Optimization of the tartrate-resistant acid phosphatase detection by histochemical method

M J Galão, A R Santos, M D Ribeiro, Anibal Ferreira, Fernando Nolasco

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Abstract

According to the new kidney disease improving global outcomes (KDIGO) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling, the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 mu m were stained to identify TRACP at different incubation temperatures (37 degrees, 45 degrees, 60 degrees, 70 degrees and 80 degrees C) for 30 min. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60 degrees C and 70 degrees C. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 min, the reaction should be carried out at 60 degrees C. As active osteoclasts are usually scarce in a bone section, the standardization of the histochemistry method is of great relevance, to optimize the identification of these cells and increase the accuracy of the histomosphometric results. Our results, allowing an increase in osteoclasts contrast, also support the use of semi-automatic histomorphometric measurements.
Original languageEnglish
Pages (from-to)1-4
JournalEuropean Journal Of Histochemistry
Volume55
Issue number1
DOIs
Publication statusPublished - 2011

Keywords

  • Bone biopsy
  • Bone resorption
  • Histochemistry
  • Osteoclasts
  • Renal osteodystrophy
  • Tartrate-resistant acid phosphatase

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