Lauren Parsons

Effects of Solid-Medium Type on Routine Identification of Bacterial Isolates by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for the identification of bacteria. Factors that may alter protein profiles, including growth conditions and presence of exogenous substances, could hinder identification. Bacterial isolates identified by conventional methods were grown on various media and identified using the MALDI Biotyper (Bruker Daltonics, Billerica, MA) using a direct smear method and an acid extraction method. Specimens included 23 Pseudomonas isolates grown on blood agar, Pseudocel (CET), and MacConkey agar (MAC); 20 Staphylococcus isolates grown on blood agar, colistin-nalidixic acid agar (CNA), and mannitol salt agar (MSA); and 25 enteric isolates grown on blood agar, xylose lysine deoxycholate agar (XLD), Hektoen enteric agar (HE), salmonella-shigella agar (SS), and MAC. For Pseudomonas spp., the identification rate to genus using the direct method was 83% from blood, 78% from MAC, and 94% from CET. For Staphylococcus isolates, the identification rate to genus using the direct method was 95% from blood, 75% from CNA, and 95% from MSA. For enteric isolates, the identification rate to genus using the direct method was 100% from blood, 100% from MAC, 100% from XLD, 92% from HE, and 87% from SS. Extraction enhanced identification rates. The direct method of MALDI-TOF analysis of bacteria from selective and differential media yields identifications of varied confidence. Notably, Staphylococci spp. from CNA exhibit low identification rates. Extraction enhances identification rates and is recommended for colonies from this medium.

Improving Detection of Metastatic Neuroblastoma in Bone Marrow Core Biopsies: A Proposed Immunohistochemical Approach

Bone marrow (BM) nvolvement is common in stage 4/M neuroblastoma patients and profoundly impacts clinical decision-making and predicts outcomes, but to our knowledge no standard exists for immunohistochemical evaluation of staging BMs. We examined the use of three immunostains—synaptophysin, tyrosine hydroxylase (TH), and PGP9.5—in detecting metastatic neuroblastoma in BM. We retrospectively selected 174 BM core biopsies from 41 neuroblastoma patients. Immunohistochemistry for synaptophysin, TH, and PGP9.5 was performed. These slides and the hematoxylin and eosin (H&E)–stained slide from each BM were randomized and independently scored by three pathologists as positive, negative, or indeterminate. Cohens κ coefficients (interobserver agreement), McNemars test (for frequencies of positive/indeterminate interpretations), and sensitivities for each stain/combination were calculated. Interobserver agreement was higher for all immunostains (synaptophysin, 78%–90%, κ = 0.54–80.787; TH, 77%–92%, κ = 0.481–0.788; and PGP9.5, 83%–90%, κ = 0.601–0.740) than for H&Es (77%–84%, κ = 0.434–0.572). Indeterminate interpretations were more frequent with H&Es (8.9%) and synaptophysin (6.0%) than with PGP9.5 (3.5%) or TH (3.3%). TH (76%) and PGP9.5 (70%) were the immunostains most likely to correctly resolve indeterminate H&E interpretation. Mean sensitivity among all three pathologists for detection of metastasis compared to the consensus diagnosis was 42.5% for H&E alone, 70.7% to 78.8% for H&E plus one immunostain, and 81.6% to 85% for H&E plus two immunostains. Immunohistochemistry enhanced sensitivity for tumor detection particularly dramatically in cases of prior chemotherapy. PGP9.5 and TH showed good interobserver agreement, fewer indeterminate interpretations, and resolved indeterminate H&E diagnoses at the highest frequencies. Therefore, we recommend H&E and two immunostains, specifically PGP9.5 and TH, for optimal detection of metastatic neuroblastoma in BM.

Intraparenchymal pulmonary lipoma: pathologic-radiologic correlation of a rare presentation of a common neoplasm

We report a rare case of pulmonary intraparenchymal lipoma. Lipomas are benign adipocytic tumors, which are ubiquitous in distribution, particularly in the subcutis and soft tissue. Visceral lipomas, in particular, pulmonary lipomas, are rarely reported. Even rarer are intraparenchymal lipomas, such as this case, of which less than 10 have been reported in the medical literature. The radiologic (computed tomographic scan) findings of pulmonary lipoma may be somewhat difficult to evaluate. In this case, on initial review, the computed tomographic findings were not diagnostic, but retrospective analysis revealed attenuation values suggestive of an adipocytic lesion. A high index of suspicion and careful attention to attenuation values are therefore required for radiologic diagnosis. Excision is necessary for histologic confirmation, which is generally relatively straight forward, although admixture with fibrous tissue and some cytologic atypia may pose diagnostic challenges.

Fine needle aspiration of alveolar soft part sarcoma in a child: Cytomorphological clues for the surgical pathologist

Alveolar soft part sarcoma (ASPS) is a rare soft tissue neoplasm generally affecting adolescents and young adults. Its unique histologic and ultrastructural features have been well-described; however, the cytopathological features of ASPS are less well-characterized, and recognition of this entitys features on cytologic preparations can ensure that the specimen adequacy and appropriate/rapid tissue allocation for additional testing. Herein we report a FNA case of ASPS with emphasis on cytomorphologic characteristics.

Book Review: Diagnostic Pediatric Cytopathology and Histopathologic CorrelationDiagnostic Pediatric Cytopathology and Histopathologic Correlation. ChouP.M.GattusoP.ReddyV.B., and Reyes-MugicaM., editors. Cambridge University Press, Cambridge, UK, 2016, ISBN 978-1-107-03391-7, 408 pp., hardcover; ISBN 978-1-107-05476-9, mixed media.

One of the most exciting—and challenging—aspects of pathology is its constant evolution. This progression occurs not only with advances in laboratory techniques but also in conjunction with innovations in other fields, such as radiology and surgery. In an age of advanced imaging modalities and highly accurate, minimally invasive biopsy techniques, cytopathology has gained popularity in the world of adult pathology as a means of “doing more with less.” It has been shown to be an effective way to obtain material for both diagnostic purposes and therapeutic testing, yet few pediatric institutions currently utilize fine-needle aspiration (FNA) cytology as a component of the diagnostic workup. Cytopathology is, however, much broader than FNA interpretation, and is performed—in one form or another—by pediatric pathologists on a daily basis in a variety of ways. To date, few textbooks address the role of cytology in pediatric pathology; Diagnostic Pediatric Cytopathology and Histopathologic Correlation, edited by Chou and colleagues, is one of the 1st books that attempts to fill this gap. The 1st 2 chapters of this book focus on specimens unique to cytopathology—exfoliative specimens from the respiratory tract and effusion fluid cytology. These topics are classical cytology fodder and yet are seen routinely by general pediatric pathologists; the respective chapters are well written and incorporate both frequently seen and uncommonly observed conditions. Subsequent chapters are organized by organ system and discuss various disease processes in a structured fashion, describing the clinical presentation, cytologic characteristics, histomorphology, important stains and ancillary techniques, a relevant differential diagnosis, and a useful summary of key points for easy reference. Diagnoses covered range from archetypal cytologic topics such as thyroid lesions to less commonly encountered cytology specimens such as bone lesions, and each chapter is tailored to discuss entities affecting pediatric patients, both benign and malignant. The text effectively caters to general pediatric pathologists both by introducing new concepts (such as FNA cytology of pediatric lesions) and by reviewing features seen in cytologic techniques commonly used by many pediatric pathologists (such as touch imprint/smear preparation cytology of brain tumors). Furthermore, the emphasis on lesions commonly encountered in the pediatric population also makes this text very useful for general cytopathologists who may be largely unfamiliar with pediatric pathology but occasionally encounter FNA specimens from younger patients. Cytopathologists often say that no talk or text is complete without high-quality cytologic pictures. There are myriad high-quality photomicrographs in Diagnostic Pediatric Cytopathology and Histopathologic Correlation—a particular challenge when dealing with certain types of cytologic specimens and preparations. Some of the pictures are small, and subsequent editions could benefit from larger photographs, as their excellent depiction of cytologic features warrants ample space on the page. However, the text provides rich descriptions that nicely supplement these sometimes small but high-quality photomicrographs, and one would be hard pressed to sacrifice this valuable aspect of the textbook. Diagnostic Pediatric Cytopathology and Histopathologic Correlation is a well-written text that bridges the gap between cytopathology and pediatric pathology, making the material relevant both to pediatric surgical pathologists who do not routinely see cytologic specimens and to cytopathologists who lack experience with pediatric lesions. As we strive to make the most out of smaller and smaller tissue specimens from our smallest patients, the information provided in this book will help in developing another skill set to make that important and challenging task a little easier.

Letter to the Editor: RE: "Importance of Phox2B Immunohistochemical Stain for Detecting Metastatic Neuroblastoma Cells in Bone Marrow Specimens"

To the Editor, We thank Warren and Shimada for their interest and thoughtful comments on our recent manuscript, “Improving Detection of Metastatic Neuroblastoma in Bone Marrow Core Biopsies: A Proposed Immunohistochemical Approach” [1]. Since we first presented these data at the 2014 combined Paediatric Pathology Society/ Society for Pediatric Pathology meeting in Birmingham, England, the most frequent question we hear—from conference attendees, journal reviewers, colleagues, and professional mentors alike—is some version of “But what about my antibody?” To this, and to the particular points proffered by Warren and Shimada, we respond. One of the major conclusions of our study was that it matters much less which antibodies one uses to evaluate bone marrow cores (BM) in patients with neuroblastoma; rather, the important point is that one should use two different antibodies and should not rely solely on hematoxylin and eosin (H&E) stains or single immunostains. The difference in detection rates between H&E alone (42.5%) and H&E plus two immunostains (81.6%–85.0% depending on antibodies) is much greater than the difference in sensitivities of the three antibodies assessed (70.7%–78.8%) [1]. We suspect that the sensitivity of any antibody currently used for these specimens falls within this range, as do the publications referenced in our Discussion. Time, tissue, and funding limit the number of antibodies that can be compared in a study such as ours. Reasonable pathologists hold differing opinions on the best approach to these specimens. There are old standbys, including chromogranin, synaptophysin, CD57, and tyrosine hydroxylase, and newcomers, such as PGP9.5, MAP2, and PHOX2B. We selected antibodies based on a review of the literature, personal experience, and practice preferences of expert pathologists. We believe the antibodies we included represent ones that are commercially available on multiple platforms, reasonably sensitive and specific, and frequently used by general pathologists and experts alike. Different laboratories may select different antibodies based on personal preference, anecdotal experience, cost, and availability, and overall utility—PGP9.5 and synaptophysin have widely accepted uses across the broad practice of pathology, whereas the published diagnostic use of Phox2B is limited to neuroblastoma [2,3]. All these factors must be weighed against the potential differences in sensitivity before selecting the ideal antibody panel for a given laboratory. Our study has one of, if not the, largest sample sizes in the published literature on immunohistochemical detection of neuroblastoma in BM, with 58 positive and 116 negative cases. The only published report on the use of Phox2B for BM evaluation was limited to 25 samples (16 positive, 9 negative) and showed 88% sensitivity compared to 100% for synaptophysin and CD57 [3]. Clearly, additional studies are needed further assessing Phox2B for this purpose, and we strongly encourage Warren and Shimada to publish their data to further this discussion and promote practice improvement. Our study specifically looked at a single scenario: BM in patients with neuroblastoma. Knowing a priori that the patient has neuroblastoma makes concerns about antibody crossreactivity with other tumor cell types a nonissue; nonspecific staining with other bone marrow elements was specifically addressed in our study. Furthermore, the immunophenotype of the primary tumor may already be known, directing immunostaining of the bone marrow cores and eliminating concerns about occasional tumors showing negativity with canonical markers. Even if a tumor is negative for a single marker, that tumor is unlikely to be negative for two immunostains highlighting again the importance of using two immunostains as previously suggested. This is a vastly different problem than initial diagnosis of a small round blue cell tumor, and may have a vastly different solution in terms of antibodies. In conclusion, we encourage pathologists to review the literature, reflect on their personal experiences, and perform their own validation studies to select the panel of at least two antibodies that is best for their laboratory and, most importantly, best for their patients.