Jan W. Gratama

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.

mRNA and microRNA expression profiles in circulating tumor cells and primary tumors of metastatic breast cancer patients

Purpose: Molecular characterization of circulating tumor cells (CTC) holds great promise. Unfortunately, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC-specific molecular characterization extremely challenging. In this study, we determined mRNA and microRNA (miRNA) expression of potentially CTC-specific genes that are considered to be clinically relevant in breast cancer. Experimental Design: CTCs were isolated with the epithelial cell adhesion molecule–based CellSearch Profile Kit. Selected genes were measured by real-time reverse transcriptase PCR in CTCs of 50 metastatic breast cancer patients collected before starting first-line systemic therapy in blood from 53 healthy blood donors (HBD) and in primary tumors of 8 of the patients. The molecular profiles were associated with CTC counts and clinical parameters and compared with the profiles generated from the corresponding primary tumors. Results: We identified 55 mRNAs and 10 miRNAs more abundantly expressed in samples from 32 patients with at least 5 CTCs in 7.5 mL of blood compared with samples from 9 patients without detectable CTCs and HBDs. Clustering analysis resulted in 4 different patient clusters characterized by 5 distinct gene clusters. Twice the number of patients from cluster 2 to 4 had developed both visceral and nonvisceral metastases. Comparing transcript levels in CTCs with those measured in corresponding primary tumors showed clinically relevant discrepancies in estrogen receptor and HER2 levels. Conclusions: Our study shows that molecular profiling of low numbers of CTCs in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs. Clin Cancer Res; 17(11); 3600–18. ©2011 AACR.

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells.

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.

Flow cytometric quantitation of immunofluorescence intensity: Problems and perspectives

Quantitation of immunofluorescence intensity serves to estimate the number of defined molecules expressed on or in cells. Clinical applications of this diagnostic tool are increasing, e.g., aberrant expression of various antigens (Ag) by leukemic blasts or lymphoma cells, intensity of CD38 expression by CD8+ T-lymphocytes to monitor activation status, and intensity of CD62P to detect platelet activation. In this report we discuss the quality-control measures required for quantitation of fluorescence intensity, and we review seven concepts that have been developed to quantify fluorescence intensity during the past 15 years. Initial work addressed the conversion of logarithmic channel numbers into units of relative fluorescence. The design and use of calibration beads labeled with predefined amounts of dye allowed instrument-independent expression of fluorescence intensity in units of molecules of equivalent soluble fluorochrome (MESF). This method was refined by the combined use of such standards with monoclonal antibodies (mAb) conjugated 1:1 with phycoerythrin (PE), allowing translation of fluorescence intensity into numbers of antibodies bound per cell. Alternatively, the use of 1:1 PE-conjugated mAb under the assumption that CD4+ lymphocytes reproducibly bind 50,000 CD4 mAb molecules was proposed to convert units of relative fluorescence intensity into units of antibodies bound per cell. The use of antibody-binding capacity as a surrogate marker for quantification of Ag expression was addressed more directly by the development of antibody-binding standards. The quantitative indirect immunofluorescence assay is based on beads labeled with various amounts of CD5 mAb that calibrate the binding of the secondary antibody in units of antibody-binding capacity. Alternatively, goat anti-mouse-labeled calibration beads have been developed. Published results obtained with the latter calibrators showed an unexpected inaccuracy. The different ways in which calibrators and cells under study bind mAb (i.e., Fab mediated versus Fc mediated) may have contributed to this variation. Recently, the use of stabilized cell populations expressing Ag in a specified range of concentrations has been proposed as an Ag-specific calibration system of mAb binding. We identify several issues on the level of instrumentation, reagents, and cells under study that should be solved to allow standardization of quantitative assessments of immunofluorescence intensity.

An uncontrolled trial of rituximab for antibody associated paraneoplastic neurological syndromes

Anti-CD20 monoclonal antibody (rituximab) is effectively used in the treatment of B-cell lymphomas. Recent reports in the literature suggest that antibody associated autoimmune disorders may respond to rituximab. We therefore treated nine patients with anti-Hu or anti-Yo associated paraneoplastic neurological syndromes (PNS) with a maximum of four monthly IV infusions of rituximab (375mg/m2). In this uncontrolled, unblinded trial of rituximab, three patients improved ≥1 point on the Rankin Scale (RS). One patient with limbic encephalitis improved dramatically (RS from 5 to 1). Further studies of rituximab in autoantibody associated PNS are warranted.

Molecular Epidemiology of Epstein–Barr Virus Infection

Publisher Summary This chapter discusses the information obtained by the molecular characterization of Epstein–Barr virus (EBV) diversity and explains its relevance for viral epidemiology, transmission, and pathogenesis. EBV is a γ-herpes virus and consists of a core containing a double-stranded DNA molecule of nearly 172,000 base pairs (bp), an icosahedral capsid, and an envelope enclosing the capsid. The two major target cell types for EBV infection are the B lymphocytes, in which the infection is largely nonproductive or latent, and stratified squamous epithelium, in which viral replication occurs. The availability and use of molecular analytical techniques at the DNA and protein levels have allowed the recognition of heterogeneity between EB viruses carried by various laboratory B-cell lines that had been derived from BL tissues. Molecular epidemiological studies of EBV have made several important contributions to the understanding of the relationship between the virus and its host in health and disease. For an uneventful symbiosis of the virus and its host, it is necessary to strike a perfect balance between various types of EBV-infected cells and the EBV-specific immune response. EBV only causes (malignant) disease in the cases that lack EBV-specific immunity, specific cytogenetic accidents, and, possibly, infection with pathogenic viral genotypes.

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.

Analysis of variation in results of CD34+ hematopoietic progenitor cell enumeration in a multicenter study

A workshop was held in The Netherlands and Belgium with the aim of investigating whether or not the use of a standard protocol vs. local protocols for flow cytometric enumeration of CD34+ hematopoietic progenitor cells would reduce interlaboratory variation. The standard protocol consisted of a three-color, whole-blood staining technique based on fluorescein isothiocyanate (FITC)-labeled CD34, and phycoerythrin (PE)-labeled CD14 and CD66e monoclonal antibodies (reactive with monocytic and myeloid cells, respectively), followed by erythrocyte lysis, washing, fixation, and selection of nucleated cells during data acquisition on the basis of their positivity for LDS-751 (staining DNA and RNA). Data analysis guidelines included the elimination of nonspecific antibody binding by monocytes and myeloid cells by gating on the CD14-, 66e- cells, followed by setting a window on a CD34 vs. sideward light scatter (SSC) plot around the CD34+, SSClow cells. The FITC-labeled isotype control was analyzed with the same gate and window settings, and the false-positive events were subtracted from CD34 result. Four samples (i.e., peripheral blood and apheresis product from two patients) were sent out. Results were received on patient 1 (2) from 36 (38) laboratories. Data obtained by 24 (26) laboratories after correct application of the standard protocol revealed that the median percentage of CD34+ cells of the four samples ranged between 1.1% and 3.7% and the CVs between 18% and 30%. Incorrect performance of the standard protocol by 12 laboratories, mainly resulting from gating errors, yielded a larger variation (CVs ranging between 50% and 82%). CD34 enumeration using local protocols by 29 (34) laboratories yielded median percentage of CD34+ cells ranging between 1.2% and 3.9% and CVs ranging between 34% and 106%. We conclude that correct application of this standard protocol was effective in reducing the interlaboratory variation in percentage of CD34+ cells assessments.

Treatment of acute graft-versus-host disease with monoclonal antibody OKT3. Clinical results and effect on circulating T lymphocytes.

Eight recipients of a bone-marrow graft from HLA-identical, MLR-nonreactive sibling donors who had developed grade II—IV acute graft-versus-host disease (aGVHD), were given 14 consecutive daily injections of 5 mg of a murine anti-T-cell monoclonal antibody (MCA) called OKT3. Four patients with grade II aGVHD showed a complete response; two patients with grade II had a partial response, and two patients (one with grade II and one with grade IV) showed no improvement at all. The main side effect was a high spiking fever after the first injection. T cells were monitored with monoclonal antibodies, indirect immunofluorescence, and flow cytometry. Circulating T3+ T cells dropped to virtually zero within 1 hr following the first injection. Low numbers of E-rosetting cells were still demonstrable during OKT3 therapy. During the second week of treatment, T-cell markers (T3, T4, T8) started to increase again, in spite of excess antibody in the circulation. At that time, T cells showed weaker fluorescence with OKT3 than before OKT3 therapy, suggesting modulation of the T3 antigen. After cessation of OKT3 therapy, T cells reached pretreatment levels within one week. None of the six patients studied developed anti-mouse-Ig antibodies. These results suggest that OKT3 therapy is effective in limited aGVHD. The absence of anti-mouse-Ig antibody formation may allow repeated courses of MCA that may add to their therapeutical potential.

Level of anti-mouse-antibody response induced by bi-specific monoclonal antibody OC/TR in ovarian-carcinoma patients is associated with longer survival

More than 60% of cancer patients injected with intact murine monoclonal antibody (MAb) develop a humoral response against the antigen even after a single dose. Analysis of a series of 35 ovarian‐cancer patients entered in phase‐I and ‐II clinical studies of T‐cells retargeted with the bi‐specific F(ab′)2 OC/TR revealed: (i) a detectable human anti‐mouse antibody (HAMA) response in 31/35 (88%) patients, with high HAMA levels (≥150 ng/ml) in 18/31 (58%) cases by the end of the treatment; (ii) no correlation between HAMA levels and the form of delivery of the mAb (OC/TR bound to T cells or bound plus soluble), time schedule or cumulative dose; (iii) an association between high HAMA levels and favorable clinical parameters and response to immunotherapy; and (iv) a significantly longer median survival probability in patients with high HAMA levels than in patients with lower HAMA levels, even when the sub‐group of non‐responder patients was considered. Evaluation of the anti‐idiotypic response in HAMA‐positive sera indicated that 11/17 sera showed high‐titer (>6000) binding of OC/TR, as evaluated by a specific radioimmunoassay, and 15/18 and 16/16 sera specifically inhibited the binding of the MOv18 and anti‐CD3 parental MAbs to ovarian‐carcinoma cells and T lymphocytes respectively. Of 7 patients evaluated for duration of the HAMA response, 5 showed stable or even increased HAMA levels. The long‐lasting HAMA response maintained an anti‐idiotypic component, directed mainly against the αCD3 idiotype of bi‐MAb OC/TR in 2 out of 3 cases tested. Int. J. Cancer (Pred. Oncol.) 84:62–68, 1999.