Reticulocyte analysis and reticulocyte maturity index.
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Anemia↗
Biomedical subjects
B H Davis
Publications and source records attributed to B H Davis.
Flow cytometric reticulocyte analysis. Multiinstitutional interlaboratory correlation study.
Reticulocyte analysis by flow cytometry offers precision and sensitivity greater than those of conventional morphologic methods and permits derivation of a reticulocyte maturity index. However, interlaboratory variability has not yet been reported. The authors analyzed 310 samples at eight sites using 11 instruments over a 4-month period to examine intermethod and interlaboratory variabilities. Stains included thiazole orange, ethidium bromide, and auramine O. Instruments included models by Coulter, Becton Dickinson, TOA Medical Electronics, and Ortho Diagnostics. The coefficient of variation (CV) among all sites and methods on these samples varied as a function of the reticulocyte percentage, ranging from a mean CV of 69% for samples with < .5% reticulocytes to 24.1% for those with > 2.5% reticulocytes. The best performance was observed with the TOA R-1000 dedicated reticulocyte analyzers, with a mean CV of 18.4% for samples with < .5% reticulocytes and 4.6% for samples with > 2.5% reticulocytes. The reticulocyte maturity index showed comparable intersite precision, with a mean CV of 16% for samples with > 2.5% reticulocytes with multipurpose flow cytometers and a mean CV of 7.3% with the TOA R-1000 instruments. Interclass correlations among all sites ranged from .79 to .99 for the reticulocyte counts and .41 to .88 for the reticulocyte maturity index. The authors conclude that flow cytometric reticulocyte analysis is more precise than manual reticulocyte analysis. With greater automation of this methodology, further interlaboratory standardization of reticulocyte counts and the reticulocyte maturity index can be achieved.
Adolescent↗
Automated reticulocyte analysis. Clinical practice and associated new parameters.
Automated reticulocyte analysis, through continued instrument, software, and reagent developments, provides improved precision for the reticulocyte count. This rapid and simple testing assists in providing a cost effective and sensitive approach to the diagnosis and therapeutic monitoring of the anemic patient. As laboratory and clinical hematologist become familiar with the benefits and expanding clinical applications of this technology, the use of reticulocyte analysis will increase.
Autoanalysis↗
1,25-Dihydroxyvitamin D3 activates Raf kinase and Raf perinuclear translocation via a protein kinase C-dependent pathway.
1,25-Dihydroxyvitamin D3's (D3) potential mitogenic mechanism of action was pursued in cultured rat hepatic Ito cells, a fibrogenic effector cell which proliferates in vivo during liver injury and fibrogenesis. D3 stimulated Ito cell DNA synthesis and potentiated platelet-derived growth factor-induced mitogenesis. D3's enhancement of [3H]thymidine incorporation was associated with nuclear Egr expression. Recent studies have causally linked the activated proto-oncogene c-Raf with downstream Egr induction. The serine-threonine kinase Raf protein is phosphorylation-activated by a large array of agonists including plasma membrane and cytoplasmic tyrosine kinases but has not previously been associated with the steroid superfamily of mediators. To consider potential prenuclear acute pathways of D3-induced stimulation, the activation of Raf was examined following D3 exposure. D3 induced Raf activation as assessed via (a) enhanced Raf phosphorylation following in vivo 32P labeling, (b) enhanced kinase function utilizing exogenous histone 1 protein as substrate, and (c) the shift in Raf physical localization changing from a diffuse cytoplasmic distribution to a perinuclear domain. A similar activation of Raf kinase was found in 3T3 cells exposed to D3 with enhanced histone phosphorylation detectable within 1 min following stimulation. The proximal cascade leading to Raf kinase activation may involve a protein kinase activity was severely attenuated by stimulated kinase activity was severely attenuated by previous phorbol ester treatment for 20 h or staurosporine pretreatment.
3T3 Cells↗
Retinoic acid suppresses the response to platelet-derived growth factor in human hepatic Ito-cell-like myofibroblasts: a post-receptor mechanism independent of raf/fos/jun/egr activation.
Activated Ito-cell-like myofibroblasts proliferate in vivo during human liver injury and subsequent fibrogenesis. To examine the associated regulatory mechanisms, human liver myofibroblasts were characterized after culture purification from mixed liver-cell isolates obtained from perfused normal human livers. The cells resembled rat Ito-cell-derived myofibroblasts expressing desmin and alpha-smooth-muscle actin filaments as well as the interstitial collagens type I and III. [3H]Thymidine incorporation was inducible with platelet-derived growth factor (PDGF) and was suppressible with retinoic acid (RAc) in a concentration-dependent fashion. RAc suppression did not alter PDGF alpha- or beta-receptor abundance or activation. In addition, RAc functioned via a pathway distal or independent of cytoplasmic raf activation (i.e. phosphorylation, kinase function and perinuclear translocation) and nuclear fos, jun and egr expression, as these steps were similarly unaffected by RAc treatment. Since normal Ito cells contain abundant amounts of vitamin A which is lost during activation, these data suggest that retinoids could contribute to the maintenance of the quiescent non-proliferative state by suppressing mitogenesis at a post-cytokine receptor step distal from or independent of fos/jun/egr [e.g. via changes in activator protein-1 (AP-1) binding].
Cell Division↗
Flow cytometric reticulocyte analysis and the reticulocyte maturity index.
Reticulocyte analysis has evolved to one of the accepted and routinely practiced clinical applications of flow cytometry technology. Similar to CD4 measurements, FCM technology has contributed to documented improvements in reticulocyte counting precision over previous microscope-based techniques. The ability to FCM instruments to quantitate fluorescence intensity has been utilized to derive a parameter of reticulocyte maturity, which we have termed the reticulocyte maturity index. The FCM-derived RMI parameter offers an additional perspective to assess the erythropoietic response in anemic patients over reticulocyte counting alone and provides further insight into the differential diagnosis of anemia. Clinical utility of the RMI has been reported in monitoring expensive, new-technology therapies, such as bone marrow transplantation and erythropoietin therapy. FCM reticulocyte analysis is still not fully mature, but is in a state of continued evolution. Needs exist for (i) improved software for data analysis, (ii) newer automation techniques to improve interlaboratory correlations, and (iii) further validation and refinement of the reticulocyte maturity index parameter.
Anemia↗
Proposal for standardization of flow cytometric reticulocyte maturity index (RMI) measurements.
Flow cytometric (FCM) reticulocyte analysis is more accurate, sensitive, and reproducible relative to previously employed manual microscopic methods in clinical laboratory hematology. FCM reticulocyte analysis using RNA binding fluorochromes additionally allows for the quantification of fluorescence intensity or population distribution of the reticulocyte RNA content. Viewed from the perspective of erythroid maturation, quantification of the fluorescence intensity distribution provides a reticulocyte maturation index (RMI). We performed a systematic study of 18 different methods to express thiazole orange stained reticulocyte fluorescence intensity, compared to standard mean fluorescence intensity quantification, using 185 anemic and non-anemic human blood samples. The method best correlating with the mean fluorescence intensity RMI on 2 different FCM instruments (R2 = 0.93 and 0.86) was a ratio of the highly fluorescent reticulocytes, defined using a normal adult population, and the total number of reticulocytes (HFR%). In contrast to mean fluorescence intensity measurements, a HFR% RMI parameter can provide similar units of expression (0.01-1.00) with good correlation between different FCM instruments (R2 = 0.76). We conclude the HFR% method of RMI expression provides a superior means of interlaboratory standardization and clinical comprehension of this useful diagnostic parameter in clinical laboratory hematology.
Anemia↗
Administration of prostaglandin E1 analog reduces rat hepatic and Ito cell collagen gene expression and collagen accumulation after bile duct ligation injury.
Recent studies suggest that prostaglandin E may have the ability to suppress cytokine responsiveness. We examined the effects of prostaglandin E administration on several parameters of acute and chronic liver injury induced by bile duct ligation. Enisoprost, a prostaglandin E1 analog, was found to suppress early hepatic and Ito cell type I collagen gene expression without diminishing the induction of the fibrogenic cytokine transforming growth factor-beta. Overall liver inflammation and cell proliferation were not altered, suggesting that prostaglandin E acts distal to the initial injurious event(s). During later phases, drug administration reduced total collagen accumulation and type I collagen periductular infiltration associated with early nodule formation.
Alanine Transaminase↗
Gamma-linolenic acid suppression of hepatic Ito cell mitogenesis: post-PDGF receptor prostaglandin-independent mechanism.
Ito cell mitogenesis occurs during liver injury and fibrogenesis in vivo. Platelet-derived-growth factor (PDGF)-induced mitogenesis was studied in cultured rat hepatic Ito cells, which resemble the myofibroblast associated with liver injury. Pretreatment with gamma-linolenic acid (GLA), an essential fatty acid prostanoid precursor, markedly suppressed the PDGF response in a dose-dependent reversible fashion. Prostaglandins E1 and E2 were found to be the predominant prostanoids formed by cultured Ito cells. GLA depressed endogenous PG production, suggesting that the antimitogenic effect was independent of GLA conversion to a prostanoid metabolite. The PDGF-induced cascade was studied with and without GLA to determine the level of regulation that induced the observed suppression. GLA caused no apparent diminution in the abundance of the surface PDGF-beta receptor nor its subsequent activation and tyrosine phosphorylation after PDGF stimulation. Raf kinase activation and Raf perinuclear translocation were also intact despite the presence of GLA. PDGF induction of nuclear Egr and Fos also occurred with or without GLA. Activation of the serine threonine kinase c-Raf has previously been found to be sufficient to activate egr and fos and to induce mitogenesis. Therefore, the GLA suppressive effect is likely to be operative at a parallel non-Raf pathway or distal to Raf-induced early gene expression.
Animals↗
Posttranslational inhibition of Ito cell type I collagen production by triiodothyronine.
Increased Ito cell collagen production occurs during in vivo liver fibrogenesis. Regulation of the overproduction of collagen was studied in cultured rat hepatic Ito cells, which resemble the myofibroblast associated with liver fibrosis. Previous studies suggest that the steroid hormones, retinoic acid, and glucocorticoids may have antifibrogenic properties in vitro and in vivo when used at pharmacological doses. Their potential roles at physiological levels are not well understood. The current study examined the potential regulation of the overproduction of type I collagen in cultured rat hepatic Ito cells by another steroid hormone, 3,5,3'-triiodo-L-thyronine (T3). T3 induced a 3.4-fold reduction in type I collagen production. The effect was dose dependent and was maximal with physiological levels of T3 (10(-9) M). The effect of T3 was independent of any suppression in total protein synthesis. The mechanism of the suppressive effect of T3 on collagen production was explored and was found to be at a posttranslational level. This study suggests that the inhibitory effects of T3 on type I collagen production are likely caused by enhanced intracellular turnover of type I collagen.
Animals↗
Pathogenesis of hepatic fibrosis and the role of cytokines.
No definitive therapy exists for the treatment of hepatic fibrosis and cirrhosis. Recent evidence suggests that hepatic lipocytes (Ito cells, fat storage cells, or stellate cells of the liver) are responsible for much of the collagen hypersecretion and nodule formation that occurs during hepatic fibrosis and cirrhosis. This review describes the cellular mechanisms of hepatic fibrogenesis emphasizing new experimental data about cytokines or growth factors to suggest potential avenues of future therapeutic design.
Cell Division↗
Hepatic fibrogenesis and its modulation by growth factors.
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Extracellular Matrix↗
Roles of the Tn21 merT, merP, and merC gene products in mercury resistance and mercury binding.
The mercury resistance (mer) operon of the gram-negative transposon Tn21 encodes not only a mercuric reductase and regulatory genes but also two inner membrane proteins (MerT and MerC) and a periplasmic protein (MerP). Although the merT, merP, and merC genes have been implicated in Hg(II) transport, the individual roles of these genes have not been established. We created in vitro precise deletion and frameshift mutations that eliminated each of the genes singly and in combination. Our results show that both merT and merP are required for Hg(II) binding but that merC is not. Both merT and merP are required for full expression of Hg(II) resistance, but loss of merP is less deleterious than loss of merT. Furthermore, mutations eliminating both merT and merP decrease resistance more than the single mutations do. In contrast, mutating merC had no effect on Hg(II) resistance. Both the merT and merP mutations increase the threshold Hg(II) concentration for induction of merA-lacZ transcriptional fusions and cause an increase in the maximal expression level. In contrast, the merC mutation had little effect on the threshold inducing concentration of Hg(II) but decreased the level of expression. Our results show that merT and merP alone are sufficient to specify a mercury transport system. The role of merC remains obscure.
Amino Acid Sequence↗
Retinoic acid and transforming growth factor beta differentially inhibit platelet-derived-growth-factor-induced Ito-cell activation.
Sinusoidal Ito cells (stellate or fat-storing cells) undergo excessive cellular proliferation before the establishment and progression of hepatic fibrosis and cirrhosis. Retinoic acid and transforming growth factor beta (TGF beta) both inhibit Ito-cell [3H]thymidine incorporation in serum-containing media. Serum-induced mitogenicity was dependent on platelet-derived growth factor (PDGF). Additionally, pre-treatment of Ito cells with retinoic acid and TGF beta blocked PDGF-induced cell proliferation. TGF beta, but not retinoic acid, diminished PDGF-receptor and smooth-muscle alpha-actin abundance.
Actins↗
Flow cytometric DNA content analysis in neuroendocrine carcinoma of the lung. Correlation with survival and histologic subtype.
The DNA content of 53 primary and locally metastatic pulmonary neuroendocrine carcinomas was retrospectively determined by flow cytometric analysis of nuclei released from paraffin-embedded tissues and compared with histologic subtypes and clinical survival. Forty-one percent (9 of 22) of tumors classified as well-differentiated neuroendocrine carcinoma (WDNC) were aneuploid. In contrast, 85% (17 of 20) of small cell neuroendocrine carcinoma (SCNC), and 73% (8 of 11) of intermediate cell neuroendocrine carcinoma (ICNC) were aneuploid. DNA content was not a significant independent predictor of patient survival within each histologic subtype (P greater than 0.05 for all subtypes). Yet, when all cases were examined as a single group, diploid cases showed better clinical survival than cases with DNA aneuploidy (P less than 0.02). However, the survival advantage for diploid DNA content did not quite achieve statistical significance when only cases of limited stage disease were analyzed (0.10 greater than P greater than 0.05). Histologic subtype was also a prognostic indicator, with WDNC cases showing a significantly longer survival than either SCNC (P less than 0.05) or ICNC (P less than 0.02), for those cases with limited-stage disease. These results indicate information on clinical staging and the histologic subtype are important parameters to compare with DNA content analysis in determining independent prognostic factors in neuroendocrine or small cell carcinoma of the lung.
Adult↗
Isodicentric chromosome 18q as sole abnormality in a histologically normal bone marrow from a patient with diffuse large-cell non-Hodgkin's lymphoma.
Cytogenetic analysis was performed on the histologically and immunophenotypically normal bone marrow (BM) of a 33-year-old woman with non-Hodgkin's lymphoma (NHL) before BM harvest. Unstimulated 24- and 48-hour cultures produced only normal metaphases. A pokeweed mitogen (PWM)-stimulated 48-hour culture, however, showed a clonal isodicentric chromosome 18q as the sole abnormality, suggesting a role for this approach in detection of submicroscopic BM involvement by B-cell NHL.
Adult↗
A morphological and phenotypic analysis of Walker 256 cells.
A detailed morphological analysis of Walker 256 cells sensitive and resistant to cis-diamminedichloroplatinum(II) has been performed. Two cell populations are identified by electron microscopy of differing differentiation corresponding structurally to cells reported in experimentally induced metastases. Phenotyping of the cells using a number of monoclonal antibodies by immunocytochemistry and flow cytometry showed the absence of epithelial cell markers: however, the cells stained intensely for markers for germ and/or hematopoietic cells. Further studies utilizing monoclonal antibodies to lymphoid, myeloid, and monocytoid cells showed the cells to be monocytoid in origin. No evidence of cell heterogeneity was evident from the phenotypic experiments (a biphasic pattern was not observed). Enzyme histochemistry showed strong focal acid phosphatase activity suggestive of cells of hematopoietic origin. Thus the concept that these cells reflect an epithelial cell of origin is not substantiated by phenotyping with two methodologies.
Animals↗
Neutrophil activation through high-affinity Fc gamma receptor using a monomeric antibody with unique properties.
The high-affinity, type I Ig Fc receptor (Fc gamma RI) for human IgG1, human IgG3, murine IgG2a, and murine IgG3 is highly expressed on monocytes, neutrophils (PMN) in certain disease states, and phagocytes treated with interferon-gamma (IFN-gamma). We studied the activation of the human PMN oxidative burst and stimulated fluid pinocytosis induced by three monoclonal antibodies (MoAbs) directed against Fc gamma RI (CD64) to study the role of this receptor in Fc-mediated cellular activation. All three MoAbs were capable of triggering PMN activation from IFN-gamma-treated PMN when cross-linked with goat antimurine Ig reagents. However, MoAb 197 alone demonstrated a concentration-dependent activation of the oxidative burst without the use of a second cross-linking antibody. The oxidative burst and stimulated fluid pinocytosis responses induced by monomeric MoAb 197 could be elicited only after the IFN-gamma induction of approximately 8,000 Fc gamma RI receptor equivalents and did not occur in freshly isolated or control-cultured PMN. Competitive blocking of Fc binding of MoAb 197 by human IgG or purified Fc fragments inhibited cellular activation. We believe the ability of MoAb 197 to activate these oxidative burst and fluid pinocytic responses was because of its murine IgG2a subclass, which allowed it to function as a trivalent anti-Fc gamma RI antibody binding through the combination of its two FAB regions and the Fc domain. This study demonstrates that the cross-linking of CD64 can activate PMN oxidative and endocytic responses and supports a role for Fc gamma RI in the human neutrophil inflammatory response.
Antibodies, Monoclonal↗