Laboratory Workup of Lymphoma in Adults.

OBJECTIVES: The diagnostic workup of lymphoma continues to evolve rapidly as experience and discovery lead to the addition of new clinicopathologic entities and techniques to differentiate them. The optimal clinically effective, efficient, and cost-effective approach to diagnosis that is safe for patients can be elusive, in both community-based and academic practice. Studies suggest that there is variation in practice in both settings. THE AIM OF THIS REVIEW IS TO: develop an evidence-based guideline for the preanalytic phase of testing, focusing on specimen requirements for the diagnostic evaluation of lymphoma. METHODS: The American Society for Clinical Pathology, the College of American Pathologists, and the American Society of Hematology convened a panel of experts in the laboratory workup of lymphoma to develop evidence-based recommendations. The panel conducted a systematic review of the literature to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach, recommendations were derived based on the available evidence, the strength of that evidence, and key judgments as defined in the GRADE Evidence to Decision framework. RESULTS: Thirteen guideline statements were established to optimize specimen selection, ancillary diagnostic testing, and appropriate follow-up for safe and accurate diagnosis of indolent and aggressive lymphoma. CONCLUSIONS: Primary diagnosis and classification of lymphoma can be achieved with a variety of specimens. Application of the recommendations can guide decisions about specimen suitability, diagnostic capabilities, and correct utilization of ancillary testing. Disease prevalence in patient populations, availability of ancillary testing, and diagnostic goals should be incorporated into algorithms tailored to each practice environment.

Laboratory Workup of Lymphoma in Adults: Guideline From the American Society for Clinical Pathology and the College of American Pathologists.

CONTEXT.­: The diagnostic workup of lymphoma continues to evolve rapidly as experience and discovery led to the addition of new clinicopathologic entities and techniques to differentiate them. The optimal clinically effective, efficient, and cost-effective approach to diagnosis that is safe for patients can be elusive, in both community-based and academic practice. Studies suggest that there is variation in practice in both settings. OBJECTIVE.­: To develop an evidence-based guideline for the preanalytic phase of testing, focusing on specimen requirements for the diagnostic evaluation of lymphoma. DESIGN.­: The American Society for Clinical Pathology, the College of American Pathologists, and the American Society of Hematology convened a panel of experts in the laboratory workup of lymphoma to develop evidence-based recommendations. The panel conducted a systematic review of literature to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation approach, recommendations were derived based on the available evidence, strength of that evidence, and key judgements as defined in the Grading of Recommendations Assessment, Development, and Evaluation Evidence to Decision framework. RESULTS.­: Thirteen guideline statements were established to optimize specimen selection, ancillary diagnostic testing, and appropriate follow-up for safe and accurate diagnosis of indolent and aggressive lymphoma. CONCLUSIONS.­: Primary diagnosis and classification of lymphoma can be achieved with a variety of specimens. Application of the recommendations can guide decisions on specimen suitability, diagnostic capabilities, and correct use of ancillary testing. Disease prevalence in patient populations, availability of ancillary testing, and diagnostic goals should be incorporated into algorithms tailored to each practice environment.

Detection of Polyclonal Increases in Immunoglobulin G4 Subclass by Distinct Patterns on Capillary Serum Protein Electrophoresis: Diagnostic Pitfalls and Clinical Observations in a Study of 303 Cases.

OBJECTIVES: Autoimmunity, hypersensitivity, and the recently recognized set of syndromes collectively termed immunoglobulin G4-related disease (IgG4-RD) may be associated with increased serum IgG4 levels. We reviewed our experience detecting increased IgG4 by distinct serum protein electrophoresis (SPEP) patterns. METHODS: We studied 303 capillary SPEP cases with dome-like anodal γ changes and increased measured serum IgG4. RESULTS: IgG4 ranged from 208 to 6,670 mg/dL (normal, <201 mg/dL). Seventeen of 91 cases evaluated by immunosubtraction appeared monotypic (16 κ, 1 λ), but all five cases further analyzed by isoelectric focusing appeared polyclonal. Six cases with markedly increased IgG4 had presumptive evidence of IgG4-RD. Sixteen of 45 assessed patients had autoantibodies. CONCLUSIONS: Increased polyclonal IgG4 has a characteristic SPEP pattern that may mimic monoclonal gammopathy, even on immunosubtraction. κ Pseudo-restriction might reflect the naturally high κ/λ ratio of the IgG4 subclass. Autoantibodies were common, and the greatest IgG4 increases had clinical features of IgG4-RD.

Building a laboratory workforce to meet the future: ASCP Task Force on the Laboratory Professionals Workforce.

OBJECTIVES: To analyze the demand for services from the nation's medical laboratories, which is predicted to dramatically increase as our citizens age and millions receive insurance coverage through the Affordable Care Act. METHODS: A systematic review of relevant publications and databases was conducted to assess the current state of the nation's medical laboratory workforce and to examine the impact of population demographics and health reform on workforce development to address the future demand for laboratory services. RESULTS: Building a Laboratory Workforce to Meet the Future, a new report from the American Society for Clinical Pathology (ASCP), provides a comprehensive strategy to address the future workforce needs of the nation's medical laboratories to meet this demand to provide timely, accurate, and safe patient care and to fully realize the benefits of personalized medicine. CONCLUSIONS: The report, from the ASCP Task Force on the Laboratory Professionals Workforce, is a comprehensive review of the myriad of factors affecting recruitment and retention of qualified laboratory professionals and provides a set of thoughtful recommendations outlining a multifaceted approach to bolster the pipeline of potential candidates for the profession as well as leadership in health care.

Immunophenotypic signatures of benign and dysplastic granulopoiesis by cytomic profiling.

BACKGROUND: The role of flow cytometry (FCM) in diagnosing myelodysplastic syndromes (MDS) remains controversial, because analysis of myeloid maturation may involve subjective interpretation of sometimes subtle patterns on multiparameter FCM. METHODS: Using six-parameter marker combinations known to be useful in evaluating the myeloid compartment in MDS, we measured objective immunophenotypic differences between non-neoplastic (n = 25) and dysplastic (n = 17) granulopoiesis using a novel method, called Fisher information nonparametric embedding (FINE), that measures information distances among FCM datasets modeled as individual high-dimensional probability density functions, rather than as sets of two-dimensional histograms. Information-preserving component analysis (IPCA) was used to create information-optimized "rotated" two-dimensional histograms for visualizing myelopoietic immunophenotypes for each individual sample. RESULTS: There was a consistent trend of segregation of higher-grade MDS (RAEB and RCMD) from benign by FINE analysis. This difference was accentuated in cases with morphologic dysgranulopoiesis and in cases with clonal cytogenetic abnormalities. However, lower grades of MDS or cases that lacked morphologic dysgranulopoiesis showed much greater overlap with non-neoplastic cases. Two cases of reactive left shift were consistently embedded within the higher-grade MDS group. IPCA yielded two-dimensional histogram projections for each individual case by relative weighting of measured cellular characteristics, optimized for preserving information distances derived through FINE. CONCLUSIONS: Objective analysis by information geometry supports the conclusions of previous studies that there are immunophenotypic differences in the maturation patterns of benign granulopoiesis and high grade MDS, but also reinforces the known pitfalls of overlap between low-grade MDS and benign granulopoiesis and overlap between reactive granulocytic left shifts and dysplastic granulopoiesis.

Statistical file matching of flow cytometry data.

Flow cytometry is a technology that rapidly measures antigen-based markers associated to cells in a cell population. Although analysis of flow cytometry data has traditionally considered one or two markers at a time, there has been increasing interest in multidimensional analysis. However, flow cytometers are limited in the number of markers they can jointly observe, which is typically a fraction of the number of markers of interest. For this reason, practitioners often perform multiple assays based on different, overlapping combinations of markers. In this paper, we address the challenge of imputing the high-dimensional jointly distributed values of marker attributes based on overlapping marginal observations. We show that simple nearest neighbor based imputation can lead to spurious subpopulations in the imputed data and introduce an alternative approach based on nearest neighbor imputation restricted to a cell's subpopulation. This requires us to perform clustering with missing data, which we address with a mixture model approach and novel EM algorithm. Since mixture model fitting may be ill-posed in this context, we also develop techniques to initialize the EM algorithm using domain knowledge. We demonstrate our approach on real flow cytometry data.

The phosphate binder equivalent dose.

Phosphate binders include calcium acetate or carbonate, sevelamer hydrochloride or carbonate, magnesium and lanthanum carbonate, and aluminum carbonate or hydroxide. Their relative phosphate-binding capacity has been assessed in human, in vivo studies that have measured phosphate recovery from stool and/or changes in urinary phosphate excretion or that have compared pairs of different binders where dose of binder in each group was titrated to a target level of serum phosphate. The relative phosphate-binding coefficient (RPBC) based on weight of each binder can be estimated relative to calcium carbonate, the latter being set to 1.0. A systematic review of these studies gave the following estimated RPBC: for elemental lanthanum, 2.0, for sevelamer hydrochloride or carbonate 0.75, for calcium acetate 1.0, for anhydrous magnesium carbonate 1.7, and for "heavy" or hydrated, magnesium carbonate 1.3. Estimated RPBC for aluminum-containing binders were 1.5 for aluminum hydroxide and 1.9 for aluminum carbonate. The phosphate-binding equivalent dose was then defined as the dose of each binder in g × its RPBC, which would be the binding ability of an equivalent weight of calcium carbonate. The phosphate-binding equivalent dose may be useful in comparing changes in phosphate binder prescription over time when multiple binders are being prescribed, when estimating an initial binder prescription, and also in phosphate kinetic modeling.

Interleukin-33 contributes to both M1 and M2 chemokine marker expression in human macrophages.

BACKGROUND: Interleukin-33 is a member of the IL-1 cytokine family whose functions are mediated and modulated by the ST2 receptor. IL-33-ST2 expression and interactions have been explored in mouse macrophages but little is known about the effect of IL-33 on human macrophages. The expression of ST2 transcript and protein levels, and IL-33-mediated effects on M1 (i.e. classical activation) and M2 (i.e. alternative activation) chemokine marker expression in human bone marrow-derived macrophages were examined. RESULTS: Human macrophages constitutively expressed the membrane-associated (i.e. ST2L) and the soluble (i.e. sST2) ST2 receptors. M2 (IL-4 + IL-13) skewing stimuli markedly increased the expression of ST2L, but neither polarizing cytokine treatment promoted the release of sST2 from these cells. When added to naïve macrophages alone, IL-33 directly enhanced the expression of CCL3. In combination with LPS, IL-33 blocked the expression of the M2 chemokine marker CCL18, but did not alter CCL3 expression in these naive cells. The addition of IL-33 to M1 macrophages markedly increased the expression of CCL18 above that detected in untreated M1 macrophages. Similarly, alternatively activated human macrophages treated with IL-33 exhibited enhanced expression of CCL18 and the M2 marker mannose receptor above that detected in M2 macrophages alone. CONCLUSIONS: Together, these data suggest that primary responses to IL-33 in bone marrow derived human macrophages favors M1 chemokine generation while its addition to polarized human macrophages promotes or amplifies M2 chemokine expression.

Immunomodulator agent-related lymphoproliferative disorders.

The recent development of inhibitors of key immune response proteins has revolutionized the therapy of autoimmune diseases; these immunomodulator agents include monoclonal antibodies and receptor antagonists. However, as with all therapies, these new agents are not without side effects and complications. In particular, anti-tumor necrosis factor alpha (TNFalpha) agents have been reported to be associated with an increased incidence of lymphoproliferative disorders, infections, and vasculitis. We evaluated the clinicopathological features of 18 cases of immunomodulator agent-related lymphoproliferative disorders (IAR-LPD) from several institutions. These included 6 cases of B-cell lymphoma, 2 cases of T-cell lymphoma, 3 cases of classical Hodgkin lymphoma, and 7 atypical lymphoid proliferations that did not fulfill diagnostic criteria for lymphoma; two of the latter regressed after discontinuation of the immunomodulator agent therapy. All eight lymphoma patients with available information had also received prior chemotherapy (methotrexate or 6-mercaptopurine). EBV was strongly associated with the B-cell and classical Hodgkin lymphomas. This case series illustrates that a broad range of lymphoid proliferations can occur after immunomodulator agent therapy and that these immunomodulator agent-related lymphoproliferative disorders have considerable overlap with other well-defined lymphoproliferative diseases associated with iatrogenic immunosuppression. Further study is warranted to evaluate how these therapies interact with other immunosuppressive agents and the underlying abnormal immune system to enhance the development of lymphomas and atypical lymphoid proliferations.

FINE: fisher information nonparametric embedding.

We consider the problems of clustering, classification, and visualization of high-dimensional data when no straightforward euclidean representation exists. In this paper, we propose using the properties of information geometry and statistical manifolds in order to define similarities between data sets using the Fisher information distance. We will show that this metric can be approximated using entirely nonparametric methods, as the parameterization and geometry of the manifold is generally unknown. Furthermore, by using multidimensional scaling methods, we are able to reconstruct the statistical manifold in a low-dimensional euclidean space; enabling effective learning on the data. As a whole, we refer to our framework as Fisher Information Nonparametric Embedding (FINE) and illustrate its uses on practical problems, including a biomedical application and document classification.

Analysis of clinical flow cytometric immunophenotyping data by clustering on statistical manifolds: treating flow cytometry data as high-dimensional objects.

BACKGROUND: Clinical flow cytometry typically involves the sequential interpretation of two-dimensional histograms, usually culled from six or more cellular characteristics, following initial selection (gating) of cell populations based on a different subset of these characteristics. We examined the feasibility of instead treating gated n-parameter clinical flow cytometry data as objects embedded in n-dimensional space using principles of information geometry via a recently described method known as Fisher Information Non-parametric Embedding (FINE). METHODS: After initial selection of relevant cell populations through an iterative gating strategy, we converted four color (six-parameter) clinical flow cytometry datasets into six-dimensional probability density functions, and calculated differences among these distributions using the Kullback-Leibler divergence (a measurement of relative distributional entropy shown to be an appropriate approximation of Fisher information distance in certain types of statistical manifolds). Neighborhood maps based on Kullback-Leibler divergences were projected onto two dimensional displays for comparison. RESULTS: These methods resulted in the effective unsupervised clustering of cases of acute lymphoblastic leukemia from cases of expansion of physiologic B-cell precursors (hematogones) within a set of 54 patient samples. CONCLUSIONS: The treatment of flow cytometry datasets as objects embedded in high-dimensional space (as opposed to sequential two-dimensional analyses) harbors the potential for use as a decision-support tool in clinical practice or as a means for context-based archiving and searching of clinical flow cytometry data based on high-dimensional distribution patterns contained within stored list mode data. Additional studies will be needed to further test the effectiveness of this approach in clinical practice.

Lanthanum carbonate reduces phosphorus burden in patients with CKD stages 3 and 4: a randomized trial.

BACKGROUND AND OBJECTIVES: Lanthanum carbonate (FOSRENOL, Shire Pharmaceuticals) is an effective noncalcium, nonresin phosphate binder for the control of hyperphosphatemia in chronic kidney disease (CKD) stage 5 patients undergoing dialysis. DESIGN, SETTING, PARTICIPANTS AND MEASUREMENTS: A Phase 2, randomized, double-blind, placebo-controlled trial evaluating the efficacy and safety of lanthanum carbonate in CKD stage 3 and 4 patients. Of 281 patients screened, 121 were randomized (2:1) to lanthanum carbonate or placebo (80 versus 41). The modified intent-to-treat population included 90 patients (56 versus 34); 71 (43 versus 28) completed the study. After run-in, when any current phosphate binders were discontinued and dietary counseling reinforced, patients with serum phosphorus >4.6 mg/dl received lanthanum carbonate (titrated up to 3000 mg/d) or matching placebo for 8 wk. RESULTS: At the end of treatment, 25 (44.6%) versus nine (26.5%) patients had serum phosphorus < or =4.6 mg/dl (difference 18.1%, P = 0.12) in the lanthanum carbonate and placebo groups, respectively. Statistically significant differences were observed between groups in change from baseline to end of treatment for serum phosphorus (P = 0.02), intact parathyroid hormone (P = 0.02), and urinary phosphorus excretion (P = 0.04). The safety profile and tolerability of lanthanum carbonate were similar to that of placebo. CONCLUSIONS: Because <1% of phosphorus is in the extracellular fluid, serum measurements may not accurately reflect total body burden in patients with CKD stages 3 and 4. However, lanthanum carbonate is an effective phosphate binder in this patient population, with a safety profile and tolerability similar to that of placebo.

Differences in germinal centre and non-germinal center phenotype in gastric and intestinal diffuse large B-cell lymphomas.

The gastrointestinal tract is the most common extranodal site of lymphoma, and the most common gastrointestinal lymphoma is diffuse large B-cell type (DLBCL). DLBCL can be separated into germinal centre (GCP) and non-germinal centre phenotypes (non-GCP) using CD10, BCL-6 and MUM1 immunohistochemistry, but primary gastrointestinal DLBCL has not been extensively studied. We investigated 48 cases of primary gastrointestinal DLBCL (33% involving the small intestine, 50% the stomach, 13% the large intestine and 4% the ileocecal junction) and found that most (88%) DLBCL in the intestines were of GCP, while only 58% of gastric DLBCL were of GCP. This difference in GCP and non-GCP in gastric vs. intestinal DLBCL may be due to variations in lymphomagenesis reflecting acquired vs. native mucosa-associated lymphoid tissue. There was no significant difference in either overall survival or disease-free survival between the germinal centre and non-germinal centre groups. The distribution of Helicobacter pylori in different gastric DLBCL phenotypes raises interesting questions about the pathogenesis of H. pylori-associated lymphomas.

Site-specific morphologic differences in extranodal marginal zone B-cell lymphomas.

CONTEXT: Lymphoepithelial lesions (LELs) are a useful diagnostic feature of extranodal marginal zone B-cell lymphoma (EMZL); however, there is scant literature comparing their frequency and morphology at various sites. OBJECTIVE: To evaluate any diagnostically useful, site-specific, morphologic patterns in EMZLs. DESIGN: In this retrospective review, we evaluated 136 EMZLs from different sites for LEL pattern and other pathologic differences, including CD43 coexpression and plasma cell component features. RESULTS: Prominent and destructive LELs were most common in salivary and thyroid gland cases, and LELs were rare to absent in breast, skin, and ocular adnexa cases. An LEL pattern with lymphocytes "stuffing" glandular lumina was seen in lung, thyroid, and salivary gland cases. Monoclonal plasma cells were most common in breast, upper aerodigestive tract, skin, and salivary gland cases. CD43 coexpression was seen in 36% of cases, most commonly in salivary gland, stomach, and upper aerodigestive tract. CONCLUSIONS: The relative importance of LEL pattern, CD43 coexpression, and clonal plasma cell component in EMZLs is site-dependent, and the differences may aid in the diagnosis of EMZLs at different anatomic sites.

Diagnostic pathology and laboratory medicine in the age of "omics": a paper from the 2006 William Beaumont Hospital Symposium on Molecular Pathology.

Functional genomics and proteomics involve the simultaneous analysis of hundreds or thousands of expressed genes or proteins and have spawned the modern discipline of computational biology. Novel informatic applications, including sophisticated dimensionality reduction strategies and cancer outlier profile analysis, can distill clinically exploitable biomarkers from enormous experimental datasets. Diagnostic pathologists are now charged with translating the knowledge generated by the "omics" revolution into clinical practice. Food and Drug Administration-approved proprietary testing platforms based on microarray technologies already exist and will expand greatly in the coming years. However, for diagnostic pathology, the greatest promise of the "omics" age resides in the explosion in information technology (IT). IT applications allow for the digitization of histological slides, transforming them into minable data and enabling content-based searching and archiving of histological materials. IT will also allow for the optimization of existing (and often underused) clinical laboratory technologies such as flow cytometry and high-throughput core laboratory functions. The state of pathology practice does not always keep up with the pace of technological advancement. However, to use fully the potential of these emerging technologies for the benefit of patients, pathologists and clinical scientists must embrace the changes and transformational advances that will characterize this new era.

Fascin expression in diffuse large B-cell lymphoma, anaplastic large cell lymphoma, and classical Hodgkin lymphoma.

CONTEXT: Fascin is an actin-bundling protein involved in the formation of dendritic processes. Fascin is a sensitive marker for classical Reed-Sternberg cells and has a high negative predictive value for diagnosis of classical Hodgkin lymphoma (CHL). Fascin has been used to distinguish CHL from non-Hodgkin lymphoma. Recently, it was shown that fascin might not help differentiate CHL from anaplastic large cell lymphoma (ALCL). Moreover, fascin has not been extensively studied in the context of other large cell lymphomas. OBJECTIVE: To analyze fascin expression in diffuse large B-cell lymphoma (DLBCL) and also reexamine its usefulness in discriminating CHL from ALCL. DESIGN: Formalin-fixed, paraffin-embedded tissue samples from 41 cases of DLBCL, 30 cases of CHL, and 30 cases of ALCL were analyzed. Fascin expression was compared across each type of lymphoma with additional correlation between fascin positivity and ALK-1 expression in ALCL performed. RESULTS: Only 6 (14.6%) of 41 cases of DLBCL stained positively for fascin, with most neoplastic large cells exhibiting a weak staining pattern. Fifteen (50%) of 30 cases of ALCL showed positivity for fascin, with most large cells staining strongly. All 30 cases of CHL demonstrated intense positive staining. Sixty percent of fascin-positive ALCLs were positive for ALK-1, while 66.7% of fascin-negative ALCLs were positive for ALK-1. CONCLUSIONS: Fascin is highly sensitive for CHL and has a very high negative predictive value (100% in this series) for distinguishing CHL from DLBCL or ALCL. However, the specificity and positive predictive value for fascin are much higher in distinguishing CHL from DLBCL than in distinguishing CHL from ALCL. Expression of fascin appears more useful in the differential diagnosis of CHL versus DLBCL than in the differential diagnosis of CHL versus ALCL.

Cytochrome P450 3A5 expression in the kidneys of patients with calcineurin inhibitor nephrotoxicity.

BACKGROUND: Nephrotoxicity secondary to calcineurin inhibitors is common in renal transplant recipients, occurring in 76-94% of patients. The role of drug transporters (P-glycoprotein) and drug metabolizing enzymes (cytochrome P450) as predisposing factors toward nephrotoxicity or its prevention has not been thoroughly examined. METHODS: The objective of this study was to analyse cytochrome P450 3A5 (CYP3A5) expression in kidneys of solid organ recipients by immunohistochemistry to determine if there is an association between expression of this enzyme and calcineurin inhibitor toxicity. Transplant recipients were compared with a control group. RESULTS: Apical tubular plasma membrane staining for CYP3A5 was present in 62% of study and 100% of control biopsies (P=0.0012). Proximal and distal tubular nuclear staining intensity was similar between groups. Cytoplasmic staining in both the proximal (2.1+/-0.9 vs 1.4+/-0.9) and distal (2.8+/-0.5 vs 1.8+/-1.1) tubules was greater in the control vs study population specimens, respectively (P=0.0093 and P=0.0005, respectively). Regression models that controlled for use of CYP3A inhibiting and inducing medications, age, gender, race and glomerular filtration rate did not predict differences between study groups with regard to staining locations and intensity, except for the cytoplasm of the distal tubule, where intensity of staining was significantly lower in the study group (0.9+/-0.3; P=0.002). CONCLUSIONS: This study showed decreased expression of CYP3A5 in nephrotoxic biopsies as compared with a control group. Our data suggest that the relationship between reduced presence of CYP3A5 in the kidney tubules and nephrotoxicity should be further explored to elucidate the role of this enzyme in mediating toxicity.