Russell A. Higgins

Whole-Genome Scanning by Array Comparative Genomic Hybridization as a Clinical Tool for Risk Assessment in Chronic Lymphocytic Leukemia

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays. Genomic changes involving loci currently interrogated by fluorescence in situ hybridization (FISH) panels were detected in 155 cases (89%) at expected frequencies: 13q14 loss (47%), trisomy 12 (13%), 11q loss (11%), 6q loss (7.5%), and 17p loss (4.6%). Genomic instability was the second most commonly identified alteration of prognostic significance with three or more alterations involving loci not interrogated by FISH panels identified in 37 CLL cases (21%). A subset of 48 CLL cases analyzed by six-probe FISH panels (288 total hybridizations) was concordant with array CGH results for 275 hybridizations (95.5%); 13 hybridizations (4.5%) were discordant because of clonal populations that comprised less than 30% of the sample. Array CGH is a powerful, cost-effective tool for genome-wide risk assessment in the clinical evaluation of CLL.

Anaplastic large cell lymphoma: twenty-five years of discovery.

CONTEXT The year 2010 commemorates the 25th year since the seminal publication by Karl Lennert and Harald Stein and others in Kiel, West Germany, describing an unusual large cell lymphoma now known as anaplastic large cell lymphoma (ALCL). Investigators at many universities and hospitals worldwide have contributed to our current in-depth understanding of this unique peripheral T-cell lymphoma, which in its systemic form, principally occurs in children and young adults. OBJECTIVE To summarize our current knowledge of the clinical and pathologic features of systemic and primary cutaneous ALCL. Particular emphasis is given to the biology and pathogenesis of ALCL. DATA SOURCES Search of the medical literature (Ovid MEDLINE In-Process & Other Non-Indexed Citations and Ovid MEDLINE: 1950 to Present [National Library of Medicine]) and more than 20 years of diagnostic experience were used as the source of data for review. CONCLUSIONS Based on immunostaining for activation antigen CD30 and the presence of dysregulation of the anaplastic lymphoma kinase gene (2p23), the diagnosis of ALCL has become relatively straightforward for most patients. Major strides have been made during the last decade in our understanding of the complex pathogenesis of ALCL. Constitutive NPM-ALK signaling has been shown to drive oncogenesis via an intricate network of redundant and interacting pathways that regulate cell proliferation, cell fate, and cytoskeletal modeling. Nevertheless, pathomechanistic, therapeutic, and diagnostic challenges remain that should be resolved as we embark on the next generation of discovery.

Application of immunohistochemistry in the diagnosis of non-Hodgkin and Hodgkin lymphoma.

CONTEXT Beginning with the immunologic classifications of Lukes and Collins and Kiel and culminating in the Revised European-American Lymphoma and World Health Organization classifications, the diagnosis of lymphoid tumors relies heavily on the determination of cell lineage, maturation, and function, based on antigen expression in addition to morphology and clinical features. Technologic advances in immunology, antibody production, genetic analysis, cloning, and the identification of new genes and proteins by microarray and proteomics have provided pathologists with many antibodies to use in routine diagnosis. OBJECTIVE To provide guidance to the practicing pathologist in the appropriate selection of an antibody panel for the diagnosis of lymphoma based on morphology and relevant clinical data and to avoid pitfalls in the interpretation of immunohistochemical data. Attention is given to some of the newer antibodies, particularly against transcription factors, that are diagnostically and prognostically useful. DATA SOURCES The information presented in this article is based on review of the literature using the OVID database (Ovid MEDLINE 1950 to present with daily update) and 20 years of experience in diagnostic hematopathology. CONCLUSIONS Immunophenotyping is required for the diagnosis and classification of lymphoid malignancies. Many paraffin-reactive antibodies are available to the pathologist but most are not specific. To avoid diagnostic pitfalls, interpretation of marker studies must be based on a panel and knowledge of a particular antigens expression in normal, reactive, and neoplastic conditions.

A prospective randomised study of a rotary powered device (OnControl) for bone marrow aspiration and biopsy

Introduction Bone marrow aspiration and biopsy is an invasive procedure associated with morbidity and mortality risk. We compared a powered bone marrow aspiration and biopsy device to the traditional method by relatively assessing pain scores, procedure times, biopsy capture rates, quality of material retrieved, and safety and operator satisfaction. Methods Two large academic medical centres participated in this trial. Patients were randomised to have procedures carried out using the powered system or the manual technique. A visual analogue scale pain score was recorded immediately following skin puncture and once again at the end of the procedure for each patient. Procedure time was measured from skin puncture to core specimen acquisition. Pathologic assessment of 30 randomised samples was carried out. Operator satisfaction with devices was measured on a scale of 0–10, with 10 as the highest rating. Results Five operators from two sites enrolled 50 patients (powered, n=25; manual, n=25). Groups were evenly matched, with no significant differences in the means for age, weight and height. The powered system was superior to the manual system with respect to patient perceived pain from needle insertion (2.6±2.0 vs 4.1±2.5, p=0.022) and procedural time (100.0±72.8 s vs 224.1±79.0 s, p<0.001). Overall pain scores at the end of both procedures were comparable (3.2±2.2 vs 3.8±3.0, p=0.438). No complications were observed in either arm of the study. Blinded pathologic analysis of the specimens retrieved revealed that cores obtained using the powered system were longer and wider than those obtained using the manual technique (25.4±12.3 mm2 vs 11.9±5.6 mm2, p=0.001). For marrow aspiration, no difference was seen between groups for clot/particle spicules or smear spicules. Operator assessment favoured the use of the powered device. Conclusions Results of this trial suggest that the use of a powered bone marrow biopsy device significantly reduces needle insertion pain and procedural time when compared to a manual technique. The superior size and overall quality of core specimens retrieved by the powered device provides more material for pathologic evaluation, thereby increasing diagnostic yield and reducing the need for repeat procedures.

Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia

Genomic aberrations have increasingly gained attention as prognostic markers in B-cell chronic lymphocytic leukemia (CLL). Fluorescence in situ hybridization (FISH) has improved the detection rate of genomic alterations in CLL from approximately 50% using conventional cytogenetics to greater than 80%. More recently, array comparative genomic hybridization (CGH) has gained popularity as a clinical tool that can be applied to detect genomic gains and losses of prognostic importance in CLL. Array CGH and FISH are particularly useful in CLL because genomic gains and losses are key events with both biologic and prognostic significance, while balanced translocations have limited prognostic value. Although FISH has a higher technical sensitivity, it requires separate, targeted hybridizations for the detection of alterations at genomic loci of interest. Array CGH, on the other hand, has the ability to provide a genome-wide survey of genomic aberrations with a single hybridization reaction. Array CGH is expanding the known genomic regions of importance in CLL and allows these regions to be evaluated in the context of a genome-wide perspective. Ongoing clinical trials are evaluating the use of genomic aberrations as tools for risk-stratifying patients for therapy, thus increasing the need for reliable and high-yield methods to detect these genomic changes. In this review, we consider the use of array CGH as a clinical tool for the identification of genomic alterations with prognostic significance in CLL, and suggest ways to integrate this test into the clinical molecular diagnostic laboratory work flow.

Autotransfusion of hemothorax blood in trauma patients: Is it the same as fresh whole blood?

BACKGROUND Autotransfusable shed blood has been poorly characterized in trauma and may have similarities to whole blood with additional benefits. METHODS This was a prospective descriptive study of adult patients from whom ≥50 mL of blood was drained within the first 4 hours after chest tube placement. Pleural and venous blood samples were analyzed for coagulation, hematology, and electrolytes. RESULTS Twenty-two subjects were enrolled in 9 months. The following measured coagulation factors of hemothorax were significantly depleted compared with venous blood: international normalized ratio (>9 in contrast to 1.1, P < .001), activated partial thromboplastin time (>180 in contrast to 28.5 seconds, P < .001), and fibrinogen (<50 in contrast to 288 mg/dL, P < .001). The mean hematocrit (26.4 in contrast to 33.9), (P = .003), hemoglobin (9.3 in contrast to 11.8 g/dL, P = .004), and platelet count (53 in contrast to 174 K/μL, P < .001) of hemothorax were significantly lower than venous blood. A hemothorax volume of 726 mL was calculated to be equivalent to 1 U of red blood cells. CONCLUSIONS Hemothorax blood contains significantly decreased coagulation factors and has lower hemoglobin when compared with venous blood.

A small amount can make a difference: a prospective human study of the paradoxical coagulation characteristics of hemothorax

BACKGROUND The evacuated hemothorax has been poorly described because it varies with time, it has been found to be incoagulable, and its potential effect on the coagulation cascade during autotransfusion is largely unknown. METHODS This is a prospective descriptive study of adult patients with traumatic chest injury necessitating tube thoracostomy. Pleural and venous samples were analyzed for coagulation, hematology, and electrolytes at 1 to 4 hours after drainage. Pleural samples were also analyzed for their effect on the coagulation cascade via mixing studies. RESULTS Thirty-four subjects were enrolled with a traumatic hemothorax. The following measured coagulation factors were significantly depleted compared with venous blood: international normalized ratio (>9 vs 1.1) (P < .001) and activated partial thromboplastin time (aPTT) (>180 vs 24.5 seconds) (P < .001). Mixing studies showed a dose-dependent increase in coagulation dilutions through 1:8 (P < .05). CONCLUSIONS An evacuated hemothorax does not vary in composition significantly with time and is incoagulable alone. Mixing studies with hemothorax plasma increased coagulation, raising safety concerns.

Primary Central Nervous System Histiocytic Sarcoma Arising After Precursor B-Cell Acute Lymphoblastic Leukemia.

Abstract Histiocytic sarcomas (HSs) are rare malignant neoplasms derived from histiocytes that may be associated with other hematolymphoid neoplasms. Histiocytic sarcomas rarely occur in the CNS and have not previously been reported in conjunction with prior B-cell lymphoblastic leukemia. We report the case of a 23-year-old man who presented with primary CNS HS 7 years after achieving remission for precursor B-cell acute lymphoblastic leukemia (B-ALL). Molecular studies revealed clonal immunoglobulin heavy-chain (IGH) gene rearrangement within the HS, suggesting linkage to his previous B-ALL. Previously reported post-ALL HSs show a strong predilection for young males (male-to-female ratio, 20:1), whereas cases of primary CNS HS without previous ALL affected older adults with balanced sex predilection. The patients survival at 60 months exceeds expectations when compared with that of other reported cases of de novo primary CNS HS (n = 18) and post-ALL HS at all sites (n = 19). In addition, we discuss the potential relationship between B-ALL and HS posed by other authors.

Isolated vaginal myeloid sarcoma in a 16-year-old girl

Involvement of the female genital tract by myeloid sarcoma as the initial presentation is extremely uncommon, especially in the vagina. The lack of specific histologic features and the unusual location can be a diagnostic challenge to both the surgical pathologist and the clinician. The very few reported cases of myeloid sarcoma occurring in the vagina have been exclusively seen in adults. We report a 16-year-old girl who presented with a vaginal mass of 4 weeks duration. The initial clinical impression was a Bartholin cyst vs an abscess. However, because of persistence of the vaginal mass after a full course of antibiotic treatment, a biopsy was performed. Immunohistochemistry supported the diagnosis of myeloid sarcoma. Peripheral blood and bone marrow studies were normal. The patient received 4 cycles of chemotherapy and remained disease free 5 months from therapy completion. The clinical course, diagnostic workup, and differential diagnosis of our patient are discussed. Reported cases of myeloid sarcoma occurring in the vagina are reviewed and summarized.

Issues Surrounding Age-Adjusted d-Dimer Cutoffs That Practicing Physicians Need to Know When Evaluating Patients With Suspected Pulmonary Embolism

This article has been corrected. The original version (PDF) is appended to this article as a Supplement. A recent best-practice advice paper from the Clinical Guidelines Committee of the American College of Physicians (ACP) provides an algorithmic approach that uses an initial clinical pretesting risk assessment (such as Wells or Geneva scores and Pulmonary Embolism Rule-out Criteria [PERC]), d-dimer testing for intermediate- and low-risk patients with positive PERC scores, and evaluation by imaging studies for high-risk patients and patients with positive d-dimer results (1). The authors recommend age-adjusted d-dimer (AADD) cutoffs based on several recently published studies (2), which demonstrated that the use of AADD cutoffs for patients older than 50 years (defined as AADD cutoff = age10 ng/mL) improved specificity for diagnosing pulmonary embolism while maintaining at least 97% sensitivity. Supplement. Original Version (PDF) We agree in principle with the approach of AADD cutoffs. However, from a laboratory perspective, widespread implementation of these cutoffs in clinical practice poses substantial concerns, including a lack of standardized d-dimer unit reporting, limitations of d-dimer testing in clinical studies, and a lack of defined strategies for clinical laboratories to adopt AADD cutoffs. Here, we elaborate on these difficulties to ensure that our clinical colleagues understand the issues surrounding d-dimer testing, but we also offer immediate and long-term strategies. The College of American Pathologists (CAP) and Clinical Laboratory and Standards Institute (CLSI) require d-dimer results to be reported with both a unit type and a unit of magnitude (3, 4). The d-dimer unit type includes either fibrinogen equivalent units (FEUs) or d-dimer units (DDUs). The FEU is a mass equivalent of the d-dimer fragments of the fibrinogen molecule from which it is derived, and the mass equivalent of the DDU is approximately half that of the FEU (5) (for example, 250 ng/mL DDU= 500 ng/mL FEU). The commonly reported d-dimer units of magnitude are milligrams per liter, micrograms per liter, micrograms per milliliter, and nanograms per milliliter. Such an array of d-dimer result formats is confusing and may result in clinical error. Based on recent CAP proficiency testing data, a small proportion but a substantial number of laboratories are reporting the incorrect d-dimer units (6). As currently written, the ACP best-practice advice does not include a unit type (that is, FEU or DDU) for the AADD cutoffs when evaluating low- and intermediate-risk patients for a possible pulmonary embolism, and the failure to report the unit type may result in misinterpretation of the d-dimer. For instance, if a cutoff of 500 ng/mL FEU were assumed for an assay calibrated in nanograms per milliliter of DDU, a d-dimer measurement of 400 ng/mL DDU (equivalent to ~800 ng /mL FEU) would be misinterpreted as a value below the cutoff for excluding pulmonary embolism. Physicians also should be mindful of the limitations of the AADD clinical studies. Differences in monoclonal antibodies used in manufactured d-dimer assays contribute to a lack of agreement among d-dimer test results (6, 7), causing differences in venous thromboembolism (VTE) exclusion cutoffs for the d-dimer kits approved or cleared by the U.S. Food and Drug Administration (FDA) (8). Most AADD clinical studies used only a few commercially available d-dimer kits. For instance, only 5 commercial, high-sensitivity, quantitative d-dimer assays (Table) were used in the most frequently quoted studyADJUST-PE (Age-Adjusted d-Dimer Cutoff Levels to Rule Out Pulmonary Embolism) (9)representing only a small portion of the commercially available assays used in U.S. clinical laboratories (8). The laboratory data from these clinical studies were insufficient to determine whether the assays performed equally or whether any assay had sufficient statistical power to avoid potential error. This concern was exemplified in an 86-year-old patient who had pulmonary embolism but a range of d-dimer values (590 to 1170 ng/mL FEU) measured by 5 commercial d-dimer assays (10). Furthermore, the numbers of patients older than 75 years or from a minority ethnic group were relatively limited among the studies (9). The d-dimer units in many studies (that is, DDU vs. FEU) were not clearly specified, and different units of magnitude were used (9). The lack of understanding of d-dimer units and assay performance in these investigations causes confusion among physicians and puts patient safety at risk. Table. d-Dimer Assays Used in Clinical Studies of AADD Cutoffs for Pulmonary Embolism Exclusion* The CLSI (guideline H59-A) and FDA requirements provide a detailed discussion of the performance characteristics that must be considered when using a d-dimer assay to exclude VTE, either pulmonary embolism or deep venous thrombosis (4). These requirements for a VTE cutoff are very strict, and any modification to the FDA-approved or -cleared cutoff value, as in the AADD cutoff recommendation, requires a laboratory or assay manufacturer to carry out the appropriate investigation. In light of the current regulatory environment, defined strategies for implementing the AADD cutoffs are not available, and if a laboratory adopts or reports AADD cutoffs on an FDA-approved or -cleared d-dimer assay, it must demonstrate assay-specific evidence of clinical performance characteristics. If literature is not available to support AADD cutoffs for a specific assay, the laboratory must perform a validation that exceeds the resources of most institutions. On the basis of our earlier discussion, we propose a short-term recommendation and a long-term strategy to help address these issues. First, we recommend clarification of the d-dimer unit type (that is, FEU or DDU) of the AADD cutoffs proposed by the ACP best-practice advice. As for the short-term strategy, for clinical laboratories interested in implementing d-dimer AADD cutoffs, we recommend that they consider only specific d-dimer assays adequately evaluated in clinical studies based on the CLSI guidelines (4). The most widely studied d-dimer assays for AADD cutoffs are listed in the Table. Each kit was examined in several studies, which combined had a total of more than 300 low- and intermediate-risk patients and provided substantial evidence that these assays have a negative predictive value of 98% or greater if the AADD cutoffs are used. Clinical laboratories should communicate with physicians regarding d-dimer assay performance, units of magnitude and type, and specific literature supporting the validity of AADD cutoffs for their d-dimer assay. As for quantitative d-dimer kits without substantial data, their validity and safety in the context of AADD cutoff implementation are uncertain. The long-term strategy is to harmonize d-dimer assays and reporting by improving assay performance and unifying reporting units. The ACP, CAP, and International Society on Thrombosis and Haemostasis should collectively advocate that the FDA and manufacturers select a uniform unit type and magnitude for reporting d-dimer. Additional d-dimer assay comparison studies in patients with pulmonary embolism are necessary to provide clinical validation data and support AADD cutoff implementation for more commercial d-dimer kits. Finally, the current vendors of d-dimer assays are urged to perform clinical validation studies and obtain FDA clearance for AADD for their existing d-dimer kits. This approach will significantly facilitate widespread implementation of AADD cutoffs in clinical practice. In conclusion, the current state of d-dimer testing and reporting is far from standardized and creates significant challenges in safely implementing AADD cutoffs. Although several d-dimer assays have data to support AADD cutoffs to exclude pulmonary embolism, assays that have not been sufficiently investigated should not be used with AADD cutoffs. Collaboration among professional organizations, regulatory bodies, and manufacturers may be necessary to improve the overall safety of AADD testing to exclude pulmonary embolism.