Series Of Research On The Detection Of Auramine O Staining Method Used For Mycobacterium Tuberculosis In Tissue Sections

【Objective】1. Comparing the sensitivity and specificity of auramine O staining and acid-fast staining in detecting of mycobacterium tuberculosis in tissue sections, exploring the viability and superiority of auramine O staining of detecting mycobacterium tuberculosis in tissue sections.2. Comparing the outcomes of auramine O staining and acid-fast staining in detecting of in tissue sections, exploring the viability and superiority of auramine O staining of detecting mycobacterium leprae in tissue sections.3. Improving auramine O staining by using tissue repair and other methods and verifying significant improvement in the positive rate of auramine O staining.Comparing sensitivity and specificity of modified auramine O staining and quantitative PCR in detecting of mycobacterium tuberculosis or nontuberculosis mycobacterium in tissue slices, verifying superiority of modified auramine O staining furthermore.【Methods】1. Collecting paraffin tissues which have been diagnosed as TB, using acid-fast staining and auramine O staining after slicing, using optical microscope and fluorescence microscope to compare the positive orientation, bacillus shape,microscopic examination time respectively,using foreign body granuloma paraffin tissue section as the control group, analyzing the results of two staining methods in statistical analysis.2. Collecting the leprosy cases of paraffin tissues from pathological diagnosis center of fuzhou general hospital from 2008 until now, using auramine O staining and acid-fast staining simultaneously after slicing, observing positive positioning of the two methods by microscope, comparative analysis of dyeing results of two methods.3. Using enzyme fixes, citric acid of high temperature and high pressure method,EDTA boiling method and other tissue repair methods to improve the auramine O staining method, looking for an optimal modified method with good modified effects and simple operation; Collecting mycobacterium tuberculosis positive paraffin tissue detected by FQPCR, using improved auramine O staining method,classical auramine O staining method and acid-fast staining method simultaneously after slicing, statistical analysis comparison of three kinds of dyeing results.4. Collecting suspected cases of granuloma infected by mycobacterium tuberculosis/nontuberculosis mycobacterium by HE microscopic examination,using modified auramine O staining and FQPCR at the same time,statistical analysis of two methods of dyeing results, exploring the application prospect of modified auramine O staining method.【Results】1.Mycobacterium tuberculosis or leprosy was bright green and thin rods or short rod by auramine O staining with fluorescent microscopy. Particularly, when the bacillus quantity was small,it was not easy to miss and microscopic examination time was shorter under the background of dark contrast where it was clear found;Mycobacterium tuberculosis or leprosy was red by acid-fast staining. When the bacillus quantity was small, it was easy to misdiagnose and microscopic examination time was longer under the background of blue contrast where it was not clear found.2.41 cases have been positive by anti-acid staining(positive rate :33.6%) and 58 cases beings positive by auramine O fluorescent staining(positive rate: 47.5%) in 122 cases of archive TB biopsy mycobacterium tuberculosis detection while the control group were negative. There was no statistical significance between acid-fast staining and auramine O fluorescent staining(P < 0.05).3. Auramine O fluorescence staining and anti-acid staining were all show the leprosy bacillus positive in 6 cases of leprosy wax piece of tissue section in the detection of mycobacterium leprae. A large number of mycobacterium lepraes were found in skin dermal granuloma tissues by auramine O staining for one of these patients with lepromatous lepros, meanwhile, a small number of single scattered mycobacterium lepraes were discovered in skin epidermis tissue; they were found in skin dermal granuloma tissues by anti-acid staining,however, a small number of single scattered mycobacterium lepraes were not discovered in skin epidermi tissues.4. EDTA repair was choosen the preferred method of improved auramine O staining after comparing several repair methods because it had the advantages of simple operation and good dyeing effect after repair;anti-acid staining positive rate was 58% on 100 cases by real-time fluorescent quantitative PCR in detecting mycobacterium tuberculosis positive TB biopsy, the traditional auramine O staining was 65%, the modified auramine O staining was 89%, positive rate difference between three groups was statistically significant(P < 0.001).【Conlusion】1. Positive rate of mycobacterium leprae by auramine O staining which have the advantages of high specificity and simple operation is higher than that by anti-acid staining on detection of mycobacterium tuberculosis in tissue biopsies so that can be used as the commonly used method instead of anti-acid staining.2. When the number of infectious bacillus is small, auramine O staining is superior to anti-acid staining in terms of analysis of leprosy bacili in tissue sections due to its simple operation.Hence,it can be used as the commonly used method instead of anti-acid staining3. When EDTA repair auramine O staining method after improvement is used to detect mycobacterium tuberculosis in tissue sections, its positive detection rate is significantly higher than anti-acid and traditional auramine O staining. The modified auramine O staining is worthy of promotion and feasible to detect tissue sections of mycobacterium tuberculosis.