Prospective assessment of AR splice variant and PSMA detection on circulating tumor cells of mCRPC patients: preliminary analysis of patients enrolled in PRIMERA trial (NCT04188275).

In our institution, a prospective observational trial testing micro-RNA (miRNA) and ARV7 mutational status in metastatic, castration resistant prostate cancer (mCRPC), is currently recruiting (PRIMERA trial, NCT04188275). A pre-planned interim analysis was performed when 50% of the planned accrual was reached. In this report, we explored the predictive value of Circulating Tumor Cell (CTC) detection in mCRPC patients undergoing 1st line therapy. Moreover, ARV7, ARFL, PSMA and PSA expression on CTC was reported to explore potential correlation with patient prognosis and response to therapy. PRIMERA is a prospective observational trial enrolling mCRPC patients undergoing standard treatment (ARTA + ADT) after I line ADT failure. Clinical and pathological features were collected. Outcomes selected for this preliminary analysis were time to castration resistance (TTCR), PSA at 8 weeks after ARTA therapy start, PSA drop at 8 weeks, Overall PSA drop, PSA nadir. Correlation between these outcomes and CTC detection was tested. Expression of ARV7, ARFL, PSA and PSMA was explored in CTC+ patients to assess their prevalence in this cohort and their impact on selected outcomes. Median TTCR was significantly shorter in CTC+ vs CTC- patients (32.3 vs 75 months, respectively, p = 0.03) and in ARFL+ vs ARFL- patients (30.2 vs 51.1 months, respectively, p = 0.02). ARV7, PSMA and PSA expression on CTC had no impact on median TTCR, nor on biochemical response to therapy. Patients in whom CTC and ARFL expression were detected had significant reduced TTCR. However, PSA response was not influenced by CTCs detection and specific biomarkers expression.

Evaluating determinants of receipt of molecular imaging in biochemical recurrent prostate cancer.

BACKGROUND: Molecular imaging with novel radiotracers is changing the treatment landscape in prostate cancer (PCa). Currently, standard of care includes either conventional and molecular imaging at time of biochemical recurrence (BCR). This study evaluated the determinants of and cost associated with utilization of molecular imaging for BCR PCa. METHODS: This is a retrospective observational cohort study among men with BCR PCa from June 2018 to May 2019. Multivariate logistic regression models were employed to analyze the primary outcome: receipt of molecular imaging (e.g. Fluciclovine PET and Prostate Specific Membrane Antigen PET) as part of diagnostic work-up for BCR PCa. Multivariate linear regression models were used to analyze the secondary outcome: overall healthcare cost within a 1-year time frame. RESULTS: The study sample included 234 patients; 79.1% White, 2.1% Black, 8.5% Asian/Pacific Islander, and 10.3% Other. The majority were 55 years or older (97.9%) and publicly insured (74.8%). Analysis indicated a one-unit reduction in PSA is associated with 1.3 times higher likelihood of receiving molecular imaging (p < 0.01). Analysis found that privately insured patients were associated with approximately $500,000 more in hospital reimbursement (p < 0.01) as compared to the publicly insured. Additionally, a one-unit increase in PSA is associated with $6254 increase in hospital reimbursement or an increase in total payments by 2.1% (p < 0.05). CONCLUSIONS: Higher PSA was associated with lower likelihood for molecular imaging and higher cost in a one-year time frame. Higher cost was also associated with private insurance, but there was no clear relationship between insurance type and imaging type.

A Monte Carlo framework for managing biological variability in manufacture of autologous cell therapy from mesenchymal stromal cells therapies.

Manufacturing processes for autologous cell therapy need to reproducibly generate in specification (quality and quantity) clinical product. However, patient variability prevents the level of control of cell input material that could be achieved in a cell line or allogeneic-based process. We have applied literature data on bone marrow-derived mesenchymal stromal cells variability to estimate probability distributions for stem cell yields given underlying truncated normal distributions in total nucleated cell concentration, stem cell percentage and plausible aspirate volumes. Monte Carlo simulation identified potential variability in harvested stem cell number in excess of an order of magnitude. The source material variability was used to identify the proportion of donor manufacturing runs that would achieve a target yield specification of 2E7 cells in a fixed time window with given proliferative rates and different aspirate volumes. A rapid, screening, development approach was undertaken to assess culture materials and process parameters (T-flask surface, medium, feed schedule) to specify a protocol with identified proliferative rate and a consequent model-based target aspirate volume. Finally, four engineering runs of the candidate process were conducted and a range of relevant quality parameters measured including expression of markers CD105, CD73, CD44, CD45, CD34, CD11b, CD19, HLA-DR, CD146 (melanoma cell adhesion molecule), CD106 (vascular cell adhesion molecule) and SSEA-4, specific metabolic activity and vascular endothelial growth factor secretion, and osteogenic differentiation potential. Our approach of using estimated distributions from publicly available information provides a route for data-poor earl- stage developers to plan manufacture with defined risk based on rational assumptions; furthermore, the models produced by such assumptions can be used to evaluate candidate processes, and can be incrementally improved with accumulating distribution understanding or subdivision by new process variables.

A decision analysis comparing 3 active surveillance protocols for the treatment of patients with low-risk prostate cancer.

BACKGROUND: Active surveillance (AS) is a viable management option for approximately 50% of men who are newly diagnosed with prostate cancer. To the authors' knowledge, no direct comparisons between the different variants of AS protocols have been conducted to date. The authors developed a microsimulation decision model to evaluate which of 3 alternative AS protocols is optimal for men with low-risk prostate cancer, and compared each of these with immediate treatment. METHODS: Men who were diagnosed with low-risk prostate cancer at age 65 years were modeled as having been treated with either immediate therapy or via each of 3 AS protocols. Modeled AS protocols represent those in the literature; a modified AS protocol was included in a sensitivity analysis. Immediate therapy included radical prostatectomy, external-beam radiotherapy, or brachytherapy. Outcome measures were quality-adjusted life-years (QALYs) and costs. Cost-effectiveness analysis and deterministic and probabilistic sensitivity analyses were performed. RESULTS: Immediate therapy produced fewer QALYs than all variants of AS. Of the AS protocols evaluated, biennial biopsy was found to be the only efficient option, with an incremental cost-effectiveness ratio of $3490 per QALY compared with immediate therapy. It delayed the need for curative therapy by a mean of 56 months, and was found to be preferred in >86.9% of cases in probabilistic sensitivity analysis. A modified version of low-intensity AS dominated all other options. CONCLUSIONS: For a 65-year-old man with low-risk prostate cancer, AS with biennial biopsy appears to be highly cost-effective compared with common alternatives. An AS protocol using triennial biopsy was found to dominate all other strategies and should be considered for men who are comfortable with a longer period between biopsies. The optimal strategy depends on a patient's tolerance for periodic biopsies and comfort with delaying radical treatment. Physicians should incorporate these patient preferences into decision making.

Assessment of sperm antigen specific T regulatory cells in women with recurrent miscarriage.

BACKGROUND AND AIMS: The prevalence of recurrent miscarriage (RM) is about 1-3% of women; the pathogenesis of RM is not fully understood yet. This study aims to assess the sperm antigen specific regulatory T cells (Treg) in women with RM. METHODS: A group of women with RM was recruited into this study. The sperm antigen was extracted from the semen samples of each woman's husband. The sperm antigen specific T cell response was assessed by flow cytometry. RESULTS: Low frequency of sperm specific Tregs and high frequency of T helper (Th)1 cells were detected in RM women as compared with women without RM. The sperm specific Tregs in RM women expressed less Ubc13. Knockdown of Ubc13 from Tregs converted the Tregs to effector T cells. CONCLUSIONS: Immune deregulation may play an important role in RM.

Assessment by electron-microscopy of recombinant Vibrio cholerae and Salmonella vaccine strains expressing enterotoxigenic Escherichia coli-specific surface antigens.

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) requires adhesion of microorganisms to enterocytes. Hence, a promising approach to immunoprophylaxis is to elicit antibodies against colonisation factor antigens (CFAs). Genes encoding the most prevalent ETEC-specific surface antigens were cloned into Vibrio cholerae and Salmonella vaccine strains. Expression of surface antigens was assessed by electron-microscopy. Whereas negative staining was effective in revealing CFA/I and CS3, but not CS6, immunolabelling allowed identification of all surface antigens examined. The V. cholerae vaccine strain CVD103 did not express ETEC-specific colonisation factors, whereas CVD103-HgR expressed CS3 only. However, expression of both CFA/I and CS3 was demonstrated in Salmonella Ty21a.

Identification and stoichiometry of glycosylphosphatidylinositol-anchored membrane proteins of the human malaria parasite Plasmodium falciparum.

Most proteins that coat the surface of the extracellular forms of the human malaria parasite Plasmodium falciparum are attached to the plasma membrane via glycosylphosphatidylinositol (GPI) anchors. These proteins are exposed to neutralizing antibodies, and several are advanced vaccine candidates. To identify the GPI-anchored proteome of P. falciparum we used a combination of proteomic and computational approaches. Focusing on the clinically relevant blood stage of the life cycle, proteomic analysis of proteins labeled with radioactive glucosamine identified GPI anchoring on 11 proteins (merozoite surface protein (MSP)-1, -2, -4, -5, -10, rhoptry-associated membrane antigen, apical sushi protein, Pf92, Pf38, Pf12, and Pf34). These proteins represent approximately 94% of the GPI-anchored schizont/merozoite proteome and constitute by far the largest validated set of GPI-anchored proteins in this organism. Moreover MSP-1 and MSP-2 were present in similar copy number, and we estimated that together these proteins comprise approximately two-thirds of the total membrane-associated surface coat. This is the first time the stoichiometry of MSPs has been examined. We observed that available software performed poorly in predicting GPI anchoring on P. falciparum proteins where such modification had been validated by proteomics. Therefore, we developed a hidden Markov model (GPI-HMM) trained on P. falciparum sequences and used this to rank all proteins encoded in the completed P. falciparum genome according to their likelihood of being GPI-anchored. GPI-HMM predicted GPI modification on all validated proteins, on several known membrane proteins, and on a number of novel, presumably surface, proteins expressed in the blood, insect, and/or pre-erythrocytic stages of the life cycle. Together this work identified 11 and predicted a further 19 GPI-anchored proteins in P. falciparum.

Nerve guide material made from fibronectin: assessment of in vitro properties.

We have previously used orientated mats of fibronectin as conduits to repair short gaps in peripheral nerves. Here we describe the in vitro properties of a new material in the form of large cables produced from a fibronectin-enriched solution with potential as a conduit for longer nerve defects. Large cables of fibronectin were made up to 14 cm long x 1.5 cm in diameter. When freeze dried, scanning electron microscopy revealed a predominant fiber orientation. Dried cables hydrated rapidly to 1.6 and 4.8 times their original length and diameter, respectively. Once hydrated these cables had pores that ranged from 10 to 100 microm through which Schwann cells and fibroblasts were able to grow in vitro and align with the axis of the fibrils by contact guidance. Furthermore, the porosity of the cable was enhanced by the natural dissolution of protein over a 3-week duration in culture with cells, such that 50- to 200-microm pores were observed. This study suggests that large fibronectin cables are a suitable alternative to the original fibronectin mats to guide components of the peripheral nerves and so to act as conduits with potential use in guiding regeneration across long nerve defects.

Identification and functional assessment of endothelial P1H12.

Monoclonal antibody P1H12 recognizes circulating endothelial cells and endothelia of all sizes of blood vessels. To identify the protein recognized by P1H12, we expressed a cDNA library in CHO cells and sequenced the cDNA from positive cells. The P1H12 sequence was identical, except at several bases, to that reported for melanoma cell surface antigen MUC18/CD146. Aggregation assays demonstrated that CD146 mediates Ca(++)-independent homotypic endothelial cell adhesion. P1H12 mAb abrogated interactions between human microvascular endothelial cells (HMVECs) but not between human umbilical vein endothelial cells (HUVECs). P1H12 mAb abrogated P1H12-positive (CHO(P1H12))-association with HMVECs or HUVECs. CD146 distribution is sparser on HUVECs than on HMVECs. These data imply that HMVECs and HUVECs express the CD146 binding partner but that CD146 is functional (or at sufficient density) only on HMVECs. HMVEC monolayers treated with soluble P1H12 mAb showed increased permeability to albumin, with accompanying changes in actin, paxillin, FAK, and caveolin distribution and changes in tyrosine phosphorylation of FAK. Stimulation with P1H12 mAb led to redistribution of NF-kappa B to the nucleus. P1H12 mAb bound to beads inhibited closure of wounded endothelial monolayers. CD146 thus joins VE-cadherin and PECAM-1 as a molecule that mediates homotypic endothelial cell adhesion. CD146 has both structural functions and signaling functions important for endothelial monolayer integrity.

Flow cytometric assessment of the reactivity of a panel of monoclonal antibodies (mAb) against two populations of human dendritic cells (DC).

BACKGROUND: The identification of antigens on human DC has been a very difficult and elusive task because of the lack of appropriate reagents. Therefore, we evaluated by flow cytometry a panel of mAb that recognize antigens on human DC, aiming to determine the kinetics of DC antigen expression at 7, 14, 21 and 28 days in (i) Dermal DC like cells (Mo-DC) and (ii) Langerhans cell like DC (Mo-LC). In addition we aimed to identify markers for DC subpopulations. RESULTS: It was found at day 7, that mAb BG6, HP-F1, BU10, RFD-1, CMRF-44 recognized <20% of Mo-DC. In contrast, 7H5, ZM3.8, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 reacted with >50% of Mo-DC. Moreover, 7H5, ZM3.8, CMRF-56, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 showed increased MFI reactivity against Mo-DC. mAb BG6, BU10 and CMRF-44 recognized <20% Mo-LC while RFD-1 reacted with 21% of Mo-LC. In contrast, HP-F1 showed 87% of Mo-LC positive. Also, 7H5, ZM3.8, RFD-7, MR15-2, CDlb/c, 55K-2, MMR1.16, MMR190.BB3 and L25 reacted with >50% of Mo-LC. The increase in % of positive cells was paralleled by MFI increases. At day 14, fourteen mAb recognized >50% of the Mo-DC, while five recognized 20-50% of Mo-DC. BG6 reacted with 7% of the Mo-DC. Nineteen mAb recognized >48% of Mo-LC while BG6 had negative reactivity. At day 21 and 28, all mAb reacted with >20% of Mo-DC and yielded a significant MFI with Mo-DC. Also nineteen mAb yielded significant MFI with Mo-LC while RFD-7 did not. CONCLUSIONS: The immunophenotyping assays demonstrated differences between the two DC populations as well as variations in the reactivity of the mAb at diverse time points, suggesting the existence of subpopulations within the Mo-DC and Mo-LC.

Quantitative assessment of the normal cerebral microvasculature by endothelial barrier antigen (EBA) immunohistochemistry: application to focal cerebral ischemia.

Cerebrovascular endothelium participates importantly in the pathophysiology of ischemic injury. Endothelial barrier antigen (EBA) is a protein located in the luminal plasma membrane of normal central and peripheral nervous-system endothelium. In this study, we assessed the sensitivity and specificity of EBA as a quantitative marker of normal endothelium and characterized alterations of EBA immunohistochemistry following focal cerebral ischemia. Anesesthetized, non-ischemic control rats (N=6) were studied. Other animals (N=5) received 90 min of middle cerebral artery occlusion (MCAo) followed by 3-day survival. Brains were prepared by perfusion-fixation and paraffin-embedding. For EBA immunohistochemistry, a monoclonal antibody (1:2000 dilution) was used. Adjacent sections were reacted for activated microglia by isolectin immunochemistry. Morphometric image-analysis was carried out in standardized microscopic fields. In control brains, pial and parenchymal blood vessels of all sizes were distinctly and selectively immunolabeled for EBA; background staining was absent. EBA-positive vascular profiles occupied 4.3+/-0.36% (mean+/-S.D.) of the microscopic field. The mean area of each identified profile was 51+/-13 micromter(2). The low coefficients of variation for both numbers of profiles (17%) and fractional areas (8%) denoted high inter-animal consistency. In brains with prior MCAo, numbers of EBA-immunoreactive vascular profiles in infarcted cortex and striatum were reduced by 39 and 46%, respectively, and their fractional areas were decreased by 63 and 76%, respectively, compared to contralateral hemisphere. Activated microglia were prominent in zones of frank infarction and in adjacent paramedian cortex; the latter region, however, showed normal-appearing EBA-immunostaining. EBA-immunohistochemistry provides a sensitive and specific index of normal cerebrovascular endothelial structures of all sizes. The technique lends itself well to quantitative morphometry and is applicable to perfusion-fixed paraffin-embedded material. EBA immunoreactivity declines in zones of ischemic infarction.

Assessment of the interaction of human complement regulatory proteins with group A Streptococcus. Identification of a high-affinity group A Streptococcus binding site in FHL-1.

Group A Streptococcus (GAS), the most frequent bacterial cause of suppurative infections in humans, expresses on the cell surface M proteins with capacity to bind factor H, FHL-1 and C4b binding protein (C4BP). This has been interpreted as a mechanism developed by this pathogen to decrease phagocytosis by macrophages and polymorphonuclear cells. We report the analysis of the capacity to bind factor H, FHL-1 and C4BP of 69 clinical isolates from 19 different serotypes. We show that strains binding complement regulators (30/69) belong to specific M serotypes. Of these, M18 strains are relatively frequent and interact with all three complement regulators simultaneously. However, the most virulent M1 and M3 strains did not bind complement regulators in our assays. The relevance of the interaction between complement regulators and S. pyogenes was analyzed using different approaches with the conclusion that under physiological conditions only FHL-1 and C4BP bind to streptococci. We show that FHL-1 presents a higher binding affinity for S. pyogenes than factor H because it carries a hydrophobic, high-affinity, GAS binding site in addition to the heparin binding site in SCR7. Using synthetic peptides we provide evidence that the high-affinity GAS binding site in FHL-1 involves the hydrophobic tail (Ser-Phe-Thr-Leu) that distinguishes FHL-1 from factor H.

Calprotectin and lactoferrin levels in the gingival crevicular fluid of children.

The purpose of this study was to determine the levels of calprotectin and lactoferrin, 2 microbiostatic proteins, in the gingival crevicular fluid (GCF) of normal children. The children represented a population, primarily underprivileged, seeking care at a regional dental health care center. GCF was collected from Ramfjord teeth (or their deciduous equivalent). GCF volume was quantified by conductance. Calprotectin and lactoferrin levels were quantified by sandwich ELISA, and found to have a mean value of 70.8+/-94.2 microg/mL and 68.2+/-108.7 microg/mL, respectively. The levels of calprotectin and lactoferrin varied directly with one another and inversely with the amount of fluid obtained in a 20-second sampling period. The mean levels were at or above the minimum inhibitory concentration determined in vitro.

Intrahepatic lymphocyte analyses and assessment of the effects of levamisole in murine hepatic metastasis model.

We used hepatic metastasis models to determine the mechanism and effect of levamisole. BALB/c mice and Colon 26 cells were used. Group I was injected with tumor cells through the portal vein. Group II was primed with tumor cells before tumor cells injecting. Group III was same as Group II, but treated with levamisole. Surface antigens of intrahepatic lymphocytes and spleen cells were determined by FACScan with Anti-CD3, anti-CD4, anti-CD8, anti-CD45, anti-NK1.1 and anti-F4/80. Nodules on the liver were greatest in Group I and fewest in Group III. Concerning intrahepatic lymphocytes, Group II, when compared with Group I, had increases of CD3+, CD4+ and CD8+ cells, and decreases of CD45+ and NK1.1+ cells. Group III when compared with Group II, showed increased CD8+ cells and decreased of NK1.1+ cells. Levamisole is considered to be effective in the prevention of liver metastasis and is suggeste to enhanced CD8+ cells.

Assessment of epidermal dendritic cell markers and T-lymphocytes in psoriasis.

Epidermal dendritic and T-cell counts have been performed in lesional and non-lesional skin from 35 psoriatic patients. The aims were to investigate absolute changes and interrelationships between these cellular elements in psoriasis and to explain apparent discrepancies between these results and reports in the literature. In non-lesional skin, the most frequently expressed dendritic cell marker was CD1a. HLA-DR+ and alpha-mannosidase+ dendritic cells were approximately 50 per cent and S100+ cells were 25 per cent as frequent. T-lymphocytes were rare, CD4+ cells predominating. In lesional psoriatic epidermis, there was a definite increase (approximately two-fold) in the absolute number of CD1a+ dendritic cells. This differs from the conclusions from the majority of previous studies. However, when cell counts were expressed per unit area of vertical section, there was a decrease in CD1a+ cells in lesional skin, which is an explanation for this discrepancy. There was a greater increase in absolute HLA-DR+ cell counts, so that the numbers of cells expressing CD1a and HLA-DR were similar in lesional skin. S100 expression increased proportionately with CD1a+, but there was no absolute increase in alpha-mannosidase+ cells, which might represent a separate sub-population of dendritic cells. The greatest cellular increase was in T-lymphocytes, particularly CD8+. In lesional skin, direct correlations have been demonstrated between epidermal thickness, HLA-DR+ dendritic cells and T-lymphocytes, particularly CD8+ cells. We would suggest that the present method of quantification is of value for the analysis of absolute changes in epidermal infiltrates, particularly psoriasis, and could be applied to other epidermal pathologies.

Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study.

This study describes methods for extraction and quantification of calprotectin (L1 protein) in feces by enzyme immunoassay. This protein is a prominent antimicrobial component of neutrophils, monocytes, macrophages, and squamous epithelia. Calprotectin was stable in feces during storage for 7 days at room temperature. Small fecal samples taken from a 24-h feces collection gave a reliable estimate of calprotectin. Within-assay precision was 1.9%, and between-assay precision 14.8%. In healthy subjects (n = 33) median fecal calprotectin was 2025 micrograms/l and in hospital controls (n = 40) 10,500 micrograms/l. Median values in patients with Crohn's disease (n = 21) was 43,000 micrograms/l and in ulcerative colitis (n = 17) 40,000 micrograms/l. Fecal calprotectin was significantly correlated to fecal alpha 1-antitrypsin in the patients with Crohn's disease. Ten of 11 patients with gastrointestinal carcinomas had calprotectin level above the suggested reference limit of 6740 micrograms/l.

Immunohistochemical demonstration of B and T-lymphocyte cell surface markers: an assessment of tissue transport/storage media.

A number of solutions were tested on lymphoid tissue with a view to devising a tissue transport/long-term storage medium which would preserve the morphology and antigenicity of lymphocytes, for demonstration of B- and T-cell surface markers using monoclonal antibodies. Some of the solutions produced total loss of any recognisable tissue structure, but one newly-devised medium gave excellent preservation of morphology and nuclear detail, together with extremely good monoclonal antibody staining. This sterilised medium, Carmichael's medium*, contains a buffer and cryoprotectant: it forms a gel at 4 degrees C, but it is easily liquefied in warm water just prior to immersion of the biopsy. Sections from tissue preserved in Carmichael's Medium were found to be of equally good quality as, and in some cases better than, those which had been fresh frozen. *International patents are currently pending on the formulation of this medium, which will be available commercially through Raymond Lamb Ltd, London, England, UK.

Humoral immunodeficiency in T-cell chronic lymphocytic leukemia. An immunologic assessment.

The humoral antibody immunodeficiency in two patients with T-cell chronic lymphocytic leukemia (T-CLL) appeared to be the result of immunoregulatory abnormalities in the leukemic T-cell populations. Both patients had CD4+ CD45R+ "virgin" or suppressor-inducer T-CLL, but Patient 1 had hypogammaglobulinemia and Patient 2, immunoglobulin (Ig) M hypergammaglobulinemia. Although, CD25+ interleukin-2 (IL-2) receptors were present on leukemic T-cells of both patient, OKT9+ (CD71) transferrin receptors and OKT10 (CD38) activation antigens were found only on Patient 2's cells. Highly elevated amounts of IL-2 was secreted from phytohemagglutinin-stimulated and concanavalin A-stimulated T-cells in both patients. In Patient 1 with hypogammaglobulinemia, immune defects involve T-cells, first an intense suppressor activity on B-cell-induced IgM and IgG synthesis and, second, deficient production of B-cell growth factor (BCGF) and B-cell differentiation factor (BCDF). In Patient 2, highly elevated BCGF and IgM-specific BCDF was secreted by T-cells, a mechanism leading to IgM hypergammaglobulinemia in this patient. These studies stress the importance of BCGF and BCDF activity of leukemic T-cells in humoral antibody immunodeficiency disorders in T-CLL cases.

Simultaneous assessment of chromatin structure, DNA content, and antigen expression by dual wavelength excitation flow cytometry.

7-aminoactinomycin D (7-AMD) efficiently discriminates between cells in the G0 and G1 phases of the cell cycle (Stokke et al., Cancer Res. 48:6708, 1988). The fluorescence and light scatter of cells stained with 7-AMD, Hoechst 33258 (H33258), and fluorescein isothiocyanate (FITC)-labeled antibodies were measured by dual wavelength excitation flow cytometry (488 nm, ultraviolet). The H33258 fluorescence was found to reflect DNA content in the presence of 7-AMD, although energy transfer caused an approximately 50% reduction in H33258 fluorescence intensity. However, energy transfer was more pronounced in dead cells, permitting exclusion of such cells during analysis. The G0, G1, S, and G2 phases of the cell cycle could be identified in the 7-AMD versus H33258 fluorescence histograms, as was demonstrated with mitogen-stimulated B lymphocytes and a mixture of unstimulated B lymphocytes and a proliferating B-cell line. One hour fixation with paraformaldehyde was compatible with prefixation labeling of surface antigens with indirectly FITC-labeled antibodies as well as postfixation labeling of intracellular antigens. Studies of expression of some surface and nuclear activation-associated antigens confirmed that cell cycle-resolved antigen expression and the time course of appearance of such antigens could be assessed accurately. Phycoerythrin could be used to label a second antigen.

Combined flow cytometric assessment of cell surface antigens and nuclear TdT for the detection of minimal residual disease in acute leukaemia.

To define more precisely the immunophenotype of lymphoid blast cells, a new flow cytometric technique for the simultaneous detection of surface antigens and nuclear terminal deoxynucleotidyl transferase (TdT) was established. After staining for the cell surface marker, mononuclear cells were treated with paraformaldehyde (1%) and methanol to permeabilize the cell membrane. Then the cells were stained by indirect immunofluorescence using a rabbit anti-human TdT antibody. Dilution experiments were performed to reveal the sensitivity of the described flow cytometric assay: 0.02% leukaemic cells could reliably be detected above background level among normal peripheral blood lymphocytes. It is concluded that the double-staining procedure described here is a sensitive tool that contributes to the detection of minimal residual disease in a substantial proportion of acute leukaemias.