Blood Flow Cytometry Research Articles

CD5 Expression in Marginal Zone Lymphoma (MZL) Predicts Worse Outcomes with Rituximab (R) but Not with Bendamustine/Rituximab (BR)

Background. CD5 expression is rare in MZL and has been historically characterized in case series of splenic (SMZL) and nodal (NMZL) MZL (Baseggio et al., haematologica, 2010; Jaso et al., Am J Clin Pathol, 2013; Salido et al., Blood 2010). These studies reported similar outcomes with CD5+ and CD5- MZL but did not examine the significance of CD5 in the context of modern immunochemotherapy. BR is a regimen increasingly used for treatment of MZL, but many patients can initially receive R monotherapy (R-mono) with no detriment to survival (Olszewski et al., ASH 2019). There are no biomarkers predictive of differential response to R-mono or BR. We examined outcomes of these treatments in MZL according to CD5 expression. Methods. We collected clinicopathologic data on patients with MZL (SMZL, NMZL, and extranodal MZL of the mucosa-associated lymphoid tissue [MALT]) diagnosed in our center between 2010 and 2020. CD5+ MZL was diagnosed by expert academic hematopathologists after excluding chronic lymphocytic leukemia and mantle cell lymphoma by immunophenotypic evaluation and cytogenetic/fluorescent in-situ hybridization studies. For prognostication, we assigned subtype-specific prognostic scores: MALT-IPI (Thieblemont et al., Blood 2017), IIL score for SMZL (Arcaini et al., Blood 2006), and standard IPI for NMZL. We examined overall response rate (ORR), progression-free (PFS) and overall survival (OS), calculated from the start of therapy, according to CD5 expression and first-line therapy. Survival was compared using log-rank tests stratified by age, MZL subtype, and stage. Results. The study included 213 patients with SMZL (N=51, 24%), NMZL (N=46, 22%), and MALT (N=116, 54%), with median age of 68 years at diagnosis, and with 52% women. Overall, 23 MZLs (10.8%) were CD5+. CD5 expression was most common in SMZL (24%), then NMZL (11%), and rare in MALT lymphoma (5%; P=.003). CD5+ cases showed del(7q) in 9%, trisomy 12 in 17%, and CD23 expression in 9%. All tested CD5+ cases had a positive peripheral blood flow cytometry (100% vs 65% of CD5- cases, P<.001) and bone marrow involvement (100% vs 64%, P=.004). Furthermore, adjusting for variable prevalence in MZL subtypes, CD5 expression was significantly associated with high lactate dehydrogenase (LDH, 56% vs 18%, adjusted odds ratio (aOR) 5.74, P=.0008), anemia (39% vs 20%, aOR 2.55, P=.044), monoclonal paraprotein (75% vs 36%, aOR 5.33, P=.016), advanced stage (83% vs 54%, aOR 4.10, P=.013), and blood involvement (78% vs 32%, aOR 7.80, P=.0001). CD5 expression also correlated with a higher IIL score in SMZL (P=.032 on test for trends of odds), higher IPI in NMZL (OR=2.84, P=.09), and higher MALT-IPI (OR=3.38, P=.10). CD5+ MZL had a numerically higher prevalence of 17p deletion (43% vs 21%, P=.35) and complex karyotype (42% vs 22%, P=.26) than CD5- MZL, when tested. Histologic transformation occurred in 2 (9%) CD5+ and 4 (2%) CD5- MZLs (P=.13). The most common first-line treatments were R-mono (29%), radiation therapy (23%), BR (20%), and surgery (15%). CD5 expression was not prognostic for PFS (stratified log-rank, P=.29, Fig. A) or OS (P=.08; Fig. B) in the entire cohort, or within the subgroup of NMZL/SMZL only (P=.46 for PFS and P=.31 for OS). However, outcomes differed according to CD5+/- status between patients treated with first-line R-mono or BR. ORR to R-mono was significantly lower for CD5+ than CD5- MZL (22% vs. 78%, P=.003), whereas ORR to BR did not differ (100% and 93%, respectively, P=.72). Similarly, PFS after R-mono was significantly worse for CD5+ than CD5- MZL (P=.036; Fig. C), while it did not significantly differ after BR (P=.64; P for interaction between treatments=.030). CD5 expression was also associated with significantly worse OS among patients receiving R-mono (P=.047) but not among those receiving BR (P=.56; Fig. D). Results were similar in the subgroup of SMZL/NMZL cases. Conclusions. To our knowledge, this is the first study examining CD5 expression as a prognostic and predictive biomarker for outcomes of R-based immunochemotherapy in MZL. CD5+ MZL was characterized by disseminated presentation with universal peripheral blood involvement, but after adjustment for other factors, CD5 expression was not independently prognostic. However, we observed worse response rates, PFS, and OS for CD5+ MZL treated with first-line R monotherapy, but not among those treated with BR, suggesting that the BR combination might be preferable in this subgroup. Disclosures Olszewski: Spectrum Pharmaceuticals: Research Funding; Genentech, Inc.: Research Funding; Adaptive Biotechnologies: Research Funding; TG Therapeutics: Research Funding.

Two Rare Cases of Severe Autoimmune Dyserythropoiesis without Any Underlying Haematological Malignancy or Autoimmune Disease

Background: Autoimmune dyserythropoiesis is a rare disorder with only 4 adult cases and 3 paediatric cases reported in the literature. These suggest an underlying cause of haematological malignancy i.e. CLL or autoimmune disorder e.g. SLE1. Two proposed mechanisms of the pathophysiology include erythroblastic synartesis, or a Fas deficiency in autoimmune conditions2. There is no standard of care, however most cases respond to immunosuppression and/or treatment of the underlying condition. This is a report of 2 rare cases of severe autoimmune dyserythropoiesis without any underlying haematological or autoimmune conditions that have both responded to immunosuppression. Methods: Investigations included a haemolytic screen (LDH, bilirubin, haptoglobin, reticulocyte count and DAT), iron, B12 and folate studies, viral screening (EBV, CMV, HBV, HCV, HIV and parvovirus serologies and parvovirus PCR), autoimmune screen (ENA, ANA, RF, anti CCP, dsDNA, ANCA, C3/4), malignancy screen (EPG/IEPG and serum light chains, peripheral blood flow cytometry and imaging including CT CAP and/or PET scans). Bone marrow aspirate and trephine were performed with cytogenetics and molecular testing for myeloid mutations using NGS panels. Results: Case 1 is a 56-year-old female who initially presented in 2006 with fevers, weight loss, night sweats and an unexplained anaemia with a haemoglobin of 67g/L and MCV of 109 fl. WCC was 6.3 x10^9/L and platelet count 495 x 10^9/L. Clinical exam revealed no signs of haematological malignancy or autoimmune disease. Reticulocyte count was low at 7x10^9/L. ESR was significantly elevated at 130 mm/hr. Bone marrow biopsy was hypercellular with increased early erythroid precursors and some erythroid islands that resembled erythroblastic synartesis. Moderate dyserythropoiesis with numerous binucleate forms was also noted. The rest of her investigations were normal. She responded briskly to low dose prednisone (25mg daily) with resolution of her anaemia, reticulocytopaenia and inflammatory markers. She was transitioned onto azathioprine in 2011 as a steroid sparing agent which was ceased in 2018 due to a diagnosis of localised breast cancer. She remained in remission off immunosuppression for 18 months. Her relapse clinical findings and investigations were identical to her initial presentation. Her NGS testing at this time was negative for myeloid genetic mutations. She again responded briskly to prednisolone. Case 2 is a 65-year-old female referred for a 4th opinion in May 2019 regarding transfusion dependent anaemia. Clinical exam revealed no signs of malignancy nor autoimmune disease. Haemoglobin averaged 60g/L with a low reticulocyte count at 11x10^9/L and elevated MCV of 117fL. Ferritin was elevated secondary to multiple prior transfusions at 1190 ug/L. ESR and CRP were elevated with an ANA titre of 1:80 (homogenous). Bone marrow was hypercellular with some minor dysplastic features and increased immature forms. Peripheral blood flow cytometry revealed a small monoclonal B cell population (0.1% of lymphocytes) that was not detected on bone marrow biopsy. The rest of her investigations were normal. She declined a trial of steroid therapy She was commenced on weekly IV rituximab 375mg/m2 for 3 weeks but was unable to tolerate mild infusion reactions and had no response. She was then treated with intravenous immunoglobulin as well as iron chelation therapy and her anaemia, reticulocytopaenia and inflammatory markers quickly resolved. Discussion: These are two rare cases of severe autoimmune dyserythropoiesis with reticulocytopaenia with no autoimmune haemolysis and no underlying autoimmune or malignant disease. Due to the rarity of this disease, there are no clear guidelines on how to manage these patients and more evidence/research is required. Both cases responded well to immunosuppressive treatment in a relapsing/remitting fashion that further reinforces the autoimmune nature of this disease.One patient did not respond to rituximab despite a small monoclonal B lymphocyte population unlike the only other case treated with rituximab reported where this was successful.

High PD-L1/CD274 Expression of Monocytes and Blood Dendritic Cells Is a Risk Factor in Lung Cancer Patients Undergoing Treatment with PD1 Inhibitor Therapy.

Simple SummaryTumor cells can evade destruction via immune cells by expressing coinhibitory membrane molecules, which suppress antitumoral immune responses. Immune checkpoint inhibitor therapy acts by blocking these inhibitory pathways. Although this type of immunotherapy has shown promising results for selected cancer patients during recent years, an important challenge remains to identify baseline characteristics of patients who will mostly benefit from such therapy. The aim of our study was to assess the expression of the coinhibitory molecule PD-L1/CD274 on different antigen-presenting cells (monocytes and dendritic cell subsets) in the peripheral blood of 35 patients with nonsmall cell lung cancer undergoing checkpoint inhibitor therapy. CD274 expression correlated with therapy response and the survival of patients. Tumor patients with high CD274 expression levels of antigen-presenting cells in blood rarely responded to checkpoint inhibitor therapy. Our results implicate that a high CD274 expression in monocytes and dendritic cell subsets is a risk factor for therapy response.The aim of this study was to investigate the expression of the coinhibitory molecule PD-L1/CD274 in monocytes and dendritic cells (DC) in the blood of lung cancer patients undergoing PD1 inhibitor therapy and to correlate data with patient’s outcome. PD-L1/CD274 expression of monocytes, CD1c+ myeloid DC (mDC) and CD303+ plasmacytoid DC (pDC) was determined by flow cytometry in peripheral blood at immunotherapy onset. The predictive value of the PD-L1/CD274-expression data was determined by patients’ survival analysis. Patients with a high PD-L1/CD274 expression of monocytes and blood DC subpopulations rarely responded to PD1 inhibitor therapy. Low PD-L1/CD274 expression of monocytes and DC correlated with prolonged progression-free survival (PFS) as well as overall survival (OS). The highest PD-L1/CD274 expression was found in CD14+HLA-DR++CD16+ intermediate monocytes. Whereas the PD-L1/CD274 expression of monocytes and DC showed a strong positive correlation, only the PD-L1/CD274 expression of DC inversely correlated with DC amounts and lymphocyte counts in peripheral blood. Our results implicate that a high PD-L1/CD274 expression of blood monocytes and DC subtypes is a risk factor for therapy response and for the survival of lung cancer patients undergoing PD1 inhibitor therapy.

NK cell markers predict the efficacy of IV immunoglobulins in CIDP.

ObjectiveTo assess whether IV immunoglobulins (IVIgs) as a first-line treatment for chronic inflammatory demyelinating polyneuropathy (CIDP) have a regulative effect on natural killer (NK) cells that is related to clinical responsiveness to IVIg.MethodsIn a prospective longitudinal study, we collected blood samples of 29 patients with CIDP before and after initiation of IVIg treatment for up to 6 months. We used semiquantitative PCR and flow cytometry in the peripheral blood to analyze the effects of IVIg on the NK cells. The results were correlated with clinical aspects encompassing responsiveness.ResultsWe found a reduction in the expression of several typical NK cell genes 1 day after IVIg administration. Flow cytometry furthermore revealed a reduced cytotoxic CD56dim NK cell population, whereas regulatory CD56bright NK cells remained mostly unaffected or were even increased after IVIg treatment. Surprisingly, the observed effects on NK cells almost exclusively occurred in IVIg-responsive patients with CIDP.ConclusionsThe correlation between the altered NK cell population and treatment efficiency suggests a crucial role for NK cells in the still speculative mode of action of IVIg treatment. Analyzing NK cell subsets after 24 hours of treatment initiation appeared as a predictive marker for IVIg responsiveness. Further studies are warranted investigating the potential of NK cell status as a routine parameter in patients with CIDP before IVIg therapy.Classification of evidenceThis study provides Class I evidence that NK cell markers predict clinical response to IVIg in patients with CIDP.

A CASE OF AUTOIMMUNE PULMONARY ALVEOLAR PROTEINOSIS WITH UNDERLYING MONOCLONAL B-CELL LYMPHOCYTOSIS

SESSION TITLE: Fellows Diffuse Lung Disease Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: October 18-21, 2020 INTRODUCTION: We describe a rare case of autoimmune pulmonary alveolar proteinosis (PAP) with a concurrent low-grade lymphoproliferative disorder. CASE PRESENTATION: A 47-year-old male nonsmoker with obesity, hypertension and type 2 diabetes with nephropathy was admitted to the ICU with fever, cough and dyspnea leading to acute hypoxemic respiratory failure requiring intubation. He had been hospitalized for “heart failure” 6 months prior to presentation, and his dyspnea had slowly progressed since then. He worked as a butcher. Three of his 1st-degree relatives had died from presumed heart failure between ages 44-54. Additional features of his presentation included Raynaud’s phenomenon, high CRP of 254 mg/L and no clinical signs of heart failure. Chest x-ray (CXR) demonstrated nonspecific patchy bilateral opacities. The CXR from 6 months ago showed similarly distributed, albeit less prominent, opacities (Fig 1). Chest CT revealed diffuse, bilateral ground-glass opacities (GGOs) with interlobular septal thickening (crazy-paving pattern), consolidation and mediastinal lymphadenopathy (Fig 2). TTE showed normal biventricular function. Rheumatologic workup was negative. BAL microbiology showed S. pneumoniae, which was treated. Periodic acid-Schiff (PAS) staining from BAL showed bright globular material consistent with PAP (Fig 3). Serum anti-GM-CSF antibody was positive. Bone marrow biopsy revealed monoclonal B-cell lymphocytosis involving 20% of the marrow with positive peripheral blood flow cytometry, mutated IGH-V, and negative TP53 mutation. The patient required bilateral whole lung lavage (WLL) on two occasions, one month apart. He was started on inhaled GM-CSF after the first WLL and rituximab was added subsequently. His course was complicated by massive pulmonary embolism requiring VA-ECMO support, acute kidney injury requiring dialysis, and critical illness myoneuropathy. He was discharged home after 84 days. DISCUSSION: PAP is classified into 3 groups: primary (autoimmune and hereditary), secondary, and congenital (1). PAP is diagnosed with positive PAS stain on BAL fluid or lung biopsy in combination with typical features on CT, including bilateral GGOs with interlobular septal thickening. The hallmark of primary, or autoimmune, PAP includes presence of autoantibodies to GM-CSF, which are not present in secondary PAP (2). Common etiologies of secondary PAP are hematologic malignancies, most commonly myeloid (3). Ascertaining the etiology of PAP is important, and guides the choice of therapy. For autoimmune PAP, treatment includes WLL and inhaled GM-CSF. For secondary PAP, treatment is directed at the underlying cause. CONCLUSIONS: This is one of the first reported cases of autoimmune PAP with a concurrent low-grade lymphoproliferative disorder. The patient improved with WLL, inhaled GM-CSF and rituximab. Reference #1: Trapnell BC, Nakata K, Bonella F, et al. Pulmonary alveolar proteinosis. Nat Rev Dis Primers. 2019 March 7;5(1):16. Reference #2: Kumar A, Abdelmalak B, Inoue Y, Culver DA. Pulmonary alveolar proteinosis in adults: pathophysiology and clinical approach. Lance Respir Med. 2018 Jul;6(7):554-565. Reference #3: Chaulagain CP, Pilichowska M, Brinckerhoff L, Tabba M, Erban JK. Secondary pulmonary alveolar proteinosis in hematologic malignancies. Hematol Oncol Stem Cell Ther. 2014 Dec; 7(4):127-35. DISCLOSURES: No relevant relationships by Simone Barreto Siqueira Parrilha T, source=Admin input No relevant relationships by Brittany Blass, source=Web Response No relevant relationships by Melanie Bois, source=Web Response No relevant relationships by Sumedh Hoskote, source=Web Response no disclosure on file for Mark Norton

S2991 Gastric Myeloid Sarcoma as a Presentation of Relapsed Acute Myelomonocytic Leukemia - A Case Report

INTRODUCTION: Myeloid sarcoma is an extramedullary proliferation of myeloid blasts that may be associated with a concurrent myeloid neoplasm involving the bone marrow, but such an association is not required. In some cases, myeloid sarcoma may herald a relapse in a patient with previously treated disease. In others, it may be the first indication of acute leukemia. CASE DESCRIPTION/METHODS: A 48-year-old male presented with nausea, vomiting and epigastric pain. Physical examination showed tachycardia, and epigastric tenderness. Nine months earlier he was diagnosed with acute myelomonocytic leukemia; cytogenetics showed 47,XY,+8,t(9;11)(p21.3;q23.3). At that time, he was treated with standard induction therapy with the 7 + 3 regimen with Cytarabine and Daunorubicin followed by 3 cycles of High-Dose Cytarabine (HiDAC). Lab results showed pancytopenia including Leukopenia with 19% blasts. Peripheral blood flow cytometry showed 18% monoblasts. CT chest and abdomen revealed diffuse lobular thickening of the gastric wall, segmental thickening of the small bowel loops, and pancreatic, paraspinal, and infiltrative right ventricular wall masses. Upper GI endoscopy showed thickened gastric folds with a nodular mucosa. Gastric mucosal resection was performed, and biopsy showed abnormal blasts infiltrating through the mucosal glands, consistent with myeloid sarcoma. A bone marrow biopsy was consistent with relapsed AML. Urgent therapy was instituted however the patient suffered a cardiac arrest and resuscitation was unsuccessful. DISCUSSION: Myeloid sarcoma (aka granulocytic sarcoma, myeloblastoma, or chloroma) may present simultaneously with, or precede bone marrow disease and may be seen in relapse or as progression of a prior myeloproliferative neoplasm. The most common sites of isolated myeloid sarcoma are the skin, followed by mucous membranes, orbits, central nervous system, and other internal organs. Myeloid sarcoma is more common in pediatric AML, although the true incidence in adults is unknown. In adults, roughly one third of myeloid sarcomas present with concurrent myeloid disease, and one third have a history of a prior myeloid neoplasm. The presence of a myeloid sarcoma is diagnostic of AML, regardless of the bone marrow status, or blast count. The diagnosis of myeloid sarcoma should be restricted to tumors that form space-occupying lesions. The approach to treatment of patients with myeloid sarcoma without evidence of AML on bone marrow biopsy is similar to that for patients with overt AML.Figure 1.: CT Abdomen showing lobular thickening of the gastric wall.Figure 2.: Upper GI endoscopy showing thickened gastric folds with nodular mucosa in the gastric body. Findings are concerning of infiltrative malignancy.Figure 3.: High power (10x) view of Hematoxylin and Eosin stain of gastric body biopsy showing abnormal monoblasts infiltrating through the mucosal glands, consistent with myeloid sarcoma.

S2492 Hemolytic Anemia: A Rare Association With Acute Hepatitis B

INTRODUCTION: We present a rare case of non-immune hemolytic anemia associated with acute hepatitis B infection in a previously healthy middle-aged female. CASE DESCRIPTION/METHODS: A 54 year old female presented to our center with symptoms of generalized weakness, pruritus and dark urine. The patient was previously healthy and had been diagnosed with acute HBV infection one month earlier. Her hemoglobin was normal at that time. Antiviral therapy was not prescribed initially as the patient manifested no evidence of acute liver failure. However, she started tenofovir disoproxil fumarate two weeks after diagnosis due to the persistence of symptoms and progressive hyperbilirubinemia with a total bilirubin of 31.5 mg/dL. On admission, her vitals were stable and the physical exam was significant for right upper quadrant abdominal pain and scleral icterus without evidence of asterixis or confusion. Laboratory work, as shown in Table 1, was significant for acute anemia and worsening bilirubin, although her liver transaminitis and viral load had improved. Renal function and coagulation studies were normal. Magnetic resonance imaging of the abdomen showed diffuse periportal edema supportive of acute hepatitis. Further work up of her anemia revealed normal manual smear, reticulocyte count 1.55 (0.60-2.71 %), lactate dehydrogenase 891 (122-222 U/L), and haptoglobin 14 (20-200 mg/dL). Additional testing included manual smear, vitamin B12, folate, iron, % saturation, disseminated intravascular coagulation panel, direct Coombs testing, cold agglutinins, glucose-6-phosphate dehydrogenase, Hgb electrophoresis, peripheral blood flow cytometry, immunoglobulins screen, which were all unremarkable. Serologic testing for autoimmune hepatitis, hepatitis A and C infections, were negative. The patient’s Hgb dropped to a nadir of 6.4 g/dL. The patient was transfused and discharged with a stable Hgb. Subsequent follow up revealed resolution of her anemia, transaminases and an undetectable HBV DNA level (Table 1). She has seroconverted with loss of HBs antigen; however the development of HBsAb, confirming resolution of infection 5 months after starting tenofovir is still pending. DISCUSSION: Non-immune hemolytic anemia is a rare extrahepatic complication of acute HBV infection. The anemia may be due to direct viral cytotoxicity and/or a heightened immune response following initiation of antiviral treatment. Treatment is predicated on suppressing HBV replication, necessitating a low threshold to start antiviral therapy.Table 1.: Laboratory trends of patient diagnosed with hemolytic anemia associated with acute hepatitis B

Flow Cytometry: An Important Diagnostic Tool in Critically Ill Preterm Neonates with Suspected Sepsis.

Sepsis is a major cause of neonatal mortality. The gold standard for diagnosis is blood culture which suffers from low sensitivity and huge turn-around time. Flow cytometry has been extensively applied to malignant disorders and is an upcoming tool for diagnosis of nonmalignant disorders due to its rapidity and accuracy in detecting cells, cell products, and their functional states. The aim of this study was to investigate the utility of flow cytometric expression of neutrophil CD64, monocyte human leukocyte antigen (HLA-DR) and CD16 in diagnosis in suspected preterm neonates. In total, 100 preterm neonates with clinical signs of sepsis were enrolled in the study. Blood culture, C-reactive protein (CRP) and flow cytometry for nCD64, mHLA-DR, and mCD16 were performed. The neonates were divided into two groups: culture positive and culture negative and CRP and flow cytometric findings compared. ROC analysis was performed to determine the best cut-off for nCD64, mHLA-DR, and mCD16 values along with estimation of sensitivity, specificity, and predictive values. Probability of <0.05 was taken as significant. Out of the 100 enrolled neonates, 34 (34%) were culture positive. CRP was not found to be significantly different in the two groups. Expression of nCD64 (p = 0.03) was significantly upregulated in the blood culture positive cases with a cut-off mean fluorescence intensity (MFI) value = 4.72 and sensitivity of 92% and specificity of 52%. Expression of mCD16 (p = 0.02) was also upregulated in the blood culture positive cases with a cut-off MFI value = 4.9, with sensitivity of 41%, specificity of 83%. The study concluded that nCD64 and mCD16 can be potential biomarkers for early diagnosis of neonatal sepsis with a high sensitivity and specificity. · Neutrophil CD64 significantly upregulated in septic neonates.. · Monocyte CD16 significantly upregulated in septic neonates.. · C-reactive protein values were not significantly different in septic versus nonseptic neonates..

Characterization of Hematopoiesis in Sickle Cell Disease by Prospective Isolation of Stem and Progenitor Cells.

The consequences of sickle cell disease (SCD) include ongoing hematopoietic stress, hemolysis, vascular damage, and effect of chronic therapies, such as blood transfusions and hydroxyurea, on hematopoietic stem and progenitor cell (HSPC) have been poorly characterized. We have quantified the frequencies of nine HSPC populations by flow cytometry in the peripheral blood of pediatric and adult patients, stratified by treatment and control cohorts. We observed broad differences between SCD patients and healthy controls. SCD is associated with 10 to 20-fold increase in CD34dim cells, a two to five-fold increase in CD34bright cells, a depletion in Megakaryocyte-Erythroid Progenitors, and an increase in hematopoietic stem cells, when compared to controls. SCD is also associated with abnormal expression of CD235a as well as high levels CD49f antigen expression. These findings were present to varying degrees in all patients with SCD, including those on chronic therapy and those who were therapy naive. HU treatment appeared to normalize many of these parameters. Chronic stress erythropoiesis and inflammation incited by SCD and HU therapy have long been suspected of causing premature aging of the hematopoietic system, and potentially increasing the risk of hematological malignancies. An important finding of this study was that the observed concentration of CD34bright cells and of all the HSPCs decreased logarithmically with time of treatment with HU. This correlation was independent of age and specific to HU treatment. Although the number of circulating HSPCs is influenced by many parameters, our findings suggest that HU treatment may decrease premature aging and hematologic malignancy risk compared to the other therapeutic modalities in SCD.

Potential antitumor effects of egg extract and purple fluid from marine Aplysia fasciata against experimental Ehrlich ascites carcinoma

Background: The wide biodiversity of marine animals provides a rich source of potential bioactive materials with biomedical applications. Aim: This study aimed to test the in vitro and in vivo anti-tumor effects of the purple fluid and egg extracts from Aplysia fasciata inhabiting the Egyptian marine water. Methods: The cytotoxicity of both the purple fluid and egg extracts were analysed in vitro against Ehrlich ascites carcinoma (EAC) cells by the trypan blue exclusion, MTT reduction assay and flow cytometry. The anti-EAC immunological effects of both extracts were assessed by blood counting and flow cytometry in mice challenged with EAC cells. Results: In vitro treatment of EAC cells with egg extract and the purple fluid caused significant decreases in the number and viability of EAC cells associated with increases in EAC apoptosis in a dose-dependent manner as compared to untreated EAC cells. In vivo, both the purple fluid extract (20μg/ mouse) and egg extract (6.7 μg/mouse) significantly reduced the numbers of EAC cells by 4.3 and 2.3 folds, respectively. However, the anti-tumour effects of the extracts either in vitro or in vivo were yet lower than that of the anti-tumor reference drug cisplatin. Both extracts induced significant increases in the levels of relative and absolute numbers of lymphocytes, mature macrophages (CD11b+ Ly6G-) while with minimal effects on immature neutrophils (CD11b+ Ly6G+). Conclusion: Both egg extract and the purple fluid of Aplysia fasciata possess potential anti-tumour effects with less toxicity, opening new avenues for further evaluation of the chemical and biological mechanisms behind these effects.

Whole blood flow cytometry protocol for the assessment of platelet phenotype, function, and cellular interactions

Platelets are a key component of the hemostatic system and their roles in inflammation via interactions with leukocytes have also gained attention in recent years. Changes in platelet phenotype and function can cause bleeding and/or thrombosis and, as such, monitoring platelet-specific changes is crucial to assessing hemostasis in the clinical setting. Currently, available platelet function tests such as platelet aggregometry and thromboelastography require a large volume of blood, which is a major limitation for the pediatric population. Whole blood flow cytometric analysis of platelets is increasingly utilized in recent years, primarily due to the sensitivity of this method, but also because it only requires a small amount of blood with minimal sample manipulation. We have developed a whole blood flow cytometry methodological approach that enables the assessment of platelet phenotype, function, and their interactions with monocytes and neutrophils.

Th2 cell markers in peripheral blood increase during an acute asthma exacerbation.

Allergic asthma is characterized by type 2 inflammation. We have shown the presence of increased type 2 inflammation in patients with severe asthma and those with frequent exacerbations. However, it is not known whether increased type 2 inflammation drives asthma exacerbations. This study aims to determine Th2 immune parameters in patients presenting to the emergency department (ED) with an acute asthma exacerbation and correlate these parameters with clinical and physiological measures of asthma. Sixteen adults presenting to the ED with acute asthma exacerbations were recruited after giving informed consent. Ten patients returned 2weeks later for follow-up. Physiological parameters, asthma control (ACQ6), asthma quality of life (AQLQ) questionnaires, and venous blood were collected during both visits. An immune cell profiling was performed by whole blood flow cytometry: CD4+ T cells, Th2 cells (CD4+ CRTh2+ T cells and % of CD4+ T cells expressing CRTh2), eosinophils and innate lymphoid cells (ILC2). During exacerbation, peripheral blood Th2 cell numbers correlated with ACQ6 and AQLQ scores, while ILC2 and eosinophil numbers did not. Subjects had higher % of CD4+ T cells expressing CRTh2 and worse FEV1 during exacerbation compared with the follow-up. The decrease in the % of CD4+ T cells expressing CRTh2 seen during the follow-up visit correlated with the improvement in lung function. These data suggest that Th2 cells in peripheral blood may be a sensitive measure of increasing symptoms in patients with asthma exacerbations and may serve as a biomarker of an asthma exacerbation.

Features of peripheral blood B-cell subset phenotype are associated with clinical outcome of widespread purulent peritonitis

The aim of the study was to investigate the phenotypic features of peripheral blood B-lymphocytes in patients with widespread purulent peritonitis (WPP) during postoperative treatment depending on the disease outcome. 52 patients with acute surgical diseases and injuries of the abdominal organs complicated by WPP were examined. Blood sampling was performed before surgery (preoperative period), as well as on day 7, 14 and 21 of the postoperative period. All patients with WPP were divided into two groups depending on the disease outcome during postoperative period: patients with favorable outcome (n = 34), patients with unfavorable outcome (n = 18). 68 healthy subjects were included into control group. Immunophenotyping of blood B-lymphocytes was assessed by flow cytometry and direct immunofluorescence of whole peripheral blood stained with monoclonal antibodies. It was found that patients with WPP in preoperative period contained lowered B1-cell subset compared to control group that was paralleled with low absolute B-lymphocyte count. Moreover, a higher number of activated (based on upregulated CD23 expression) peripheral blood B1-lymphocytes was observed in the patients with unfavorable than in patients with favorable WPP outcome. In addition, dynamics of changes in frequency of B-lymphocytes during postoperative period (day 7—21) varied profoundly depending on the disease outcome. In particular, patients with favorable WPP outcome during postoperative period were found to contain decreased percentage of the most B-cell subsets (including activated cells) examined, whereas patients with unfavorable disease outcome had virtually unaltered B-cell composition in the postoperative period. Moreover, count of total B-cells, naive B-cells and B2-cells negative or positive for CD23 expression was higher in patients with unfavorable outcome than in patients with favorable WPP outcome throughout entire postoperative period. Percentage of some other B-cell subsets in patients with unfavorable than with favorable outcome was also higher only at certain stages of postoperative treatment. It is assumed that such features in peripheral blood B-cell subset composition were closely linked to the disease outcome so that patients with unfavorable WPP outcome turned out to have disturbed B-cell maturation and migration in developing immune response that might be due to lowered total sensitivity of host body to postoperative antibiotic therapy.

Recombinant ling zhi-8 enhances Tregs function to restore glycemic control in streptozocin-induced diabetic rats.

To explore the effect of recombinant LZ-8 (rLZ-8) on streptozocin (STZ)-induced diabetic rats and further illustrate its underlying mechanism. Rats were intraperitoneally injected with single-dose STZ 50mg/kg for induction of type 1 diabetes (T1D), and then, the diabetic rats were treated with rLZ-8 for 3months. The clinical symptoms, fasting blood glucose, insulin, cytokines, histopathology, flow cytometry and immunofluorescence were used to evaluate the therapeutic effect and underlying mechanism of rLZ-8 on alleviating diabetes mellitus (DM). Treatment with rLZ-8 obviously alleviated the clinical symptoms of T1D and dose-dependently reduced the levels of blood glucose, blood lipid and haemoglobin A1c (HbA1c) in diabetic rat model. Meanwhile, rLZ-8 markedly increased insulin secretion and protected against STZ-induced pancreatic tissue injury. Additionally, rLZ-8 dramatically inhibited the levels of TNF-α and IL-1β, and obviously increased the level of IL-10 in serum and pancreas. Further investigation indicated that rLZ-8 treatment significantly increased the number of regulatory T cells (Tregs) and up-regulated the expression of Foxp3 to restore balance between anti-inflammatory and inflammatory cytokines. These data suggest that rLZ-8 can antagonize STZ-induced T1D, and its mechanism may be related to inhibit inflammation and enhance Tregs generation.

Association of CD4+ CD25+ FOXP3+ Regulatory T-cells and HumanPapilloma Virus Infection in Egyptian Women with Breast Cancer

Several subsets of regulatory CD4+ T cells (CD4+ Tregs) have been described in peripheral blood and tumor microenvironment of breast cancer (BC) patients and may play a key role in the progression of BC. High-risk human papilloma virus (HPV) have a causal role in a significant proportion of cervical, and head, and neck tumors and may play an important role in evoking neoplasia in BC. In this study we assessed the prevalence of CD4+Tregs (CD4+ CD25+ FOXP3+ cells) and CD3+ CD8+ T cells by flow cytometry in peripheral blood from a total of 55 Egyptian women, including 20 treatment-naive BC, 15 with breast benign lesions (BBL) and 20 healthy volunteers (HV). Highrisk HPV genotype type 16, 18, and 31 was investigated in breast tissue from all BC and BBL patients using Real- Time PCR. HPV was detected in 4 BC, but in none of BBL patients. The frequency of CD4+ Tregs was significantly higher in BC compared to BBL and HV, (p < 0.001). In addition, we observed a significantly higher frequency of CD3+ CD8+ T cells in peripheral blood of patients with late stage III BC compared to early stage I and II BC (p = 0.011). However, there was no significant association between the ratio of CD8+ T cell to CD4+ Tregs frequencies and the expression of Estrogen Receptor (ER), Progesterone Receptor (PR), and Human Epidermal Growth Factor Receptor 2 (HER2). The potential role CD4+ Tregs as a prognostic or predictive parameter should be analyzed in a larger longitudinal study with sufficient follow-up time.

Morbidity and Mortality Associated With Pediatric Critical Mediastinal Mass Syndrome.

ObjectiveThe critical mediastinal mass syndrome (CMMS) is a life-threatening condition and is challenging for physicians. We analyse the clinicopathological profile and outcome of CMMS from a large tertiary-care pediatric oncology center in Pakistan.MethodsWe retrospectively reviewed the medical record of a tertiary-care hospital in Pakistan from April 2017 to September 2019 for all children (1 month-16 years) who presented with an anterior mediastinal mass (AMM). A CMMS case is defined as a child with an AMM presenting with cardiorespiratory compromise and needing intensive care support. Demographic data, clinical profile, pathological diagnosis, and outcome of all such children were recorded. Descriptive statistics were applied using the Statistical Package for the Social Sciences (SPSS), version 22 (IBM Corp., Armonk, NY).ResultsOf the total 221 mediastinal masses, 61 children were diagnosed as CMMS and enrolled in the study. The mean age was 9 ± 3.3 years, and 68.9%% were male; 65.6% of patients had a weight for age less than the fifth percentile. A total of 49.2% of patients had a duration of illness of more than one month before diagnosis. Fever (97.6%) and lymphadenopathy (82%) were the most common findings, along with respiratory and cardiovascular signs and symptoms; 9.8% had superior vena cava syndrome. The pericardial effusion was present in 54.6% and 27.9% had pleural effusion. Peripheral blood flow cytometry made the diagnosis in 59%, peripheral lymph node biopsy in 13%, mediastinal core biopsy in 5%, and pleural fluid flow cytometry in one case; 62.3% had a white blood cell count of >100,000/mm3. A total of 72.1% (n=44) cases were diagnosed as T-cell acute lymphoblastic leukemia in our cohort. Clinical and laboratory tumor lysis syndrome developed in 10% and 73% of cases, respectively. Mechanical ventilation was required in 9.8% of the cohort. Mortality was reported in 10 (16.4%) patients.ConclusionWe found that the 100% fatality rate with controlled positive pressure ventilation and spontaneous breathing is ideal. Tumour lysis syndrome was the most common morbidity in our cohort.

Myeloid Cells in Circulation and Tumor Microenvironment of Colorectal Cancer Patients with Early and Advanced Disease Stages.

Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of cells that have been implicated in the development of an immunosuppressive environment, which promotes tumorigenesis and tumor progression. Numerous studies have reported expansion of MDSCs in circulation and the tumor microenvironment (TME) of cancer patients. However, due to the heterogenic nature of MDSCs and the different approaches for their identification, their detailed characterization and impact on disease progression in cancer patients are warranted. In this study, we investigated the levels of different myeloid cell subsets and antigen-presenting cells (APCs) using flow cytometry in unfractionated whole blood (WB), peripheral blood mononuclear cells (PBMCs), tumor tissue (TT), and adjacent normal tissue (NT) of colorectal cancer (CRC) patients. We found high levels of granulocytic myeloid cells (GMCs) in whole blood, but their levels were significantly lower in PBMCs. Importantly, we found significantly higher levels of GMCs in the TME compared to NT. In addition, monocytic myeloid cells (MMCs) showed significantly higher levels in PBMCs of CRC patients, compared to healthy donors (HDs). Notably, patients with advanced disease stages showed significantly higher levels of GMCs compared to early stages in whole blood, but PBMCs and tumor-infiltrating myeloid cells did not show any significant differences. Lastly, we found that levels of GMCs decreased, while IMCs increased in the TME with tumor budding. Our results highlight the importance of investigating the levels of different myeloid cell subsets in PBMCs versus whole blood of cancer patients and improve current knowledge on the potential prognostic significance of myeloid cells in CRC patients.

The Bluegreen Algae (AFA) Consumption over 48 Hours Increases the Total Number of Peripheral CD34+ Cells in Healthy Patients: Effect of Short-Term and Long-Term Nutritional Supplementation (Curcumin/AFA) on CD34+ Levels (Blood).

Several active principles from plants could trigger the release of stem cells from the bone marrow. Stem cell mobilizers have shown side effects in patients. Thus, the purpose of this paper is to find the natural products from plants (curcuminoids, glycosinolate of sulforaphane, AFA bluegreen algae), which could be potential stem mobilizes without adverse side effects. The antioxidant curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-2,5-dione], glycosinolate of sulforaphane (broccoli) or AFA (Aphanizomenon flos) extract promote beneficial effects in patients. The number of circulating stem cells were monitored by HSC marker-CD34 by flow cytometry in peripheral blood from healthy subjects. CD34 is a hematological stem cells (HSC) marker. A double-blind study was conducted in 22 healthy subjects. We have evaluated whether short-term AFA—Aphanizomenon flos aquae—algae or curcuminoids consumption (powder or liquid formulation) over 48 consecutive hours could increase the total number of peripheral CD34+ blood cells (n = 22, n = 5 subjects/group). The total number of circulating CD34+ cells were quantified after short-term and long-term nutritional supplementation; their levels were compared with their own basal levels (n = 5/group, controls: before taking any supplement) or placebo-treated patients (n = 7); their average age was 54 years old. We also evaluated whether long-term nutritional supplementation with several nutraceuticals could enhance HSC mobilization by increasing the total number of peripheral CD-34+ cell after seven or 38 consecutive days of administration (n = 5, with seven placebo-treated patients). The long-term administration take place with these doses/day [curcuminoids: 2000 mg/day, equivalent to 120 mg of curcuminoids/day), glycosinolate of sulforaphane (66 mg/day), plus AFA Algae bluegreen extract (400 mg/day)]. On the last day (10 a.m.) of treatment, blood samples were collected six hours after taking these supplements; the average age was 54 years old. Notably, the blue green AFA algae extract consumption over 48 h enhances HSC mobilization by increasing the total number of peripheral CD34+ cells. The long-term administration with curcuminoids, glycosinolate of sulforaphane, and AFA bluegreen algae extract also increased the total number of CD34-HSC cells after seven or 38 days of consecutive of administration in healthy subjects.

Efficacy of low dose rituximab in treatment-resistant CIDP with antibodies against NF-155

We herein to describe the response and the potential treatment mechanism of low dose rituximab in patients with treatment-resistant chronic inflammatory demyelinating polyneuropathy (CIDP) with antibodies against neurofascin-155 (NF-155). Patients received 100 mg rituximab once weekly for 4 weeks followed by 100 mg per month for 2 additional doses. Clinical function scores, Fahn- Tolosa-Marin Tremor Rating Scale (FTMTRS) and flow cytometry of peripheral blood were scheduled before and at 1, 3, 6 months after rituximab treatment. All clinical function score including MRC, INCAT, Hughes, mRS, ODSS and FTMTRS scores showed obvious improvement at the post-treatment follow-up 1,3,6 months in comparison with baseline values. The proportion of CD19 + CD27+, CD19 + CD38+ and CD138 in lymphocytes of all patients declined at 1,3,6 month and the proportion of CD19 + CD24hiCD38hi in one patient was increased at 6 months after rituximab treatment. Low dose rituximab can significant improve disease severity and disabling tremor of CIDP patients with anti-NF155 antibody by the powerful role of B cell depletion within six months and subsequent reestablishment of B-cell subsets including increasing regulatory B cells, inhibiting memory B cells and reducing plasmablasts.

Mitochondrial energetics and contents evaluated by flow cytometry in human maternal and umbilical cord blood

Background: Mitochondrial dysfunction may relate to metabolic disorders. The relation between maternal and fetal mitochondrial function needs attention due to heritage.Objectives: To evaluate the use of the staining methods TetraMethylRhodamine Methyl Ester (TMRM) and Mitotracker Green (MTG) for flow cytometric measurements of umbilical cord blood mitochondrial function. Methods: 53 euthyroid at-term pregnant women and their offspring were included by blood collections. The offspring had blood drawn from the clamped umbilical cord. Flow cytometry with MTG, TMRM and Propidium Iodide were performed the following day. A cell count (antibody coating and flow cytometry) was performed for 9 maternal and cord samples. As a quality control, blood of 32 healthy donors was evaluated by flow cytometric analyzes same day as sampling and the following day to test stability of the measurements.Results: Cord mitochondrial measurements were lower than maternal. Maternal and cord mitochondrial function were positively correlated, especially reflected by MTG fluorescence-intensity (FI). Samples stored presented with very changed fluorescence patterns. However, the fluorescence intensity ratios MTG/TMRM of stained white blood cells were related within same day measurements, depicting an extensive and common bioenergetic cellular change.Conclusion: Cord blood flow cytometry by MTG- and TMRM- staining is possible with fluorescence intensity positively correlated to maternal fluorescence intensity. Storage of blood triggers mitochondrial dynamics. The methods are applicable with certain reservations, and they benefit from their non-invasive character compared to mitochondrial evaluation by muscle-biopsies.