Aspiration Smears Research Articles

Cytological Features of "Non-Invasive Follicular Tumour with Papillary like Nuclear Features" - A Single Institutional Experience in India.

The term noninvasive follicular tumor with papillary-like nuclear features (NIFTP) was introduced as a surrogate for noninvasive encapsulated follicular variant of papillary thyroid carcinoma with a defined set of histopathologic criteria. There are very few studies depicting the cytological cues for the diagnosis of NIFTP. The objective of the study was to determine the spectrum of cytological features in fine needle aspiration cytology (FNAC) smears of cases histopathologically diagnosed as NIFTP. This was a retrospective cross-sectional study conducted over a duration of four years between January 2017 and December 2020. All surgically resected cases (n-21), who met the diagnostic criteria of NIFTP on histopathology and who underwent preoperative FNAC were included and reviewed in the study. Out of a total of 21 cases, at FNAC, diagnosis of benign, suspicious for malignancy, follicular variant of papillary thyroid carcinoma, and classic papillary thyroid carcinoma (PTC) was rendered in 14 (66.6%), 2 (9.5%), 2 (9.5%), and 3 (14.28%), respectively. Scanty cellularity was noted in 12 (57.1%) cases. Papillae, sheets, and microfollicles were seen in 1 (4.7%), 10 (47.6%), and 13 (61.9%) cases, respectively. Nucleomegaly, nuclear membrane irregularities, nuclear crowding, and overlapping were seen in 7 (33.3%), 9 (42.8%), and 9 (42.8%), respectively. Nucleoli, nuclear grooving, and inclusions were seen in 3 (14.2%) 10 (47.6%), and 5 (23.8%) cases, respectively. At FNAC, NIFTP can be found in every category of The Bethesda System for Reporting Thyroid cytopathology (TBSRTC). Nuclear membrane irregularities, nuclear grooving, mild nuclear crowding, and overlapping were noted in a modest number of cases. However, the absence or rare occurrence of features such as papillae, inclusions, nucleoli, and metaplastic cytoplasm may help prevent an overdiagnosis of malignancy.

Clinico-cytomorphological Spectrum of Calcinosis Cutis.

The deposition of calcium in the skin is known as calcinosis cutis. It can affect any part of the body and can mimic soft tissue or bony lesions clinically. To describe the clinical and cytomorphologic features of calcinosis cutis on fine needle aspiration cytology smears. A total of 17 cases reported as calcinosis cutis on fine needle aspiration cytology were reviewed for the available clinical and cytological details. The cohort included both adult and pediatric patients. Clinically, the lesions appeared as painless swellings of variable sizes. The common sites affected were the scrotum, iliac region, scalp, pinna, neck, axilla, elbow, arm, thigh, and gluteal region. Aspirate was chalky white, paste-like in all the cases. The cytologic evaluation revealed amorphous crystalline deposits of calcium along with histiocytes, lymphocytes, and multinucleated giant cells. Calcinosis cutis has a wide spectrum of clinical presentations. Fine needle aspiration cytology is a minimally invasive approach for diagnosing calcinosis cutis, thus eliminating the need for more extensive biopsy procedures.

Positive Rate of Malignant Cells in Endometrial Cytology Samples of Ovarian Cancer, Fallopian Tube Cancer, and Primary Peritoneal Cancer Patients: A Systematic Review and Meta-Analysis.

To estimate the feasibility of diagnosing ovarian cancer, fallopian tube cancer, and primary peritoneal cancer through endometrial cytology, we performed a systematic review and meta-analysis to calculate the pooled positive rate of malignant cells in endometrial cytology samples. We queried PubMed, EMBASE, Medline, and Cochrane Central Register of Controlled Trails from inception to November 12, 2020 for studies estimating positive rates of malignant cells in endometrial cytology samples from patients with ovarian cancer, fallopian tube cancer, and primary peritoneal cancer. The positive rates of the included studies were calculated as pooled positive rate through meta-analyses of proportion. Subgroup analysis based on different sampling methods was conducted. Seven retrospective studies involving 975 patients were included. Pooled positive rate of malignant cells in endometrial cytology specimens of ovarian cancer, fallopian tube cancer, and primary peritoneal cancer patients was 23% (95% CI: 16% - 34%). Statistical heterogeneity between the included studies was considerable (I2 = 89%, P < 0.01). The pooled positive rates of the group of brushes and the group of aspiration smears were 13% (95% CI: 10% - 17%, I2 = 0, P = 0.45) and 33% (95% CI: 25% - 42%, I2 = 80%, P < 0.01), respectively. Although endometrial cytology is not an ideal diagnostic tool for ovarian cancer, fallopian tube cancer, and primary peritoneal cancer, it is a convenient, painless, and easy-to-implement adjunct to other tools. Sampling method is one of the factors that affect the detection rate.

Plasmacytoid Cells in Aspiration Smears of the Breast: Every Time a New Surprise.

Plasmacytoid Cells in Aspiration Smears of the Breast: Every Time a New Surprise.

MASC Gets UNMASKED: Visiting a Rare Tumor with Emphasis on Cytomorphological Features.

Secretory carcinoma (SC) is a newly described entity which has been often misdiagnosed earlier as acinic cell carcinoma on cytology. Diagnosing SC was initially based upon identifying the ETV6:NTRK3 fusion gene with the help of fluorescence in situ hybridization (FISH). Lately, with more knowledge of the reliable histomorphology, cytology, and immunohistochemistry features, definitive diagnosis can be confidently made without the help of FISH in almost every case. Six histologically confirmed cases of SC were studied. The cytology slides of all the six cases were retrieved and reviewed to identify the characteristic features which could have helped in raising the possibility of SC on fine needle aspiration cytology itself. Cell blocks were also studied, wherever available. Patients were all male with average age of 35.2 years. The six cases in the current study demonstrated at least focal cytoplasmic vacuolization of varying sizes, papillae formations, and bland nuclear features on fine needle aspirate smears. It was also seen that S-100 and mammaglobin immunohistochemistry (IHC) are very helpful in confirming the diagnosis. The results of the current study highlight the cytomorphological features which may help in clinching the diagnosis SC on cytology itself. They also highlight certain cytological features which help to rule out the other differential diagnoses.

Risk assessment of sydney classification categories in lymph node aspiration smears

Risk assessment of sydney classification categories in lymph node aspiration smears

Cytologic diagnostic approach of pleuropulmonary blastoma: a case report

Pleuropulmonary blastoma (PPB) is a very rare pediatric lung tumor that arises in the pulmonary parenchyma, mediastinum, and pleura. The tumor has rapid disease progression and therefore the prognosis is remarkably poor. We reported a 4-year-old girl who complained of high fever and shortness of breath for the past 8 weeks. The patient was referred from the previous hospital with a pulmonary mass. CT scan of the chest with contrast showed a solid cystic mass with necrotic areas in the 1st, 2nd, and 3rd segments of the left lung with sized 4.8 x 8.1 x 6.6 cm3. As the tumor mass was inoperable, an ultrasound-guided fine-needle aspiration biopsy (FNAB) was conducted to diagnose the pulmonary lesion. We concluded that the lung tumor was a PPB based on FNAB cytology and immunocytochemistry staining. The histopathology feature of PPB appeared similar to fetal lung tissue. Cytologic features obtained from fine-needle aspiration cytology smears and cell blocks followed by immunocytochemistry assay could provide a proper and accurate diagnosis in an inoperable surgical pathology case.

Cytomorphology of Human Bone Marrow Subjected to Prolonged Standing That Mimic Features of Myelodysplasia

Examination of bone marrow (BM) aspirate smears plays an important role in the diagnosis of patients with various hematological disorders particularly patients with myelodysplastic syndromes (MDS). In current practice, bone marrow samples are often collected by the clinical staff and sent to the laboratory after a variable period of delay. As a result bone marrow aspirate smears prepared from such samples are prone to show morphological changes which may mimic changes observed in MDS. To ascertain the type and extent of morphological changes that may occur due to prolonged standing we have studied BM aspirate specimens from patients diagnosed with various hematological disorders which have been left standing for 30 minutes to 24 hours. Bone marrow aspirate samples were obtained from the right or left posterior iliac crest from ten patients (seven men and three women, age range 50-70 years) presenting with various hematological disorders who underwent BM aspiration and BM trephine biopsy procedures as part of their diagnostic work-up. Clinical indications included pancytopenia (n=3), leukopenia (n=2), thrombocytopenia (n=2), thrombocytosis (n=1), lymphocytosis (n=2). The first draw of 2.0 ml of BM aspirate sample was obtained using a conventional BM aspiration needle and placed in a tube containing appropriate concentrations of dipotassium ethylenediamine tetraacetic acid (EDTA). A drop of marrow was then placed on each of the three glass slides and smears were made. The subsequent smears (three slides from each patient) were made from the collected samples at intervals of 30 minutes, one hour, two hours, four hours, eight hours, 12 hours, 18 hours, and 24 hours. The slides were stained with Romanowsky stain and examined under a light microscope. Five hundred consecutive cells in each slide were assessed and the number of cells exhibiting dysplastic changes was recorded. Striking morphological changes were observed between the smears made of fresh marrow as compared to marrows left standing for a "lengthy" period. No noticeable changes in the morphology of hematopoietic cells were observed in the films made from the marrow which was left standing for not more than an hour at room temperature and were not easily distinguishable from the films made immediately after its collection (fresh marrow). By two hours changes were discernible and by 18-24 hours they became striking, affecting the morphology of all the hematopoietic cells. Myeloid cells were affected uniformly; their nuclei stained more homogeneously than in the fresh marrow, the nuclear lobes became separated and the cytoplasmic margins appeared ragged or less well defined. Small vacuoles appeared in the cytoplasm and they became more prominent and more numerous and the quantity of cytoplasmic granules decreased the longer the samples stood. Nuclear hypolobulation (pseudo-Pelger-Huet anomaly) and bizarre nuclear shapes were also observed. Erythroid precursors were also affected by prolonged standing which led to progressive degenerative changes such as nuclear lobulation and budding, nuclear-cytoplasmic asynchrony, karyorrhexis, cytoplasmic vacuolation, and cytoplasmic blebs. Megakaryocytes were also adversely affected and showed morphological changes in the form of multi-nuclearity and less granulation. Some of the lymphocytes also showed similar changes, including cytoplasmic vacuolation. Some of the nuclei showed major budding giving the appearance of a nucleus with two or three lobes (clover leaf shape). Examination of a BM aspirate smear is a highly informative hematological tool at the hematologist's disposal. However, the preparation of a good quality BM aspirate smear and the aberrations in the morphology of hematopoietic cells caused by a delay in smear preparation have not been reported. Our findings demonstrate that the morphology of hematopoietic cells in the BM aspirate deteriorates considerably during prolonged standing. Some of the changes that were noted were akin to the alterations seen in patients with MDS. We recommend that whenever possible the smears should be made immediately after the sample is withdrawn from the patient. This approach helps ensure the ideal morphology of hematopoietic cells and their accurate analysis for hematological diagnosis and may even prevent misdiagnosis of conditions such as myelodysplastic syndromes.

Study of micronuclei as a potent biomarker in breast cytology aspirates

: Micronuclei scoring can be used as a bio-marker of genotoxic and chromosomal damage. Fine needle aspiration cytology (FNAC) is applied as the primary tool for diagnosis in breast masses because of its ease and rapidity. Micronucleus (MN) scoring is carried out in benign (fibroadenoma) and malignant (infiltrating ductal carcinoma) breast lesions to evaluate the role of Micronuclei as a biomarker in breast carcinomas.: To study Micronuclei (MN) scores as a biomarker on breast cytology aspirated smears. This was a prospective study done for duration of two years in the Department of Pathology, A.C.S Medical College and Hospital, Chennai. The features of micronuclei in 60 breast aspirate smears were studied and compared in benign conditions, proliferative and malignant conditions.The most common diagnosis was of fibroadenoma seen in 38 (63.3%) cases. Adenosis was seen in 10 (16.6%) cases. Usual ductal hyperplasia in 6.6% cases and Invasive ductal carcinomas in 6 (10%) cases. The Average Micronucleus score/1000 cells and the range of micronucleus score was higher in malignancy as compared to benign conditions. : Micronuclei can be used as a biomarker on fine needle aspiration cytology smears of breast lesions. An increase in micronuclei is usually seen in malignant conditions as compared to benign tumours. Attention to features of micronuclei can give a clue to the presence of malignancy.

The Morphologic Spectrum of Myelokathexis in WHIM Syndrome and Germline CXCR4 Variants: New Insights into Cellular Changes in the Bone Marrow and Peripheral Blood

Introduction WHIM syndrome (WHIMs) is a rare disorder caused by germline CXCR4 mutations, classically characterized by warts, hypogammaglobulinemia, infection, and neutrophil retention in the bone marrow (BM), with distinctive morphology termed myelokathexis. Demonstration of myelokathexis by BM evaluation plays a key role in disease recognition and diagnosis. However, the spectrum of BM morphologic features and correlation with specific CXCR4 mutations are not well described. Moreover, diagnostically useful peripheral blood (PB) smear findings and morphologic changes after CXCR4 antagonist therapy are largely unknown. Methods We conducted a multinational, retrospective review of clinicopathologic features of patients with established WHIMs (n=20) or germline CXCR4 variants considered likely pathogenic or of uncertain significance (n=3), including 14 BM specimens and 10 PB smears. Assessments to date were performed by 2 observers in 6 cases and a single observer in the remainder. BM morphology was compared to other neutropenia disorders (Shwachman-Diamond syndrome, n=5; GATA2 deficiency, n=5; severe congenital neutropenia with ELANE variants, n=4) and patients without hematologic disorders (n=10), together "controls." Correlations and contingency analysis (2-tailed Fisher exact test) were performed using Prism v.9.4.0 (GraphPad Software). Results Median age at WHIMs diagnosis was 18.5 years (range, 0.1-53 years), with female:male ratio of 1.1:1. Recurrent infections, warts, and hypogammaglobulinemia were present in 100%, 53%, and 40% of cases, respectively. BM aspirate smears contained neutrophils with long intranuclear chromatin strands and hypermature condensed chromatin ("myelokathectic neutrophils") in all WHIMs cases, making up 32% to 80% (median 66%) of total neutrophils (Table). In comparison, neutrophils with myelokathectic changes were seen in 0% to 4% of control BMs. Myeloid hyperplasia (myeloid:erythroid ratio >4:1) was present in 50% of WHIMs and 4% of controls (P=.0028); vacuolated neutrophils in 92% of WHIMs (≤29% of total neutrophils) and 17% of controls (P<.0001); apoptotic neutrophils in 58% of WHIMs (1%-4% of total neutrophils) and 8% of controls (P=.0025); and nonparatrabecular neutrophil clusters on core biopsy in 3/3 WHIMs and 5% of controls (P=.0017). Vacuolated eosinophils and myeloid shift toward hypermaturity were present in WHIMs. There was no correlation between percentage of BM myelokathectic neutrophils and degree of CXCR4 truncation. BM in 2 patients with CXCR4 p.S341Y showed myeloid hyperplasia, increased neutrophil apoptosis, and infrequent myelokathectic neutrophils (5%-10%). Neutropenia was present in all untreated WHIMs. Myelokathectic neutrophils were present in PB of all patients with known WHIMs and accounted for 8% to 30% (median, 18%) of total neutrophils. Percentage of PB myelokathectic neutrophils showed an inverse correlation with absolute neutrophil count (ANC) (r=-0.783; P=.0074). Neutrophil apoptosis, neutrophil vacuolization, dysmorphic (hypogranular, hyperlobated, vacuolated, or apoptotic) eosinophils, and/or dysmorphic basophils were present in 80%, 30%, 70%, and 60% of WHIMs cases, respectively (Figure). PB in 1 patient with CXCR4p.D84H showed neutrophil hypolobation without myelokathectic morphology. CXCR4 antagonist therapy with plerixafor (n=4) [Dale et al. Blood. 2011] or mavorixafor (n=1) (interval, 4-12 h) resulted in increased white blood cell (WBC) count and ANC, with a similar proportion of myelokathectic neutrophils as before therapy. Conclusions In WHIMs BM specimens, the proportion of myelokathectic neutrophils is variable and may not be prominent; the classic finding of myeloid hyperplasia is only present in a subset of cases, which may result in underrecognition of the disease and delayed diagnosis. Neutrophil vacuolization and apoptosis, eosinophil vacuolization, and prominent neutrophil clusters may also prompt disease recognition. We establish characteristic PB morphologic features which, in a patient with unexplained neutropenia, may suggest a diagnosis of WHIMs, including myelokathectic neutrophils, neutrophil vacuolization or apoptosis, and dysmorphic eosinophils and basophils. CXCR4 antagonist therapy led to an increase of WBC/ANC, while pretreatment morphologic changes persisted after a short interval. Analysis of additional cases is ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pediatric Acute Myeloid Leukemia with Co-Occurring BCR::ABL and CBFA2T3::GLIS2 Dual Fusion with Deep Response to FOLR1-Targeting Antibody Drug Conjugate Stro-002 and Tyrosine Kinase Inhibitor

Introduction: Pediatric acute myeloid leukemia (AML) is characterized by significant cytogenetic and molecular heterogeneity. AML with CBFA2T3::GLIS2 fusion (CBF::GLIS) involving infants and very young children is a rare but uniformly refractory disease with high mortality. Laboratory data demonstrates that this fusion is sufficient for leukemic transformation without recurrent secondary variants. These studies have also linked CBF::GLIS fusion to induction of folate receptor alpha (FOLR1) expression in transformed cells (Meshinchi et al, 2020). Incidence of Ph+ de novo (BCR::ABL minor breakpoint, p190 hybrid) disease ranges from 0.5-3% of all AML cases across the age spectrum (Soupir et al, 2017). We, for the first-time, report dual existence of both fusions in a child with AML and discuss novel interventions including combination of a BCR::ABL tyrosine kinase inhibitor (TKI) and antifolate therapy consisting of methotrexate (MTX) and FLOR1-specific antibody-drug conjugate (ADC) STRO-002 (Sutro Biopharma) based on available data regarding its in-vitro preclinical activity in CBF::GLIS disease (Meshinchi et al, ASH abstract, 2021). Case: A 2-year-old girl of Vietnamese descent received a bone marrow examination for bicytopenia and macrocytosis. Flow cytometry confirmed the poor prognostic RAM immunophenotype AML (CD45 dim, HLA-DR negative, CD38 dim, CD56 bright) and FOLR1 expression. Marrow aspirate smears showed numerous hypercellular spicules and blasts with scant cytoplasm and some blebbing but no granules or Auer rods. Marrow exhibited 100% cellularity. CNS was not involved. Review of diagnostic genomic profile mid induction showed a BCR::ABL minor breakpoint fusion as well as CBF::GLIS fusion. The child began AML induction on COG AAML1831 trial randomized to the experimental arm with CPX-351 and gemtuzumab ozogamicin (GO). Due to lack of response to AML therapy at end of Induction (EOI) I, she was taken off protocol and started on modified ALL regimen for Induction II consisting of cytarabine, low dose weekly methotrexate and bimonthly peg-asparaginase with addition of an oral TKI, dasatinib. EOI II marrow examination showed improvement with reduction in blasts (11%) and detectable BCR::ABL but undetectable CBF::GLIS by Q-RT-PCR. Induction III included low dose weekly methotrexate, cytarabine, GO, and dasatinib. EOI III marrow examination showed complete morphologic remission (CR) with no evidence of minimal residual disease (MRD) or fusion transcripts of either clone. Due to availability of a matched sibling donor, the child underwent hematopoietic stem cell transplantation (HSCT); however, was noted to have 3% blasts on day +83 marrow examination despite previously commencing dasatinib post-transplant prophylaxis. BCR::ABL and CBF::GLIS transcripts were again detectable at low levels. Dasatinib dosage was maximized and weekly MTX and bimonthly peg-asparaginase were reintroduced, but the marrow disease burden increased significantly to 78% blasts positive only for CBF::GLIS fusion. Given availability of FOLR1 directed ADC on single-patient compassionate use basis, patient received single agent STRO-001 on a bi-monthly basis. With rising ANC, evaluation of marrow after 2nd STRO-002 dose remarkably showed CR with MRD negative remission by flow and molecular studies. This time remission was consolidated with monthly STRO-002 and donor lymphocyte infusions (DLI). Currently, patient remains in MRD-negative remission after the 3 of 4 planned STRO-002/DLI maintenance. Patient has maintained a 100% Lansky play score and tolerated the regimen with only prolonged initial cytopenia requiring supportive care as a toxicity. Plan is to continue TKI and monthly STRO-002 as maintenance therapy. Conclusion: Complete genomic sequencing has revolutionized pediatric AML categorization. It is quite conceivable that (BCR::ABL, CBF::GLIS) dual fusion disease will become a more common finding in future. Although CBF::GLIS AML is uniformly refractory to conventional therapy, targeted therapy with FOLR1-directed STRO-002 was highly effective in eliminating CBF::GLIS clone in this patient, and combination with DLI appears to be feasible. Long-term follow-up is required to assess durability of remission. Additional testing of this approach in a larger patient population is needed to determine the role of STRO-002 in this high-risk pediatric AML population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bone Marrow Biopsy, Does CT Guidance Add Value?

Background: Bone marrow aspirate and biopsies (BMAB) remain a corner stone in the diagnosis and management of various hematologic disorders. Specialized testing, such as cytogenetics, immunophenotypic and molecular analysis performed on these specimens, plays a crucial role in diagnosis and staging of Leukemias/Lymphomas, as well as in finding out molecular targets for potential therapeutic benefits. Thus, inadequate, and poor-quality specimens can limit making a precise diagnosis and providing optimal treatment. At our institution, we recognized that a substantial number of non-CT guided BMAB were being reported as suboptimal, although diagnosis was being made. Also, recently more CT guided (CTG) BMAB are being implemented. A drawback with CTG BMAB is scheduling delays, that can potentially result in treatment delays, and fatal outcomes especially in conditions like Acute Leukemias. Additionally, there is concern of higher cost associated with CT guided BMAB. Therefore, we performed a retrospective analysis on BMAB procedures performed at our institution between January 2019 and April 2022. Purpose:Evaluate and compare the quality of BMAB procedures performed with and without CT guidance (blind procedure) at our institution, and their respective waiting times.Identify potential areas for improvement.Provide data to help guide in choosing the most appropriate procedure. Methods: We retrospectively reviewed all adult BMAB procedures performed at our institution between January 2019 and April 2022, including patient demographics. Pathology reports were reviewed for adequacy of aspirate smears and core biopsies. Samples were deemed inadequate even if one of bone marrow aspirate or core or clot specimens were described by the pathologist as "suboptimal", "inadequate", "fragmented", "crushed", "hemodiluted", "no spicules", or "scant". Waiting period for the procedure was defined as the duration of time (in days) from the date of placing an order for biopsy (CTG or blind) to the date of procedure. CTG BMAB are typically performed by intervention radiologists (IR) while bind BMAB are usually performed by Oncologists (Fellows or attendings) at our institutions. Results: A total of 469 posterior iliac crest bone marrow aspirate and biopsy procedures were performed. More patients underwent BMAB procedure under CT guidance (61% vs 39% for non-CTG). More CTG BMAB samples were categorized as adequate (69%), compared to the non CTG (55%), (p=0.02). No statistical difference was noted in patient's age (p=0.639) or gender (p=.22) or Body mass index (BMI) (p=2.05) of CTG and non CTG groups (see table). The mean waiting period was longer for CTG procedure (27 days +/- 20 (0-135), compared to non CTG (15 days +/- 17 (0-105) (p=0.031). Although our pathologist described a sample as "suboptimal", a definitive diagnosis was still able to be made in all cases using the available bone marrow clot, aspirate and core specimens. Conclusion/Interventions: CTG BMAB procedures were more likely to result in adequate samples when compared to non-CTG procedures, however with longer waiting period and higher costs. So, we propose that obtaining non-CTG BMAB will improve cost effectiveness and reduce the wait times. To improve the adequacy of non CTG BMAB, we propose some interventions: 1. Provide an organized training during fellowship that includes theory followed by simulation, and practice-based training under supervision. 2. Discuss ICSH (International Council for Standardization in Hematology) guidelines for bone marrow adequacy criteria with the providers. For example, the length of a core biopsy from an adult should be at least 2cm. As our study suggests significant scheduling delays with CTG BMAB, such orders when requested, may be considered as urgent procedures to avoid potential treatment delays and poor outcomes. Alternately, when an aggressive hematologic disorder like acute leukemia is suspected, blind approach may be undertaken for prompt diagnosis. Interestingly, BMI was not significantly different in the two groups. However, if locating physical landmarks is impaired by body habitus, then CT guided BMAB should be strongly considered. We would like to highlight the importance of having adequate personnel including nurse navigators, technicians who play a vital role in obtaining timely insurance clearance, and procedure scheduling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Case of Ambiguous Lineage Acute Leukemia Posing Diagnostic Challenge

Abstract Introduction/Objective N/A. Methods/Case Report Acute leukemias with ambiguous lineage encompass those leukemias that show no clear evidence of differentiation along a single lineage and account for less than 4% of all cases of acute leukemias and often poses diagnostic dilemma. We present a challenging case of an ambiguous lineage acute leukemia in a previously healthy 27-year-old male, who presented to the ED with acute fever, dyspnea on exertion, nausea and vomiting. A complete blood count (CBC) showed a white blood cell count of 323 × 109/L, Hemoglobin of 3.3 g/dl, Platelet count of 44 × 109/L and a differential count of Neutrophil: 11, Lymphocyte: 31, Monocyte: 02, Band Neutrophil:1, Myelocyte :1, Blast: 54. Bone marrow aspirate smears revealed a heterogenous population of small to large sized blasts comprising 83% of cellularity. They expressed scant to moderate basophilic cytoplasm, high N/C ratio, round to irregular nuclear contour, finely granular chromatin and some with nucleoli. Other lineages were markedly suppressed. Flow cytometric analysis revealed an abnormal blast population (~80% of total) expressing CD34, HLA-DR, Tdt (subset), CD71 (dim) and CD38. In addition, the blasts expressed myeloid associated antigens CD13, MPO (smaller subset), as well as B-cell associated antigens CD19 (dim), cytoplasmic CD22 and cytoplasmic CD79a. They also expressed T-cell associated antigens, CD2, CD3 (smaller subset), CD5 (subset) and CD7 (subset) but negative for cytoplasmic CD3. This unusual pattern of expression posed a challenge as to what type of mixed phenotype leukemia it should be with variable expressions from all three lineage of myeloid, B- and T-cell. Further work-up showed, scattered blasts are also positive for MPO by cytochemical (on aspirate smear) and immunohistochemical stain (on core). The latter also showed negativity for CD3. Based on all these findings, this mixed phenotype acute leukemia (MPAL) was best categorized as MPAL (B/myeloid) over so called “Triphenotypic (B/T/myeloid) acute leukemia”. We considered expression of myeloid and B cell markers as more lineage defining in this case as compared to small subset expressing T lineage marker CD3. This case exemplifies the importance of multimodal comprehensive analysis of acute leukemia, especially in the setting of MPAL with expression of multi- lineage markers posing a diagnostic challenge. Results (if a Case Study enter NA) N/A. Conclusion N/A.

An Automated Pipeline for Cell Differentials on Whole-Slide Bone Marrow Aspirate Smears

Abstract Current pathologic diagnosis of benign and neoplastic bone marrow disorders relies in part on the microscopic analysis of bone marrow aspirate (BMA) smears and manual counting of nucleated cell populations to obtain a cell differential. This manual process has significant limitations, including the limited sample of cells analyzed by a conventional 500-cell differential compared to the thousands of nucleated cells present, as well as the inter-observer variability seen between differentials on single samples due to differences in cell selection and classification. To address these shortcomings, we developed an automated computational platform for obtaining cell differentials from scanned whole-slide BMAs at 40x magnification. This pipeline utilizes a sequential process of identifying BMA regions with high proportions of marrow nucleated cells that are ideal for cell counting, detecting individual cells within these optimal regions, and classifying cells into one of 11 types within the differential. Training of convolutional neural network models for region and cell classification, as well as a region-based convolutional neural network for cell detection, involved the generation of an annotated training data set containing 10,948 BMA regions, 28,914 cell boundaries, and 23,609 cell classifications from 73 BMA slides. Among 44 testing BMA slides, an average of 19,209 viable cells per slide were identified and used in automated cell differentials, with a range of 237 to 126,483 cells. In comparing these automated cell differential percentages with corresponding manual differentials, cell type-specific correlation coefficients ranged from 0.913 for blast cells to 0.365 for myelocytes, with an average coefficient of 0.654 among all 11 cell types. A statistically significant concordance was observed among slides with blast percentages less or greater than 20% (p=1.0x10-5) and with plasma cell percentages less or greater than 10% (p=5.9x10-6) between automated and manual differentials, suggesting potential diagnostic utility of this automated pipeline for malignancies such as acute myeloid leukemia and multiple myeloma. Additionally, by simulating the manual counting of 500 cells within localized areas of a BMA slide and iterating over all optimal slide locations, we quantified the inter-observer variability associated with limited sample size in traditional BMA cell counting. Localized differentials exemplify an average variance ranging from 24.1% for erythroid precursors to 1.8% for basophils. Variance in localized differentials of up to 44.8% for blast cells and 36.9% for plasma cells was observed, demonstrating that sample classification based on diagnostic thresholds of cell populations is variable even between different areas within a single slide. Finally, pipeline outputs of region classification, cell detection, cell classification, and localized cell differentials can be visualized using whole-slide image analysis software. By improving cell sampling and reducing inter-observer variability, this automated pipeline has potential to improve the current standard of practice for utilizing BMA smears in the diagnosis of hematologic disorders.

Cytopathology Practice in the Covid-19 Pandemic, During the Lockdown and Post-lockdown Period: A Tertiary-Care Center Experience

Background: Covid-19 pandemic has changed the healthcare delivery and cytopathology practice worldwide. We evaluated our cytopathology laboratory workload during the lockdown and post-lockdown period and compared it with the same period in 2019, to see the impact of the Covid-19 pandemic. Methods: The cytological reports issued during the lockdown (10 March - 31 May 2020) and the post-lockdown period (1 June -31 August 2020); and the corresponding periods in 2019 were retrospectively reviewed from the database. Sample type, sampling site, and diagnostic categories were recorded. Results: During the Covid-19 lockdown period, the total number of cytological specimens, was reduced from n=3197 to n=745, with a rate of 76,7%. The most reduction was in thyroid fine-needle aspiration (FNA) and cervicovaginal smears. Relative increases were observed for soft tissue, lung, and liver FNA samples (p&lt;0.05) and cerebrospinal fluid, peritoneal and pleural fluid samples (p&lt;0.05). In the post-lockdown period, the total number reduced from n=2461 to n=2032 with a rate of 17.4%. Significant reduction continued for thyroid FNAs, but other samples have nearly reached the pre-covid levels. During the total six months period, the rate of the malignant category increased while the negative for malignancy category decreased compared to 2019. Conclusion: During the Covid-19 lockdown period, the reduction was primarily observed in the samples taken for screening purposes, and high-risk oncological patients continued to receive healthcare services. In the initial phase of the post-lockdown period, health services and cytopathology practice have rapidly reached almost the levels of the pre-pandemic period.

Automated Blasts and Plasma Cells Counting on Manually Prepared Bone Marrow Aspirate Smears, Closest Deep Learning Pipeline for Generalizability

Abstract Introduction/Objective Medical errors, liability and reduced productivity in Hematopathology labs stem from fatigue, stress and shortage. 5-35% of complete blood count specimens (at least 3 billion tests worldwide annually) compared to 100% of bone marrow aspirates require visual examination using microscopes or digital scanners. The deep learning technologies and growing digital scanners market empower for developing work-fit automated visual examination to assist the Hematopathologist. Methods/Case Report 2.6K+ manually smeared and stained bone marrow aspirate slides were scanned using integrated cameras on microscopes with electronic/mechanical control units. On top of the intracellular and background staining/dyeing variations, new scanning-related variations in exposure, resolution/focus and stitching were noted inter-patches and intra-patches. This led to consensus loss and "carry over" during cells identification. A convolutional autoencoder with skip connection for restoring/normalizing the stain and resolution was developed using data simulating the entire variations and artifacts. Generation of dimensions and ground truths was performed just by drawing on cells borders using pens, this enabled to localize and segment 5K+ cells per day with almost 100% accuracy. To help with labelling, intracellular colors split and quantification were applied. The processed 1.72+ million cells for 14 different WBCs classes/types were used to train a U-net architecture for segmentation, a transfer learning with pre-trained network EfficientNetB0 with ImageNet for classification and YOLO for detection. A previously developed pipeline for RBCs counting was used to select the proper areas for counting on smears. New diverse datasets were used for testing. Results were validated against confirmatory tests including immunohistochemistry and flow cytometry. Results (if a Case Study enter NA) The five-by-two cross validations revealed minor performance fluctuations (+/- 1.05%) with significant averages normalization SSIM, detection mAP, segmentation IoU, and classification F1-score exceeded 0.96, 0.97, 0.98 and 0.97, respectively. This beta prototype finds the proper areas for counting, calculates the M:E ratio, collects and counts the differential. Sufficient qualitative abnormalities assessment needs more work. Conclusion Without normalization and at least 100K cell images for each class, there is no generalizability.

Deep learning application of the discrimination of bone marrow aspiration cells in patients with myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of hematologic neoplasms accompanied by dysplasia of the bone marrow hematopoietic cells with cytopenia. Detecting dysplasia is important in the diagnosis of MDS, but it takes considerable time and effort. Also, since the assessment of dysplasia is subjective and difficult to quantify, a more efficient tool is needed for quality control and standardization of bone marrow aspiration smear interpretation. In this study, we developed and evaluated an algorithm to automatically discriminate hematopoietic cell lineages and detect dysplastic cells in bone marrow aspiration smears using deep learning technology. Bone marrow aspiration images were acquired from 34 patients diagnosed with MDS and from 24 normal bone marrow slides. In total, 8065 cells were classified into eight categories: normal erythrocytes, normal granulocytes, normal megakaryocytes, dysplastic erythrocytes, dysplastic granulocytes, dysplastic megakaryocytes, blasts, and others. The algorithm demonstrated acceptable performance in classifying dysplastic cells, with an AUC of 0.945–0.996 and accuracy of 0.912–0.993. The algorithm developed in this study could be used as an auxiliary tool for diagnosing patients with MDS and is expected to contribute to shortening the time required for MDS bone marrow aspiration diagnosis and standardizing visual reading.

A simplified molecular method to detect high-risk HPV using the Aptima HPV assay on head and neck FNA smears.

Fine-needle aspiration is used as a diagnostic tool in head and neck oropharyngeal squamous cell carcinoma and its metastases. Prognosis and treatment rely on the presence or absence of the human papilloma virus. The purpose of this study was to validate the performance of the Aptima HPV assay using Hema-Diff stained fine-needle aspiration smears in the diagnosis of human papilloma virus-related oropharyngeal squamous cell carcinoma using a simplified method to obtain tumor cells for testing. Patients with a diagnosis of squamous cell carcinoma and positive p16 immunohistochemical staining were identified. Aptima Specimen Transport Media was used to remove tumor cells from the Hema-Diff stained slides using a moistened swab. The selected cells were tested for high risk-human papilloma virus using the Aptima HPV assay and Aptima HPV 16 18/45 genotype assay. The results were compared with the p16 immunohistochemical staining of the related cell block and surgical specimens. Twenty-one of the 21 (100%) p16-positive cases were found to be positive for high risk-human papilloma virus, whereas 20 of 21 (95%) negative cases were found to be negative for high risk-human papilloma virus using the Aptima HPV assay. The Aptima HPV assay can be used to detect high-risk human papilloma virus in Hema-Diff stained fine-needle aspiration smears of oropharyngeal squamous cell carcinoma with a sensitivity of 100% and a specificity of 95%. This provides a valuable alternative to p16 immunohistochemical staining of cell block sections that often lack appropriate numbers of tumor cells.

Evaluation of Fine-needle Aspiration Cytology (FNAC) Sensitivity Compared to PCR for Diagnosing Tuberculosis Lymphadenitis

Background: Tuberculosis (TB) is a major healthcare burden in Sudan and other developing countries, it is considered the second most common cause of death from infectious diseases after those due to AIDS. In Sudan, TB lymphadenitis (TBLA) remains one of the major health problems. This descriptive cross-sectional study was conducted at the University of Medical Sciences and Technology (UMST) and Total Labcare Diagnostic Center (TDC). The study aims to compare the sensitivity of Fine Needle Aspiration Cytology (FNAC) smears with that of the Polymerase Chain Reaction (PCR) for the diagnosis of TBLA.&#x0D; Materials and Methods: Fifty-five dry smears were obtained using fine-needle aspiration (FNA) from an enlarged lymph node. PCR was applied to detect the target gene (IS6110). May-Grunwald-Giemsa (MGG) or Diff quick stains were used.&#x0D; Results: Two (4%) patients with TBLA were non-necrotic, while fifty-three of them (96%) were necrotic. Moreover, 17 (30%) fine-needle lymph node aspiration specimens were confirmed by PCR to be positive for Mycobacterium tuberculosis complex (MTB complex) while 38 (70%) of them were negative.&#x0D; Conclusion: There was no significant difference between the sensitivity of PCR and that of FNAC (P-value = 0.33).

Classification of endoscopic ultrasound guided fine needle aspiration cytology of pancreatic space occupying lesionsby Papanicolaou Society of Cytopathology System: A five year study.

Majority of the pancreatic cancer patients present at an advanced stage and have poor 5year survival rate. Thus, there is a need for early detection of pancreatic cancer with the initiation of the therapy. This is a retrospective study including all the endoscopic ultrasound guided (EUS) guided pancreatic FNAs from 2016 to 2020. The aspirate smears were analyzed and classified according to The Papanicolaou Society of Cytopathology System for Reporting Pancreaticobiliary Cytology (PSCPC). A total of 245 EUS guided FNAs from pancreatic lesions were included. Cyto-histological correlation was done wherever available. Category I (non diagnostic) accounted for 40 cases (16%) cases, Category II (negative) comprised of 44 cases (18%); and Category III (Atypical) had 5 cases (2%). Category IV neoplastic-benign category included 3 cases of serous cystadenoma, while neoplastic-others category included pancreatic neuroendocrine tumors (n=21), solid pseudo-papillary neoplasms (SPEN) (n=12) and mucinous cystic neoplasms (n=4). A total of 7 cases (2.8%) were reported in Category V (Suspicious). A diagnosis of adenocarcinoma (Category VI) was rendered in 105 cases (42.8%) cases. Rarer types included non Hodgkins lymphoma (n=3) and one case of primary undifferentiated carcinoma with osteoclastic giant cells. Cyto-histological correlation in all categories was available in 58 cases with 8 false negative cases. Thus overall sensitivity of EUS guided FNAC was found to be 87.8% with a diagnostic yield of 83.6% while sensitivity in diagnosing adenocarcinoma was 96.9%. The present study highlights the spectrum of EUS guided FNA of pancreatic lesions in a subset of North Indian population and classified them according to PSCPC. EUS guided FNAC is a sensitive investigation which plays a crucial role in confirming the diagnosis of pancreatic space occupying lesions (SOLs)in advanced stage.