Revisión del intervalo de referencia del recuento plaquetario: ¿son adecuados los valores que estamos utilizando? / Review of the platelet count reference range: are the values we are using appropriate?

Los intervalos de referencia (IR) dependen de la población y de las características metrológicas del procedimiento de medida utilizado. A pesar de las recomendaciones internacionales, son pocos los laboratorios que establecen sus propios IR para cada magnitud por la dificultad para conseguir voluntarios de referencia y el elevado costo económico asociado. La International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) acepta la adopción de IR bibliográficos o su cálculo por métodos indirectos dado su bajo costo y fácil obtención. Existen varias fuentes confiables de IR bibliográficos para el hemograma. No obstante, para el recuento plaquetario, es una práctica común de los laboratorios emplear el rango de valores de 150-450.109 /L independiente de la metodología utilizada y grupo etario. El objetivo de este trabajo fue revisar los IR bibliográficos disponibles para el recuento plaquetario y estimarlo empleando el método indirecto de Hoffmann a partir de nuestra población. Los métodos indirectos se basan en aplicar criterios de exclusión y cálculos matemáticos sobre los resultados de una base de datos de laboratorio. Nuestros IR para el recuento plaquetario se comparan con los bibliográficos, que han sido establecidos por técnicas de muestreo directo. Por este motivo y dado que no existen estudios poblacionales que lo avalen, sería apropiado reemplazar el rango de 150-450.109 / L. Estos límites podrían seguir empleándose como puntos de corte o niveles de decisión médica para definir, según la clínica y otros resultados de laboratorio, los pacientes que ameritan un seguimiento posterior (AU)

Reference ranges (RR) depend on the population and the metrological characteristics of the measurement procedure used. Despite international recommendations, few laboratories establish their own RRs for each magnitude because of the difficulty in obtaining reference volunteers and the associated high economic cost. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) accepts the use of literaturebased RRs or RRs calculated by indirect methods because of their low cost and easy collection. There are several reliable sources of literature-based RRs for the Cell Blood Count. However, for platelet count, it is common laboratory practice to use the range of 150-450,109 /L regardless of the methodology used and age group. The aim of this study was to review the available literature regarding RRs for platelet count and to establish it using the indirect Hoffmann method in our population. Indirect methods are based on applying exclusion criteria and mathematical calculations on the results of a laboratory database. Our RRs for platelet counts are compared with those in the literature, which have been established by direct sampling techniques. Therefore, and given that there are no population studies to support these findings, it would be appropriate to replace the 150-450,109 /L range. These limits may continue to be used as cut-off points or medical decision levels to define, according to clinical manifestations and other laboratory results, patients who warrant further follow-up (AU)

Genotoxicity of AMPA, the environmental metabolite of glyphosate, assessed by the Comet assay and cytogenetic tests.

Formulations containing glyphosate are the most widely used herbicides in the world. AMPA is the major environmental breakdown product of glyphosate. The purpose of this study is to evaluate the in vitro genotoxicity of AMPA using the Comet assay in Hep-2 cells after 4h of incubation and the chromosome aberration (CA) test in human lymphocytes after 48h of exposition. Potential in vivo genotoxicity was evaluated through the micronucleus test in mice. In the Comet assay, the level of DNA damage in exposed cells at 2.5-7.5mM showed a significant increase compared with the control group. In human lymphocytes we found statistically significant clastogenic effect AMPA at 1.8mM compared with the control group. In vivo, the micronucleus test rendered significant statistical increases at 200-400mg/kg. AMPA was genotoxic in the three performed tests. Very scarce data are available about AMPA potential genotoxicity.

In vitro inhibition of murine hematopoietic progenitors and stromal cells by vinorelbine.

Hematopoietic progenitor colony assays were used to establish the effects of the vinca alkaloid vinorelbine (VRB) on murine bone marrow. The in vitro growth of colony-forming units-granulocyte/macrophage (CFU-GM), burst forming units-erythroid (BFU-E) and colony-forming units-mix (CFU-mix) was dose-dependently inhibited by VRB. The highest dose assayed (0.02 microg/ml) suppressed all of the different progenitor cells by 100%. A comparison of the dose-response curves showed that CFU-GM, BFU-E, and CFU-mix exhibited similar-patterns of sensitivity to the cytotoxic action of VRB. Long-term bone marrow cultures have provided a valuable in vitro model for studying the role of the microenvironment of bone marrow. Cellularity of stromal layers was reduced with increasing doses of VRB. The appearance of these layers was altered minimally with the lowest dose used; a gradual loss of cellularity was seen in cultures exposed to 0.05 and 0.075 microg/ml; and a marked loss at the dose of 0.1 microg/ml. Our results show that VRB has an important effect on hematopoietic progenitors at the highest dose tested, while the stromal cells were not affected at a similar dose (0.025 microg/ml), suggesting that the stroma is more resistant to this drug.

Comparison of the aneugenic effect of vinorelbine and vincristine in cultured human lymphocytes.

Vinca alkaloids are used clinically against a variety of hematological and solid tumors. These compounds interact with tubulin subunits to prevent microtubule assembly, inducing abnormal chromosome segregation in dividing cells and causing aneuploidy. The vinca alkaloid vincristine sulfate (VCR) and the semisynthetic analog vinorelbine (VRB) were studied by analysis of micronuclei (MN) in cultured human lymphocytes using the cytokinesis block method. Furthermore, fluorescence in situ hybridization with a human alphoid satellite pancentromeric DNA probe was used to detect centromeres in isolated MN of VRB- or VCR-treated lymphocytes. At all the doses tested, both chemicals induced a significant increase in MN frequencies in binucleated (BN) cells (P < 0.001). The maximal effect was reached at a dose of 0.50 microgram/ml. At this dose, VRB produced an approximately 5-fold increase with respect to the control frequency of MN, while with VCR, this frequency increased 10-fold. Both drugs produced a slowing of the cell cycle, causing a decrease in the percentage of BN cells. This effect was lower with VRB. The percentages of centromere-positive MN were 89.79 and 87.60% in VRB- and VCR-treated cultures, respectively (control 27.03%). The high percentage of positive-signals in treated cultures (P < 0.001) indicates that the MN contained whole chromosomes. Our results confirm the aneugenic mode of action of these chemicals, VRB having less genetic effect.

Mitotic arrest and anaphase aberrations induced by vinorelbine in hamster cells in vitro.

Aneugenic effects of the Vinca alkaloid vinorelbine (VRB) were evaluated in vitro, measuring sister chromatid exchanges (SCE), cell proliferation kinetics and anaphase telophase aberrations in Chinese hamster ovary (CHO) cells. The highest dose of VRB (0.50 microg/ml) arrested cells at the first metaphase. An increase in abnormal anaphases was seen at 0.05-0.50 microg/ml of VRB, containing chiefly lagging chromosomes and multipolar spindles. No increase in SCE was found. These results indicate that VRB does not directly damage DNA, but acts on spindle microtubules, altering chromosome movement and causing aneuploidy.

Vinorelbine: cell cycle kinetics and differential sensitivity of human lymphocyte subpopulations.

Antiproliferative activity induced by vinorelbine (VRB) was studied in whole blood cultures (WBC) and in lymphocyte subpopulations evaluated by the MAC (morphology-antibody-chromosome) method. In WBC a significant delay in cell cycle kinetics in VRB-treated cultures compared with controls (P < 0.001) was observed. The highest dose (1.0 microg/ml) arrested all cells at the first metaphase. Both WBC and isolated lymphocyte cultures showed a significant increase in the mitotic index (MI) in VRB-treated cultures compared with controls (P < 0.001). Moreover, a significant increase in the MI was found in VRB-treated CD4 cells compared with VRB-treated CD8 (P < 0.01) and B-lymphocytes (P < 0.001).

Chromosome damage induced by carboplatin (CBDCA).

The chromosome damage induced by carboplatin (CBDCA) was evaluated in vitro. In human lymphocytes 5 micrograms/ml CBDCA produced a 7-fold increase in the frequency of sister chromatid exchanges (SCE) and a 3-fold increase in the number of cells with structural abnormalities compared with the control. Likewise, at this highest dose a significant increase was induced in the value of micronuclei (MN) and an important delay in the lymphocyte cycle progression was observed. In Chinese hamster ovary (CHO) cells, CBDCA showed a significant increase in chromosome aberrations (CA) at the doses assayed. The increase of SCE, CA and MN by CBDCA remained much lower than that produced by mitomycin C (positive control). The results suggest that CBDCA is a DNA-damaging drug with similar behaviour as an alkylating agent.

Genotoxic activity of azidothymidine (AZT) in in vitro systems.

The genotoxic activity of azidothymidine (AZT) was evaluated in vitro, measuring cytogenetic parameters in two cell systems. In human lymphocytes AZT induced a statistically significant increase in chromosome breakage at 100 micrograms/ml and in micronucleated cells at the highest dose assayed (500 micrograms/ml). Sister-chromatid exchanges (SCE) showed a two-fold increase over control values at 50 micrograms/ml. Lymphocyte cycle kinetics showed an important delay at 500 micrograms/ml. In Chinese hamster ovary (CHO) cells, AZT produced a significant increase in chromosome aberrations and SCE at 1000 and 500 micrograms/ml, respectively. At 2500 micrograms/ml the drug produced a delay in cell cycle progression. These results suggest that AZT is a DNA-damaging agent in both cell systems assayed. Moreover, human lymphocytes seem to be more sensitive to AZT than CHO cells.

Mutagenic bioassay of certain pharmacological drugs: III. Metronidazole (MTZ).

The genotoxic activity of MTZ was evaluated in vitro with the anaphase-telophase test in a CHO cell line, chromosomal aberration and micronucleus test in lymphocyte cultures, and in vivo using the micronucleus test in mouse bone marrow cells. The In vivo test was performed using clinical trial doses (23, 70 and 160 mg/kg). A significant increase in micronucleated cells (p < 0.02) was observed in the three assayed doses with a linear dose response (r = 0.91). In vitro studies showed a significant increase in the percentage of abnormal anaphases (p < 0.05), in chromosome aberrations (p < 0.01) and in the frequency of micronuclei (p < 0.02) at all the concentrations assayed (0.1, 1 and 10 micrograms/ml). These findings demonstrate the clastogenic effect of this drug which should be taken into account considering its wide human consumption.

Chromosome instability in lymphocytes from patients with celiac disease.

Cytogenetic studies were performed in celiac disease (CD) patients to determine if the presence of chromosome instability is related to the predisposition to cancer. Chromosome aberrations (CA) and sister chromatid exchange (SCE) frequencies in peripheral blood lymphocyte cultures from untreated CD patients and healthy controls were analyzed. Patients showed aberrations in 23% of cells, while only 3% were detected in the control group (p < 0.0001). The mean frequencies of gaps, breaks and total CA were found to be higher in CD patients compared to controls (p < 0.0001). Breakpoint distribution was nonrandom among chromosomes from celiac patients (p = 0.01), but not among controls (p = 0.04). The frequency of SCE/cell showed a mean value of 6.9 +/- 0.6 in CD patients and 7.3 +/- 0.2 in controls. No statistical differences were found. Breakpoints involved in CD patients presented a strong coincidence with the location of fragile sites (78.6%) and sites of cancer chromosome rearrangements (57.1%), most of them (75%) associated with malignant non-Hodgkin lymphomas. These results suggest that CD is a condition with increased chromosome instability characterized by a high level of CA and normal SCE frequencies, probably related to the increased incidence of cancer.

Leather tanning workers: chromosomal aberrations in peripheral lymphocytes and micronuclei in exfoliated cells in urine.

A cytogenetic study was performed on workers of a leather tanning industry. Two different approaches for the biological monitoring of the individuals were used: chromosomal aberration analysis in peripheral lymphocytes and the frequency of micronucleated cells exfoliated in urine samples. 26 men working in the sections considered to present a greater risk were included in the study. Controls were 20 men that were not exposed to chemicals. The percentage of abnormal cells was higher in workers than in controls. Smokers showed higher values of chromosome breaks than non-smokers in both groups. These differences were not statistically significant. The percentage of cells with chromatid and chromosome gaps in workers and controls was different (p less than 0.01). A slight but not significant increase in the mean percentage of micronuclei was observed in the exposed group. We conclude that exposure to chemicals during leather tanning did not produce genotoxic effects measured by chromosomal aberrations in peripheral lymphocytes and micronuclei in urine in this group of workers.

Genotoxicity of the pesticide propoxur and its nitroso derivative, NO-propoxur, on human lymphocytes in vitro.

The aim of this work was to investigate whether the pesticide propoxur and its nitroso derivative nitroso-propoxur increased the frequencies of sister-chromatid exchanges and micronuclei in human lymphocytes in vitro. The results show that both chemicals were genotoxic in the tested system.

Nitroso-aldicarb induces sister-chromatid exchanges in human lymphocytes in vitro.

Nitroso-aldicarb was tested for its ability to induce sister-chromatid exchanges (SCE) and cell-cycle delay in human peripheral blood lymphocytes in vitro. This derivative of aldicarb induced a dose-dependent increase in SCE values per cell. In addition, a slight decrease in the successive mitotic progression of cells in culture was observed.

Chromosomal aberrations in cultured human lymphocytes treated with aldicarb, a carbamate pesticide.

Aldicarb was tested for its ability to induce chromosomal aberrations in human peripheral lymphocytes in vitro, in the presence of an exogenous metabolic activation system. The pesticide induced an increase in the number of chromatid and chromosome breaks. The increase was higher in the presence of S9 mix. A positive linear association between frequencies of abnormal cells and dose of aldicarb was observed.

Sister-chromatid exchanges and chromosomal aberrations in a population exposed to pesticides.

Sister-chromatid exchanges (SCE) and chromosomal aberrations were studied in a population of floriculturists occupationally exposed to organophosphorus, carbamate and organochlorine pesticides. Blood samples from 36 individuals from a community of 154 persons of asiatic origin were obtained. Among the group sampled, 21 individuals exhibited at least one symptom of chronic intoxication. SCE analysis was performed in 14 symptomatic and 13 asymptomatic persons. The asymptomatic group showed a SCE frequency of 5.47 +/- 1.03 and the symptomatic group a frequency of 6.45 +/- 1.19. Comparison between both groups with the Mann-Whitney 'U' test revealed a significant difference (p 0.0409). Case-control analysis of 9 pairs matched by sex and age also showed significant differences between both groups (p 0.0104). In contrast, the frequencies of chromosomal aberrations were not correlated with intoxication symptomatology, though a significant increment of exchange-type aberrations in relation to a group of non floriculturists was observed in the population studied.

Intercambio de cromatidas hermanas en trabajadores de una industria quimica. / Sister chromatid exchange in workers of a chemical industry

Hay evidencias de que la exposicion ocupacional a ciertas sustancias quimicas puede inducir danos a nivel cromosomico. En este trabajo se eligio el intercambio de cromatidas hermanas (ICH) en linfocitos perifericos humanos como tecnica sensible ya que permite medir el efecto de sustancias que actuan sobre la estructura cromosomica a dosis que no producen otras alteracoes detectables. Se estudiaron 22 trabajadores de una industria quimica que formula pesticidas (malation, diazinon y heptacloro), produce curtientes (a partir de taninos sinteticos) y blanqueadores (utilizando etildiamina, dietanolamina, anilazina). Como controles se eligieron 20 individuos sanos no expuestos. Los valores de ICH/ celula hallados entre los trabajadores estan dentro de un rango de 6.1 a 10.8 con un promedio de 7.9 + ou - 1.2. Esta cifra no difiere estadisticamente de los valores del grupo control lo que indicara que las sustancias utilizadas no produjeron efectos genotoxicos sobre la poblacion estudiada. No hubo sinergismo con el humo de cigarillo. Las frecuencias de ICH en los fumadores expuestos no difieren estadisticamente de las de los fumadores control

Intercambio de cromatidas hermanas en trabajadores de una industria quimica. / Sister chromatid exchange in workers of a chemical industry

Hay evidencias de que la exposicion ocupacional a ciertas sustancias quimicas puede inducir danos a nivel cromosomico. En este trabajo se eligio el intercambio de cromatidas hermanas (ICH) en linfocitos perifericos humanos como tecnica sensible ya que permite medir el efecto de sustancias que actuan sobre la estructura cromosomica a dosis que no producen otras alteracoes detectables. Se estudiaron 22 trabajadores de una industria quimica que formula pesticidas (malation, diazinon y heptacloro), produce curtientes (a partir de taninos sinteticos) y blanqueadores (utilizando etildiamina, dietanolamina, anilazina). Como controles se eligieron 20 individuos sanos no expuestos. Los valores de ICH/ celula hallados entre los trabajadores estan dentro de un rango de 6.1 a 10.8 con un promedio de 7.9 + ou - 1.2. Esta cifra no difiere estadisticamente de los valores del grupo control lo que indicara que las sustancias utilizadas no produjeron efectos genotoxicos sobre la poblacion estudiada. No hubo sinergismo con el humo de cigarillo. Las frecuencias de ICH en los fumadores expuestos no difieren estadisticamente de las de los fumadores control