An Automated Pipeline for Differential Cell Counts on Whole-Slide Bone Marrow Aspirate Smears.

The pathologic diagnosis of bone marrow disorders relies in part on the microscopic analysis of bone marrow aspirate (BMA) smears and the manual counting of marrow nucleated cells to obtain a differential cell count (DCC). This manual process has significant limitations, including the analysis of only a small subset of optimal slide areas and nucleated cells, as well as interobserver variability due to differences in cell selection and classification. To address these shortcomings, we developed an automated machine learning-based pipeline for obtaining 11-component DCCs on whole-slide BMAs. This pipeline uses a sequential process of identifying optimal BMA regions with high proportions of marrow nucleated cells, detecting individual cells within these optimal areas, and classifying these cells into 1 of 11 DCC components. Convolutional neural network models were trained on 396,048 BMA region, 28,914 cell boundary, and 1,510,976 cell class images from manual annotations. The resulting automated pipeline produced 11-component DCCs that demonstrated a high statistical and diagnostic concordance with manual DCCs among a heterogeneous group of testing BMA slides with varying pathologies and cellularities. Additionally, we demonstrated that an automated analysis can reduce the intraslide variance in DCCs by analyzing the whole slide and marrow nucleated cells within all optimal regions. Finally, the pipeline outputs of region classification, cell detection, and cell classification can be visualized using whole-slide image analysis software. This study demonstrates the feasibility of a fully automated pipeline for generating DCCs on scanned whole-slide BMA images, with the potential for improving the current standard of practice for utilizing BMA smears in the laboratory analysis of hematologic disorders.

Three patients highlighting potential pitfalls in platelet refractory testing.

BACKGROUND: Platelet-transfusion refractory (PR) patients do not achieve expected post-transfusion platelet counts. We investigate suspected PR patients with post-transfusion platelet counts, indirect platelet antibody screens (ind-PAS), Class I HLA antibody tests (HLA-Scr), and physical platelet crossmatch (PXM) studies. STUDY DESIGN AND METHODS: The three following cases describe possible pitfalls of laboratory tests used in PR workup and management. RESULTS: Case #1: Antibody testing detected antibodies to only HLA-B13, corresponding to a 4% calculated panel reactive antibodies (CPRA; 96% predicted donor compatibility). However, PXM showed the patient compatible with 11/14 (79%) donors; two of the PXM-incompatible units were ABO-incompatible. Case #2: PXM revealed compatibility with 1/14 screened donors; however, the patient did not respond to the product from the compatible donor. The patient did respond to HLA-matched product. Dilution studies provided evidence of the prozone effect, which caused negative PXM despite clinically relevant antibodies. Case #3: There was a discrepancy between the ind-PAS and HLA-Scr. Ind-PAS was negative for HLA antibodies, while HLA-Scr was positive and specificity testing corresponded to 38% CPRA. Per the package insert, the sensitivity of ind-PAS is ~85% compared to HLA-Scr. DISCUSSION: These cases highlight the importance of investigating incongruent results. Cases #1 and #2 demonstrate PXM pitfalls: ABO incompatibility can result in positive PXM and false-negative PXM can occur in the setting of the prozone effect. Case #3 reveals the importance of knowing a test's sensitivity. Centers that only perform ind-PAS may fail to detect HLA antibodies.

Plasma cells and lymphoid aggregates in sleeve gastrectomy specimens: Normal or gastritis?

Lymphoid follicles/aggregates in gastric biopsies have been traditionally linked to Helicobacter pylori gastritis, and less commonly to other inflammatory and neoplastic conditions. The frequency of such aggregates in normal stomachs has yet to be adequately evaluated. This is especially relevant when it comes to diagnosing non-specific chronic gastritis in biopsy specimens with chronic inflammation but no evidence of H pylori infection. Sleeve gastrectomies represent an opportunity to study adequately preserved gastric mucosa in patients who are otherwise asymptomatic and lack a history of gastric disease.To study sleeve gastrectomy specimens to quantify the amount of lymphoid follicles/aggregates and lymphocytic infiltration in normal stomachs.Sixty-eight bariatric sleeve gastrectomies and 13 control specimens from Whipple resections were examined for multiple histologic features including type, quantity, and distribution of chronic inflammation and lymphoid follicles/aggregates. Presence of H pylori was documented by both Hematoxylin and eosin-stained (H&E) and immunohistochemistry (IHC). Clinical information including age, sex, medication intake, prior endoscopy, and/or H pylori infection was recorded. The patient population was divided in 2 groups, H pylori negative versus H pylori positive, and statistical analysis was performed by a biostatistician.Two hundred sixty three fundic sections from 68 bariatric patients were examined. Fifty three patients were found to be H pylori-negative, compared with 15 who were positive for H pylori. Among the H pylori-negative group, the average number of lymphoid aggregates was 3.33, compared with an average of 6.26 in the H pylori positive group (the difference was statistically significant with a P-value of .008). The average number of plasma cells per high power field was 2.15 in the H pylori negative group, compared and average of 5.07 in the H pylori positive group (the difference was also statistically significant with a P-value <.001). Clinically, 10 of the 53 H pylori-negative patients had esophagogastroduodenoscopy (EGD) that showed endoscopic mild non-erosive gastric erythema. The remaining had no documentation of symptoms or medication intake, including Non-steroidal anti-inflammatory drugs (NSAIDs) and Proton Pump Inhibitors (PPI).Our results suggest that the presence of lymphoid aggregates and plasma cells infiltration can be a normal finding in otherwise normal gastric mucosa, though more pronounced in H pylori infected patients.

Shortcomings of ultrasound-guided fine needle aspiration in the axillary management of women with breast cancer.

BACKGROUND: Ultrasound, along with ultrasound-guided fine needle aspiration, is currently used for the axillary evaluation of breast cancer patients in order to identify candidates for axillary lymph node dissection. The aim of this study is to evaluate the accuracy of this tool in correctly identifying patients who may or may not benefit from axillary clearance in light of the ACOSOG Z0011 trial recommendations. METHODS: One hundred one patients (65 with positive US-FNA with corresponding axillary lymph node dissection (ALND), and 36 with negative US-FNA with corresponding ALND/sentinel lymph node biopsy) were studied for the number of involved axillary lymph nodes, tumor clinicopathologic features, and axillary radiologic findings. RESULTS: From the positive US-FNA group, 43% of patients had two or fewer positive lymph nodes upon ALND pathologic examination. In the US-FNA negative group, the negative predictive value for detecting axillary disease was 72.7%. With both groups combined, the sensitivity, specificity, PPV, and NPV of US-FNA for selecting patients based on axillary disease burden were 86%, 51.7%, 57%, and 83.3%, respectively. CONCLUSION: Based on Z0011 guidelines, US-FNA is not a reliable tool in triaging patients in need for ALND and leads to overtreatment of 43% patients when positive, while depriving a small but significant percentage of patients from necessary therapy, when negative.

Lack of expression of ALK and CD30 in breast carcinoma by immunohistochemistry irrespective of tumor characteristics.

CD30 is a member of the tumor necrosis factor family of cell surface receptors normally expressed in lymphocytes, as well as some lymphomas, but has been described in other malignancies. Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that belongs to the insulin receptor superfamily, and is normally expressed in neural cells, but has been detected in several malignancies. There is conflicting data in the literature that describes the expression of these receptors in breast cancer, and the aim of this study is to test the expression of CD30 and ALK in a cohort of Middle Eastern patients with breast carcinoma.Cases of invasive breast cancer from the archives of AUBMC were reviewed over a period of 9 years, and the blocks that were used for immunohistochemical staining for ER, PR, Her-2/neu were selected. Immunohistochemical staining for CD30 (JCM182) and ALK (5A4 and D5F3) was performed.Two hundred eighty-four cases were identified (2 cases were male), with a mean age of 55 ±â€Š12. CD30 and ALK expression was not seen in any of the cases.Our cohort showed complete negativity to both CD30 and ALK, adding to the conflicting data available in the literature, and more studies are needed to reliably identify a trend of expression of CD30 and ALK in breast carcinoma, especially in the Middle East.

Carriage trends and fitness cost of MDROs in Lebanese nursing homes.

INTRODUCTION: Nowadays, medical treatments efficiency is challenged by multi drug resistant organisms (MDROs). Lebanese nursing homes' residents revealed high fecal carriage rates of MDR Enterobacteriacea. Previous studies claim that bacteria with resistant genes experience fitness cost. This study assesses the competitive growth of MDR Escherichia coli and Klebsiella pneumoniae. METHODOLOGY: Fecal swabs were collected, during six consecutive months, from ten elderly residing in a Lebanese nursing home. All isolates were subject to API 20E (bioMerieux, Marcy L' Etoile, France) and antimicrobial susceptibility (Kirby-Bauer method) testing. Phenotypically, ESBL (extended spectrum ß-lactamase), MBL (metallo ß-lactamase), AmpC and KPC (Klebsiella pneumoniae carbapenemase) were detected using EDTA, PBA, cloxacillin, and DDSTs (Biorad, Hercules, USA). Selected ESBL producing E. coli and K. pneumoniae underwent multiplex PCR analysis. Intra and inter-species in-vitro competition assays were conducted in multiple combinations. RESULTS: Among 117 collected isolates, E. coli was predominant (71.8%); 7.7% were ESBL and 5.1% AmpC producers. With E. coli intra-species assays, sensitive isolates out-competed all others, followed by ESBL, AmpC, and OXA-48 (oxacillin) producers. Inter-species assays, demonstrated a decreased fitness of ESBL producing K. pneumoniae in presence of sensitive E. coli. While out-competing ESBL producing E. coli required 2 sensitive K. pneumoniae isolates. CONCLUSION: This study highlights resistant E. coli and K. pneumoniae frequency decrease in presence of sensitive isolates, endorsing the fitness cost hypothesis.  Hence, competing supplementary species reproducing gut flora, would ensure further steps in the fight against MDROs.

Fecal carriage of MDROs in a population of Lebanese elderly: Dynamics and impact on bacterial fitness.

Muti-Drug Resistant Organisms (MDROs) are problematic all over the world, especially in Lebanon. High fecal carriage rates of MDR Enterobacteriaceae were reported from Lebanese nursing homes. Some studies show that MDROs have a fitness cost as compared to sensitive isolates. In this study, the competitive growth of MDR Escherichia coli obtained from fecal samples from elderly is assessed. Fecal swabs from ten elderly patients from a Lebanese nursing home were obtained between June and December, 2015. Isolates were identified by API 20E and antimicrobial susceptibilities were determined. Production of ESBL (extended spectrum ß lactamase), MBL (metallo ß lactamse), AmpC and KPC (Klebsiella pneumonia carbapenemase) was detected phenotypically by the use of EDTA, PBA, cloxacillin, and DDSTs. In-vitro competition assays were performed using E. coli isolates with different combinations of bacterial resistance. A total of 117 isolates was obtained with 71.8% E. coli, 7.7% of which were ESBL and 5.1% AmpC producers. Sensitive E. coli isolates out-competed all other isolates when in competition, followed sequentially by ESBL, AmpC, and OXA-48 (oxacillin) producers. This study shows an advantage of sensitive E. coli strains obtained from fecal samples to out-compete resistant strains in specific in-vitro conditions. This ability could be exploited in the elimination of MDR organisms from the gut flora, after further investigation.