To normalize or not?: Dilute Russell viper venom time testing.

OBJECTIVES: We conducted a comparison between the nonnormalized dilute Russell viper venom time (dRVVT) screen/confirm ratio (SCR) in patient plasma and the normalized SCR obtained using reference pooled plasma. The aim was to assess the impact of normalization on the lupus anticoagulant (LA) status in our patient population. METHODS: In our retrospective analysis, we included a total of 464 patients who underwent dRVVT testing. For those with positive screens, mixing studies were performed, followed by confirmatory testing. Additionally, the dRVVT of reference pooled plasma was measured. A positive conventional (nonnormalized) or normalized SCR was defined as an SCR greater than or equal to 1.2. RESULTS: In total, 5.6% (26) of the 464 samples tested were confirmed positive for LA by both methods, out of which 12 had a clinical history of thrombosis. Although a statistically significant difference between the 2 groups (P = .0096) was found, the magnitude of absolute mean SCR differences (bias) was 0.04 (2.51%). There was 100% concordance of testing results between the 2 groups. CONCLUSIONS: The lupus anticoagulant status by the dRVVT assay was not changed based on normalization. Normalization was of no clinical benefit in our patient population; therefore, there was no need for the extra calculation step. Normalization may be useful for intermethod and interlaboratory studies and not for within-method LA detection.

Comparative analysis of platelet depleted plasma prepared on the Roche 8100 automation line and manually centrifuged platelet poor plasma for routine coagulation assays.

Objectives: To evaluate whether the routine coagulation tests can be performed using platelet depleted plasma (PDP, residual platelet count <40000/µL) to achieve maximum efficiency of the automated workflow and compare results of these tests performed with platelet poor plasma (PPP residual platelet count <10,000/µL) prepared manually 'offline'. Design and Methods: The PDP was obtained first following 'on line' centrifugation at 4150 RPM (3000g) for 7 min. The routine coagulation tests, Prothrombin Time (PT), Activated Partial Thromboplastin Clotting Time (aPTT), D-dimer (DD), Antithrombin III (AT3) and Fibrinogen (FBG) were performed. The PPP was obtained from an aliquot of PDP samples with additional 'manual off line' centrifugation at 7700 RPM (3314g) for 3 min (total 10 min, online + offline) and the same tests were performed. The statistical analysis was carried out using EP Evaluator v11 to compare results from both methods. Results: The results from both PPP and PDP samples demonstrated strong correlation. For example, PT (R = 0.9989; N = 55, and of Bias -0.12 (-0.67%), aPTT(R = 0.9957; N = 60, Bias 0.26 (0.58%)), AT3(R = 0.9800; N = 49, Bias -2.0 (-2.2%)), FBG (R = 0.9956; N = 57, Bias -1.9 (-0.5%)) and DD (R = 0.9981; N = 38, Bias 0.005 (0.373%)) with insignificant bias. Conclusions: The utilization of the Roche cobas® 8100 automated 'online' centrifugation helps achieve optimal workflow efficiency without impacting analytical performance of the PT, aPTT, DD, AT3 and FBG assays. The use of PDP can be superior method to PPP for routine coagulation tests.

AACC Guidance Document on Cervical Cancer Detection: Screening, Surveillance, and Diagnosis.

BACKGROUND: Persistent genital infection with high-risk human papilloma virus (hrHPV) causes the vast majority of cases of cervical cancer. Early screening, ongoing surveillance, and accurate diagnosis are crucial for the elimination of cervical cancer. New screening guidelines for testing in asymptomatic healthy populations and management guidelines for managing abnormal results have been published by professional organizations. CONTENT: This guidance document addresses key questions related to cervical cancer screening and management including currently available cervical cancer screening tests and the testing strategies for cervical cancer screening. This guidance document introduces the most recently updated screening guidelines regarding age to start screening, age to stop screening, and frequencies of routine screening as well as risk-based management guidelines for screening and surveillance. This guidance document also summarizes the methodologies for the diagnosis of cervical cancer. Additionally, we propose a report template for human papilloma virus (HPV) and cervical cancer detection to facilitate interpretation of results and clinical decision-making. SUMMARY: Currently available cervical cancer screening tests include hrHPV testing and cervical cytology screening. The screening strategies can be primary HPV screening, co-testing with HPV testing and cervical cytology, and cervical cytology alone. The new American Society for Colposcopy and Cervical Pathology guidelines recommend variable frequencies of screening and surveillance based on risk. To implement these guidelines, an ideal laboratory report should include the indication for the test (screening, surveillance, or diagnostic workup of symptomatic patients); type of test (primary HPV screening, co-testing, or cytology alone); clinical history of the patient; and prior as well as current testing results.

Assessment of serum total 25-hydroxyvitamin D assays for Vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.

Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 1 liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays - impact of 3-epi-25-hydroxyvitamin D3 on assay performance.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25-hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3). A set of 50 single-donor samples was assigned target values for concentrations of 25(OH)D2, 25(OH)D3, 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3), and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) using isotope dilution liquid chromatography - tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 1 includes results from 14 laboratories using 14 custom LC-MS/MS assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 53% of the LC-MS/MS assays met the VDSP criterion of mean % bias ≤ |±5%|. For the LC-MS/MS assays not meeting the ≤ |±5%| criterion, four assays had mean % bias of between 12 and 21%. Based on multivariable regression analysis using the concentrations of the four individual vitamin D metabolites in the 50 single-donor samples, the performance of several LC-MS/MS assays was found to be influenced by the presence of 3-epi-25(OH)D3. The results of this interlaboratory study represent the most comprehensive comparison of LC-MS/MS assay performance for serum total 25(OH)D and document the significant impact of the lack of separation of 3-epi-25(OH)D3 and 25(OH)D3 on assay performance, particularly with regard to mean % bias.

Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and Vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2.

An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials® (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] using reference measurement procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D (i.e., the sum of 25(OH)D2 and 25(OH)D3) determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D2 below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D2 were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (level 4), which has high exogenous concentration of 3-epi-25(OH)D3, was deemed non-commutable for 50% of the LC-MS/MS assays.

A smartphone-based chloridometer for point-of-care diagnostics of cystic fibrosis.

Chloride in sweat is an important diagnostic marker for cystic fibrosis (CF), but the implementation of point-of-care systems for diagnosis is hindered by the prohibitive costs of existing chloride sensors. To enable low cost diagnostic solutions, we recently established a citrate-derived synthesis platform for the development of new fluorescence sensors with high selectivity for chloride. As a next step, we herein designed a smartphone operated chloridometer that optimizes the analytical performance of the citrate-derived sensor materials for the detection of chloride in sweat. The sensor material demonstrated a wide linear range of 0.8-200mM chloride and a diffusion-limited response time; sweat chloride levels corresponded to measurable changes in fluorescence emission that was captured by a smartphone. Clinical validation was performed with sweat from individuals with and without CF, demonstrating convenient sweat diagnostics with reliable detection of cystic fibrosis. To our knowledge, this is the first clinical study of a smartphone-based chloride sensor, paving the way for point-of-care diagnostic systems for CF.

Citrate-based fluorescent materials for low-cost chloride sensing in the diagnosis of Cystic Fibrosis.

Chloride is an essential electrolyte that maintains homeostasis within the body, where abnormal chloride levels in biological fluids may indicate various diseases such as Cystic Fibrosis. However, current analytical solutions for chloride detection fail to meet the clinical needs of both high performance and low material or labor costs, hindering translation into clinical settings. Here we present a new class of fluorescence chloride sensors derived from a facile citrate -based synthesis platform that utilize dynamic quenching mechanisms. Based on this low-cost platform, we demonstrate for the first time a selective sensing strategy that uses a single fluorophore to detect multiple halides simultaneously, promising both selectivity and automation to improve performance and reduce labor costs. We also demonstrate the clinical utility of citrate-based sensors as a new sweat chloride test method for the diagnosis of Cystic Fibrosis by performing analytical validation with sweat controls and clinical validation with sweat from individuals with or without Cystic Fibrosis. Lastly, molecular modeling studies reveal the structural mechanism behind chloride sensing, serving to expand this class of fluorescence sensors with improved chloride sensitivities. Thus citrate-based fluorescent materials may enable low-cost, automated multi-analysis systems for simpler, yet accurate, point-of-care diagnostics that can be readily translated into clinical settings. More broadly, a wide range of medical, industrial, and environmental applications can be achieved with such a facile synthesis platform, demonstrated in our citrate-based biodegradable polymers with intrinsic fluorescence sensing.

Clinical Implications of CD30 Expression in Aggressive B-Cell Lymphomas.

BACKGROUND: US Food and Drug Administration approval of brentuximab vedotin for treatment of CD30-positive relapsed/refractory lymphomas, including classical Hodgkin lymphoma and anaplastic large cell lymphoma, initiated significant interest in researching CD30 expression in other therapy-resistant or relapsed lymphomas. We evaluated CD30 expression in 116 cases of aggressive B-cell lymphomas diagnosed at Penn State Milton S. Hershey Medical Center between 2000 and 2012 with the purpose of assessing the benefit of treatment with brentuximab. PATIENTS AND METHODS: We studied CD30 expression in types of aggressive B-cell lymphomas not previously studied, including Burkitt lymphoma, high-grade (grade III) follicular lymphoma, mixed grade III follicular lymphoma/diffuse large B-cell lymphoma (DLBCL), posttransplantation lymphoproliferative disease large B-cell lymphoma, and primary mediastinal large B-cell lymphoma. RESULTS: CD30 expression was found in 37.5% of DLBCL and 46.2% of other non-DLBCL aggressive B-cell lymphomas. CONCLUSION: Expression of CD30 in patients with both DLBCL and other aggressive B-cell lymphomas and the absence of MYC oncogene-driven proliferation in the majority of these tumors suggests that brentuximab may be a particularly effective form of targeted therapy in the subset of patients with high CD30 expression.

Estimation of Daily Proteinuria in Patients with Amyloidosis by Using the Protein-To-Creatinine ratio in Random Urine Samples.

Measurement of daily proteinuria in patients with amyloidosis is recommended at the time of diagnosis for assessing renal involvement, and for monitoring disease activity. Renal involvement is usually defined by proteinuria >500 mg/day. We evaluated the accuracy of the random urine protein-to-creatinine ratio (Pr/Cr) in predicting 24 hour proteinuria in patient with amyloidosis. We compared results of random urine Pr/Cr ratio and concomitant 24-hour urine collections in 44 patients with amyloidosis. We found a strong correlation (Spearman's ρ=0.874) between the Pr/Cr ratio and the 24 hour urine protein excretion. For predicting renal involvement, the optimal cut-off point of the Pr/Cr ratio was 715 mg/g. The sensitivity and specificity for this point were 91.8% and 95.5%, respectively, and the area under the curve value was 97.4%. We conclude that the random urine Pr/Cr ratio could be useful in the screening of renal involvement in patients with amyloidosis. If validated in a prospective study, the random urine Pr/Cr ratio could replace the 24 hour urine collection for the assessment of daily proteinuria and presence of nephrotic syndrome in patients with amyloidosis.

Current role of radiation therapy for multiple myeloma.

BACKGROUND: Radiation therapy (RT) is a treatment modality traditionally used in patients with multiple myeloma (MM), but little is known regarding the role and effectiveness of RT in the era of novel agents, i.e., immunomodulatory drugs and proteasome inhibitors. METHODS: We retrospectively reviewed data from 449 consecutive MM patients seen at our institute in 2010-2012 to assess indications for RT as well as its effectiveness. Pain response was scored similarly to RTOG 0631 and used the Numerical Rating Pain Scale. RESULTS: Among 442 evaluable patients, 149 (34%) patients and 262 sites received RT. The most common indication for RT was palliation of bone pain (n = 109, 42%), followed by prevention/treatment of pathological fractures (n = 73, 28%), spinal cord compression (n = 26, 10%), and involvement of vital organs/extramedullary disease (n = 25, 10%). Of the 55 patients evaluable for pain relief, complete and partial responses were obtained in 76.4 and 7.2%, respectively. Prior RT did not significantly decrease the median number of peripheral blood stem cells collected for autologous transplant, even when prior RT was given to both the spine and pelvis. Inadequacy of stem cell collection for autologous stem cell transplant (ASCT) was not significantly different and it occurred in 9 and 15% of patients receiving no RT and spine/pelvic RT, respectively. None of the three cases of therapy-induced acute myelogenous leukemia/MDS occurred in the RT group. CONCLUSION: Despite the introduction of novel effective agents in the treatment of MM, RT remains a major therapeutic component for the management in 34% of patients, and it effectively provides pain relief while not interfering with successful peripheral blood stem cell collection for ASCT.

Heparin-induced thrombocytopenia and cerebral venous sinus thrombosis: case report and literature review.

BACKGROUND: Heparin-induced thrombocytopenia (HIT)-related cerebral venous sinus thrombosis (CVST) has been described in 10 prior case reports in the English language medical literature. We report the first case of low molecular weight HIT-related CVST with detailed clinical course and novel therapeutic approach. METHODS: A 69-year-old woman presented with a focal seizure after total hip replacement. Enoxaparin for venous thromboembolism prophylaxis had been initiated 8 days prior to the seizure. RESULTS: The patient experienced progressive neurologic deterioration, and MRI and CT angiography were consistent with cerebral sinus thrombosis (CVST). The new onset of thrombocytopenia, thrombosis, and positive heparin ELISA (enzyme-linked immunosorbent assay) and SRA (serotonin release assay) assays confirmed HIT. In spite of aggressive management of HIT-related CVST, including argatroban therapy and endovascular mechanical thrombolysis, the patient expired. CONCLUSIONS: A review of the previous 10 case reports in the literature confirms that HIT-related CVST is often a fatal condition, particularly when diagnosed in comatose patients. Because the diagnosis is rare and often delayed relative to initial presentation, prevention is the key to improve patient outcomes. Newer anticoagulants with different mechanism of action than heparin are currently under review by the FDA; they will facilitate prevention of HIT-related CVST and other HIT-related neurological complications.

Human cord blood progenitors with high aldehyde dehydrogenase activity improve vascular density in a model of acute myocardial infarction.

UNLABELLED: Human stem cells from adult sources have been shown to contribute to the regeneration of muscle, liver, heart, and vasculature. The mechanisms by which this is accomplished are, however, still not well understood. We tested the engraftment and regenerative potential of human umbilical cord blood-derived ALDH(hi)Lin(-), and ALDH(lo)Lin(-) cells following transplantation to NOD/SCID or NOD/SCID beta2m null mice with experimentally induced acute myocardial infarction. We used combined nanoparticle labeling and whole organ fluorescent imaging to detect human cells in multiple organs 48 hours post transplantation. Engraftment and regenerative effects of cell treatment were assessed four weeks post transplantation. We found that ALDH(hi)Lin(-) stem cells specifically located to the site of injury 48 hours post transplantation and engrafted the infarcted heart at higher frequencies than ALDH(lo)Lin(-) committed progenitor cells four weeks post transplantation. We found no donor derived cardiomyocytes and few endothelial cells of donor origin. Cell treatment was not associated with any detectable functional improvement at the four week endpoint. There was, however, a significant increase in vascular density in the central infarct zone of ALDH(hi)Lin(-) cell-treated mice, as compared to PBS and ALDH(lo)Lin(-) cell-treated mice. CONCLUSIONS: Our data indicate that adult human stem cells do not become a significant part of the regenerating tissue, but rapidly home to and persist only temporarily at the site of hypoxic injury to exert trophic effects on tissue repair thereby enhancing vascular recovery.

Factors influencing cord blood viability assessment before cryopreservation.

BACKGROUND: Cord blood (CB) viability determines product quality and varies with time and temperature of exposure before cryopreservation. Global viability assessment may not reflect viability of white blood cell (WBC) subsets, CD34+ cell viability, or hematopoietic stem/progenitor cells function. STUDY DESIGN AND METHODS: We compared trypan blue (TB) and acridine orange/propidium iodide (AO/PI) staining with flow-cytometric (7-aminoactinomycin D [7-AAD]) viability in total WBCs (Tot-AAD), granulocytes, monocytes, lymphocytes, and CD34+ cells and total nucleated cell, CD34+, and colony-forming cell (CFC) recovery as a function of time and temperature (4, 24, and 37 degrees C) before cryopreservation. RESULTS: TB, AO/PI, and Tot-AAD viability was concordant up to 72 hours (4 degrees C) and 48 hours (24 degrees C) postcollection; however, CD34+ viability was significantly higher due to loss of viable granulocytes. In contrast, at "physiologic" temperature (37 degrees C), the decline in TB, AO/PI, and Tot-AAD viability was significantly lower than the rate of viable CD34+ and CFC loss. At all times and temperatures, CFC recovery correlated best with CD34+ viability and recovery. CONCLUSIONS: CB cell populations exhibit differential time- and temperature-dependent susceptibility to in vitro cell death; consequently, global viability measurements using TB, AO/PI, or 7-AAD (Tot-AAD) significantly underestimate (4-24 degrees C) or overestimate (24-37 degrees C) CD34+ viability and CFC recovery. Our results demonstrate the limitations of global viability assessment with TB, AO/PI, and total AAD; endorse the routine use of CD34+ cell viability measurements; emphasize the importance of temperature control during shipment; and have implications with regard to establishing acceptable "cutoff" values for viability measurements and CB collection through processing time.

Polymorphonuclear leukocytes isolated from umbilical cord blood as a useful research tool to study adherence to cell monolayers.

One of the initial steps in the inflammatory process involves the adherence and transmigration of circulating polymorphonuclear leukocytes (PMN) across the endothelial cell monolayer. One of the main constituents of the neutrophil phagosome that contributes to bacterial killing is myeloperoxidase (MPO) which can be measured spectrophotometrically, using hydrogen peroxide as a substrate, and hence can be used as an index to quantify neutrophil adherence. To evaluate whether PMN isolated from umbilical cord blood could be used for in vitro experiments to monitor neutrophil adherence, we compared the adherence to confluent endothelial and epithelial cell monolayers using PMN isolated from umbilical cord and adult peripheral blood. The extent of PMN adherence was assessed by measuring MPO activity. In initial experiments, we isolated PMN from umbilical cord and adult peripheral blood and measured MPO activity with respect to cell number and assay incubation times. Our data demonstrate that PMN obtained from either source had similar MPO activity and similar adherence to endothelial or epithelial cells. In conclusion, our data suggest that umbilical cord blood is a suitable source of leukocytes to examine PMN adherence in the setting of inflammation in a variety of disease processes.

Building an immune-mediated coagulopathy consensus: early recognition and evaluation to enhance post-surgical patient safety.

Topical hemostats, fibrin sealants, and surgical adhesives are regularly used in a variety of surgical procedures involving multiple disciplines. Generally, these adjuncts to surgical hemostasis are valuable means for improving wound visualization, reducing blood loss or adding tissue adherence; however, some of these agents are responsible for under-recognized adverse reactions and outcomes. Bovine thrombin, for example, is a topical hemostat with a long history of clinical application that is widely used alone or in combination with other hemostatic agents. Hematologists and coagulation experts are aware that these agents can lead to development of an immune-mediated coagulopathy (IMC). A paucity of data on the incidence of IMC contributes to under-recognition and leaves many surgeons unaware that this clinical entity, originating from normal immune responses to foreign antigen exposure, requires enhanced post-operative vigilance and judicious clinical judgment to achieve best outcomes.Postoperative bleeding may result from issues such as loosened ties or clips or the occurrence of a coagulopathy due to hemodilution, vitamin K deficiency, disseminated intravascular coagulation (DIC) or post-transfusion, post-shock coagulopathic states. Other causes, such as liver disease, may be ruled out by a careful patient history and common pre-operative liver function tests. Less common are coagulopathies secondary to pathologic immune responses. Such coagulopathies include those that may result from inherent patient problems such as patients with an immune dysfunction related to systemic lupus erythrematosus (SLE) or lymphoma that can invoke antibodies against native coagulation factors. Medical interventions may also provoke antibody formation in the form of self-directed anti-coagulation factor antibodies, that result in problematic bleeding; it is these iatrogenic post-operative coagulopathies, including those associated with bovine thrombin exposure and its clinical context, that this panel was convened to address.The RETACC panel's goal was to attain a logical consensus by reviewing the scientific evidence surrounding IMC and to make recommendations for the clinical recognition, diagnosis and evaluation, and clinical management of these complications. In light of the under-recognition and under-reporting of IMC, and given the associated morbidity, utilization of health care resources, and potential economic impact to hospitals, the panel engaged in a detailed review of peer-reviewed reports of bovine thrombin associated IMC. From that clinical knowledge base, recommendations were developed to guide clinicians in the recognition, diagnosis, and management of this challenging condition.