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* Context.--In 2018 the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee designed and implemented a new plasma cell neoplasia flow cytometry proficiency testing program--PCNEO--to allow clinical flow cytometry laboratories to monitor and assess their performance compared with a peer group. Objective.--To report the results from the first 4 years of the PCNEO program. Design.--Program participants were sent 2 sets of challenges per year, each including 1 wet challenge and 2 dry challenges, with associated clinical and laboratory findings. The wet challenges were composed of myeloma cell line specimens (with or without dilution in preserved whole blood) for flow cytometric analysis. The dry (paper) challenges were composed of clinical case summaries and images of flow cytometric test results from various flow cytometry laboratories of committee members. Results.--A total of 116 to 145 laboratories from 17 countries enrolled in the proficiency testing program. For the wet challenges, almost all participants (97%-100%; cumulative, 98.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of undiluted myeloma cell lines. Slightly fewer participants (89.0%-97.4%; cumulative, 95.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of diluted myeloma cell lines (10% or 50% dilutions into peripheral blood) intended to better represent a typical clinical sample. There was generally agreement among 80% or more of participants for positive or negative staining for CD38, CD138, CD19, CD20, and surface and cytoplasmic k and X light chains. Similarly, 84% to 100% of participants were able to correctly identify the presence of neoplastic plasma cell populations in paper challenges, including the presence of small, neoplastic plasma cell populations (0.01%-5.0% clonal plasma cells) and the presence of nonneoplastic plasma cell populations (correctly identified by 91 %-96% of participants). Conclusions.--Participant performance in the new proficiency testing program was excellent overall, with the vast majority of participants able to perform flow cytometric analysis and identify neoplastic plasma cell populations and to identify small plasma cell clones or expanded populations of reactive plasma cells in dry challenge flow cytometry results. This program will allow laboratories to verify the accuracy of their testing program and test interpretations for the assessment of patients suspected of having a plasma cell neoplasm. (Arch Pathol Lab Med. 2024;148:699-704; doi: 10.5858/arpa. 2023-0035-CP), The Diagnostic Immunology and Flow Cytometry Committee (DIFCC) acts as an expert scientific and educational resource for the College of American Pathologists in diagnostic immunology and flow cytometry clinical testing. [...]

* Context.--Misdiagnosis of antiphospholipid syndrome can occur owing to the wide diversity of antiphospholipid (aPL) assays and a lack of international calibrators and harmonized reference intervals. Objective.--To assess laboratory practices regarding reporting and establishing reference intervals for immunoglobulin (Ig) G/IgM anti-cardiolipin (aCI) and anti-beta-2 glycoprotein I (anti-[[beta].sub.2]GPI) assays. Design.--Supplemental questions related to reporting and establishing reference ranges for aPL assays were sent as part of the Antiphospholipid Antibody (ACI)-B 2019 College of American Pathologists (CAP) proficiency testing survey. The response rate and methods assessment details were determined, as well as qualitative and quantitative results for 3 test samples. Results.--The number of participants reporting results for IgG aCL (n = 489), IgM aCL (n = 476), IgG anti-[[beta].sub.2]GPI (n = 354), and IgM anti-[[beta].sub.2]GPI (n = 331) varied by antibody type. The enzyme-linked immunosorbent assay (ELISA) (up to 58.6%, 260 of 444) was the most used method; others included multiplex (from 18.9% to 23.9%), fluorescence enzyme immunoassay (14.4%-17.6%), and chemiluminescence immunoassay (6.5%-9.0%). More respondents reported quantitative than qualitative results, and manufacturer cutoff ranges were used by 92.9% and 94.2% of respondents for aCL and anti-[[beta].sub.2]GPI, respectively. Despite variation in the use of semiquantitative ranges, qualitative negative/positive reporting of the test samples achieved almost 100% consensus. Qualitative consensus was met in contrast to the wide range of quantitative results obtained for each analyte across different kits. Conclusions.--ELISA remains the most used method for detecting aPL antibodies, with most laboratories reporting quantitative results based on manufacturers' suggested reference ranges. The categorization of quantitative results as equivocal, weak positive, or positive for responders using kits from the same manufacturer was variable. (Arch Pathol Lab Med. 2024;148:686-693; doi: 10.5858/arpa.2023-0095-CP), The 2006 revised Sapporo laboratory criteria for antiphospholipid syndrome (APS) recommend testing for specific antiphospholipid (aPL) antibodies, namely, lupus anticoagulant as well as immunoglobulin (Ig) G and IgM antibodies to [...]

The study objectives were to quantify the time- and magnitude-dependence of flow-induced alignment in vascular smooth muscle cells (SMC) and to identify pathways related to the orientation process. Using an intensity gradient method, we demonstrated that SMC aligned in the direction perpendicular to applied shear stress, which contrasts with parallel alignment of endothelial cells under flow. SMC alignment varied with the magnitude of and exposure time to shear stress and is a continuous process that is dependent on calcium and cycloskeleton based mechanisms. A clear understanding and control of flow-induced SMC alignment will have implications for vascular tissue engineering. [DOI: 10.1115/1.1427697] Keywords: Shear Stress, Smooth Muscle Cell, Cell Mechanics, Cell Alignment

Laboratory evaluation for the presence of antineutrophil cytoplasmic antibodies (ANCAs) is used diagnostically in the evaluation of patients suspected of having systemic vasculitis. (1-4) The main utility in ANCA testing [...], Context.--Variability in testing for antineutrophil cytoplasmic antibodies (ANCAs) contributes to confusion and controversy related to testing for vasculitis and other ANCA-associated diseases. Objectives.--To survey laboratory testing practices regarding ANCA testing and to investigate differences in testing algorithms. Design.--Supplemental questions were sent to the 333 laboratories participating in the College of American Pathologists proficiency testing program for ANCA as part of the Special Immunology S2 Survey. Results.--A total of 315 laboratories submitted responses to the supplemental questions. Only 88 of 315 participants (28%) reported using a combination of indirect immunofluorescence (IFA) and enzyme immunoassay (EIA) techniques as recommended by current guidelines, with a few additional labs using IFA and multiplex bead assay as an acceptable alternative to EIA. Other labs reported using only IFA, EIA, or multiplex bead assays. Conclusions.--A wide variety of testing algorithms are in use for ANCA testing despite evidence to suggest that a combination of IFA and EIA testing provides the most comprehensive information. Laboratories should inform clinicians clearly about testing practices and utility of testing in specific disease states. (Arch Pathol Lab Med. 2016; 140:524-528; doi: 10.5858/arpa.2015-0221-CP)

A moderately detailed 3-D finite element model of the conductive anatomy of a canine thorax was used to examine the sensitivity of the results obtained during simulated transthoracic defibrillation to variations in skeletal muscle anisotropy and differing degrees of model inhomogeneity. Our results suggest that the myocardial current density distribution is not particularly sensitive to the method used to model skeletal muscle anisotropy. However, anisotropy variations caused defibrillation parameters such as paddle to paddle impedance and threshold current to change by as much as 50%. We found a greater sensitivity in the myocardial current density and the defibrillation parameters to variations in model inhomogeneity. The changes observed in both depended substantially on paddle placement. This sensitivity to paddle placement highlights the difficulty in predicting how a reduction in anatomical detail will affect the myocardial current density distribution. In general, we found the defibrillation parameters to be more sensitive than the myocardial current density distribution to the variations in anatomical detail we examined.

A realistic three-dimensional finite element model of the conductive anatomy of a canine thorax was constructed for use in the study of transthoracic electrical defibrillation. The model was constructed from a series of 21 cross-sectional CT scans of a 14.5 kg beagle, each separated by 0.82 cm. The electrical conductive properties of 8 distinct tissues were incorporated, including the anisotropic properties of skeletal muscle. Current density distributions were obtained for six paddle pairings and two paddle sizes. A quantitative basis for comparing the resulting distributions was formulated. Our results suggest that placing one or both of the paddles near the heart delivers a higher fraction of current to the heart. However, such paddle placements also produce a less uniform myocardial current density distribution and thus have a higher potential for causing damage. We found that some paddle positions can produce myocardial current densities close to the threshold for damage in successful defibrillations. Results obtained for 8 and 12 cm paddles indicate that 12 cm paddles may offer modest advantages over 8 cm paddles in clinical defibrillation. Comparison of our results to available in vivo experimental data confirm the validity of the finite element method for examining continuum field variables pertinent to electrical defibrillation.

Although multicolor flow cytometry was originally introduced into the field of diagnostic hematopathology as an ancillary or companion diagnostic test, this powerful tool is becoming a primary diagnostic modality, independent [...], Context.--Flow cytometry is often applied to minimal residual disease (mRd) testing in hematolymphoid neoplasia. Because flow-based MRD tests are developed in the laboratory, testing methodologies and lower levels of detection (LODs) are laboratory dependent. Objectives.--To broadly survey flow cytometry laboratories about MRD testing in laboratories, if performed, including indications and reported LODs. Design.--Voluntary supplemental questions were sent to the 549 laboratories participating in the College of American Pathologists (CAP) FL3-A Survey (Flow Cytometry--Immunophenotypic Characterization of Leukemia/ Lymphoma) in the spring of 2014. Results.--A total of 500 laboratories (91%) responded to the supplemental questions as part of the FL3-A Survey by April 2014; of those 500 laboratories, 167 (33%) currently perform MRD for lymphoblastic leukemia, 118 (24%) for myeloid leukemia, 99 (20%) for chronic lymphocytic leukemia, and 91 (18%) for plasma cell myeloma. Other indications include non-Hodgkin lymphoma, hairy cell leukemia, neuroblastoma, and myelodysplastic syndrome. Most responding laboratories that perform MRD for lymphoblastic leukemia reported an LOD of 0.01%. For myeloid leukemia, chronic lymphocytic leukemia, and plasma cell myeloma, most laboratories indicated an LOD of 0.1%. Less than 3% (15 of 500) of laboratories reported LODs of 0.001% for one or more MRD assays performed. Conclusions.--There is major heterogeneity in the reported LODs of MRD testing performed by laboratories subscribing to the CAP FL3-A Survey. To address that heterogeneity, changes to the Flow Cytometry Checklist for the CAP Laboratory Accreditation Program are suggested that will include new requirements that each laboratory (1) document how an MrD assay's LOD is measured, and (2) include the LOD or lower limit of enumeration for flow-based MRD assays in the final diagnostic report. (Arch Pathol Lab Med. 2015; 139:1276-1280; doi: 10.5858/arpa.2014-0543-CP)

Karlon, William J., Andrew D. McCulloch, James W. Covell, John J. Hunter, and Jeffrey H. Omens. Regional dysfunction correlates with myofiber disarray in transgenic mice with ventricular expression of ras. Am. J. Physiol. Heart Circ. Physiol. 278: H898-H906, 2000.--A hallmark of certain cardiac diseases such as familial hypertrophic cardiomyopathy is focal myofiber disarray. Regional ventricular dysfunction occurs in human subjects with hypertrophic cardiomyopathy; however, no direct evidence exists to correlate regional dysfunction with myofiber disarray. We used a transgenic mouse, which exhibits regional myofiber disarray via ventricular expression of the human oncogene ras, to investigate the relationship between myofiber disarray and septal surface strain. An isolated ejecting mouse heart preparation was used to record deformation of markers on the septal surface and to determine nonhomogeneous septal surface strain maps. Myofiber disarray made in histological tissue sections was correlated with gradients in surface systolic shortening. Significantly smaller maximum principal shortening was associated with disarray located near the right ventricle (RV) septal surface. There was also significantly smaller surface shear strain associated with disarray located either near the RV surface or at the midwall. Because surface shear is a local indicator of torsion, we conclude that myofiber disarray is associated with reduced septal torsion and reduced surface shortening. hypertrophic cardiomyopathy; fiber angle; surface strain; ventricular torsion

Zimmerman, Scott D., William J. Karlon, Jeffrey W. Holmes, Jeffrey H. Omens, and James W. Covell. Structural and mechanical factors influencing infarct scar collagen organization. Am. J. Physiol. Heart Circ. Physiol. 278: H194-H200, 2000.--Although large collagen fibers in myocardial infarct scar are highly organized, little is known about mechanisms controlling this organization. The preexisting extracellular matrix may act as a scaffold along which fibroblasts migrate. Conversely, deformation within the ischemic area could guide fibroblasts so new collagen is oriented to counteract the stretch. To investigate these potential mechanisms, we infarcted three groups of pigs. Group 1 served as infarct controls. Group 2 had the endocardium slit longitudinally to alter local systolic deformation. Group 3 had a plug sectioned from ischemic tissue and rotated 90 [degrees]. The slit altered systolic deformation in the infarcted tissue, changing circumferential strain from expansion to compression and increasing radial strain and shears and the variability of collagen fiber angles but not the mean angle. In the plug pigs, when deformation, matrix orientation, and continuity are altered in the infarct area, the result is complete disarray in the organization of collagen within the infarct scar. cardiac strain; infarct healing; myocardial infarction; collagen fiber angles

Identifies the structural and mechanical factors that controls the organization of collagen fibers in myocardial infarct scar. Fibroblasts in deformed ischemic area; Alterations of systolic deformation in the infarcted issue; Increase in radial strain and shears; Facts about fibroblasts.

Despite the success of BCMA-targeting CAR-Ts in multiple myeloma, patients with high-risk cytogenetic features still relapse most quickly and are in urgent need of additional therapeutic options. Here, we identify CD70, widely recognized as a favorable immunotherapy target in other cancers, as a specifically upregulated cell surface antigen in high risk myeloma tumors. We use a structure-guided design to define a CD27-based anti-CD70 CAR-T design that outperforms all tested scFv-based CARs, leading to >80-fold improved CAR-T expansion in vivo. Epigenetic analysis via machine learning predicts key transcription factors and transcriptional networks driving CD70 upregulation in high risk myeloma. Dual-targeting CAR-Ts against either CD70 or BCMA demonstrate a potential strategy to avoid antigen escape-mediated resistance. Together, these findings support the promise of targeting CD70 with optimized CAR-Ts in myeloma as well as future clinical translation of this approach., Competing Interests: Competing interests Patent application filed related to CD27-based CAR design described here (A.P.W., C.K., A.K, R.D., T.K.). A.P.W. honoraria from Sanofi and AstraZeneca, equity holder in Indapta Therapeutics.

The following abstracts are compiled from Check Sample exercises published in 2008. These peer-reviewed case studies assist laboratory professionals with continuing medical education and are developed in the areas of clinical chemistry, cytopathology, forensic pathology, hematology, microbiology, surgical pathology, and transfusion medicine. Abstracts for all exercises published in the program will appear annually in AJCP.