clinical relapses (9.6% vs. 16.3%, p = 0.338), magnetic resonance imaging activity (9.8% vs. 14.1%, p = 0.374) or disability progression (11.3% vs. 18.4%, p = 0.103). Similar findings were observed in PPMS patients. In the pooled EID group, treatment delay correlated with CD19-positive relative (r = 0.530, p < 0.001) and absolute (r = 0.491, p < 0.001) cell counts, without implications on disease activity. Sporadic ocrelizumab administration delay granted sustained treatment efficacy in our cohort. Prospective data should be obtained to confirm these observations and set up systematic extended-interval regimens.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Background: Cancer is a recognized condition of secondary immunosuppression. Lymphoma patients often show a decrease in absolute lymphocyte counts and in peripheral blood gamma-globulin levels. However, less is known about how NK cells are altered in lymphoma patients. The aim of this study was to evaluate the patterns of circulating NK cell dysfunction in lymphoma patients. Methods: We performed a cross-sectional study of patients with a recent diagnosis of nodal lymphoproliferative disorder. Patients with active anticancer treatment or steroid use were excluded. Peripheral blood samples in EDTA were analyzed by multicolor flow cytometry. A custom panel of 30 markers was applied to all samples to test for major subpopulations, maturation, and functional markers. Results were compared to healthy controls. Clinical data were obtained from the electronic database. This study was approved by the institutional IRB and Clinical Ethics Committee. Results: A total of 30 patients were examined between October 2021 and November 2022. The median age was 62 years (range 15–88) and 45.7% were female (16). Patients had the following subtypes: large B-cell lymphoma (LBCL; 9), follicular lymphoma (FL; 9), Hodgkin lymphoma (HL; 5), mantle cell lymphoma (MCL; 4), marginal zone lymphoma (MZL; 2), Waldenström macroglobulinemia (WM; 1). Most patients had advanced stage disease (20). Compared to healthy controls (n = 10), lymphoma patients showed a significant decrease in absolute NK cell counts (ANKC, 319.7 vs. 131.1 cells/mm3; p < 0.0001). MCL patients had the lowest ANKC (68.8) and Hodgkin patients the highest (165.6). Advanced stage disease correlated with lower ANKC compared to localized disease (99.85 vs. 148.9; p = ns). The pattern of maturation was altered in lymphoma patients compared to controls (p = 0.0092). Patients had a higher proportion of immature CD56-bright NK cells (4.97% vs. 2.88%) and lower levels of CD16 expression in the mature CD56-dim subpopulation (68.12% vs. 85.94%). Lymphoma patients also showed dysfunctional expression of receptors and activation markers (Figure). There was a significant increase in the levels of FAS-L, IFNg, CD49b, CD57, CD96, and CD107a, while the levels of TIGIT and NKG2D were significantly decreased. Differences in the expression of immune checkpoints (PD-1, LAG-3, TIM-3), as well as additional activating, inhibitory, and chemokine receptors were not significant. 22 of 30 patients (73.3%) achieved a complete response. All patients with ANKC above 135 cells/mm3 at diagnosis achieved sustained complete remission. The research was funded by: Fondecyt 11200805 Clínica Alemana de Santiago ID1025 Keyword: diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.