Michael Keeney

Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines

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 > 0.05). Linearity of the absolute counting method generated an R2 value of 1.00 over the range of 0-250/microl. Precision of the absolute counting method measured at three concentrations of CD34+-stabilised KG1 a 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.

Diagnosing PNH with FLAER and multiparameter flow cytometry.

PNH is an acquired hematopoietic stem cell disorder leading to a partial or absolute deficiency of all glycophosphatidyl‐inositol (GPI)‐linked proteins. The classical approach to diagnosis of PNH by cytometry involves the loss of at least two GPI‐linked antigens on RBCs and neutrophils. While flow assays are more sensitive and specific than complement‐mediated lysis or the Hams test, they suffer from several drawbacks. Bacterial aerolysin binds to the GPI moiety of cell surface GPI‐linked molecules and causes lysis of normal but not GPI‐deficient PNH cells. FLAER is an Alexa488‐labeled inactive variant of aerolysin that does not cause lysis of cells. Our goals were to develop a FLAER‐based assay to diagnose and monitor patients with PNH and to improve detection of minor populations of PNH clones in other hematologic disorders.

Detection and quantification of circulating tumor cells in mouse models of human breast cancer using immunomagnetic enrichment and multiparameter flow cytometry

Circulating tumor cells (CTCs) in the peripheral blood of breast cancer patients may be an important indicator of metastatic disease and poor prognosis. However, the use of experimental models is required to fully elucidate the functional consequences of CTCs. The purpose of this study was to optimize the sensitivity of multiparameter flow cytometry for detection of human tumor cells in mouse models of breast cancer.

A comparison of three rapid D-dimer methods for the diagnosis of venous thromboembolism.

We compared three rapid d‐dimer methods for the diagnosis of venous thromboembolism. Patients presenting to four teaching hospitals with the possible diagnosis of deep vein thrombosis or pulmonary embolism were investigated with a combination of clinical likelihood, d‐dimer (SimpliRED) and initial non‐invasive testing. Patients were assigned as being positive or negative for deep vein thrombosis or pulmonary embolism based on their three‐month outcome and initial test results. The three d‐dimer methods compared were: (a) Accuclot d‐dimer (b) IL‐Test d‐dimer (c) SimpliRED d‐dimer. Of 993 patients, 141 had objectively confirmed deep vein thrombosis or pulmonary embolism. The sensitivity of SimpliRED, Accuclot and IL‐Test were 79, 90 and 87% respectively. All three d‐dimer tests gave similar negative predictive values. The SimpliRED d‐dimer was found to be less sensitive than the Accuclot or IL‐Test. When combined with pre‐test probability all three methods are probably acceptable for use in the diagnosis of venous thromboembolism.

Isotype controls in the analysis of lymphocytes and CD34+ stem and progenitor cells by flow cytometry—time to let go?

The isotype control has long been considered a useful part of both microscopic and flow cytometric immunologic assays, and, consequently, is still routinely used in clinical laboratories. In flow cytometry, the isotype control has traditionally been used to distinguish between fluorescent positive and fluorescent negative cell populations. Additionally, it has been used to estimate the number of cells reacting non-specifically with the target antibody under investigation. Over the past 10 years, the widespread use of directly conjugated monoclonal antibodies (mAb) and multiparameter analysis in clinical flow cytometry has reduced the need for a separate ‘‘negative control’’ tube. This tendency has materialized in guidelines recommending that the isotype control is irrelevant and potentially misleading in commonly used flow cytometric assays (3, 14). This perspective summarizes the rationale for omitting isotype control staining for surface membrane marker analysis, focusing on lymphocyte and CD341 hematopoietic stem/progenitor cell analyses. Consequently, these points also pertain to the immunophenotyping of leukemia/lymphoma samples (14). Prior to the development of directly conjugated mAb, pre-immune sera were used in microscopic and flow cytometric studies to estimate the level of ‘‘non-specific staining’’ of the specific antibody to its target cell, i.e., the binding of that specific antibody by mechanisms other than specific antibody-to-antigen interactions. Such nonspecific binding is usually, but not exclusively, mediated by receptors that bind the Fc portion of the various immunoglobulin subclasses (19). In flow cytometry, an estimate of the number of cells reacting non-specifically is typically determined by placing a cursor at the foot of the isotype control negative population on a fluorescence (FL) histogram such that less than 2% of events are assessed as positive. This cursor position is maintained to determine the ‘‘percent positive cells’’ in the experimental stainings. Currently, many isotype controls are produced by fusion of antibody producing cells with a myeloma-derived cell line to form a hybridoma. By the very nature of mAb production, antibodies produced by hybridomas will differ structurally from each other, even within the same immunoglobulin subclass or isotype. Thus mAb that ‘‘specifically’’ bind to the same antigen on the cells under study might each additionally bind ‘‘non-specifically’’ to other leukocytes, platelets, etc. in an unpredictable manner. Other issues to consider in the use of monoclonal isotypes include differences in protein concentration and FL to protein (F/P) ratio between test antibody and isotype control. Different manufacturers use different protocols to produce, purify and chemically conjugate antibodies with a variety of fluorochromes which almost certainly impact the reliability with which experimental and isotype control mAb can be used to distinguish specific from nonspecific binding. A compounding problem is that in a panel with several surface markers each would need their own isotype control matched for the above criteria. This is rarely done in the clinical laboratory.

Use of a FLAER-Based WBC Assay in the Primary Screening of PNH Clones

Diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) with flow cytometry traditionally involves the analysis of CD55 and CD59 on RBCs and neutrophils. However, the ability to accurately detect PNH RBCs is compromised by prior hemolysis and/or transfused RBCs. Patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS) can also produce PNH clones. We recently described a multiparameter fluorescent aerolysin (FLAER)-based flow assay using CD45, CD33, and CD14 that accurately identified PNH monocyte and neutrophil clones in PNH, AA, and MDS. Here, we compared the efficiency of this WBC assay with a CD59-based assay on RBCs during a 3-year period. PNH clones were detected with the FLAER assay in 63 (11.8%) of 536 samples tested, whereas PNH RBCs were detected in only 33 (6.2%), and always with a smaller clone size. The FLAER assay on WBCs is a more sensitive and robust primary screening assay for detecting PNH clones in clinical samples.

A new model for lymphatic metastasis : Development of a variant of the MDA-MB-468 human breast cancer cell line that aggressively metastasizes to lymph nodes

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.

Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry

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.

ISHAGE Protocol: Are We Doing It Correctly?

Flow cytometric CD34+ stem cell enumeration is routinely performed to optimize timing of peripheral blood stem cell collections and assess engraftment capability of the apheresis product. While a number of different flow methodologies have been described, the highly standardized ISHAGE protocol is currently the most widely employed, with 204/255 (81%) international participants in the UK NEQAS CD34+ stem cell enumeration program indicating their use of this method. Recently, two laboratories were identified as persistent poor performers, a fact attributed to incorrect ISHAGE protocol usage/setup. This prompted UK NEQAS to question whether other laboratories were making similar errors and, if so, how this might affect individual EQA performance.

Quality assurance of stem cell enumeration by flow cytometry

The enumeration of CD34+ cells by flow cytometry is commonly employed to assess progenitor/stem cell numbers in peripheral blood, cord blood, and apheresis products used for peripheral blood stem cell transplantation (PBSCT). Until recently, the enumeration of CD34+ cells has evolved in the absence of any procedural guidelines or quality assurance programs. We established a voluntary quality assurance for stem cell enumeration (QASE) program for Canadian laboratories involved in PBSCT. The goals of this study were 1) to survey current flow cytometric procedures for CD34 enumeration; 2) to evaluate inter-institutional reproducibility of currently employed assays; and 3) to evaluate the impact of adoption of a common method on inter-laboratory variability. Fourteen to nineteen laboratories participated in this program by enumerating CD34+ cells in shared cord blood samples. The survey showed a wide variation in flow cytometric methodology which in part contributed to the wide range in results obtained between different centres. Major differences in methodology included the number of events counted, use of isotype control and gating strategy. Participation in this program and adoption of a common methodology increased level of agreement for any given result although this trend did not reach statistical significance (P = 0.13). The results of this survey emphasize the need for a quality assurance program for stem cell enumeration by flow cytometry.