Histochemistry is used to localize the specific substances present in the tissue and one of numerous fields utilized in cancer research (Leuchtenberger, 1949; Cohen, 1951, Pirozynski et al., 1952: Willghagen et al., 1963; Ishihara et al., 1964).
Determination of histochemical differences between normal and malignant tissues in histologic preparation or vaginal secretions in recent years has led to many investigations with special attention directed to the normal and malignant uterine cervix (McManus et al., 1949; Foraker, 1956; Gross et al., 1957, 1959, 1960; Hopman, 1960, Fishman, 1963; Thiery, 1966).
For these investigations, the study was conducted to obtain histochemical differences among chronic cervicitis, basal cell hyperplasia, leukoplakia, preinvasive carcinoma(carcinoma in situ) and invasive carcinoma of the uterine cervix, by special staining methods, hoping that morphological alteration may become more easily noted and interpreted than routine hematoxylin-eosin stain.
Materials Methods :
cervical material was obtained by punch biopsies of 40 cases and hysterectomies of 11 cases including proved cases of chronic cervicitis, basal cell hyperplasia, leukoplakia, carcinoma in situ, and in-vasive carcinoma by hematoxylin-eosin stains, and parts of each cervical material were used for frozen sections. In each instance normal cervical epithelium was studied as control. Special stains include periodic acid Schiff's reaction (PAS) for polysaccharides, PAS with saliva digestion method, Best carmine stains for glycogen, methyl green pyronin method for DNA and RNA combined, Feulgen reaction for DNA and Oil red O stain for lipids.
PAS reaction: This reaction shows that in normal cervical squamous epithelium the basal layer has little or no polysacharides, but in the superfical epithelial layer the PAS positive materials are increased. No appreciable difference is noted between chronic cervicitiv and normal cervix. In cases of basal cell hyperplasia PAS positive materials are decreased particularly at the hyperplastic area but superficial layer shows moderate to marked PAS positive materials remaining. In carcinoma in-situ PAS positive materials are markedly decreased or variably absent. In epithelial dysplasia PAS positive materials are moderately decreased but slight PAS positive materials are seen in the superficial layer, and there is no remarkable difference between carcinoma in situ and marked epithelial dysplasia. In
invasive carcinoma the PAS positive materials are markedly diminished or absent, but focally strong positive reaction it seem particularly in the highly differentiated area. In leukoplakia the PAS positive materials are generally decreased, moderately and markedly, from basal to superficial layer.
Best carmine reaction: In normal cervical squamous epithelium, the basal layer has little or no reaction. The positive reaction (glycogen) is gradually increased from the basal to the superficial epithelial layer. No appreciable difference is noted between chronic cervicitis and normal cervix. In basal cell hyperplasia the reaction is slight or absent. In dysplasia the reaction is markedly decreased but is focally prominent in the superficial layer. In carcinoma in situ the reaction is absent or slightly positive and slightly more negative reaction than dysplasia. In invasive carcinoma the reaction is markedly decreased or absent, but in highly differentiated area moderate positive reaction is seen. In leukoplakia the reaction is diffusely and variably decreased or absent.
Methyl green pyronin reaction: In normal cervical epithelium the basal cell prickle cell layers show moderate intensity of staining reaction. In chronic cervicitis the pyronin reaction shows no particular difference from the normal control, but in basal cell hyperplasia the reaction is variably increased.
In epithelia dysplasia the pyronin reaction is moderately to markedly increased. In carcinoma in situ pyronin reaction is more markedly increased than of epithelial dysplasia. In invasive carcinoma the pyronin reaction is marked, particularly in poorly differentiated cells, but slight to moderate reaction in highly
differentiated areas. In leukoplakia, no appreciable change in comparison with normal control is seen. The methyl green reaction to nucleus is also increased to proportion to the pyronin reaction to cytoplasm.
Feulgen reaction: In normal cervical epithelium the basal layer shows moderate to marked reaction but in the superficial layer the reaction is decreased. In chronic cervicitis and basal cell hyperplasia no appreciable different reaction than normal is seen but in epithelial dysplasia moderate increased reaction is noted. In carcinoma in situ and invasive carcinoma both show moderate to markedly increased reaction, and poorly differentiated malignant cells show more marked reaction of intensity than highly differentiated malignant cells. In leukoplakia, the Feulgen
reaction shows no appreciable difference compared with normal cervical epithelium.
Oil red O stain: In normal cervical epithelium, only slight, fine red granules are seen in basal cell layer and other layers are almost negative in reaction.
There are no differences among chronic cervicitis, basal hyperplasia, epithelial dysplasia, carcinoma in situ, invasive carcinoma, and leukoplakia.
A total of fifty five uterine cervical lesions comprising 11 cases of chronic cervicitis, 8 cases of leukoplakia, 8 cases of basal cell hyperplasia, 9 cases of epithelial dysplasia, 10 cases of carcinoma in situ and 9 cases of invasive carcinoma was subjected to histochemical study with special staining methods
including periodic acid Schiff's reaction (PAS) for polysaccharides, Best's carmine stain for glycogen, methyl green pyronin method for RNA, Feulgen reaction for DNA and Oil red O stain for lipids.
1. The amount of glycogen and polysaccharides are slight or absent in immature or poorly differentiated malignant squamous cells of uterine cervix and are proportionatly high as the cells differentiate.
2. Ribonucleic acid (RNA) and Desoxyribonucleic acid (DNA) are moderate to markedly increased in immature, precancerous, and malignant epithelial cells and these were inversely proportional to the differentiation of the cells.
3. Lipids in squamous epithelium are almost absent, and no differences among the uterine cervical lesions.