Remedy Methods And Major Reasons For The Poor HE Staining Of Tissue Slices

Research objective:Hematoxylin and Eosin staining (HE staining) is the foundation of all kinds of pathology department. Pathological technologist put the tissue samples from patients or animal experiments through fixation, dehydration, transparency, paraffin, embedding, slicing, stalls, patch, roast slice, and a series of operation, which is known as HE staining technology. The quality of HE staining affect the pathologist to make correct pathological diagnosis. Under a microscope, a cleared contrast can be a high quality HE dyeing slices specimens. But it is not easy to produced a large number of high-quality HE staining slices. During the process of routine pathological work, we usually counter all sorts of unexpected conditions. For example, the reasons which caused by dehydration, transparent, lack in paraffin slice grey staining, such as tissues structure is not clear, causes the condition which was hard to diagnose. Especially, for small specimens, such as gastroscope biopsy specimens, puncture biopsy tissues, these tissues are very small, once appear aforementioned problems, mostly tissues unable to be selected again, and the mistaken problems were mostly unable to remedy. Therefore, we have to look for reasons for the failure and make sure that the same mistakes not to recurrence. In this work, we found out the causes of failure which is improper dehydrator processing tissues. We find another ways to solve the problem, Based on this thing, we studied the remedial measures of staining failure specimens ①:Reprocessing of the stained the gray slice, ②:Tissues improper handling of wax block slice again reprocessing restaining, ③:The improper handling tissues of wax block, by many times of xylene dewaxing, and enter into gradient ethanol until hydration, then it will be treated as soft tissues to dehydration. The results show that, remedial measures ③ accord with the requirement of the basic pathological diagnosis, but there is a big risk of this approach, which be affected by any carelessness led to tissues brittle harden and production difficulties, while the remedial measures ① and ② can significantly improve the quality of pathological slices, it is satisfactory with the requirements of pathological diagnosis.Methods:1. Find the Major reasons for the poor HE staining of tissue slicesCollect 20 cases of poor HE staining of tissue slices specimens, including gastroscope biopsy specimens of 5 cases, endoscopic biopsy specimens of 5 cases, endometrial specimens of 5 cases, pulmonary biopsy specimens of 5 cases. At the same time, collecting recently staining of normal gastric biopsy paraffin blocks of 5 cases, endoscopic biopsy specimens of 5 cases, endometrial specimens of 5 cases, pulmonary biopsy specimens of 5 cases. And then collected the immunohistochemical staining of HE slices compared with observation. Design of experiment is divided into seven groups:The poor HE staining of tissue slices as a failure group; The wax block which have poor HE staining of tissue slices using four kinds of methods for processing, treatment group 1:cases of improper treatment of specimens were stained, after the adjustment of the paraffin section of the staining machine procedures restaining (extended bake time); treatment group 2:cases of improper handling of specimens were stained, the staining machine program regulation after staining (after replacement of hematoxylin and eosin); treatment group 3:cases of improper handling of specimens were stained, the staining machine program regulation after staining (after prolonged staining dewaxing and dewatering machine time); treatment group 4: Paraffin sections after the normal process of staining staining machine, and adjusting the concentration of neutral balsam. At the same time,set up two control groups. Control group of normal HE staining:normal specimens were treated with dehydration, and the paraffin sections were stained with normal staining machine. Control group of immunohistochemistry:the poor HE staining of tissue slices were stained with immunohistochemical staining procedure.2. Study of the poor HE staining of tissue slices of remedy methodsMake use of the above failure of 20 specimens, respectively using three methods for processing. Remedial group 1:the day has been made into the gray staining failed HE slices in xylene for 30 min; in the tissue overlying coverslip removed during this period; the slices were then moved to the 100%,95%,85%,70% ethanol 3 min, slices washing 3 min. Then washed slices in a 500ml beaker which containing 300 ml of distilled water, in a water bath heating,and the beaker in the stainless steel pot water, heating furnace or oven, boiling water to cook for 30 min, the beaker is cooled to room temperature after removing the slices, make slices restaining. Remedial group 2: The wax block sliced, baking 30 min with the temperature of 65℃, conventional dewaxing to water. The slices into the stainless steel pot of boiling water bath heating tap water containing 30 min, conventional HE staining again. Remedial group 3:the wax block by xylene dewaxing repeatedly, until the water into the gradient ethanol, then the weak tissues of retreatment, the wax block inverse procedure to the origin, update the reagent again after treatment. At the same time, put the poor HE staining of tissue slices as the control group.Research results:1. Find the reasons for the poor HE staining of tissue slicesCompared with the failure group,treatment group 1,2 and 4 slices staining grey, the tissue’s microscopic structure was not clear, can’t reached the diagnostic criteria. The effect was not quite different from the original HE slices. It’s pathological score had no obvious difference (p>0.05). Although the pathological grading of group 3 are significant difference in statistics (p<0.05), it still can’t completely reached the diagnostic criteria, the normal HE staining of tissue slices show the nuclear mass clear, bright colorred, it is clear between red and blue colour, clear contrast. Thereby precluding the steps of roasting, staining and sealing. Thereby eliminated the reasons of roasting, staining, sealing slices, and so on. In addition, control group of immunohistochemistry is equivalent to control group of normal HE staining. Through excluding these causes gradually, finally we found the major reasons for the poor HE staining of tissue slices:automatic dewaterer improper handling of dehydration.2. The evaluation of remedy methodsAccording to the pathological grading after staining, this study found that three remedy methods staining effects were better, completely reached to the diagnostic standard. At the same time, compared with the normal HE slices. It’s pathological grading have obvious difference in statistics (p<0.05). But the remedial group 3 applies only to larger tissue specimens, and have some risk, probably cause tissues damage. The staining effect of remedy methods 1 and 2 is satisfactory, and can’t cause any impact to tissue samples, so we recommend remedy methods 1 and 2 to deal with the similar problem in the future work.Conclusions:The influencing factors of paraffin slices HE staining throughout the tissues process and staining process, from the resected tissues after fixation, dehydration process and staining process of each step will affect HE staining. Specimens were fixed and staining process caused by the adverse problem can be given lead to adverse factors of staining, the corresponding solutions and remedy methods, the problem is easy to solve.In this study, we found the major reasons for the poor HE staining of tissue slices: automatic dewaterer improper handling of dehydration. Meanwhile, we use 3 different measures on the original biopsy and improper handling wax blocks for processing, found two kinds of remedy methods to solve the failure HE staining, offer a scientific basis for clinical pathology workers to solve such similar problems in the future.