Development of Novel Protective Polyvalent Irradiated Pseudomonas aeruginosa Vaccine for Immuno-Compromised Patients

Pseudomonas aeruginosa is an opportunistic pathogen affecting immuno-compromised patients; however, no effective vaccine is currently available in the market. Here, we developed novel polyvalent irradiated P. aeruginosa vaccine using cobalt 60 that inhibited pathogen viability but retained antigenic expression functionally. Mice were vaccinated by the developed vaccine by intranasal, intramuscular and subcutaneous route of administration followed by challenge test. The protective efficacy of the novel vaccine reached up to 95%. This significant protection was mainly associated with measurable antiserum opsonic killing activity. In conclusion, the novel vaccine provides a promising strategy of both prophylactic and therapeutic approaches for immuno-compromised patients against MDR P. aeruginosa.

Studying Immunophenotypic Modulation at Different Time Points of Chemotherapy : A Walk through the Disease Course in 121 Pediatric B Lymphoblastic Leukemia Cases; Providing Implications for MRD Detection

Introduction Disease monitoring in management of acute leukemia is essentially important in terms of risk stratification and assessing response to chemotherapy throughout the disease course for which minimal residual disease (MRD) testing is the most reliable tool. Measurement of MRD by flow cytometry is based on the principle of studying difference in antigenic expression of the leukemic cells, hematogones and residual normal B cells. Modulation of antigen expression is an established fact that occurs on leukemic blasts during treatment under the influence of chemotherapeutic drugs, which may cause difficulty in analysis and detection of MRD [H.Burnusuzov et al. Folia Medica 2016;58(1):28-35]. Literature has reported antigen expression modulation, particularly during the glucocorticoid phase of induction therapy [Dworzak et al. Cytometry Part B 2010; 78B: 147-153]. For MRD assessment, such phenotypic shifts pose a particular challenge since they can cause misinterpretations and false results. However to our knowledge there hasn't been a large cohort studied to map antigenic modulation and analyze similar antigenic trends in comparison between leukemic blasts and non-leukemic B cells which may coexist during different phases of chemotherapy. Key words: B-lymphoblastic leukemia, Flowcytometry, Antigen modulation, Minimal residual disease, Immunophenotype Methods: The study is conducted at the Hematology department of The Indus Hospital from April 2018 to March 2020. Among 445 pediatric B-lymphoblastic leukemia patients (1 month-18 years); we studied 121 patients who were MRD positive in bone marrow throughout the evaluation period. All patients received Berlin-Frankfurt-Munster (BFM) chemotherapy protocol as per National Cancer Institute (NCI) risk stratification criteria. To observe immunophenotypic modulation (IM) in antigen expression of Tdt, CD34, CD10, CD20, CD45, CD13, CD33 and CD66, compared from the time of diagnosis (day 0), post induction chemotherapy (day 35), post consolidation chemotherapy (day 52) and during maintenance chemotherapy (day78). Using eight color Flowcytometry (BD FACS CANTO-II; DIVA software version 8.0.2), the IM was assessed by comparative analyses of the changes in the mean flourescence intensity (MFI) of leukemic and non-leukemic B cells. The Wilcoxon signed rank test is used to compare median of MFI values at diagnosis and subsequent time points of MRD. Independent sample T-test/Mann-Whitney U test was applied as appropriate to determine significant differences in expression of antigens. A p-value of < 0.05 was considered as significant. Box and whisker plots and run charts will be used for descriptive purposes. Results: Mean age of the patients was 5.7years ±3.7 with male to female 1.7:1.We analyzed forward scatter and side scatter properties and antigen expression of the leukemic and normal residual B-cells. We observed statistically significant differences in MFI levels of CD10, CD20, CD19, CD45, CD66 and Tdt of leukemic cells showing trends at different phases. Down modulation of CD10 was noticed from day 0-day 35 however up modulation was observed in subsequent phases. CD20 and TdT both showed significant and stable up modulation from day 0-day78. A steep down modulation was observed in CD45 and CD19 from day 0 to day 52 and thereafter remained stable. CD34 expression was variable and showed up modulation at day 35 followed by down modulation and then again a reincrease at day 78. CD66, CD33 and CD13 showed similar varying trends exhibiting up modulation at day 35 and then down modulation at day 52 and day 78. Residual normal B cells showed substantial decrease in antigenic expression for CD19, CD20 and CD45 till day 52 followed by an ascent at day 78. Conclusion: Immunophenotypic modulation occurred to different extents in all analyzed samples. Our results confirm the presence of transient immunophenotypic changes for CD10, CD34, CD13, CD33 and CD66 at induction phase of chemotherapy whereas CD20 and TdT showed irreversible gain in expression that is exploitable for MRD detection. The MFI of the different antigens expressed by the leukemic blasts should be taken into consideration and cautiously analyzed for MRD detection. Figure 1 Disclosures No relevant conflicts of interest to declare.

Bone Marrow Aspirate Clot: A Useful Technique in Diagnosis and Follow-Up of Hematological Disorders

Bone marrow biopsy is a diagnostic tool largely used in the evaluation of a broad number of disorders that could affect the hematopoietic system. Differently, bone marrow aspirate clot technique is rarely performed even though it has been described in literature. Here, we highlight the utility of the bone marrow aspirate clot, exemplifying through the discussion of three clinical cases in which this technique was used for diagnosis and follow-up purposes: megaloblastic hemopathy, multiple myeloma, and chronic lymphocytic leukemia. Bone marrow clot analysis increases sensitivity to diagnose hemopathies and offers the possibility of morphological evaluation and anatomopathological study, with the advantage of not needing decalcification processes, hence improving antigenic expression in immunohistochemical and FISH techniques. It is an easy-to-perform technique, offering a quick, reliable, and more comfortable procedure for patients.

Improved Recognition of Hematogones from B Lymphoblastic Leukemia By 5-Color Flow Cytometric Analysis

Background: Hematogones (HG) are benign precursor B-cells that are seen in increased numbers in the bone marrow during childhood, following chemotherapy or bone marrow transplant, in certain immune deficiencies, and in autoimmune disorders. Flow cytometry typically shows expression of TdT, CD10, CD19, variable CD20, dim CD22, CD34 (early HG), and dim CD45. As this phenotype is also seen in patients with B lymphoblastic leukemia (B-ALL), it causes a significant problem in distinguishing leukemic blasts from HG, particularly in a regenerating marrow. Furthermore, hematogones are usually more numerous at baseline in younger patient populations, the same age group with a relatively higher incidence of B-ALL. Despite guidance by earlier studies using the above markers for differentiating HG from B-ALL, these markers are not always sufficient and may hinder correct interpretation. Previous studies demonstrate CD58 is commonly expressed in B-ALL but not in hematogones; however, CD58 as a single marker is somewhat limited when expressed at lower levels. In order to improve diagnostic accuracy, we generated a five color antibody panel including CD10, CD19, CD45, CD38, and CD58 to assess the utility of a single tube panel in distinguishing B-ALL from HG. Design: A total of 35 cases with immature B-cell populations, 16 B-ALL (diagnostic and residual/relapsed cases) and 19 HG, were analyzed by 5-color flow cytometry. 32/35 cases were bone marrow aspirates and 3/35 cases were peripheral blood. A single tube containing CD10 FITC/CD58 PE/CD19 ECD/CD38 PC5/CD45 PC7 was analyzed together with the standard acute leukemia panels. To eliminate technical and fluorochrome variability in expression level analysis, relative antigenic expression was determined through comparison with appropriate internal controls. Antigen expression, as measured by the geometric mean fluorescent intensity (MFI), was then compared between B-ALL and HG using the Mann-Whitney U-test to assess for significant difference. Results were correlated with the morphologic, immunohistochemical, cytogenetic, and molecular findings for precise diagnostic classification as HG or leukemia. Results: HG demonstrated significantly brighter expression of CD38 (p<0.01) than that seen in B-ALL. In contrast, B-ALL expressed significantly brighter CD58 (p <0.01) than HG, which showed dim to no expression of the antigen. HG also showed significantly bright expression of CD10 relative to internal control granulocytes; however, this level of expression was similar to that seen in B-ALL. Median antigen expression. Hematogones show bright CD38, but dim to no CD58. Conversely, B-ALL expresses very dim CD38 and variable CD58. CD10 expression, though, demonstrates overlap between the two populations. B-ALL = B lymphoblastic leukemia, MFI = mean fluorescent intensity Comparative antigenic expression levels in hematogones and B-ALL. Select representative histograms showing HG and B-ALL blasts for the antigens CD38, CD58, and CD10 were selected from various patients studied based on those with the closest relative MFI to the overall median detected for that population in the study. The far right column shows the distribution in MFI of relative antigen expression exhibited the populations studied. HG show significantly brighter CD38 expression than B-ALL does (p<0.01). While B-ALL generally expresses brighter CD58 relative to internal controls, expression levels are variable. HG, though, show significantly dimmer CD58 to essentially no CD58 expression, compared to B-ALL (p<0.01). Similar to CD38, HG demonstrate significantly bright CD10, while B-ALL shows overall bright CD10 but variable expression levels amongst studied cases. These expression levels for CD10 overlap between HG and B-ALL and show no real statistical difference. Conclusions: The combination of CD38 and CD58 in a single tube increases the diagnostic accuracy in differentiation of HG from B-ALL. Without utilizing both antigens together, certain cases would have been difficult to interpret. Based on this analysis, we recommend that these markers be utilized in the routine evaluation for acute lymphoblastic leukemia. This is especially critical in post treatment cases in order to avoid misdiagnosis. Furthermore, the use this single tube panel would cut costs while at the same time improve patient care. Table Table. Figure Figure. Disclosures No relevant conflicts of interest to declare.