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Official Journal of the Italian Sports Medicine Federation
Indexed/Abstracted in: BIOSIS Previews, EMBASE, Science Citation Index Expanded (SciSearch), Scopus
Impact Factor 0,163
Online ISSN 1827-1863
Roi G. S. 2, Ferrara F. 1, Makovec E. 1, Lamonaca M. G. 1, Buselli P. 2, Banfi G. 1
1 Servizio Integrato di Medicina di Laboratorio, IRCCS H San Raffaele, Milano;
2 Marathon Sport Medical Center, Brescia
In this study, urine sediments of 10 athletes were analysed during the first two days of an international cross country skiing competition. Analysis was performed by the traditional microscopic method and flow cytometry. The latter, using only light scatter parameters related to biophysical characteristics of the particles (erythrocytes, leukocytes and epithelial cells), allows a rapid, automated and not expansive method for urine sediments analysis.
We have used the two methods in order to evaluate the correlation between manual and automated method and the qualitative and quantitative modification in the urine sediments during prolonged heavy exercise.
Urine specimens were collected from 10 volunteer donors before and immediately after each competition for two consecutive days. Specimens were aliquoted in two samples. An aliquote was fixed in alcohol 70% and stored at -20°C for flow cytometry. The second aliquot was fixed in alcool 50% and stored at 4°C for routinary microscopic analysis performed after centrifugation for 10 minutes at 3000 rpm.
Flow cytometric analysis was performed on samples at room temperature: samples were centrifuged three times at 2500 rpm for 10 minutes. After each centrifugation, samples were resuspended in 1 mL of Phosphate Buffer Solution pH 7.0. Samples were analysed using a BRYTHE-HS flow cytometer (BioRad, Segrate, Italy) equipped with a Xe-Hg arc-lamp. At least 20.000 events were acquired during each sample analysis, using only Forward Angle Light Scatter (FALS) and Large Angle Side Scatter (LALS) parameters: photomultiplier were adjusted at 310 mV and 370 mV respectively. Flow cytometric evaluation of urine sediments permitted us to define the cellular component in samples with a negative microscopic anlysis. In fact flow cytometry was characterized by higher sensibility than observed by the traditional method in 43% percent of the samples (p<0.001) in evaluating erythrocytes. Flow cytometric evaluation of leukocytes and epitelial cells showed higher sensibility than microscopic method in 38% of the samples (p<0.001).
44% of the samples that showed absence of erythrocytes by the microscopic method, were positive for the same cellular subset evaluation performed by the flow cytometric analysis. The presence of an enriched cellular component in athletes urine sediments seems to be related to the exercise, which is higher in not well trained athletes.
In fact flow cytometry permitted us to detect with high accuracy the modifications in urine sediments which are related to the kidney function and to the training level of each athlete during prolonged heavy exercise.