T cell receptor V gene usage by human T cells stimulated with the superantigen streptococcal M protein.

M proteins, the major virulence factor of group A streptococci, have been implicated in the pathogenesis of acute rheumatic fever (ARF) and other streptococcal related autoimmune diseases. A 22-kD fragment of M type 5 protein is a potent stimulant of human T cells and has recently been shown by our laboratory to belong to the newly designated family of superantigens. Using flow cytometry and the polymerase chain reaction, we demonstrate that this molecule reacts with subsets of human T cells expressing specific T cell receptor (TCR) V beta elements, namely V beta 2, 4, and 8. We employed similar techniques to analyze the TCR V alpha usage of pep M5-stimulated T cells. These studies revealed that the preferential usage of particular V alpha elements is not specific for the superantigen; rather, it may reflect the repertoire of the individual being tested. The expansion of a large number of T cells bearing specific TCR V beta sequences by M protein may account for its role in mediating the pathogenesis of post-streptococcal diseases. Furthermore, the preferential usage of TCR V alpha elements in certain individuals may be an important factor that predisposes them to development of self-reactivity.

Membrane skeletal alterations during in vivo mouse red cell aging. Increase in the band 4.1a:4.1b ratio.

We have examined membrane protein profiles for alterations during red blood cell aging. To obtain populations of in vivo-aged red cells, we maintained mice in a state of continuous erythropoietic suppression for up to 8 wk using serial hypertransfusion. The circulating t1/2 of red cells from mice which had been erythropoietically suppressed for 8 wk was less than 1 d compared with a t1/2 of 15 d for red cells from normal animals. The most obvious alteration in membrane proteins was an increase in the ratio of the membrane skeletal components 4.1a:4.1b from 0.3 for the normal red cell population to greater than 1 for these old cells. The 4.1a:4.1b ratio thus appears to be a useful index of red cell age. Analyses of the density profile of cells aged in the hypertransfused mice disclosed that these old cells had a density range similar to that of controls, suggesting that cell density does not increase significantly with red cell age in the mouse.

Quantitative variation of the common acute lymphoblastic leukemia antigen (gp100) on leukemic marrow blasts.

Marrow blasts from children with B cell precursor acute lymphoblastic leukemia (ALL) were studied for differences in quantitative expression of the common ALL antigen (CALLA). Of 42 untreated patients, 35 had detectable amounts of CALLA by flow cytometric (FCM) analysis of J-5 monoclonal antibody binding. Using an FCM technique that provides correlated measurements of a given cell surface antigen, cell size, and DNA content, we detected increased CALLA expression as lymphoblasts moved from G0/G1 phase through S phase of the cell cycle. The density of the antigen (per unit of blast surface area) remained relatively constant over the same interval, indicating that the change was not due to S phase-specific enhancement of CALLA expression. Eight cases had hyperdiploid cellular DNA content and in seven of these, only cells with clonal abnormalities of DNA content expressed the CALLA marker. Mean amounts of CALLA for each patient ranged widely within the study group, from very high to marginally detectable. This variation had no discernible relation to cell size, stem-line DNA content, percentage of cells in S phase, or the presence or absence of cytoplasmic immunoglobulin. Results of a univariate proportional hazards analysis showed that both quantitative level of CALLA for S phase cells (P = 0.048) and white blood cell count (P = 0.012) had made significant contributions to treatment outcome. Patients with relative amounts of CALLA less than the median value for the entire CALLA+ group had a higher rate of failure, which was virtually identical to that for the seven HLA-DR+ patients whose blasts lacked detectable CALLA. The observed interpatient variation in quantitative expression of CALLA is consistent with recognized steps in B cell precursor differentiation and may be useful in distinguishing patients with a less favorable prognosis.

Resistance to N-benzyladriamycin-14-valerate in mouse J774.2 cells: P-glycoprotein expression without reduced N-benzyladriamycin-14-valerate accumulation.

N-Benzyladriamycin-14-valerate (AD 198) is a highly lipophilic analogue of Adriamycin with novel cytotoxic mechanisms, greater in vivo antitumor activity, and the ability to circumvent multidrug resistance due to P-glycoprotein-mediated drug efflux or decreased topoisomerase II activity. To identify the mechanism(s) which may confer AD 198 resistance, J774.2 mouse macrophage-like cells were selected for growth in cytotoxic levels of AD 198 (AD 198R). AD 198R cells exhibited over-expression of the mdr1b (P-glycoprotein) gene, cross-resistance to Adriamycin and vinblastine, and potentiation of drug cytotoxicity by verapamil. However, net intracellular accumulation of AD 198 in AD 198R cells was unchanged compared to parental cells, while Adriamycin and vinblastine accumulations were reduced 40% and 95%, respectively. AD 198 was localized in the perinuclear region of the cytoplasm in both parental and AD 198R cells, with additional vesicular compartmentalization in AD 198R cells. Verapamil-induced reversal of AD 198 resistance coincided with some drug redistribution from cytoplasmic vesicles, but without redistribution of AD 198 into the nucleus. These results suggest that AD 198 resistance was not conferred through a P-glycoprotein-mediated reduction in intracellular drug accumulation but through other cytoplasmic mechanisms, including, but not limited to, drug compartmentalization.

Evidence against an abnormal hepatic microsomal lipid matrix as the primary genetic defect in the jaundiced Gunn rat.

The congenitally jaundiced Gunn rat does not conjugate bilirubin but does conjugate bilirubin dimethyl diester. Partial defects in conjugating p-nitrophenol and demethylating aminopyrine are also evident. A proposed mechanism to explain this combination of findings is a defective microsomal membrane. To examine the 'matrix' of Gunn microsomal membranes, hepatic microsomes were isolated from Gunn (jj) and outbred Wistar (JJ) rats and were studied by electron paramagnetic resonance spectroscopy of 7-doxylstearic and 12-doxylstearic acid probes, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, glucose-6-phosphatase activity vs. temperature, and lipid analysis. The data indicate several factors related to lipid bilayer order do not differ in microsomes from jj and JJ.

Calcitonin inhibits thyrotropin-releasing hormone-induced increases in cytosolic Ca2+ in isolated rat anterior pituitary cells.

Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells. Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect. Secretogogue-induced elevations of cytosolic free Ca2+ ([Ca2+]i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry. AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 microM Indo-1 AM for 30 min. Indo-1-loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of [Ca2+]i, was determined for each cell. Under basal conditions, AP cells showed stable Indo-1 ratios during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio. Approximately 25-30% of the AP cells responded to a 1 microM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in [Ca2+]i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA. In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 microM TRH. In EGTA-pretreated AP cells, the initial EGTA-insensitive phase of the TRH-induced [Ca2+]i increase was also abolished by prior exposure to sCT. These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in [Ca2+]i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system.

Neuropeptide-Y enhances luteinizing hormone (LH)-releasing hormone-induced LH release and elevations in cytosolic Ca2+ in rat anterior pituitary cells: evidence for involvement of extracellular Ca2+ influx through voltage-sensitive channels.

The present studies were designed to investigate the mechanism by which neuropeptide-Y (NPY) augments the effect of LHRH to stimulate the release of LH from cultured rat anterior pituitary cells. Anterior pituitary cells from ovariectomized rats were enzymatically dispersed, cultured for 3 days, and then exposed to various secretagogues during 3-h incubations. As reported by this laboratory previously, NPY alone (100 nM) did not affect LH release, but significantly enhanced the LH response to 1 nM LHRH. This facilitatory action of NPY was mimicked by the dihydropyridine Ca2+ channel agonist Bay K 8644 (1 microM), and the enhancement of LHRH-induced LH release by either NPY or Bay K 8644 was prevented by the dihydropyridine antagonist nitrendipine (1 microM). Nitrendipine alone reduced the response to LHRH by approximately 25%, but did not affect basal LH release. In contrast, NPY failed to amplify the release of PRL in response to TRH, another Ca2(+)-mobilizing hormone. To test whether NPY also enhances the increase in cytosolic Ca2+ induced by LHRH, anterior pituitary cells were acutely dispersed into single cell suspensions, loaded with the fluorescent Ca2+ probe Indo-1 AM, and analyzed with a UV laser in an EPICS-753 flow cytometer at a rate of 500 cells/sec for 200 sec. The ratio of intracellular fluorescence resulting from Ca2+ bound to the Indo-1 to the fluorescence from Indo-1 alone (Indo-1 ratio), which is an index of the concentration of free cytosolic Ca2+, was determined for each cell. Approximately 7% of anterior pituitary cells responded to LHRH (1 or 10 nM) with significant increases in Indo-1 ratios, indicative of an increase in the concentration of free cytosolic Ca2+. EGTA (2.5 mM) reduced the basal Indo-1 ratios and attenuated, but did not abolish, the initial increase in response to LHRH, consistent with the initial extracellular Ca2+ influx-independent phase of the response to LHRH. NPY alone (100 nM) did not affect the Indo-1 ratios in anterior pituitary cells, but pretreatment with the peptide for 10 min before the scans significantly augmented the Indo-1 ratio response to 10 nM LHRH. This effect of NPY was also blocked by EGTA. Taken together, these biochemical and pharmacological studies suggest that NPY enhances the release of LH stimulated by LHRH by increasing extracellular Ca2+ entry, possibly by selectively affecting that component of the response involving dihydropyridine-sensitive L-type voltage-sensitive Ca2+ channels during the initial stages of the cellular response to LHRH.

Isolation and characterization of lactose-binding lectins from the venoms of the snakes Lachesis muta and Dendroaspis jamesonii.

Two lectins have been isolated: one from the venom of Lachesis muta (bushmaster lectin) and one from Dendroaspis jamesonii venom (Jameson's mamba lectin). The lectin from bushmaster venom (BML) is similar to the lactose-binding lectins previously isolated from snake venoms (Gartner et al. (1980) FEBS Lett. 117, 13-16; Gartner & Ogilvie (1984) Biochem. J. 224, 301-307) in that it is calcium-dependent, lactose inhibitable, and is a dimer of molecular weight 28,000. In contrast, the lactose-blockable lectin from Jameson's mamba venom (JML) has an apparent molecular weight of 26,000 and agglutinates erythrocytes in the presence of EDTA. The absorption spectra of BML were affected by the binding of calcium, or calcium and lactose to the lectin. However, JML spectra were not affected by these conditions. While the hemagglutination activity of each of the previously described lactose-binding snake venom lectins is inhibited by reducing agent, the activities of BML and JML are not affected by reducing agent. Antiserum against bushmaster lectin cross-reacts with thrombolectin, cottonmouth lectin (CML), rattlesnake lectin (RSL), and copperhead lectin (CuHL) but not lectin from Jameson's mamba venom. This evidence plus a comparison of atomic absorption spectra, isoelectric points and amino acid analyses of the lectins demonstrate that JML and BML are different from thrombolectin, CML, RSL, and CuHL.

Flow cytometry provides rapid and highly accurate detection of antisperm antibodies.

OBJECTIVE: Immunobead testing (IBT), the current standard for antisperm antibody detection, is time consuming and somewhat subjective. To overcome these limitations and maintain accuracy, we studied an immunofluorescent assay using flow cytometry. DESIGN: A validation study comparing flow cytometry to IBT in the detection of serum antisperm antibodies. SETTING: Flow cytometry laboratory. PATIENTS: Sera from 37 men after vasectomy (test) and sera from 35 fertile men (control). MAIN OUTCOME MEASURE: Test serum with and without immunoglobulin (Ig)G, IgA, and IgM antisperm antibodies as defined by IBT were analyzed by flow cytometry. Sensitivity and specificity of flow cytometry was calculated by defining the IBT as the true result. RESULTS: Flow cytometry identified 22 of 22 sera that were IgG positive (100% sensitivity), 12 of 14 sera that were IgA positive (86% sensitivity), and 4 of 4 sera that were IgM positive (100% sensitivity). Overall, 22 of 37 men were positive for antisperm antibodies. The flow cytometry correctly identified 71 of 71 negative sera (100% specificity). Fluorescence intensity values from the 37 study patients significantly correlated with immunobead binding to the head region and to the entire (more than one) region. CONCLUSIONS: Detection of IgG, IgA, and IgM antisperm antibodies by flow cytometry is highly sensitive and specific. In addition, flow cytometry is able to assess thousands of sperm rapidly and accurately, reducing sampling error and technical time.

Secreted platelet thrombospondin binds monovalently to platelets and erythrocytes in the absence of free Ca2+.

Washed human platelets suspended in Ca2+-free buffer bind thrombospondin secreted in response to stimulation by the calcium ionophore A23187. Under these conditions, the secreted thrombospondin binds to the surface of the platelets monovalently, that is, the thrombospondin does not agglutinate the platelets. In addition, the secreted thrombospondin can bind monovalently to the surface of the erythrocytes used to assay the endogenous lectin of human platelets. These findings resolve the contradictions resulting from the apparent requirement for free Ca2+ in the binding of secreted thrombospondin to the plasma membranes of platelets, the behavior of purified thrombospondin in hemagglutination assays and the characteristics of the endogenous lectin (thrombospondin) expressed by platelets stimulated with A23187 or gamma-thrombin.

External Na+ is not required for Ca2+ mobilization in platelets stimulated with rattlesnake lectin or alpha-thrombin.

The effects of extracellular sodium on platelet aggregation and calcium mobilization in platelets stimulated with either rattlesnake (Crotalus atrox) lectin (RSL) or alpha-thrombin were compared. The absence of extracellular sodium had no effect on platelet aggregation or calcium mobilization in response to all levels of RSL tested. In contrast platelet aggregation was sodium-dependent in response to less than or equal to .2 units/ml alpha-thrombin. Surprisingly, calcium mobilization occurred in platelets treated with a threshold level of alpha-thrombin in the absence of external sodium. Thus sodium-dependent platelet aggregation in response to a low dose of thrombin apparently is not the result of sodium-dependent calcium mobilization.

Fluorescent photochemical surface labeling of intact human erythrocytes.

A photolabile nitrene precursor, 3-azido-(2,7)-naphthalene disulfonate (ANDS), has been synthesized and used as a membrane-impermeable probe. The aryl azide was nonfluorescent. When activated by light, a highly reactive nitrene was generated which was capable of nonspecific covalent modifications of hydrophilic regions of cell surfaces. The products of the photolysis were highly fluorescent and modified proteins could be identified by their characteristic fluorescence after electrophoresis on sodium dodecyl sulfate polyacrylamide gels. When intact human erythrocytes were labeled with ANDS, Protein 3, the major membrane protein, and the sialoglycoproteins were modified. No proteins of apparent molecular weight greater than Protein 3 were labeled by ANDS, suggesting that none of these membrane components was exposed to the hydrophilic external surface of the red blood cell. When open erythrocyte stroma were labeled with ANDS, virtually all protein bands detectable by Coomassie blue staining could be shown to contain some fluorescence label. The significance of these findings are discussed with relation to the use of various aryl azides as surface labels of membranes.

Paroxysmal nocturnal hemoglobinuria erythrocytes are of two distinct types: positive or negative for acetylcholinesterase.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder. Erythrocytes isolated from PNH patients show increased sensitivity to complement and decreased acetylcholinesterase (AChE) activity. In this study, indirect immunofluorescence analysis of a monoclonal antibody specific for a surface epitope of human erythrocyte AChE is used to quantitate the content of this enzyme at the single-cell level. Flow-cytofluorimetric analysis of erythrocytes from normal donors indicates that all erythrocytes contain detectable levels of the surface epitope with a strong correlation between cell size and enzyme content. In contrast, erythrocytes from PNH patients show two distinct populations of erythrocytes; namely, those containing a normal content of AChE and a second population containing no detectable AChE. The AChE-negative population of cells is quantitatively complement-sensitive. These data support suggestions that PNH is a clonal disorder resulting in two distinct types of circulating erythrocytes. The abnormal clone produces cells that are both surface-AChE-negative and complement-sensitive. In addition, the method described provides an attractive alternative for the diagnosis and quantitative evaluation of abnormal erythrocytes in PNH patients.

Membrane lateral phase separations and chlortetracycline transport by Bacillus megaterium.

Chlortetracycline, a fluorescent probe of its own active transport, has been used to study lateral phase separations of membrane lipid in Bacillus megaterium cells. Arrhenius plots of initial accumulation rates are triphasic, with transitions or characteristic temperatures of 20 degrees and 9.5 degrees . At the higher temperature, the mobility of the chloretracycline, as measured by fluorescence polarization, is markedly altered. Chlortetracycline transport exhibits saturation kinetics, and fluorescence energy transfer from protein to bound antibiotic can be observed. N-Phenyl-1-naphthylamine, a lipophilic fluorescent probe, responds to changes in the hydrophobic regions of the membrane that are distinct from membrane protein. The fluorescent properties of N-phenylnaphthylamine in partitioning and polarization experiments are altered most significantly at the lower characteristic temperature. No fluorescence energy transfer between N-phenylnaphthylamine and membrane protein or bound tetracycline can be detected. In correlative electron spin resonance experiments on the partitioning of a lipid-soluble spin label, the same characteristic temperatures detected in the fluorescence studies were measured. These data suggest that different probes may respond to either or both of the characteristic temperatures describing the lateral phase separation. Between these characteristic temperatures the chlortetracycline transport system is most intimately associated with relatively immobile lipids that are surrounded by a more mobile lipid phase.

Quantitation of protein 3 content of circulating erythrocytes at the single-cell level.

The density and size of human erythrocytes has been roughly correlated with cell age, with the denser and smaller cells being older. Observations of this type have led to a hypothesis that the membranes of circulating erythrocytes are dynamic with respect to composition and that material is lost from the membrane during cell maturation and circulation. In this study, flow cytofluorimetry was used to investigate the distribution of the human erythrocyte anion transport protein (protein 3) in heterogeneous samples of circulating red cells. We verified that protein 3 can be specifically and quantitatively labeled in intact human erythrocytes with eosin-5-maleimide, a luminescent probe. Individual cells were accordingly analyzed for size by forward light scattering and for protein 3 content by quantitation of eosin fluorescence. Initial results indicated that the smallest erythrocytes had a protein 3 content equal to that of the largest circulating erythrocytes. This result was independently verified by light scatter-activated cell sorting; direct measurement of cell diameters by microscopy verified that the cell sizes of erythrocytes showing the 10% greatest and 10% smallest light-scattering signal were indeed distinct. Independent analysis of the size-sorted erythrocytes for protein 3 content was accomplished by gel electrophoresis of stroma from 150,000 large and small erythrocytes. Quantitative scanning densitometry of silver-stained gels of prepared stroma showed that protein 3 content of each set of fractionated cells was equal and did not vary as a function of cell size. Taken in combination with the reported correlation between increasing red blood cell age and decreasing cell size, these results indicate that any loss of membranous material during the cell aging process is not random.

Proliferative verrucous leukoplakia. Four cases with flow cytometric analysis.

Proliferative verrucous leukoplakia is a slow-growing but highly aggressive precancerous form of leukoplakia of unknown cause. Proliferative verrucous leukoplakia is though to possess a continuous spectrum of clinical and histopathologic expression, ranging from simple hyperkeratosis to invasive squamous cell carcinoma. Early diagnosis is difficult because of an initial innocuous character, but multiple and rapid multifocal warty recurrences are common. This article reports four additional archival cases of proliferative verrucous leukoplakia to determine if flow cytometric analysis can be useful in the early diagnosis of proliferative verrucous leukoplakia. Flow cytometric analysis was performed on available formalin-fixed paraffin-embedded specimens (N = 27). Flow cytometric analysis results showed DNA aneuploid cell lines in each proliferative verrucous leukoplakia case studied (DNA index range, 1.1 to 2.6). In all four patients the abnormal cell line DNA index appeared to be maintained throughout the sampling period. The results suggest flow cytometric analysis could be a possible aid in early recognition of proliferative verrucous leukoplakia and might enable aggressive therapy at an earlier stage.

Evaluation of lymphocyte phenotype and phytohemagglutinin response in healthy very low birth weight infants.

Sixteen healthy very low birth weight infants (VLBWI) were studied in a serial fashion over a 3-week period. Subjects were evaluated for lymphocyte phenotype, phytohemagglutinin (PHA) response, and metabolic status including weight, blood urea nitrogen, creatinine, albumin, pH, calcium, phosphorus, and ammonia. Lymphocyte phenotype determination showed a decreased proportion of CD3+ cells (66.8 +/- 10.4 vs 75.9 +/- 6.1, P less than 0.02) in VLBWI. When subsets of the CD4+ population were examined, VLBWI had a lower proportion of CD4+/CD29+ cells (8.2 +/- 5.8% vs 23.5 +/- 8.0%, P less than 0.0001) than adults and a higher proportion of CD4+/CD45R+ cells (35.6 +/- 12.4% vs 22.2 +/- 7.4%, P less than 0.03). The CD4+ subsets in VLBWI were similar to those seen in term infants. The peak PHA response in VLBWI was greater than that of adults (P less than 0.01). There was little change in the immune measurements over the 3-week study period. There were no strong correlations between any of the immunological measurements and the metabolic measurements except that the proportion of CD8+ cells increased with birth weight. Our findings demonstrate that immune measurements in healthy VLBWI differ from values found in adults but are similar to those of full-term infants. Lower proportions of the CD4+/CD29+ cells (the helper/inducer subset for antibody production) may contribute to some of the differences in immune function reported in neonates.

Analysis of human platelet glycoproteins IIb-IIIa and Glanzmann's thrombasthenia in whole blood by flow cytometry.

Antibodies that bind to human platelet membrane glycoproteins IIb and IIIa were used to develop methods for analyzing platelet membrane components by flow cytometry. Platelets were tentatively identified by their low-intensity light scatter profiles in whole blood or platelet-rich plasma preparations. Identification of this cell population as platelets was verified by using platelet-specific antibodies and fluorescein-conjugated antiimmunoglobulin. Two-parameter analysis of light scatter versus fluorescence intensity identified greater than 98% of the cells in the "platelet" light scatter profile as platelets due to their acquired fluorescence. Both platelet-rich plasma and whole blood were used to study platelet membrane glycoproteins IIb and IIIa on a single cell basis in an unwashed system. Prostacycline was included in these preparations as a precautionary step to inhibit platelet aggregation during analysis. Flow cytometry is a successful technique for rapid detection of platelet membrane defects such as Glanzmann's thrombasthenia. Platelets from Glanzmann's thrombasthenic individuals were readily distinguished from platelets with normal levels of glycoprotein IIb and IIIa and from platelets with glycoprotein levels characteristic of heterozygote carriers of this disorder. This technique provides a sensitive tool for investigating platelet functional defects due to altered expression or deficiency of platelet surface proteins.

Comparing flow cytometric analysis and nucleolar organizer region enumeration in archival oral premalignant lesions.

Flow cytometric analysis (FCA) and silver colloidal nucleolar organizer region-associated protein staining (AgNOR) have been used individually in assessing the histopathologic nature of various human tumors. However, few researchers have investigated the relationship between the two techniques in a single series. In a retrospective study, we examined 36 premalignant lesions of the oral cavity by FCA and AgNOR on formalin-fixed, paraffin-embedded tissue submitted to the University of Tennessee, Memphis, oral pathology laboratory. Three categories of epithelial dysplasia were represented (9 mild, 9 moderate, 6 severe), as well as four epithelial hyperplasias without dysplasia, three squamous cell carcinomas, and five fibrous nodules as controls. Parameters recorded for each case included age, race, gender, site, light microscopic diagnosis (LMD), DNA index (DI), total proliferative index (TPI), S-phase (S), range of nucleolar organizer regions (RNOR), and mean number of nucleolar organizer regions (MNOR). The average maximum nucleolar organizer region count (AMXNOR) for each LMD category was also calculated. The objective of the study was to determine if FCA or AgNOR aided in the subjective LMD of oral premalignant lesions and if the parameters recorded for the specimens exhibited any positive correlation. The FCA results indicated an abnormal DI in 6 of the 24 dysplastic lesions. A positive partial correlation was seen between DI and MNOR (r = 0.434; P < 0.012) and TPI and S (r = 0.774; P < 0.0001), holding gender and race constant. Additionally, the AMXNOR exhibited a slight tendency to increase for each increasing grade of dysplasia but this could not be confirmed statistically.(ABSTRACT TRUNCATED AT 250 WORDS)