Flow cytometry, molecular analysis, and other special techniques (in Serous Fluid Cytopathology).

Morphological and architectural pattern evaluations play a major role in the rpretation of hematopoietic neoplasms. However, confirmation of diagnosis, classification, prognosis, and risk stratification are highly dependent on the utilization of multiple ancillary studies. The importance of these ancillary studies increases in evaluating serous fluid samples, as these samples lack architecture and patterns. Likewise, the morphology can be disturbed by sample preparation. The most common ancillary studies utilized are flow cytometry, immunohistochemistry for immunophenotyping, Fluorescent In Situ Hybridization (FISH), cytogenetics for structural and gene rearrangements, and molecular studies for mutational analysis. Among them, flow cytometry analysis is the handiest test to perform with high diagnostic yield on serous fluid specimens. In this article we will discuss the use, caveat, and role of the most common ancillary studies on serous fluid specimen evaluation. This review article will be incorporated finally as one of the chapters in CMAS (CytoJournal Monograph/Atlas Series) #2. It is modified slightly from the chapter by the initial authors (Choladda Vejabhuti, MD and Chung-Che (Jeff) Chang, MD, PhD) in the first edition of Diagnostic Cytopathology of Serous Fluids.

Serous fluids and hematolymphoid disorders.

Diagnosing hematolymphoid neoplasm by evaluating fine-needle aspiration (FNA) cytology sample is controversial and requires experience and clinical skills. This concept becomes more challenging when evaluating hematolymphoid neoplasm in body fluid. Differentiating between low-grade lymphoma and reactive lymphocytes is often difficult by morphology alone as reactive lymphoid cells may acquire activation morphology from being exposed to different cytokines within the body fluid. However, in most cases there are specific features that may aid in differentiating small reactive from non-reactive lymphocytes including the round shape of the nucleus, the absence of visible nucleoli and the presence of fine clumped chromatin. In large cell lymphoma and leukemia cells involvement of body fluid this concept becomes less challenging. Large cell lymphoma and leukemia cells tend to have large size nuclei, less mature chromatin, and visible nucleoli with and without cytoplasmic vacuoles. However, to reach accurate diagnosis and subclassification, the utilizing of flow cytometry, to confirm monoclonality, and other ancillary studies such immunocytochemistry, cytogenetics and molecular studies is needed. This review article will be incorporated finally as one of the chapters in CMAS (CytoJournal Monograph/Atlas Series) #2. It is modified slightly from the chapter by the initial authors in the first edition of Diagnostic Cytopathology of Serous Fluids.

Cell-blocks and hematolymphoid lesions.

Cell-blocks are an important component for evaluation for hematolymphoid lesions. They are especially critical for immunocharacterization of the lymphoid population especially when flow cytometry is not available or cannot be performed. In addition, cell-blocks allow various molecular pathology tests including gene rearrangement studies and FISH, proteomics analysis, and microbiology/histochemical special stains. Fine-needle aspiration (FNA) for mass lesions, lymphadenopathy, and effusion fluids are common cytopathology specimens which are frequently cell-blocked. The differential diagnosis of enlarged lymph nodes (LNs) and mass lesions is broad and includes reactive processes, granulomatous lesions and malignancies including solid tumor metastases and various types of hematological malignancies, of which lymphoma would be most common. Depending on the patient population, most lymphomas may be diagnosed with immunocharacterization on cell-block or/and flow cytometry in concert with excellent cytomorphology in Diff-Quik stained FNA aspirate smears. However, a proportion of lymphoma cases (up to 12-30%) may still require an excisional LN biopsy to evaluate architectural parameters. Similarly, various effusion fluids suspicious for lymphoma can be immunocharacterized by immunostaining of cell-block sections (or/and by flow cytometry). Availability of quantitatively and qualitatively optimum cell-blocks of specimens to be evaluated for hematolymphoid processes is critical for immunohistochemistry, polymerase chain reaction, in situ hybridization (FISH), and gene expression profiling studies.

Pleural effusion in hematological pathology.

A 51-year-old male with a history of chronic myelomonocytic leukemia-2 (CMML-2) presented with fatigue, night sweats, dyspnea, and right-sided chest pain exacerbated by deep breath. Computed tomography scan demonstrated right-sided pleural effusion with atelectasis. Pleural fluid cytology showed reactive mesothelial cells mixed with atypical cells [Figure 1]. The immunostains are performed using the SCIP approach.[1] The atypical cells were immunoreactive for vimentin, CD68, and CD163, while non-immunoreactive for cytokeratin, calretinin, BerEP4, and MOC31.

Platelets and renal failure in the SARS-CoV-2 syndrome.

The coronavirus disease 19 (COVID-19) is a highly transmittable viral infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 utilizes metallocarboxyl peptidase angiotensin receptor (ACE) 2 to gain entry into human cells. Activation of several proteases facilitates the interaction of viral spike proteins (S1) and ACE2 receptor. This leads to cleavage of host ACE2 receptors. ACE2 activity counterbalances the angiotensin II effect, its loss may lead to elevated angiotensin II levels with modulation of platelet function, size and activity. COVID-19 disease encompasses a spectrum of systemic involvement far beyond respiratory failure alone. Several features of this disease, including the etiology of acute kidney injury (AKI) and the hypercoagulable state, remain poorly understood. Here, we show that there is a high incidence of AKI (81%) in the critically ill adults with COVID-19 in the setting of elevated D-dimer, elevated ferritin, C reactive protein (CRP) and lactate dehydrogenase (LDH) levels. Strikingly, there were unique features of platelets in these patients, including larger, more granular platelets and a higher mean platelet volume (MPV). There was a significant correlation between measured D-dimer levels and MVP; but a negative correlation between MPV and glomerular filtration rates (GFR) in critically ill cohort. Our data suggest that activated platelets may play a role in renal failure and possibly hypercoagulability status in COVID19 patients.

A rare case of primary cardiac Hodgkin lymphoma in a child.

Primary pediatric cardiac tumors are extremely rare. We report a 14-year-old girl with primary cardiac Hodgkin lymphoma. The large right atrial tumor extended upward and occluded the superior caval vein and left innominate vein.

Isolation and characterization of a CD34+ sub-clone in B-cell lymphoma.

Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy in the US. Many types remain incurable despite response to initial therapy and achievement of complete remission (CR). Advanced laboratory techniques like multicolor flow cytometry (FCM) and polymerase chain reaction (PCR) have demonstrated persistence of rare malignant cell population post therapy. However, the functional and biological characteristics of this population have not been elucidated. Established B-lymphoma cell lines (B-NHL) and patient-derived samples (PDS) were analyzed using 8-color FCM. CD34+ sub-population was enriched using in vitro exposure to 2-chlorodeoxyadenosine (2-CdA) and by CD34 magnetic beads. Genetic analysis of cell fractions was done by karyotyping and array comparative genomic hybridization (aCGH). Sensitivity to chemotherapy was assayed by short-term in vitro exposure to chemotherapy. Clonogenicity was determined by soft agar colony formation assay, and proliferation was determined using DNA staining with propidium iodide and FCM. FCM demonstrated the presence of a minute sub-clone of monotypic B-cells that express CD34 in B-NHL cell lines (3 of 3) and in PDS (8 of 8). This sub-population enriched up to 50 fold in vitro by exposure to 2-CdA and up to 80% purity by CD34 magnetic bead column isolation. Except for CD34 expression, this population expressed identical phenotype and genotype to parent cells, but was more proliferative, Hoechst 33342-positive, clonogenic, and resistant to chemotherapy compared with the CD34- population. The isolated CD34+ monotypic B-cells may contribute to resistance of certain NHL to treatment and should be targeted by potential new drugs for NHL.

Eosinophilia With Hepatic Mass and Abnormal Liver Function Tests: An Unusual Presentation of Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a chronic myeloproliferative neoplasm characterized by excess granulocytes at different stages of maturation and the presence of BCR-ABL1 fusion gene or Philadelphia chromosome. Absolute eosinophilia, basophilia, and monocytosis are not uncommon in CML. However, a rare entity called eosinophilic variant of CML (eoCML) can present with eosinophilia without excess neutrophils or basophils. Here, we report a rare and unusual case of eoCML presenting as a liver mass with abnormal liver function tests, which has not been reported in the literature so far.

High Ki67 proliferation index but not cell-of-origin subtypes is associated with shorter overall survival in diffuse large B-cell lymphoma.

BACKGROUND: CD10, BCL6, and MUM1 are commonly used immunohistochemical stains for classifying diffuse large B-cell lymphoma (DLBCL), which is useful in predicting outcome. Conflicting reports of the prognostic value of other markers such as BCL2, CD23, and Ki67 proliferation index have been reported. Our objective was to correlate these immunostains and Hans classification with response to therapy and overall survival. MATERIALS AND METHODS: A retrospective study of patients diagnosed with DLBCL from 2008-2014 at a tertiary-care cancer hospital. The slides with the IHC stains were reviewed by two independent pathologists. The clinical outcomes--assessed independently--were response to therapy and overall survival. The treatment response evaluation was based on the new Lugano classification. Statistical analyses were conducted using the Fisher's exact test and Kaplan-Meier survival curves. Significance was set at P < 0.05. RESULTS: Forty-one patients were included in the study with a known Hans classification, available clinical data, and at least 5-year follow-up. CD10 immunostain was reported in all patients, whereas CD23 was the least reported in only four patients. No significant association was observed between CD10, BCL6, MUM1, BCL2, and both Response to therapy and overall survival. Owing to few cases reported CD23 immunostain, further analysis of association is not reported. High Ki67 proliferative index of >80% was statistically significantly associated with shorter overall survival and not statistically significant associated with no response to therapy. Hans classification subtypes were not predictive in regard to therapy response. CONCLUSION: High Ki67 expression (>80%) was associated with shorter overall survival in DLBCL. Hans classification subtypes were not predictive.

Practical Approaches on CD30 Detection and Reporting in Lymphoma Diagnosis.

While our understanding of the biology of CD30 in lymphoma continues to evolve, our need to detect and measure its expression at the protein level remains critically important for diagnosis and patient care. In addition to its diagnostic and prognostic utility, CD30 has emerged as a vehicle for drug targeting through the antibody-drug conjugate brentuximab-vedotin (BV). Given the numerous ways that CD30 is utilized and its emergence as a predictive/prognostic biomarker, pathologists must come to a general consensus on the best reporting structure and methodology to ensure appropriate patient care. In this manuscript, we review the indications for testing, various modalities for testing, technical challenges, pitfalls, and potential standards of reporting. The following questions will try to be addressed in the current review article: What defines a "POSITIVE" level of CD30 expression?; How do we evaluate and report CD30 expression?; What are the caveats in the evaluation of CD30 expression?

Comparative assessment of conventional chromosomal analysis and fluorescence in situ hybridization in the evaluation of suspected myelodysplastic syndromes: a single institution experience.

BACKGROUND: Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic neoplasms, roughly half of which harbor cytogenetic abnormalities with diagnostic, prognostic, and therapeutic significance. Fluorescence in situ hybridization (FISH) for the most commonly seen abnormalities (5/5q, -7/7q, +8, and -20/20q-) is routinely performed alongside conventional cytogenetics (CC) in the evaluation of suspected MDS despite conflicting reports of its relative contribution compared to CC alone. OBJECTIVES: To assess the additional diagnostic and prognostic value of performing concurrent FISH versus CC alone in cases of suspected MDS. MATERIALS AND METHODS: A total of 127 bone marrow samples submitted to our cytogenetic laboratory with a presumptive diagnosis of MDS were evaluated by concurrent CC and an MDS FISH panel. RESULTS: CC was used as the gold standard method with 100% sensitivity in detecting suspected MDS-associated cytogenetic abnormalities. FISH alone had a sensitivity of 76%, whereas CC alone achieved a sensitivity of 97%. The addition of FISH did not change the diagnosis nor change the Revised International Prognostic Scoring System score in any patient. Moreover, in 12 cases identified as positive by both CC and FISH, CC identified multiple chromosomal aberrations of clinical significance not interrogated by the FISH probe panel. CONCLUSION: CC alone is sufficiently sensitive in detecting suspected MDS-associated cytogenetic abnormalities that influence clinical decision-making. Routine FISH testing does not provide a significant increase in test sensitivity when an adequate karyotype is obtained. Therefore, FISH testing is best reserved for suspected MDS cases lacking sufficient metaphases.

Molecular Diagnosis of Hematopoietic Neoplasms: 2018 Update.

Diagnosis of hematologic malignancies have matured to encompass molecular as well as phenotypic characteristics. Cytogenetic abnormalities are considered common events in this regard. These abnormalities generally consist of structural chromosomal abnormalities or gene mutations, which often are integral to the pathogenesis and subsequent evolution of an individual malignancy. Improvements made in identifying and interpreting these molecular alterations have resulted in advances in the diagnosis, prognosis, monitoring, and therapy for cancer. As a consequence of the increasingly important role of molecular testing in hematologic malignancy management, this article presents an update on the importance and use of molecular tests, detailing the advantages and disadvantages of each test when applicable.

Green neutrophilic inclusions: current understanding and review of literature.

PURPOSE OF REVIEW: Green cytoplasmic inclusions in neutrophils are an uncommon finding on the peripheral blood smear. A comprehensive review of the literature is presented here with the goal of summarizing knowledge to date and increasing awareness. RECENT FINDINGS: A total of 41 cases have been reported in the literature, indicating the rarity of these green neutrophilic inclusions. Clinically, the inclusions have most consistently been associated with elevated transaminases, hepatic failure, and a high early mortality rate. The precise composition of the inclusions has not been confirmed. SUMMARY: Clinicians, hematologists, and laboratory technicians should be educated and have a high level of awareness of green neutrophilic inclusions. A more comprehensive analysis of a larger number of cases will be needed to clarify the true prevalence, associated conditions, biochemical nature, and clinical significance of this unusual finding.

Flow cytometric false myeloperoxidase-positive childhood B-lineage acute lymphoblastic leukemia.

BACKGROUND: Flow cytometric intracellular myeloperoxidase (MPO) staining of leukemic blasts is a useful tool in diagnosis of leukemia subtype. Interpretation of high MPO-positivity can be a diagnostic challenge in B-lineage acute lymphoblastic leukemia (B-ALL). While very few such cases have been reported, high MPO positive B-ALL cases without additional myeloid antigen positivity are suspect and require further investigation. METHODS: Three pediatric cases of B-ALL with strong MPO staining (clone 8E6; Invitrogen) at diagnosis and three others with negative MPO staining were studied by flow cytometry and immunohistochemistry. In-vitro drug cytotoxicity, oxidative stress generation, and immunophenotyping using other MPO clones were performed to further investigate MPO presence. RESULTS: Expectedly, normal myeloid cells in all six samples were positive and mature lymphocytes negative for MPO staining. However, MPO monoclonal antibody (mAb) obtained from clones other than Invitrogen and other myeloid-specific mAbs gave negative results suggesting false positivity of the initial MPO staining. Immunohistochemistry for MPO was also negative on all six cases tested. Furthermore, in-vitro vincristine cytotoxicity was greater in leukemic cells from MPO false-positive cases compared with MPO-negative B-ALL samples, demonstrating indirect lack of MPO activity. Moreover, drug treatment did not lead to generation of reactive oxidative species, also reflective of lack of significant MPO presence. CONCLUSIONS: The cause of false-positive MPO staining remains unknown in these three cases; a cross reactivity could be the culprit. Caution should be given to similar phenomena and detailed investigation may contribute to the understanding of altered protein expression in such outlier cases. © 2017 International Clinical Cytometry Society.

Malignant acrospiroma: a case report in the era of next generation sequencing.

BACKGROUND: Malignant acrospiroma is a rare tumor of the eccrine sweat glands accounting for around 6% of all malignant eccrine tumors. Typically, it presents as large ulcerated nodules, and diagnosis can be challenging as it has great overlap with its benign counterpart. CASE PRESENTATION: We herein report a case of acral malignant acrospiroma, initially treated with surgical excision and adjuvant radiotherapy. After metastatic disease was confirmed, subject received multiple lines of chemo- as well as targeted therapy. Genomic testing was also done using next generation sequencing. CONCLUSION: To the best of our knowledge, this is the first case of acral malignant acrospiroma with reported next generation sequencing results.

Simultaneous hepatosplenic T-cell lymphoma and myelofibrosis.

Hepatosplenic T-cell lymphoma (HSTL) is a rare T-cell neoplasm of the lymphoid system. This type of lymphoma is characterized by sinusoidal infiltration of spleen, liver, bone marrow and lymph nodes by neoplastic lymphocytes. Here, we discuss a patient who had a left axillary lymph node biopsy with characteristic histological and immunohistochemical features of HSTL. In addition, infiltrating neoplastic T-cells and simultaneous characteristic features of myelofibrosis (MF) were also present in the bone marrow biopsy specimen. In contrast to secondary MF, primary MF is a progressive disease and may significantly affect the prognosis of coexisting HSTL. There are few reports in the literature talking about mild bone marrow fibrosis in association with T cell lymphoma, however marked increase in bone marrow fibrosis and HSTL never being reported. This case is shedding light on HSTL and marked increase in bone marrow fibrosis.