Michael Keeney - Academia.edu (original) (raw)

Papers by Michael Keeney

Cytometry. Part B, Clinical cytometry, Jan 8, 2015

Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an in... more Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an invasive procedure. Reticulated platelets (RP) are immature RNA containing platelets, accurate RP enumeration has yet to be achieved, partially due to the lack of a robust reference method. To refine previous work and gating strategies distinguishing RP from mature platelets while incorporating accurate platelet enumeration into the analysis. After reviewing previously published studies on Thiazole Orange (TO) staining of RP, we systematically evaluated CD41/CD61 in combination with a commercial source of TO (BDBiosciences). Previous RP methods have not taken advantage of platelet enumeration therefore our goal was to incorporate the ICSH platelet enumeration protocol into our method. TO concentration, incubation and fixation method were determined to be 10% of stock concentration, 30min, and 1% formaldehyde respectively. Gating strategy to determine RP fraction used an unstained control t...

Methods in Cell Biology, 2011

The majority of cancer-related deaths are as a result of metastatic disease, which has been corre... more The majority of cancer-related deaths are as a result of metastatic disease, which has been correlated with the presence of circulating tumor cells (CTCs) in the bloodstream. Therefore the ability to reliably enumerate and characterize these cells could provide useful information about the biology of the metastatic cascade; facilitate patient prognosis; act as a marker of therapeutic response; and/or aid in novel anticancer drug development. Several different techniques have been utilized for the enrichment and detection of these rare CTCs, each having their own unique advantages and disadvantages. In this chapter we will briefly discuss each of these techniques as well as the pros and cons of each approach. In particular, we will provide a comprehensive examination of two image cytometry approaches for CTC analysis that are in routine use in our laboratory; the iCys Laser Scanning Cytometer (Compucyte, Cambridge, MA), and the CellSearch® system (Veridex, North Raritan, NJ). The ability to detect, enumerate, and characterize CTCs is an important tool for the study of the metastatic cascade and the improved clinical management of cancer patients. These rare cells could shed light on the basic biology behind this highly lethal process and ultimately change current patient treatment guidelines.

Laboratory Hematology, 2004

The challenge for modern hematology laboratories is to provide accurate and reproducible results,... more The challenge for modern hematology laboratories is to provide accurate and reproducible results, with seamless performance between facilities, in a cost-effective manner. Beckman Coulter recently developed the Coulter LH 500 to meet the needs of smaller laboratories or serve as a backup in larger laboratories. The principal goal of this study was to validate all parameters and performance specifications of the LH 500 compared to the Coulter LH 750 predicate analyzer. A total of 245 spent clinical samples from the London Health Sciences Centre (LHSC) and 251 from the University of Pittsburgh Medical Center Health System (UPMCHS) were analyzed during the study. The samples were selected to include 75% abnormal and 25% normal blood samples. According to the results of a rank sum test, there was no significant difference between the LH 500 and LH 750 for all complete blood count parameters (P > .05) except the red cell distribution width, which showed a slight negative bias on the LH 500. Differential parameters comparing the LH 500 to a 400-cell manual differential showed correlation coefficients (r2) from 0.75 to 0.99 for all parameters except basophils. Of the samples run on the LH 500 at LHSC, the false-positive differential flagging rate was 17.32% and the false-negative rate was 3.03%. Sensitivity was 82.93%, specificity 78.95%, and efficiency 79.65%. At UPMCHS, the false-positive differential flagging rate was 13.37% and false-negative rate 2.97%. Sensitivity was 91.89%, specificity 78.91%, and efficiency 83.66%. Overall, the LH 500 performed accurately and reproducibly compared to the LH 750 and the reference procedures. It would be an excellent second instrument for larger laboratories concerned with harmonization of instrumentation and reagents or as a primary instrument for smaller hematology laboratories with limited space.

Cytometry Part A, 2009

The inability to sensitively detect metastatic cells in preclinical models of cancer has created ... more The inability to sensitively detect metastatic cells in preclinical models of cancer has created challenges for studying metastasis in experimental systems. We previously developed a flow cytometry (FCM) method for quantifying circulating tumor cells (CTCs) in mouse models of breast cancer. We have adapted this methodology for analysis of tumor dissemination to bone marrow (BM) and lymph node (LN), and for analysis of these samples by laser scanning cytometry (LSC). Our objective was to implement these methodologies for characterization of tumor cell dissemination in preclinical models of cancer metastasis. Human cancer cells were injected into mice via mammary fat pad (MFP; spontaneous metastasis), tail vein (TV; targets lung), or intracardiac (IC; targets bone) routes. At several time points postinjection (4 h to 8 weeks), mice were sacrificed and blood, LNs, and BM were collected. Samples were immunomagnetically enriched and labeled with human leukocytic antigen-fluorescein isothiocyanate and CD45-PE antibodies (FCM/LSC), and propidium iodide (FCM) prior to quantitative analysis. Following MFP injection, CTCs increased over time, as did disseminated cells to the LN. Interestingly, tumor cells also spontaneously disseminated to BM, peaking at 2 weeks postinjection. Following TV injection, CTCs were initially high but decreased rapidly by 1 week before increasing to peak at endpoint. Combined with an observed concurrent increase in disseminated cells to LN and BM, this suggests that tumor cells may shed into the circulation from lung metastases that establish following initial cell delivery. Following IC injection, CTCs increased over time, peaking at 4 weeks. Tumor cells in the BM (most prevalent site of metastasis after IC injection) remained at moderate levels until peaking at endpoint. Combined use of FCM and LSC allows sensitive quantification of disseminated tumor cells in preclinical models of metastasis. These methods will be valuable for future studies aimed at testing new therapeutics in the metastatic setting. ' 2008 International Society for Advancement of Cytometry Key terms breast cancer; metastasis; preclinical animal models; tumor cell dissemination; flow cytometry; laser scanning cytometry BREAST cancer is a leading cause of morbidity and mortality in women (1,2), primarily because of the failure of effective clinical detection and management of metastatic disease in distant sites such as lymph node (LN), bone, lung, liver, and brain (3,4). The metastatic process is comprised of a series of sequential steps, and cancer cells must successfully complete each step in order to give rise to a metastatic tumor. These steps include dissemination of cancer cells from the primary tumor into the bloodstream (intravasation), survival in the circulation, arrest and extravasation into the secondary site, and initiation and maintenance of growth to form clinically detectable metastases (3-7). Breast cancer cells may also disseminate from the primary tumor through the lymphatic system, although the lack of direct flow from the lymphatic system to other organs means that tumor cells escaping via this route must still enter the vascular system in order to be distributed to distant organs .

Clinical & Experimental Metastasis, 2005

Breast cancer often spreads from the primary tumor to regional lymph nodes. Lymph node status pro... more Breast cancer often spreads from the primary tumor to regional lymph nodes. Lymph node status provides clinically important information for making treatment decisions. Spread via lymphatics is also important for the biology of breast cancer, as tumor cells in lymph nodes may provide a reservoir of cells leading to distant, lethal metastases. Improved understanding of the biology of lymphatic spread thus is important for improved breast cancer survival. Advances towards understanding the interactions between tumors cells and lymphatic vessels have in part been limited by the lack of suitable cell lines and experimental models. We have addressed this need by developing a new model of lymphatic metastasis. Here we describe the establishment of 468LN cells, a variant of the MDA-MB-468 human breast adenocarcinoma cell line, which produces extensive lymph node metastasis following orthotopic injection of nude mice. 468LN cells are also more aggressive in vitro, produce more osteopontin and express different surface integrins compared to the parent line. The dramatic in vitro and in vivo phenotypic and molecular differences of 468LN and parental 468GFP cells make this pair of cell lines a unique model for the specific study of lymph node metastasis of breast cancer.

British Journal of Haematology, 2001

The need for quality control of leucoreduction of blood products has led to the development of va... more The need for quality control of leucoreduction of blood products has led to the development of various methods to count low levels of residual leucocytes. We compared five platforms side-by-side: the Nageotte haemocytometer and four based on fluorescent staining of nuclei: two flowcytometers (Beckman Coulter, BD Biosciences) with methods based on counting beads, a volumetric flow cytometer (Partec) and the microvolumic fluorimeter imagn2000 (BD Biosciences), all according to their manufacturers' recommended methods. Analysis of doublefiltered red cell concentrates (RCCs) and platelet concentrates (PCs), spiked with various numbers of leucocytes, revealed good linearity for all methods over the range of 1´6±32´7 leucocytes/ml, all with r 2 . 0´99. At the rejection level of leucocyte-reduced blood components, i.e. 1 Â 10 6 per unit corresponding with approximately 3´3 leucocytes/ml, the Nageotte haemocytometer had low accuracy (0% for RCCs, 56% for PCs), and was relatively imprecise [coefficient of variance (CV) of 34% and 30% respectively]. The Partec flow cytometer gave good results for RCCs (accuracy 67%, CV 22%), but not for PCs (accuracy 0%, CV 25%). The imagn2000 had an accuracy of 44% for RCCs and 89% for PCs, but the precision was variable (CV 32% for RCCs, 15% for PCs). The best results were obtained with the Beckman Coulter (RCCs: accuracy 86%, CV 13%, PCs: accuracy 67%, CV 16%), and BD Biosciences platforms (RCCs: accuracy 100%, CV 10%; PCs: accuracy 89%, CV 11%). We conclude that, at the rejection level of 1 Â 10 6 leucocytes per unit, the widely used Nageotte haemocytometer performs poorly in terms of inaccuracy and imprecision, and that both counting-bead-based, flow cytometric methods performed best.

American Journal of Clinical Pathology, 2008

External quality assessment (EQA) of D-dimer assays has been limited by a lack of standardized hu... more External quality assessment (EQA) of D-dimer assays has been limited by a lack of standardized human-derived testing material. In 2006 and 2007, the Quality Management Program--Laboratory Services, Toronto, Canada, investigated the use of commercially prepared lyophilized human plasma spiked with human-derived D-dimer components manufactured by Affinity Biologicals, Hamilton, Canada. Four surveys were performed. Participants reported the level or presence of D-dimer using quantitative or qualitative methods. Participants performing quantitative testing provided their unit of measure and reference interval. Results were considered correct if they fell within the range appropriate for each sample (normal/negative or abnormal/positive). Overall, survey results were excellent, with 4.0% (95% confidence interval [CI], 1.3%-9.1%), 0.8% (CI, 0.0%-1.5%), 2.3% (CI, 0.5%-6.6%), and 2.3% (CI, 0.4%-6.6%) of participants reporting an incorrect result in the first, second, third, and fourth surveys, respectively. A commercially prepared D-dimer is a suitable material for EQA testing.

Cytometry Part B: Clinical Cytometry, 2015

We previously developed a 10-color 11-antibody combination including a viability dye, to screen T... more We previously developed a 10-color 11-antibody combination including a viability dye, to screen T-, B-, and natural killer (NK)-cell populations in blood, bone marrow, tissue, and body fluids. Recently, Beckman Coulter has introduced a line of dried reagents that, unlike liquid reagents and cocktails, require no refrigeration, titration, or manipulation before using. We evaluated custom tubes based on our standard lymphocyte screening panel, focusing on comparative analysis, ease of use, and advantages compared with our liquid reagent set. We tested 42 samples from blood (n = 15), bone marrow (n = 17), and tissue (n = 10) with the combination CD4/CD8/KAPPA/LAMBDA/CD19/CD56/CD5/CD20/CD10/CD3/CD45 and a vital dye by both methods and compared positivity and staining intensity for each antigen. Of the 42 samples, 5 were normal samples, 3 were red cell disorders, 20 were B-cell malignancies, 5 T-cell malignancies, 4 myeloid malignancies, and the remaining 5 were other diagnoses. Dried reagents gave equivalent staining intensity results to our standard panel in a variety of sample types, with diagnoses including reactive lymphocytosis, chronic lymphocytic leukemia, and various lymphomas. Our standard panel for evaluation of mature lymphoid malignancies allows rapid assessment of any sample type while providing direct assessment of viability. The dried reagent tube reduces preanalytical work, with simple addition of sample and the viability dye to the tube, saving time, reducing potential errors, and obviating need to titrate and monitor individual antibodies. With a shelf life of at least 12 months, the reagents also offer potential savings in reagent costs by reducing wastage due to expiration or tandem breakdown in standard liquid formulation. © 2015 International Clinical Cytometry Society.

Thrombosis Research, 2015

Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon disorder characterized by hemolysis, thr... more Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon disorder characterized by hemolysis, thrombosis and marrow failure. Whereas venous and arterial thrombosis is a very common symptom of the disease, the frequency of PNH clones in patients with unexplained venous thromboembolism, including deep vein thrombosis and pulmonary embolism, has not been studied. We conducted a cross sectional study evaluating the presence of PNH clones in patients with prevalent venous thromboembolism using a high sensitivity flow cytometry assay for erythrocytes and neutrophils. Among the 388 patients enrolled in the study one patient had a detectable PNH clone of 0.02% in the neutrophil population (0.26%; 95% CI 0.05 to 1.45) and no detectable erythrocyte clone. We conclude that the presence of PNH clones in patients with idiopathic venous thrombosis is rare. Screening for PNH clones among VTE patients might be better reserved for patients with signs of hemolysis.

Cytometry. Part B, Clinical cytometry, Jan 8, 2015

Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an in... more Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an invasive procedure. Reticulated platelets (RP) are immature RNA containing platelets, accurate RP enumeration has yet to be achieved, partially due to the lack of a robust reference method. To refine previous work and gating strategies distinguishing RP from mature platelets while incorporating accurate platelet enumeration into the analysis. After reviewing previously published studies on Thiazole Orange (TO) staining of RP, we systematically evaluated CD41/CD61 in combination with a commercial source of TO (BDBiosciences). Previous RP methods have not taken advantage of platelet enumeration therefore our goal was to incorporate the ICSH platelet enumeration protocol into our method. TO concentration, incubation and fixation method were determined to be 10% of stock concentration, 30min, and 1% formaldehyde respectively. Gating strategy to determine RP fraction used an unstained control t...

Cytometry, Jan 15, 1998

The isotype control has long been considered a useful part of both microscopic and flow cytometri... more The isotype control has long been considered a useful part of both microscopic and flow cytometric immuno- logic assays, and, consequently, is still routinely used in clinical laboratories. In flow cytometry, the isotype control has traditionally been used to distinguish between fluores- cent positive and fluorescent negative cell populations. Additionally, it has been used to estimate the number of cells

Clinical Immunology Newsletter, 1997

T he increased use of peripheral blood stem cells (PBSC) to reconstitute hematopoiesis in both au... more T he increased use of peripheral blood stem cells (PBSC) to reconstitute hematopoiesis in both autoand, more recently, allo-transplant settings has not been associated with a consensus means to assess the engraftment potential of the PBSC product. Since the small population of bone marrow cells bearing the CD34 antigen are responsible for sustained multi-lineage engraftment, flow cytometric quantitation of CD34+ cells should provide a rapid, reliable, and reproducible means to assess the engraftment potential of PBSC collections, To date, however, the lack of a standardized method has led to the generation of widely divergent data. To address this problem, we were asked by the International Society of Hema-totherapy and Graft Engineering (ISHAGE) to develop clinical guidelines for the quantitation of CD34+ cells in peripheral blood based upon a previously published flow cytometric methodology (FCM). We also sought to establish the method's utility on a variety of flow cytometers in clinical laboratories and its reproducibility between transplant centers. Here we describe the basic four-parameter flow protocol adopted by ISHAGE and compare this method with others currently in use. We also describe how this very flexible methodology can be used to analyze the qualitative composition of the CD34+ cell fraction, and, by incorporating fluorescent beads in the analysis, how the absolute CD34+ content of apheresis collections can be determined on a single instrument platform.

Cytometry, 1998

In concert with the International Society of Hematotherapy and Graft Engineering (ISHAGE), we pre... more In concert with the International Society of Hematotherapy and Graft Engineering (ISHAGE), we previously described a set of guidelines for detection of CD34؉ cells based on a four-parameter flow cytometry method (CD45 FITC/CD34 PE staining, side and forward angle light scatter). With this procedure, an absolute CD34؉ count is generated by incorporating the leukocyte count from an automated hematology analyser (two-platform method). In the present study, we modified the basic ISHAGE method with the addition of a known number of Flow-Count fluorospheres. To reduce errors inherent to sample washing/centrifugation, we implemented ammonium chloride lyse, no-wash no-fix sample processing. These modifications convert the basic protocol into a single-platform method to determine the absolute CD34 count directly from a flow cytometer and form the basis of the Stem-Kit from Coulter/Immunotech. A total of 72 samples of peripheral blood, apheresis packs, and cord blood were analysed and compared using the ISHAGE protocol with or without the addition of fluorescent microspheres. Comparison of methods showed a high correlation coefficient (r ‫؍‬ 0.99), with no statistically significant difference or bias between methods (P G 0.05). Linearity of the absolute counting method generated an R 2 value of 1.00 over the range of 0-250/l. Precision of the absolute counting method measured at three concentrations of CD34؉-stabilised KG1a cells (Stem-Trol, COULTER) generated a coefficient of variation (C.V.) ranging from 4% to 9.9%. In a further modification of the single-platform method, the viability dye 7-amino actinomycin D was included and demonstrated that both viable and nonviable CD34؉ cells could be identified and quantitated. Together, these modifications combine the accuracy and sensitivity of the original ISHAGE method with the ability to produce an absolute count of viable CD34؉ cells. It is the accurate determination of this value that is most clinically relevant in the transplant setting. These modifications may improve the interlaboratory reproducibility of CD34 determinations due to the reduction in sample handling and calculation of results. Cytometry (Comm. Clin. Cytometry) 34:61-70, 1998.

Cytometry, 2002

Background: Enumeration of CD4 ؉ and CD8 ؉ T-cell subsets provides relevant information for diagn... more Background: Enumeration of CD4 ؉ and CD8 ؉ T-cell subsets provides relevant information for diagnosis and monitoring of patients with cellular immunodeficiencies. As a result, an external quality assurance scheme was implemented in Belgium, The Netherlands, and Luxemburg in 1995. A workshop was held to train the participants in state-of-the art technology for assessment of absolute T-cell subset counts (i.e., a three or four-color, single-platform assay with lymphocyte gating based on CD45 and sideward light scatter) with the aim to achieve between-site coefficients of variation (CVs) <10% and within-site CVs <5% for >75% of the participants. Methods: Three send-outs of stabilized blood from a healthy donor were distributed to 55 laboratories, each with the request to perform the standard assay on three occasions. For comparison, each laboratory performed its local technique in parallel. Results: With the standard technique, between-site CVs of ϳ8% (CD3 ؉ T cells), ϳ9% (CD4 ؉ T cells), and ϳ10% (CD8 ؉ T cells) were achieved. Within-site CVs were <5% for 82% (CD3 ؉ T cells) and ϳ70% (CD4 ؉ and CD8 ؉ subsets) of the participants. Local techniques yielded between-site CVs of 13%-17% for CD3 ؉ , CD4 ؉ , and CD8 ؉ T cells. Conclusions: The state-of-the-art technology for T-cell subset enumeration was implemented successfully among 55 Belgian-Dutch laboratories and resulted in significant reductions of between-site variation of absolute CD3 ؉ , CD4 ؉ , and CD8 ؉ T-cell counts. Cytometry (Clin. Cytometry) 50:92-101, 2002.

Journal of Visualized Experiments, 2014

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. ... more The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Cytometry, 1998

The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and ... more The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/microl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in the CD34+ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34+ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34+ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies.

Transfusion Medicine and Hemotherapy, 2004

SummaryOver the last few years, the number of clinical applications for autologous and allogeneic... more SummaryOver the last few years, the number of clinical applications for autologous and allogeneic stem cell transplantation has expanded considerably. Concurrently, stem cell collections have been obtained from increasingly diverse sources, including bone marrow, peripheral blood and umbilical cord blood. Various ex vivo manipulations have been developed to process stem cell transplants for specific clinical requirements (e.g. positive selection techniques

Thrombosis Research, 1998

event rate this study does not support the need to monitor anti-Xa levels or adjusting the dose E... more event rate this study does not support the need to monitor anti-Xa levels or adjusting the dose Enoxaparin after joint arthroplasty is effective prophylaxis against venous thromboembolism. This is according to weight.

Thrombosis and Haemostasis, 2005

Many patients with venous thromboembolism are being treated with low molecular weight heparin for... more Many patients with venous thromboembolism are being treated with low molecular weight heparin for extended periods of time. It is not certain if it is necessary to assess anti-Xa levels for extended treatment periods. This study is a prospective assessment of anti-Xa levels in patients on long-term therapy for acute venous thromboembolism who have active cancer. Consecutive consenting patients from one center in a multicenter trial that compared 6 months of low molecular weight heparin with oral anticoagulant therapy were treated with therapeutic doses of dalteparin (200 IU per kilogram) subcutaneously daily. Anti-Xa levels were assessed at the end of weeks 1 and 4,4-6 hours after injection of dalteparin. Patients were followed for bleeding and recurrent venous thromboembolism. There were 24 patients who had anti-Xa levels measured at weeks 1 and 4. Two other patients had week 1 measurements performed but died before the week 4 sample was collected due to their underlying cancer. The mean anti-Xa levels at weeks 1 and 4 were 1.11 and 1.03 anti-Xa units/ml respectively (P=0.13). These results suggest that for patients with active cancer receiving extended duration therapy with low molecular weight heparin (dalteparin) there is no accumulation of anti-Xa effect over the first month of therapy. Monitoring of anti-Xa levels in this situation is usually not required.

Cytometry. Part B, Clinical cytometry, Jan 8, 2015

Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an in... more Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an invasive procedure. Reticulated platelets (RP) are immature RNA containing platelets, accurate RP enumeration has yet to be achieved, partially due to the lack of a robust reference method. To refine previous work and gating strategies distinguishing RP from mature platelets while incorporating accurate platelet enumeration into the analysis. After reviewing previously published studies on Thiazole Orange (TO) staining of RP, we systematically evaluated CD41/CD61 in combination with a commercial source of TO (BDBiosciences). Previous RP methods have not taken advantage of platelet enumeration therefore our goal was to incorporate the ICSH platelet enumeration protocol into our method. TO concentration, incubation and fixation method were determined to be 10% of stock concentration, 30min, and 1% formaldehyde respectively. Gating strategy to determine RP fraction used an unstained control t...

Methods in Cell Biology, 2011

The majority of cancer-related deaths are as a result of metastatic disease, which has been corre... more The majority of cancer-related deaths are as a result of metastatic disease, which has been correlated with the presence of circulating tumor cells (CTCs) in the bloodstream. Therefore the ability to reliably enumerate and characterize these cells could provide useful information about the biology of the metastatic cascade; facilitate patient prognosis; act as a marker of therapeutic response; and/or aid in novel anticancer drug development. Several different techniques have been utilized for the enrichment and detection of these rare CTCs, each having their own unique advantages and disadvantages. In this chapter we will briefly discuss each of these techniques as well as the pros and cons of each approach. In particular, we will provide a comprehensive examination of two image cytometry approaches for CTC analysis that are in routine use in our laboratory; the iCys Laser Scanning Cytometer (Compucyte, Cambridge, MA), and the CellSearch® system (Veridex, North Raritan, NJ). The ability to detect, enumerate, and characterize CTCs is an important tool for the study of the metastatic cascade and the improved clinical management of cancer patients. These rare cells could shed light on the basic biology behind this highly lethal process and ultimately change current patient treatment guidelines.

Laboratory Hematology, 2004

The challenge for modern hematology laboratories is to provide accurate and reproducible results,... more The challenge for modern hematology laboratories is to provide accurate and reproducible results, with seamless performance between facilities, in a cost-effective manner. Beckman Coulter recently developed the Coulter LH 500 to meet the needs of smaller laboratories or serve as a backup in larger laboratories. The principal goal of this study was to validate all parameters and performance specifications of the LH 500 compared to the Coulter LH 750 predicate analyzer. A total of 245 spent clinical samples from the London Health Sciences Centre (LHSC) and 251 from the University of Pittsburgh Medical Center Health System (UPMCHS) were analyzed during the study. The samples were selected to include 75% abnormal and 25% normal blood samples. According to the results of a rank sum test, there was no significant difference between the LH 500 and LH 750 for all complete blood count parameters (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; .05) except the red cell distribution width, which showed a slight negative bias on the LH 500. Differential parameters comparing the LH 500 to a 400-cell manual differential showed correlation coefficients (r2) from 0.75 to 0.99 for all parameters except basophils. Of the samples run on the LH 500 at LHSC, the false-positive differential flagging rate was 17.32% and the false-negative rate was 3.03%. Sensitivity was 82.93%, specificity 78.95%, and efficiency 79.65%. At UPMCHS, the false-positive differential flagging rate was 13.37% and false-negative rate 2.97%. Sensitivity was 91.89%, specificity 78.91%, and efficiency 83.66%. Overall, the LH 500 performed accurately and reproducibly compared to the LH 750 and the reference procedures. It would be an excellent second instrument for larger laboratories concerned with harmonization of instrumentation and reagents or as a primary instrument for smaller hematology laboratories with limited space.

Cytometry Part A, 2009

The inability to sensitively detect metastatic cells in preclinical models of cancer has created ... more The inability to sensitively detect metastatic cells in preclinical models of cancer has created challenges for studying metastasis in experimental systems. We previously developed a flow cytometry (FCM) method for quantifying circulating tumor cells (CTCs) in mouse models of breast cancer. We have adapted this methodology for analysis of tumor dissemination to bone marrow (BM) and lymph node (LN), and for analysis of these samples by laser scanning cytometry (LSC). Our objective was to implement these methodologies for characterization of tumor cell dissemination in preclinical models of cancer metastasis. Human cancer cells were injected into mice via mammary fat pad (MFP; spontaneous metastasis), tail vein (TV; targets lung), or intracardiac (IC; targets bone) routes. At several time points postinjection (4 h to 8 weeks), mice were sacrificed and blood, LNs, and BM were collected. Samples were immunomagnetically enriched and labeled with human leukocytic antigen-fluorescein isothiocyanate and CD45-PE antibodies (FCM/LSC), and propidium iodide (FCM) prior to quantitative analysis. Following MFP injection, CTCs increased over time, as did disseminated cells to the LN. Interestingly, tumor cells also spontaneously disseminated to BM, peaking at 2 weeks postinjection. Following TV injection, CTCs were initially high but decreased rapidly by 1 week before increasing to peak at endpoint. Combined with an observed concurrent increase in disseminated cells to LN and BM, this suggests that tumor cells may shed into the circulation from lung metastases that establish following initial cell delivery. Following IC injection, CTCs increased over time, peaking at 4 weeks. Tumor cells in the BM (most prevalent site of metastasis after IC injection) remained at moderate levels until peaking at endpoint. Combined use of FCM and LSC allows sensitive quantification of disseminated tumor cells in preclinical models of metastasis. These methods will be valuable for future studies aimed at testing new therapeutics in the metastatic setting. ' 2008 International Society for Advancement of Cytometry Key terms breast cancer; metastasis; preclinical animal models; tumor cell dissemination; flow cytometry; laser scanning cytometry BREAST cancer is a leading cause of morbidity and mortality in women (1,2), primarily because of the failure of effective clinical detection and management of metastatic disease in distant sites such as lymph node (LN), bone, lung, liver, and brain (3,4). The metastatic process is comprised of a series of sequential steps, and cancer cells must successfully complete each step in order to give rise to a metastatic tumor. These steps include dissemination of cancer cells from the primary tumor into the bloodstream (intravasation), survival in the circulation, arrest and extravasation into the secondary site, and initiation and maintenance of growth to form clinically detectable metastases (3-7). Breast cancer cells may also disseminate from the primary tumor through the lymphatic system, although the lack of direct flow from the lymphatic system to other organs means that tumor cells escaping via this route must still enter the vascular system in order to be distributed to distant organs .

Clinical & Experimental Metastasis, 2005

Breast cancer often spreads from the primary tumor to regional lymph nodes. Lymph node status pro... more Breast cancer often spreads from the primary tumor to regional lymph nodes. Lymph node status provides clinically important information for making treatment decisions. Spread via lymphatics is also important for the biology of breast cancer, as tumor cells in lymph nodes may provide a reservoir of cells leading to distant, lethal metastases. Improved understanding of the biology of lymphatic spread thus is important for improved breast cancer survival. Advances towards understanding the interactions between tumors cells and lymphatic vessels have in part been limited by the lack of suitable cell lines and experimental models. We have addressed this need by developing a new model of lymphatic metastasis. Here we describe the establishment of 468LN cells, a variant of the MDA-MB-468 human breast adenocarcinoma cell line, which produces extensive lymph node metastasis following orthotopic injection of nude mice. 468LN cells are also more aggressive in vitro, produce more osteopontin and express different surface integrins compared to the parent line. The dramatic in vitro and in vivo phenotypic and molecular differences of 468LN and parental 468GFP cells make this pair of cell lines a unique model for the specific study of lymph node metastasis of breast cancer.

British Journal of Haematology, 2001

The need for quality control of leucoreduction of blood products has led to the development of va... more The need for quality control of leucoreduction of blood products has led to the development of various methods to count low levels of residual leucocytes. We compared five platforms side-by-side: the Nageotte haemocytometer and four based on fluorescent staining of nuclei: two flowcytometers (Beckman Coulter, BD Biosciences) with methods based on counting beads, a volumetric flow cytometer (Partec) and the microvolumic fluorimeter imagn2000 (BD Biosciences), all according to their manufacturers' recommended methods. Analysis of doublefiltered red cell concentrates (RCCs) and platelet concentrates (PCs), spiked with various numbers of leucocytes, revealed good linearity for all methods over the range of 1´6±32´7 leucocytes/ml, all with r 2 . 0´99. At the rejection level of leucocyte-reduced blood components, i.e. 1 Â 10 6 per unit corresponding with approximately 3´3 leucocytes/ml, the Nageotte haemocytometer had low accuracy (0% for RCCs, 56% for PCs), and was relatively imprecise [coefficient of variance (CV) of 34% and 30% respectively]. The Partec flow cytometer gave good results for RCCs (accuracy 67%, CV 22%), but not for PCs (accuracy 0%, CV 25%). The imagn2000 had an accuracy of 44% for RCCs and 89% for PCs, but the precision was variable (CV 32% for RCCs, 15% for PCs). The best results were obtained with the Beckman Coulter (RCCs: accuracy 86%, CV 13%, PCs: accuracy 67%, CV 16%), and BD Biosciences platforms (RCCs: accuracy 100%, CV 10%; PCs: accuracy 89%, CV 11%). We conclude that, at the rejection level of 1 Â 10 6 leucocytes per unit, the widely used Nageotte haemocytometer performs poorly in terms of inaccuracy and imprecision, and that both counting-bead-based, flow cytometric methods performed best.

American Journal of Clinical Pathology, 2008

External quality assessment (EQA) of D-dimer assays has been limited by a lack of standardized hu... more External quality assessment (EQA) of D-dimer assays has been limited by a lack of standardized human-derived testing material. In 2006 and 2007, the Quality Management Program--Laboratory Services, Toronto, Canada, investigated the use of commercially prepared lyophilized human plasma spiked with human-derived D-dimer components manufactured by Affinity Biologicals, Hamilton, Canada. Four surveys were performed. Participants reported the level or presence of D-dimer using quantitative or qualitative methods. Participants performing quantitative testing provided their unit of measure and reference interval. Results were considered correct if they fell within the range appropriate for each sample (normal/negative or abnormal/positive). Overall, survey results were excellent, with 4.0% (95% confidence interval [CI], 1.3%-9.1%), 0.8% (CI, 0.0%-1.5%), 2.3% (CI, 0.5%-6.6%), and 2.3% (CI, 0.4%-6.6%) of participants reporting an incorrect result in the first, second, third, and fourth surveys, respectively. A commercially prepared D-dimer is a suitable material for EQA testing.

Cytometry Part B: Clinical Cytometry, 2015

We previously developed a 10-color 11-antibody combination including a viability dye, to screen T... more We previously developed a 10-color 11-antibody combination including a viability dye, to screen T-, B-, and natural killer (NK)-cell populations in blood, bone marrow, tissue, and body fluids. Recently, Beckman Coulter has introduced a line of dried reagents that, unlike liquid reagents and cocktails, require no refrigeration, titration, or manipulation before using. We evaluated custom tubes based on our standard lymphocyte screening panel, focusing on comparative analysis, ease of use, and advantages compared with our liquid reagent set. We tested 42 samples from blood (n = 15), bone marrow (n = 17), and tissue (n = 10) with the combination CD4/CD8/KAPPA/LAMBDA/CD19/CD56/CD5/CD20/CD10/CD3/CD45 and a vital dye by both methods and compared positivity and staining intensity for each antigen. Of the 42 samples, 5 were normal samples, 3 were red cell disorders, 20 were B-cell malignancies, 5 T-cell malignancies, 4 myeloid malignancies, and the remaining 5 were other diagnoses. Dried reagents gave equivalent staining intensity results to our standard panel in a variety of sample types, with diagnoses including reactive lymphocytosis, chronic lymphocytic leukemia, and various lymphomas. Our standard panel for evaluation of mature lymphoid malignancies allows rapid assessment of any sample type while providing direct assessment of viability. The dried reagent tube reduces preanalytical work, with simple addition of sample and the viability dye to the tube, saving time, reducing potential errors, and obviating need to titrate and monitor individual antibodies. With a shelf life of at least 12 months, the reagents also offer potential savings in reagent costs by reducing wastage due to expiration or tandem breakdown in standard liquid formulation. © 2015 International Clinical Cytometry Society.

Thrombosis Research, 2015

Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon disorder characterized by hemolysis, thr... more Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon disorder characterized by hemolysis, thrombosis and marrow failure. Whereas venous and arterial thrombosis is a very common symptom of the disease, the frequency of PNH clones in patients with unexplained venous thromboembolism, including deep vein thrombosis and pulmonary embolism, has not been studied. We conducted a cross sectional study evaluating the presence of PNH clones in patients with prevalent venous thromboembolism using a high sensitivity flow cytometry assay for erythrocytes and neutrophils. Among the 388 patients enrolled in the study one patient had a detectable PNH clone of 0.02% in the neutrophil population (0.26%; 95% CI 0.05 to 1.45) and no detectable erythrocyte clone. We conclude that the presence of PNH clones in patients with idiopathic venous thrombosis is rare. Screening for PNH clones among VTE patients might be better reserved for patients with signs of hemolysis.

Cytometry. Part B, Clinical cytometry, Jan 8, 2015

Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an in... more Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an invasive procedure. Reticulated platelets (RP) are immature RNA containing platelets, accurate RP enumeration has yet to be achieved, partially due to the lack of a robust reference method. To refine previous work and gating strategies distinguishing RP from mature platelets while incorporating accurate platelet enumeration into the analysis. After reviewing previously published studies on Thiazole Orange (TO) staining of RP, we systematically evaluated CD41/CD61 in combination with a commercial source of TO (BDBiosciences). Previous RP methods have not taken advantage of platelet enumeration therefore our goal was to incorporate the ICSH platelet enumeration protocol into our method. TO concentration, incubation and fixation method were determined to be 10% of stock concentration, 30min, and 1% formaldehyde respectively. Gating strategy to determine RP fraction used an unstained control t...

Cytometry, Jan 15, 1998

The isotype control has long been considered a useful part of both microscopic and flow cytometri... more The isotype control has long been considered a useful part of both microscopic and flow cytometric immuno- logic assays, and, consequently, is still routinely used in clinical laboratories. In flow cytometry, the isotype control has traditionally been used to distinguish between fluores- cent positive and fluorescent negative cell populations. Additionally, it has been used to estimate the number of cells

Clinical Immunology Newsletter, 1997

T he increased use of peripheral blood stem cells (PBSC) to reconstitute hematopoiesis in both au... more T he increased use of peripheral blood stem cells (PBSC) to reconstitute hematopoiesis in both autoand, more recently, allo-transplant settings has not been associated with a consensus means to assess the engraftment potential of the PBSC product. Since the small population of bone marrow cells bearing the CD34 antigen are responsible for sustained multi-lineage engraftment, flow cytometric quantitation of CD34+ cells should provide a rapid, reliable, and reproducible means to assess the engraftment potential of PBSC collections, To date, however, the lack of a standardized method has led to the generation of widely divergent data. To address this problem, we were asked by the International Society of Hema-totherapy and Graft Engineering (ISHAGE) to develop clinical guidelines for the quantitation of CD34+ cells in peripheral blood based upon a previously published flow cytometric methodology (FCM). We also sought to establish the method's utility on a variety of flow cytometers in clinical laboratories and its reproducibility between transplant centers. Here we describe the basic four-parameter flow protocol adopted by ISHAGE and compare this method with others currently in use. We also describe how this very flexible methodology can be used to analyze the qualitative composition of the CD34+ cell fraction, and, by incorporating fluorescent beads in the analysis, how the absolute CD34+ content of apheresis collections can be determined on a single instrument platform.

Cytometry, 1998

In concert with the International Society of Hematotherapy and Graft Engineering (ISHAGE), we pre... more In concert with the International Society of Hematotherapy and Graft Engineering (ISHAGE), we previously described a set of guidelines for detection of CD34؉ cells based on a four-parameter flow cytometry method (CD45 FITC/CD34 PE staining, side and forward angle light scatter). With this procedure, an absolute CD34؉ count is generated by incorporating the leukocyte count from an automated hematology analyser (two-platform method). In the present study, we modified the basic ISHAGE method with the addition of a known number of Flow-Count fluorospheres. To reduce errors inherent to sample washing/centrifugation, we implemented ammonium chloride lyse, no-wash no-fix sample processing. These modifications convert the basic protocol into a single-platform method to determine the absolute CD34 count directly from a flow cytometer and form the basis of the Stem-Kit from Coulter/Immunotech. A total of 72 samples of peripheral blood, apheresis packs, and cord blood were analysed and compared using the ISHAGE protocol with or without the addition of fluorescent microspheres. Comparison of methods showed a high correlation coefficient (r ‫؍‬ 0.99), with no statistically significant difference or bias between methods (P G 0.05). Linearity of the absolute counting method generated an R 2 value of 1.00 over the range of 0-250/l. Precision of the absolute counting method measured at three concentrations of CD34؉-stabilised KG1a cells (Stem-Trol, COULTER) generated a coefficient of variation (C.V.) ranging from 4% to 9.9%. In a further modification of the single-platform method, the viability dye 7-amino actinomycin D was included and demonstrated that both viable and nonviable CD34؉ cells could be identified and quantitated. Together, these modifications combine the accuracy and sensitivity of the original ISHAGE method with the ability to produce an absolute count of viable CD34؉ cells. It is the accurate determination of this value that is most clinically relevant in the transplant setting. These modifications may improve the interlaboratory reproducibility of CD34 determinations due to the reduction in sample handling and calculation of results. Cytometry (Comm. Clin. Cytometry) 34:61-70, 1998.

Cytometry, 2002

Background: Enumeration of CD4 ؉ and CD8 ؉ T-cell subsets provides relevant information for diagn... more Background: Enumeration of CD4 ؉ and CD8 ؉ T-cell subsets provides relevant information for diagnosis and monitoring of patients with cellular immunodeficiencies. As a result, an external quality assurance scheme was implemented in Belgium, The Netherlands, and Luxemburg in 1995. A workshop was held to train the participants in state-of-the art technology for assessment of absolute T-cell subset counts (i.e., a three or four-color, single-platform assay with lymphocyte gating based on CD45 and sideward light scatter) with the aim to achieve between-site coefficients of variation (CVs) <10% and within-site CVs <5% for >75% of the participants. Methods: Three send-outs of stabilized blood from a healthy donor were distributed to 55 laboratories, each with the request to perform the standard assay on three occasions. For comparison, each laboratory performed its local technique in parallel. Results: With the standard technique, between-site CVs of ϳ8% (CD3 ؉ T cells), ϳ9% (CD4 ؉ T cells), and ϳ10% (CD8 ؉ T cells) were achieved. Within-site CVs were <5% for 82% (CD3 ؉ T cells) and ϳ70% (CD4 ؉ and CD8 ؉ subsets) of the participants. Local techniques yielded between-site CVs of 13%-17% for CD3 ؉ , CD4 ؉ , and CD8 ؉ T cells. Conclusions: The state-of-the-art technology for T-cell subset enumeration was implemented successfully among 55 Belgian-Dutch laboratories and resulted in significant reductions of between-site variation of absolute CD3 ؉ , CD4 ؉ , and CD8 ؉ T-cell counts. Cytometry (Clin. Cytometry) 50:92-101, 2002.

Journal of Visualized Experiments, 2014

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. ... more The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Cytometry, 1998

The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and ... more The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/microl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in the CD34+ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34+ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34+ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies.

Transfusion Medicine and Hemotherapy, 2004

SummaryOver the last few years, the number of clinical applications for autologous and allogeneic... more SummaryOver the last few years, the number of clinical applications for autologous and allogeneic stem cell transplantation has expanded considerably. Concurrently, stem cell collections have been obtained from increasingly diverse sources, including bone marrow, peripheral blood and umbilical cord blood. Various ex vivo manipulations have been developed to process stem cell transplants for specific clinical requirements (e.g. positive selection techniques

Thrombosis Research, 1998

event rate this study does not support the need to monitor anti-Xa levels or adjusting the dose E... more event rate this study does not support the need to monitor anti-Xa levels or adjusting the dose Enoxaparin after joint arthroplasty is effective prophylaxis against venous thromboembolism. This is according to weight.

Thrombosis and Haemostasis, 2005

Many patients with venous thromboembolism are being treated with low molecular weight heparin for... more Many patients with venous thromboembolism are being treated with low molecular weight heparin for extended periods of time. It is not certain if it is necessary to assess anti-Xa levels for extended treatment periods. This study is a prospective assessment of anti-Xa levels in patients on long-term therapy for acute venous thromboembolism who have active cancer. Consecutive consenting patients from one center in a multicenter trial that compared 6 months of low molecular weight heparin with oral anticoagulant therapy were treated with therapeutic doses of dalteparin (200 IU per kilogram) subcutaneously daily. Anti-Xa levels were assessed at the end of weeks 1 and 4,4-6 hours after injection of dalteparin. Patients were followed for bleeding and recurrent venous thromboembolism. There were 24 patients who had anti-Xa levels measured at weeks 1 and 4. Two other patients had week 1 measurements performed but died before the week 4 sample was collected due to their underlying cancer. The mean anti-Xa levels at weeks 1 and 4 were 1.11 and 1.03 anti-Xa units/ml respectively (P=0.13). These results suggest that for patients with active cancer receiving extended duration therapy with low molecular weight heparin (dalteparin) there is no accumulation of anti-Xa effect over the first month of therapy. Monitoring of anti-Xa levels in this situation is usually not required.