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J W Gratama

Publications and source records attributed to J W Gratama.

At least 19 recordsLinked to original sources
PubMed2006

T-lymphocyte reconstitution following rigorously T-cell-depleted versus unmodified autologous stem cell transplants.

We compared the kinetics of T-cell recovery after extensive ex vivo and in vivo T-cell depleted autologous stem cell transplantation (SCT) for multiple sclerosis (MS; n=8) with unmodified SCT for hematological malignancies (HM; n=39). Both patient group showed a very protracted recovery of 'naive' CD4(+), 45R0(-) ( approximately CD45RA(+)) T-cells. Within the 'primed' CD4(+), 45R0(+) T-cells, the 'central memory' cells expressing the CD62L and CD27 markers were the slowest to recover. The repopulating T-cells were highly activated, as shown by increased expression of HLA-DR and the apoptosis marker CD95. The capability of CD4(+) and CD8(+) T-cells to produce IFN-gamma, IL-2 and TNF-alpha had reached normal ranges from 2 months post SCT onwards. Unexpectedly, the kinetics of T-cell recovery between 3 and 12 months post transplant was similar in T-depleted and unmodified SCT. Before SCT, the HM patients showed lymphopenia of all T-cell subsets, upregulated HLA-DR and CD95 expression and increased cytokine responses. We suggest that the similar kinetics of T-cell recovery in the two patient groups may be explained by the susceptibility to apoptosis of the activated CD4(+) T-cells in the autografts of the HM patients. This susceptibility to apoptosis would interfere with a swift and sustained CD4(+) T-cell regeneration post SCT.

Adult↗
PubMed2006

Phoenix-ampho outperforms PG13 as retroviral packaging cells to transduce human T cells with tumor-specific receptors: implications for clinical immunogene therapy of cancer.

We have designed a transgene that encodes a scFv(G250) chimeric receptor, which is specific for carboxyanhydrase IX (G250-ligand, G250L), a molecule overexpressed by renal cell cancer (RCC). Retroviral transduction of this transgene into primary human T lymphocytes confers these cells with specific functional responses towards G250L-positive RCC cells. In preparation of a clinical phase (I/II) study in RCC patients, we set up a protocol for gene transduction and expansion of primary human T cells. For this purpose, we directly compared two packaging cell lines, that is, the GALV-pseudotyped MLV producing cell line PG13, and the MLV-A-producing cell line Phi-NX-Ampho (a.k.a. Phoenix-A). We generated and characterized stable scFv(G250)-positive clones of both PG13 and Phoenix cells and optimized the retrovirus production conditions. Transductions of primary human T cells yielded 30-60% scFv(G250)+ T cells using PG13-derived retrovirus versus up to 90% scFv(G250)+ T cells using Phoenix-derived retrovirus. The median number of transgene integrations per scFv(G250)+ T cell differed only 1.5-fold as determined by real-time PCR (mean number of integrations per T cell 2.6 and 3.7 for PG13 and Phoenix-based transductions, respectively). In addition, T cells transduced with Phoenix-derived retrovirus showed, on a per cell basis, 10-30% higher levels of scFv(G250)-mediated TNFalpha production and cytolysis of G250L+ RCC cells than T cells transduced with PG13-derived retrovirus. The improved functional transduction efficiency together with a limited increase in the number of integrations per recipient cell, made us select Phoenix clone 58 for our clinical immunogene therapy study.

Antigens, Neoplasm↗
PubMed2006

Process validation and clinical evaluation of a protocol to generate gene-modified T lymphocytes for imunogene therapy for metastatic renal cell carcinoma: GMP-controlled transduction and expansion of patient's T lymphocytes using a carboxy anhydrase IX-specific scFv transgene.

BACKGROUND: Adoptive transfer of autologous T cells that are gene-transduced to express Ag-specific receptors represents an experimental strategy to provide tumor-specific immunity to cancer patients. We studied this concept in patients with metastatic renal cell cancer (RCC) using retroviral transduction of T cells with a single-chain Ab-G250 chimeric receptor [scFv(G250)]. We describe the validation of our clinical protocol for gene transduction and expansion of human T lymphocytes. METHODS: A batch of scFv(G250) transgene-containing retrovirus was produced under conditions of good manufacturing practice (GMP). In addition to quality control and safety testing of the virus batch, extensive potency testing was performed, i.e. assessment of its functional transduction efficiency in primary human T cells. Subsequently, the clinical gene transduction and cell-expansion protocol was subjected to a series of process validations and a clinical evaluation using T cells obtained from healthy donors and three RCC patients. RESULTS: The clinical batch of scFv(G250) transgene-containing retrovirus met the quality and safety control criteria. Small-scale transductions yielded 62-92% scFv(G250)+ T cells and, at a clinical scale, 50-84% transduction efficiencies were obtained. Patient and healthy donor T cells showed similar expansion potencies, and also yielded similar levels of scFv(G250)-mediated immune functions, i.e. specific cytolysis of G250-ligand expressing RCC cells and production of IFN-gamma upon stimulation with such cells. All T cell cultures were free of replication competent retroviruses. DISCUSSION: We have shown that the validated batch of scFv(G250) transgene-containing retrovirus in combination with our GMP T-cell transduction and expansion protocol successfully generates clinically relevant numbers of functional scFv(G250) gene-modified T cells for patient treatment.

Adoptive Transfer↗
PubMed2005

Parallel detection of transduced T lymphocytes after immunogene therapy of renal cell cancer by flow cytometry and real-time polymerase chain reaction: implications for loss of transgene expression.

We have started a phase I/II immunogene therapy study of metastatic renal cell cancer (RCC), using autologous T lymphocytes transduced ex vivo with a gene encoding a single-chain receptor based on the monoclonal antibody (mAb) G250 [scFv(G250)]. G250 recognizes carbonic anhydrase IX, which is overexpressed by RCC cells. We have developed and validated flow cytometric and real-time polymerase chain reaction (PCR) assays to quantitatively detect transduced T cells in patient blood. The flow assay was based on staining with the anti-G250 idiotype mAb NuH82 and showed a sensitivity of 0.06% scFv(G250)(1) cells within CD3(1) T cells. The real-time PCR method showed a sensitivity of 14 copies of scFv(G250) DNA per 100 ng of total DNA, which enabled detection of 0.008% scFv(G250)(1) T cells within leukocytes. Both assays were further validated for their specificity and reproducibility. When applied to blood samples from three RCC patients treated with intravenous infusions of scFv(G250)(1) T cells, the kinetics of scFv(G250)(1) T cell counts as detected by flow cytometry were similar to those detected by real-time PCR, although PCR allowed detection of transduced T cells over a longer period of time (i.e., for patient 3, 7 versus 32 days, respectively). Interestingly, follow-up studies of patient 3 demonstrated that the number of circulating scFv(G250)(1) T cells remained fairly constant during the first 7 days posttreatment, whereas the number of gene copies increased during the same period of time. These results suggest loss of scFv(G250) membrane expression on adoptive transfer, which would have important implications for the antitumor efficacy of this form of immunogene therapy.

Antibodies, Monoclonal↗
PubMed2004

Visual scoring versus histogram subtraction of in vivo binding of immunoglobulins against platelets after transfusion.

BACKGROUND: We developed a technique, in vivo binding of immunoglobulins in the platelet immunofluorescence test (IVBI-PIFT), that detects immunoglobulins bound in vivo to transfused platelets. The visually scored results of this technique, however, are susceptible to interobserver variation. We describe a more objective method to generate results in IVBI-PIFT. METHODS: We studied 201 samples in 120 patients with hematologic malignancies in the IVBI-PIFT. Histogram subtraction, i.e., fluorescence (anti-immunoglobulin G and fluorescein isothiocyanate) histogram before platelet transfusion subtracted from the histogram after platelet transfusion, was compared with visual scoring (pattern 1: no enhanced fluorescence before and after transfusion; pattern 2: enhanced fluorescence before and after platelet transfusion; pattern 3: enhanced fluorescence before transfusion; pattern 4: enhanced fluorescence after transfusion, interpreted as alloimmunization). After histogram subtraction, the number of remaining events (events post substraction, EPS) and the mean amount of fluorescence of these remaining events (mean channel post substraction, MCPS) were used and compared with the visual scoring and with platelet survival after transfusion. RESULTS: In 26 (13%) of the 201 samples studied in the IVBI-PIFT, fewer than three of five observers agreed on the visually scored pattern. In the 175 (87%) remaining samples, histogram subtraction showed a significant differentiation between pattern 4 and patterns 1 and 2 by using EPS, whereas patterns 4 and 3 were distinguished by using MCPS. The combination of EPS and MCPS differentiated best between pattern 4 and patterns 1, 2, and 3 (73% sensitivity, 96% specificity, 79% positive predictive value, and 95% negative predictive value). In contrast, the predictive value for platelet recovery after 1 and 16 h of pattern 4 from the visual scoring method and the results of histogram subtraction were poor. CONCLUSION: This objective method of histogram subtraction correlated well with the visual scoring method of IVBI-PIFT.

Animals↗
PubMed2004

The value of crossmatch tests and panel tests as a screening tool to predict the outcome of platelet transfusion in a non-selected haematological population of patients.

BACKGROUND AND OBJECTIVES: Alloantibodies against platelets can be detected by using different laboratory tests. Most of these tests, which use panel cells or antigens as a target, perform poorly in non-selected haematological patients. In relation to these tests, a crossmatch test of transfused platelets and patient's serum may be viewed as the standard and may be superior in predicting donor platelet destruction by alloimmunization. MATERIALS AND METHODS: In 95 randomly selected thrombocytopenic patients with haematological malignancies, who were receiving leucodepleted blood products, 184 serum samples were studied in an in vitro crossmatch test by using the technique of the platelet immunofluorescence test (crossmatch-PIFT), in an in vivo crossmatch test detecting in vivo binding of immunoglobulins to transfused platelets according to the PIFT technique (in vivo-PIFT), in the indirect PIFT using five random donors as a target (panel-PIFT) and in an enzyme linked immunosorbent assay using immobilized human leucocyte antigens (HLAs) of 100 standardized donors (ELIHLA). The results of all these methods were related to the recovery at 1 and 16 h post-transfusion. RESULTS: The results of the crossmatch-PIFT were not associated with platelet recovery at 1 and 16 h after transfusion. Even in a subgroup of patients, in whom predefined clinical factors were excluded, no association with platelet recovery was found. The results of the crossmatch-PIFT correlated with those of the in vivo-PIFT (P = 0.02); however, 35 (19%) discrepant results were identified between these tests. The results of the crossmatch-PIFT were not related to the panel-PIFT (P = 0.25), but did relate to those of the ELIHLA (P = 0.02), still revealing 36 (20%) discrepant results. None of the in vivo-PIFT, the panel-PIFT or the ELIHLA was associated with platelet recovery after 1 h, whilst only a positive panel-PIFT was associated with poor platelet recovery at 16 h after transfusion (P = 0.03). CONCLUSIONS: In a population at low risk for alloimmunization, the correlation of test outcome and platelet recovery is poor. None of these crossmatch tests or screening tests was identified as superior to any other in this population.

Antigen-Antibody Reactions↗
PubMed2003

Crossover study of three methods for low-level white blood cell counting on two types of flow cytometer.

BACKGROUND: Flow cytometric methods were previously shown to be preferable to microscopic and volumetric methods for counting residual white blood cells (WBCs). In this study, three flow cytometric, low-level WBC counting methods were cross compared using two flow cytometers. METHODS: Double-filtered red cell and platelet concentrates were spiked with different amounts of WBC to obtain panels of unspiked and 0.3, 1.0, 3.3, and 10.0 WBC/microl. The methods of BD Biosciences (BDB), Beckman-Coulter (BC), and an in-house method were performed on flow cytometers from BDB and BC. Samples were measured in ninefold. We required that (a) r(2) be at least 0.98 (linearity), (b) at least 80% of observations fell within 20% of expected values (accuracy), and (c) the coefficients of variation be at least 20% (precision) for samples containing at least 3.3 WBC/microl. RESULTS: For the red cell panel, our requirements were met by the BDB method on both flow cytometers and by the BC and in-house methods on the BDB flow cytometer only. For the platelet panel, our requirements were met on all combinations of methods and flow cytometers, except for the in-house method on the BDB flow cytometer. Intra-assay variation was lowest for the BDB method, irrespective of the type of flow cytometer used. CONCLUSION: Based on accuracy and precision, the BDB method on the BDB flow cytometer produced the best results for counting low-level WBCs.

Cell Separation↗
PubMed2003

Diagnostic potential of tetramer-based monitoring of cytomegalovirus-specific CD8+ T lymphocytes in allogeneic stem cell transplantation.

Cytomegalovirus (CMV) infection remains a significant problem in allogeneic stem cell transplant (SCT) recipients despite the availability of effective antiviral drugs. This problem concerns patients which are unable to mount an effective T-lymphocyte response against CMV. Therefore, the development and use of tetramer technology to enumerate CMV-specific T cells will be valuable to identify these patients as early as possible. Here, we review clinical studies in which CMV-specific CD8(+) T cells have been monitored in allogeneic SCT recipients using tetramers in the context of similar studies that are based on functional assays of CMV-specific T cells. The results thus far warrant the further development of tetramer technology as a diagnostic tool to monitor CMV-specific T cells in SCT recipients and other groups of immunocompromised patients threatened by CMV.

Alleles↗
PubMed2003

Validation of the single-platform ISHAGE method for CD34(+) hematopoietic stem and progenitor cell enumeration in an international multicenter study.

BACKGROUND: Flow cytometric enumeration of CD34+ hematopoietic sterm and progenitor cells (HPC) is the reference point for undertaking apheresis and evaluation of adequacy for PBSC engraftment. An external quality assurance (EQA) scheme for CD34+ HPC enumeration has been operational in Belgium, Netherlands and Luxemburg (Benelux) since 1995. Within this group, a multicenter survey was held to validate the state-of-the-art methodology, i.e., multiparametric definition of HPC based on light scatter, expression of CD34 and CD45, and counting beads (i.e., 'single platform ISHAGE' method). METHODS: 'Real-time' EQA was used to monitor the application of the single-platform ISHAGE method by 36 participants. Three send-outs of stabilized blood with CD34+ cell counts 35-60 cells/microl were distributed to 36 participants, who were required to assay the samples on three occasions using the standard assay and their local techniques. These results were compared with thosed obtained by 111-116 UK NEQAS participants testing the same specimens. RESULTS: Using the single platform ISHAGE methods, between-laboratory coefficients of variations (CVs) as low as 10% were achieved. Intra-laboratory CVs were < 5% for approximately 50% of the participants. Local single-platform techniques yielded between-laboratory CVs as low as 9% in both Benelux and UK NEQAS cohorts. In contrast, the lowest between-laboratory CVs using dual-platform techniques were 17% (Benelux) and 21% (UK NEQAS), respectively. CONCLUSION: The single-platform ISHAGE method for CD34+ cell enumeration has been validated by an international group of 36 laboratories. The observed varation between laboratories allows a meaningful comparison of CD34+ cell enumeration.

Antigens, CD34↗
PubMed2002

Increased levels of soluble CD27 in the cerebrospinal fluid are not diagnostic for leptomeningeal involvement by lymphoid malignancies.

Soluble CD27 (sCD27) reportedly is a sensitive and specific marker for leptomeningeal involvement (LI) of CD27-expressing lymphoproliferations such as B-cell non-Hodgkin's lymphoma (B-NHL) or chronic B-lymphocytic leukemia (B-CLL). Because morphological analysis of cerebrospinal fluid (CSF) in patients suspected of LI is false negative in one-third of patients, a diagnostic marker for LI by B-NHL or B-CLL would be very valuable. sCD27 was determined in the first CSF sample from each of 102 unselected patients submitted for (immuno)morphologic detection of malignant cells. The patients were considered to have LI if either (immuno)morphologic analyses showed tumor cells or if neuroradiological evaluation showed typical abnormalities consistent with LI. Patients were suspected of having LI if CSF samples revealed atypical lymphocytes and/or if clinical symptoms and signs suggestive of LI were present, but clinical follow-up was shorter than 3 months because of deterioration of the patient. LI was considered absent if (immuno)morphologic analyses of CSF samples were negative without evidence for LI during 3 months of clinical follow-up. In patients with chronic lymphoproliferative disorders [mainly B-non-Hodgkin's lymphoma (NHL)], sCD27 concentrations were significantly higher in the CSF samples of 16 patients with confirmed or suspected LI than in those of 46 patients without LI. However, sCD27 was also increased in a variety of other predominantly inflammatory neurological disorders including herpes simplex and zoster infections. The positive predictive value of sCD27 determination for LI was only 54%, but the negative predictive value was 92%. Normal sCD27 concentrations in CSF samples of patients with chronic lymphoproliferation makes LI unlikely, but the determination of CSF sCD27 is not sufficiently specific to serve as a reliable tumor marker.

Biomarkers, Tumor↗
PubMed2001

Tetramer-based quantification of cytomegalovirus (CMV)-specific CD8+ T lymphocytes in T-cell-depleted stem cell grafts and after transplantation may identify patients at risk for progressive CMV infection.

Recovery of cytomegalovirus (CMV)-specific T-cell-mediated immunity after allogeneic hematopoietic stem cell transplantation (SCT) is critical for protection against CMV disease. The study used fluorochrome-conjugated tetrameric complexes of HLA-A2 molecules loaded with the immunodominant NLVPMVATV (NLV) peptide derived from the CMV protein pp65 to quantify A2-NLV-specific CD8+ T cells in partially T-cell-depleted grafts administered to 27 HLA-A*0201+ patients and to monitor recovery of these T cells during the first 12 months after SCT. None of the 9 CMV-seronegative patients became infected with CMV, whereas 14 of 18 CMV-seropositive patients developed CMV antigenemia after SCT. CMV-seropositive recipients of grafts from CMV-seronegative donors required more preemptive treatment with ganciclovir (GCV) than those of grafts from CMV-seropositive donors (3 [1-6] versus 1 [0-3] courses, respectively; P =.009). The number of A2-NLV-specific CD8+ T cells in the grafts correlated inversely with the number of preemptive GCV courses administered (r = -0.61; P =.01). None of the 9 CMV-seronegative patients mounted a CMV-specific immune response as measured by monitoring A2-NLV-specific CD8+ T cells after SCT. Thirteen of 14 CMV-seropositive patients without CMV disease recovered these T cells. In spite of preemptive GCV treatment, CMV disease developed in 4 patients, who all failed to recover A2-NLV-specific CD8+ T cells after SCT (P =.002). Thus, enumeration of HLA-restricted, CMV-specific CD8+ T cells in the grafts and monitoring of these T cells after SCT may constitute a rapid and sensitive tool to identify SCT recipients at risk for developing CMV disease.

Adolescent↗
PubMed2001

Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation (SCT) and quantitatively predicts EBV-lymphoproliferative disease following T-cell--depleted SCT.

Reactivation of the Epstein-Barr virus (EBV) after allogeneic stem cell transplantation (allo-SCT) may evoke a protective cellular immune response or may be complicated by the development of EBV-lymphoproliferative disease (EBV-LPD). So far, very little is known about the incidence, recurrence, and sequelae of EBV reactivation following allo-SCT. EBV reactivation was retrospectively monitored in 85 EBV-seropositive recipients of a T-cell--depleted (TCD) allo-SCT and 65 EBV-seropositive recipients of an unmanipulated allo-SCT. Viral reactivation (more than 50 EBV genome equivalents [gEq]/mL) was monitored frequently by quantitative real-time plasma polymerase chain reaction until day 180 after SCT. Probabilities of developing viral reactivation were high after both unmanipulated and TCD-allogeneic SCT (31% +/- 6% versus 65% +/- 7%, respectively). A high CD34(+) cell number of the graft appeared as a novel significant predictor (P =.001) for EBV reactivation. Recurrent reactivation was observed more frequently in recipients of a TCD graft, and EBV-LPD occurred only after TCD-SCT. High-risk status, TCD, and use of antithymocyte globulin were predictive for developing EBV-LPD. Plasma EBV DNA quantitatively predicted EBV-LPD. The positive and negative predictive values of a viral load of 1000 gEq/mL were, respectively, 39% and 100% after TCD. Treatment-related mortality did not differ significantly between TCD and non-TCD transplants, but the incidence of chronic graft-versus-host disease was significantly less in TCD patients. It is concluded that EBV reactivation occurs frequently after TCD and unmanipulated allo-SCT, especially in recipients of grafts with high CD34(+) cell counts. EBV-LPD, however, occurred only after TCD, and EBV load quantitatively predicted EBV-LPD in recipients of a TCD graft. (Blood. 2001;98:972-978)

Adolescent↗
PubMed2001

Flow cytometric enumeration and immunophenotyping of hematopoietic stem and progenitor cells.

Flow cytometric enumeration of CD34(+) hematopoietic stem and progenitor cells (HPC) is widely used to evaluate the adequacy of peripheral blood stem cell grafts and is also useful for planning the apheresis sessions needed to obtain these grafts. A state-of-the-art method to enumerate CD34(+) cells has been developed that makes use of a multiparameter definition of HPC, based on their light scatter characteristics and dim expression of CD45, utilizing fluorescent counting beads. This approach allows the absolute CD34(+) cell count to be determined directly from a flow cytometer. The method can be extended with a viability stain and additional markers for further immunologic characterization of CD34(+) cells, and has been successfully implemented in multicenter trials. Using such a standardized assay, it should be possible to define more accurately the lower threshold for a safe HPC graft in terms of short- and long-term hematopoietic reconstitution. Semin Hematol 38:139-147.

Animals↗
PubMed2001

Molecular quantification of viral load in plasma allows for fast and accurate prediction of response to therapy of Epstein-Barr virus-associated lymphoproliferative disease after allogeneic stem cell transplantation.

Epstein-Barr virus lymphoproliferative disease (EBV-LPD) following allogeneic stem cell transplantation (allo-SCT) has a poor prognosis. We used a sensitive real-time polymerase chain reaction (PCR) assay for quantitative detection of EBV-DNA in plasma and serially measured EBV-DNA levels to assess the response to treatment in allo-SCT recipients with EBV-LPD. Fourteen allo-SCT recipients with EBV-LPD who received a T cell-depleted (TCD) sibling (n = 5) or matched unrelated donor (n = 9) graft were monitored from the time of EBV-LPD diagnosis, during therapy and assessment of clinical response. Seven patients had complete responses of EBV-LPD to therapy, of whom 21% (3 out of 14) survived beyond 6 months from EBV-LPD diagnosis. Clinically responding patients showed a rapid decline of EBV-DNA plasma levels within 72 h from the start of therapy. In contrast, all clinical non-responders showed an increase of EBV-DNA levels. Absolute EBV-DNA levels at the time of EBV-LPD diagnosis did not predict for response, but the pattern of EBV-DNA levels within 72 h from the start of therapy (> 50% decrease versus increase) strongly predicted for clinical response (P = 0.001). Quantitative monitoring of EBV-DNA levels from the start of and during therapy for EBV-LPD rapidly and accurately predicts for response to therapy as early as within 72 h. It may thus provide a powerful tool to adjust and select treatment in individuals with EBV-LPD following allo-SCT.

Acute Disease↗
PubMed2001

Comparison of five platforms for enumeration of residual leucocytes in leucoreduced blood components.

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 double-filtered 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/microl, all with r(2) > 0.99. At the rejection level of leucocyte-reduced blood components, i.e. 1 x 10(6) per unit corresponding with approximately 3.3 leucocytes/microl, 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 x 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.

Flow Cytometry↗
PubMed2000

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.

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.

Antigens, CD34↗
PubMed2000

Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a. Interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols.

Detection of intracellular myeloperoxidase (MPO), CD79a and CD3 has become the most specific tool for the assignment of myeloid, B- and T-lymphoid lineages in acute leukemias. In order to establish the best combination of monoclonal antibody reagent and sample preparation technique for the intracellular detection of these three markers, we compared six different cell fixation-permeabilization kits (Cytofix/Cytoperm, Fix and Perm, Intraprep, Intrastain, Permeacyte and Permeafix) using 12 fluorochrome conjugates derived from seven monoclonal antibody (mAb) clones. A total of 21 samples corresponding to normal peripheral blood (n=4), normal bone marrow (n=3), acute myeloblastic leukemia (AML, n=6), precursor B-acute lymphoblastic leukemia (ALL, n=6) and T-ALL (n=2) cases, were analysed in two centers. All fixation/permeabilization methods resulted in decreased side scatter and mostly increased forward scatter as compared to erythrocyte-lyse-washed and 1% paraformaldehyde fixed samples. The autofluorescence levels of the leukocyte populations was only significantly increased with use of the Cytofix/Cytoperm kit and mildly with the other techniques. In addition, non-specific staining increased significantly for combinations of any anti-MPO mAb with the Cytofix/Cytoperm kit and for the CD3 clone S4.1 combined with any intracellular method. Anti-MPO antibodies gave a stronger fluorescence signal when conjugated to PE than when coupled to FITC. In conclusion, MPO-7-PE, UCHT-1-PE (CD3) and any HM57-PE conjugate (CD79a) in combination with Fix and Perm, Intraprep, Intrastain or Permeafix, provided specific staining of the respective markers in sufficient intensities. Thus, combined selection of fixation/permeabilization kits and monoclonal antibody reagents against CD3, CD79a and MPO is required for obtaining optimal cytoplasmic detection of these antigens.

Antibodies, Monoclonal↗