Initial flow cytometric evaluation of the Clearllab lymphoid screen.

INTRODUCTION: Flow cytometric immunophenotyping (FCI) is an integral part in the diagnosis and classification of hematologic malignancies. FCI results also influence therapeutic decisions and disease prognosis. ClearLLab LS is a 12-antibody 10-color cocktail provided in dry format designed as a screen for patients suspected of having hematolymphoid disease. METHODS: A blinded comparison between ClearLLab LS, (CD8-FITC, Kappa-FITC,CD4-PE, Lambda-PE, CD19-ECD, CD56-PE-Cy5.5, CD10-PE-Cy7, CD34-APC, CD5-APC-A700, CD20-APC-A750, CD3-PB, and CD45-KrO), ClearLLab Reagents (five-color, 17-antibodies) and individual Laboratory Developed Tests (LDTs), was conducted at four laboratories. Evaluation of ClearLLab LS was performed on 210 specimens, compared to the five-color ClearLLab Reagents (IVD and CE-IVD), and a subset (n = 167) to LDTs. RESULTS: ClearLLab LS showed good agreement to ClearLLab Reagents in detecting the absence (104/104) or presence (106/106) of abnormal populations. Of specimens with abnormal populations the ClearLLab LS agreed with the ClearLLab Reagent for neoplasm maturity assessment (70/70 mature and 36/36 immature). Out of 167 specimens with LDTs results, 86 contained abnormal population(s), ClearLLab LS detected 82 (95.3%) of cases. Of the 4 cases not detected by ClearLLab LS, 3 were plasma cell neoplasms and 1 was a mature T cell malignancy. Eighty-one samples with no hematological malignancy as analyzed by LDT were also negative by ClearLLab LS (100% agreement). ClearLLab LS agreed with LDTs assessment of neoplasms' maturity (55/55 mature and 27/27 immature). CONCLUSION: ClearLLab LS screening tube showed excellent agreement between ClearLLab Reagents and with LDT's. The presence of CD34 and CD10 in the tube allowed the detection of blast populations in several acute leukemias and myeloid neoplasms that were tested. © 2017 International Clinical Cytometry Society.

Flow cytometry-Recognizing unusual populations in leukemia and lymphoma diagnosis.

Flow cytometry is an invaluable technology in the examination of blood, bone marrow, tissue and body fluids for the presence or absence of hematological disease. It is used in both diagnostic and follow-up testing, with an increasingly important role in the detection of very small residual disease populations (Minimal Residual Disease, MRD) However, flow cytometry immunophenotyping of leukemia and lymphoma is highly dependent on interpretation of results and with the increased complexity of 8-10 color instruments routinely used in clinical laboratories, knowledge of disease-defining populations is increasingly important as is recognizing normal and reactive patterns. This manuscript presents case studies with flow cytometric patterns encountered in routine screening of samples sent for leukemia and lymphoma immunophenotyping, focusing mainly on B-cell disorders which may be missed or incorrectly interpreted by the laboratory (including a hematopathologist) performing the test. Case studies are used to illustrate our laboratory's standardized approach to the interpretation of flow cytometric data. In addition to a standardized approach, these cases emphasize the importance of interpretative skills of technologist and hematopathologists in recognizing abnormal patterns in detecting hematological malignancies.

Adenoid cystic carcinoma of the lacrimal gland is frequently characterized by MYB rearrangement.

PurposeAdenoid cystic carcinoma (ACC) represents ~10-15% of salivary neoplasms and almost universally exhibits a lethal clinical course. ACC is also known to occur in the lacrimal gland. ACC is characterized by its heterogeneous morphology and may demonstrate tubular, cribriform, and/or solid architectural patterns. Unfortunately, these histopathological features are not specific to ACC and can be seen in other salivary gland-type neoplasms, introducing a diagnostic dilemma. The discovery of fusion transcripts has revolutionized the diagnosis, surveillance, and treatment of epithelial malignancies. In several anatomic subsites ACC is frequently characterized by a fusion transcript involving genes MYB and NFIB; more specifically, t(6;9)(q22-23;p23-24). This study explores the incidence of MYB rearrangement in cases of lacrimal gland ACC using fluorescent in situ hybridization.Materials and methodsRetrospective clinical and histopathological review of 12 cases of lacrimal gland ACC seen at Mayo Clinic over a 25-year period (1990-2015) was performed. Demographic and clinical data were obtained from medical records. Surgical pathology archival material including H&E slides and immunostains was re-examined. Formalin-fixed paraffin-embedded material was further evaluated using immunohistochemistry when appropriate. Fluorescent in situ hybridization (FISH) using a MYB break-apart probe was applied to all histologically confirmed cases of ACC and benign salivary gland parenchyma.ResultsThe median patient age was 53.6 years (range 12-64) and distributed equally by gender (six male and six female). Rearrangement of MYB was identified using FISH in seven cases (58%). Twenty-five sections of benign salivary gland parenchyma showed no evidence of MYB rearrangement. Primary surgical resection was most common treatment, and 78% of the patient received adjuvant radiation therapy. Median overall survival (OS) was 11 years. Rearrangement of MYB did not affect OS.ConclusionsIn summary, our results indicate that the MYB rearrangement defines a significant subset of lacrimal gland ACCs. Importantly, FISH for MYB rearrangement may be used as a diagnostic tool during pathological examination of lacrimal gland neoplasms. Our results showed no relationship between rearrangement status and clinical outcome. Lastly, the presence of t(6;9) in ACC may provide a platform for molecular-targeting strategies in the future.

Nanocoating for biomolecule delivery using layer-by-layer self-assembly.

Since its introduction in the early 1990s, layer-by-layer (LbL) self-assembly of films has been widely used in the fields of nanoelectronics, optics, sensors, surface coatings, and controlled drug delivery. The growth of this industry is propelled by the ease of film manufacture, low cost, mild assembly conditions, precise control of coating thickness, and versatility of coating materials. Despite the wealth of research on LbL for biomolecule delivery, clinical translation has been limited and slow. This review provides an overview of methods and mechanisms of loading biomolecules within LbL films and achieving controlled release. In particular, this review highlights recent advances in the development of LbL coatings for the delivery of different types of biomolecules including proteins, polypeptides, DNA, particles and viruses. To address the need for co-delivery of multiple types of biomolecules at different timing, we also review recent advances in incorporating compartmentalization into LbL assembly. Existing obstacles to clinical translation of LbL technologies and enabling technologies for future directions are also discussed.

Novel biological strategies for treatment of wear particle-induced periprosthetic osteolysis of orthopaedic implants for joint replacement.

Wear particles and by-products from joint replacements and other orthopaedic implants may result in a local chronic inflammatory and foreign body reaction. This may lead to persistent synovitis resulting in joint pain and swelling, periprosthetic osteolysis, implant loosening and pathologic fracture. Strategies to modulate the adverse effects of wear debris may improve the function and longevity of joint replacements and other orthopaedic implants, potentially delaying or avoiding complex revision surgical procedures. Three novel biological strategies to mitigate the chronic inflammatory reaction to orthopaedic wear particles are reported. These include (i) interference with systemic macrophage trafficking to the local implant site, (ii) modulation of macrophages from an M1 (pro-inflammatory) to an M2 (anti-inflammatory, pro-tissue healing) phenotype in the periprosthetic tissues, and (iii) local inhibition of the transcription factor nuclear factor kappa B (NF-κB) by delivery of an NF-κB decoy oligodeoxynucleotide, thereby interfering with the production of pro-inflammatory mediators. These three approaches have been shown to be viable strategies for mitigating the undesirable effects of wear particles in preclinical studies. Targeted local delivery of specific biologics may potentially extend the lifetime of orthopaedic implants.

Initial performance evaluation of the UniCel® DxH slide maker/stainer Coulter® cellular analysis system.

INTRODUCTION: Despite the advancements in instrumentation within hematology laboratories, there is still a need for review of a peripheral blood film (PBF). For a thorough PBF evaluation, it is critical that a well spread and stained film is available. METHODS: In this study, we evaluated an automatic slide maker/stainer (DxH-SMS, Beckman Coulter) compared with manually prepared blood films on 124 normal and abnormal samples. The primary goal of the study was to determine whether or not the DxH-SMS was able to consistently and reproducibly prepare and stain blood films of exemplary quality, without carryover between specimens. Additionally, repeatability of white blood cell distribution, comparability of morphology to reference methodologies, and grading of acceptance criteria outlined in the CLSI document H20-A2 were assessed. RESULTS: Carryover was not an issue and repeatability was within expected limits. There was excellent agreement of the 5-part differential between the automated blood films made by the DxH-SMS compared with the manually prepared reference blood film. There was no difference in identification and enumeration of blasts, variant lymphocytes, or nucleated red blood cells (P < 0.05). Red cell morphology showed excellent agreement. CONCLUSION: Blood films prepared by the DxH-SMS are of excellent quality, reproducible, and compare well with manually prepared slides. Introduction to our laboratory has improved and standardized slide quality.

Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection.

BACKGROUND: Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH. METHODS: CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15, and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages. RESULTS: Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R(2) >0.99) with predicate values over a range (0.06%-99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples. CONCLUSIONS: While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs.

Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection.

Background: Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH. Methods: CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15 and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages. Results: Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R2 >0.99) with predicate values over a range (0.06% - 99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples. Conclusion: While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs. © 2013 Clinical Cytometry Society.

Technical issues: flow cytometry and rare event analysis.

Flow cytometry has become an essential tool for identification and characterization of hematological cancers and now, due to technological improvements, allows the identification and rapid enumeration of small tumor populations that may be present after induction therapy (minimal residual disease, MRD). The quantitation of MRD has been shown to correlate with relapse and survival rates in numerous diseases and in certain cases, and evidence of MRD is used to alter treatment protocols. Recent improvements in hardware allow for high data rate collection. Improved fluorochromes take advantage of violet laser excitation and maximize signal-to-noise ratio allowing the population of interest to be isolated in multiparameter space. This isolation, together with a low background rate, permits for detection of residual tumor populations in a background of normal cells. When counting such rare events, the distribution is governed by Poisson statistics, with precision increasing with higher numbers of cells collected. In several hematological malignancies, identification of populations at frequencies of 0.01% and lower has been attained. The choice of antibodies used in MRD detection facilitates the definition of a fingerprint to identify abnormal populations throughout treatment. Tumor populations can change phenotype, and an approach that relies on 'different from normal' has proven useful, particularly in the acute leukemias. Flow cytometry can and is used for detection of MRD in many hematological diseases; however, standardized approaches for specific diseases must be developed to ensure precise identification and enumeration that may alter the course of patient treatment.

Enhanced flagging and improved clinical sensitivity on the new DxH 300(TM) Coulter(®) cellular analysis system.

INTRODUCTION: The Beckman Coulter DxH 300™ is a hematology analyzer that performs a CBC and 3-part WBC differential and incorporates new electronic, algorithm and mechanical design. METHODS: This instrument was compared with the predicate analyzer (Coulter(®) A(c) ·Tdiff2) and the larger format Coulter LH780 analyzer. Of interest were flagging rates, clinical sensitivity, and accuracy of the WBC in the presence of interfering particles. The total sample set (n = 404) consisted of morphologically normal and hematologically abnormal patients. RESULTS: Correlation of the DxH 300 with A(c) ·Tdiff2™ showed good agreement with all directly measured parameters. When compared with the LH780, WBC and platelets (PLT) counts showed good agreement with small biases. Importantly in the low range (PLT <50 × 10(9) /L), there was a small positive bias of only 2 × 10(9) /L PLT. Interfering particles did not affect the DxH 300 WBC count (P > 0.05) with strong correlations to the LH780 (r(2) values >0.95). Importantly, overall and specific flagging rates as well as false-negative and false-positive rates were significantly reduced on the DxH 300 compared with the A(c) ·Tdiff2 (27% and 41% reduction, respectively, P < 0.001). CONCLUSIONS: The DxH 300 offers significant improvement over the predicate Coulter analyzer in flagging rates and improved correlation with larger format analyzers for WBC and PLT counts. Reduced false negatives and false positives significantly improved sensitivity and specificity compared to the predicate analyzer. The 28% improvement in flagging efficiency together with numerous software and data handling enhancements should translate into reduced need to perform follow-up analysis on a significant number of samples.

Preferential cell response to anisotropic electro-spun fibrous scaffolds under tension-free conditions.

Anisotropic alignment of collagen fibres in musculoskeletal tissues is responsible for the resistance to mechanical loading, whilst in cornea is responsible for transparency. Herein, we evaluated the response of tenocytes, osteoblasts and corneal fibroblasts to the topographies created through electro-spinning and solvent casting. We also evaluated the influence of topography on mechanical properties. At day 14, human osteoblasts seeded on aligned orientated electro-spun mats exhibited the lowest metabolic activity (P < 0.001). At day 5 and at day 7, no significant difference was observed in metabolic activity of human corneal fibroblasts and bovine tenocytes respectively seeded on different scaffold conformations (P > 0.05). Osteoblasts and corneal fibroblasts aligned parallel to the direction of the aligned orientated electro-spun mats, whilst tenocytes aligned perpendicular to the aligned orientated electro-spun mats. Mechanical evaluation demonstrated that aligned orientated electro-spun fibres exhibited significant higher stress at break values than their random aligned counterparts (P < 0.006) and random orientated electro-spun fibres exhibited significant higher strain at break values than the aligned orientated scaffolds (P < 0.006). While maintaining fibre structure, we also developed a co-deposition method of spraying and electro-spinning, which enables the incorporation of microspheres within the three-dimensional structure of the scaffold.

Initial performance evaluation of the UniCel® DxH 800 Coulter® cellular analysis system.

The Beckman Coulter UniCel® DxH 800 is a hematology analyzer incorporating new electronic and mechanical design with advanced algorithm technology to perform CBC, white blood cell (WBC) differential, nucleated red blood cell (NRBC), and reticulocyte analysis. Evaluation of this instrument was performed in our 800-bed tertiary care hospital and specifically centered upon the correlation of WBC, NRBC, and platelet (PLT) enumeration when compared to a predicate analyzer, the Coulter® LH 780, and flow cytometry (FCM) reference methods. Of particular interest were those samples with morphologically confirmed interference and extreme leukocytosis (evaluated with respect to red blood cell parameter correction). The sample set (n=272) consisted of morphologically normal and hematologically abnormal patients. Correlation of the WBC, PLT, and NRBC showed r(2) values of 0.994, 0.985, and 0.910 for the DxH 800 vs. FCM, respectively. The presence of interfering particles did not affect the accuracy of the DxH 800 with respect to WBC counts. The DxH 800 showed accurate PLT and NRBC counts in the clinically significant low range when compared to FCM. Compared to the LH 780, flagging rates were significantly reduced (NRBC flag), or equivalent (WBC, PLT flag) on the DxH 800. The DxH 800 demonstrated higher sensitivity and specificity for PLTs and NRBCs and achieved a lower NRBC false negative rate compared to the LH 780. The UniCel® DxH 800 represents a significant improvement to previous impedance analyzers in accurately detecting the presence of NRBCs at counts >1/100 WBC. Furthermore, it provides accurate PLT and WBC counts in the presence of interference and improved NRBC flagging efficiency when compared to the LH 780. Correction of red blood cell parameters is appropriate and accurate in cases of extreme leukocytosis.

A reliable method for detecting complexed DNA in vitro.

Quantification of eluted nucleic acids is a critical parameter in characterizing biomaterial based gene-delivery systems. The most commonly used method is to assay samples with an intercalating fluorescent dye such as PicoGreen®. However, this technique was developed for unbound DNA and the current trend in gene delivery is to condense DNA with transfection reagents, which interfere with intercalation. Here, for the first time, the DNA was permanently labeled with the fluorescent dye Cy5 prior to complexation, an alternative technique hypothesized to allow quantification of both bound and unbound DNA. A comparison of the two methods was performed by quantifying the elution of six different varieties of DNA complexes from a model biomaterial (collagen) scaffold. After seven days of elution, the PicoGreen® assay only allowed detection of three types of complexes (those formed using Lipofectin™ and two synthesised copolymers). However, the Cy5 fluorescent labeling technique enabled detection of all six varieties including those formed via common transfection agents poly(ethylene imine), poly-L-lysine and SuperFect™. This allowed reliable quantification of the elution of all these complexes from the collagen scaffold. Thus, while intercalating dyes may be effective and reliable for detecting double-stranded, unbound DNA, the technique described in this work allowed reliable quantification of DNA independent of complexation state.

Effect of activated recombinant human factor 7 (Niastase) on laboratory testing of inhibitors of factors VIII and IX.

Activated recombinant human factor VIIa (rFVIIa) has been used as a hemostatic agent in patients with hemophilia and acquired inhibitors. Other indications for rFVIIa may include liver disease, warfarin sodium (Coumadin) overdose, or trauma. Monitoring patients on this treatment with standard laboratory testing is problematic. Bleeding risk does not correlate well with the prothrombin time (PT) or the activated partial thromboplastin time (aPTT) during therapy with rFVIIa. In addition, there is no identifiable literature on the effect of rFVIIa on assays of inhibitors in this patient group. Monitoring inhibitors may be important during interventions aimed at acutely reducing inhibitor levels, such as during plasma exchange or protein adsorption. We performed factor assays and evaluated inhibitor levels in plasma from 3 patients with deficiencies in FVIII (2 patients) or FIX (1 patient) and inhibitors (titer range, 5.8-17.4 Bethesda units) before and after adding rFVIIa (range, 0.25-8 microg/mL) in vitro. Additionally, we performed assays of factors of both intrinsic and extrinsic systems to determine the impact of rFVIIa on these tests. We found that both factor levels and inhibitor titers from patients with hemophilia A or B could be measured accurately, even in the presence of suprapharmacologic doses of rFVIIa (8 microg/mL). We also obtained accurate measurements for other assays of the intrinsic coagulation system (FXI and FXII) based on the aPTT. Conversely, we found that assays of the extrinsic system based on the PT (FII, FV, and FX) produced results that were unreliable. FVII results were very high but reproducible. These results suggest that assays based on the PT are inaccurate and should be avoided during FVIIa treatment. Conversely, FVIII and FIX levels and inhibitor titers can be accurately monitored in hemophilia patients receiving rFVIIa according to results of aPTT-based coagulation tests.

Impact of standardization on clinical cell analysis by flow cytometry.

The evolution of flow cytometry from a research tool to a pivotal technology for clinical diagnostic purposes has required significant efforts to standardize methods. The great advantage of flow cytometry is that it's applications are highly amenable to standardization. Here, we review the efforts that have been made for flow cytometric applications in four major fields of clinical cell analysis: CD4+ T-cell enumeration, CD34+ hematopoietic stem and progenitor cell enumeration, screening for the HLA-B27 antigen and leukemia/lymphoma immunophenotyping. These standardization efforts have been parallelled by the establishment of external quality assessment (EQA) schemes in many countries worldwide. The goal of these EQA exercises has been primarily educa-tional, but their results will increasingly serve as a basis for laboratory accreditation. This important development requires that the EQA schemes, in particular the quality of the distributed samples and the procedures for evaluating the results, meet the highest standards.