Diagnostic value of Gram stain for assessment of vaginal smears during pregnancy

The main aim of this study was to compare the diagnostic value of Giemsa stained method with Gram stained method for the evaluation of vaginal smears among pregnant women. A study population comprised 111 pregnant between 6 and 30 weeks of gestation. The vaginal smears from every subject was diagnosed according to Giemsa and Gram stained method and micro-organisms were isolated by culture. In 29.3% cases diagnosed as normal flora (2a) on the basis of Giemsa method bacterial vaginosis was detected in Gram stains according to Spiegel's criteria and pathological microflora in concentration > or = 10(5) CFU/ml was cultured among 75.9% of them. Among 31.7% women who had grade 3a (abnormal) in Giemsa stains method normal flora was diagnosed on the basis on Gram's method and from 17.1% pregnant women from this group we did not isolated any pathogens. For evaluation of vaginal smears during pregnancy the Giemsa method should be replaced by Gram stained method.     

An indirect immunofluorescence method for detection of Mycoplasma hominis in vaginal smears.

We developed a novel method for the detection of Mycoplasma hominis from vaginal swabs using an indirect immunofluorescence technique. It is a rapid and simple method that can be finished in only 5 hr and is more sensitive than the usual culture isolation method. The indirect immunofluorescence method was applied to vaginal smears from 193 healthy women and 33.7% gave a positive test. This value was much higher than that (11.4%) obtained from the same specimens by the culture method. When vaginal smears were subjected to Papanicolaou staining after the indirect immunofluorescence method, the specific immunofluorescence of the epithelial cells was located exactly at the sites of granular aggregates stained with Papanicolaou stain. A histological examination by Papanicolaou staining showed that the incidence of inflammation seems to be slightly higher in M. hominis-carriers than in non-carriers.     

Mucosal cytology in the determination of the pubertal status in girls

AIM: To assess and validate the diagnostic value of buccal and vaginal smears in the ambulatory care of girls with potential disorders of puberty. METHODS: Smears were obtained from 77 girls who presented for assessment of their pubertal status, stained as described by Papanicolaou, examined for signs of estrogenization according to the method of Schmitt, and compared with the clinical status. RESULTS: Vaginal but not buccal smears reflect accurately the changes of sexual maturation, even more sensitive than single serum hormone measurements. CONCLUSIONS: Vaginal smears represent a valid, reproducible, and well-tolerated diagnostic tool in the ambulatory care of peripubertal girls to identify their estrogen status with high sensitivity and specificity. The decision to perform confirmative but invasive diagnostic procedures can be based on auxology, physical examination, bone age determination, and analysis of vaginal smears.     

Comparison between Gram stain and culture for the characterization of vaginal microflora: Definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora

Background  

The microbiological diagnosis of bacterial vaginosis is usually made using Nugent's criteria, a useful but rather laborious scoring system based on counting bacterial cell types on Gram stained slides of vaginal smears. Ison and Hay have simplified the score system to three categories and added a fourth category for microflora with a predominance of the Streptococcus cell type. Because in the Nugent system several cell types are not taken into account for a final score, we carried out a detailed assessment of the composition of the vaginal microflora in relation to standard Gram stain in order the improve the diagnostic value of the Gram stain. To this purpose we compared Gram stain based categorization of vaginal smears with i) species specific PCR for the detection of Gardnerella vaginalis and Atopobium vaginae and with ii) tDNA-PCR for the identification of most cultivable species.     

Standardized Heat Fixation for Blood Smears

Because a technique was needed for good fixation of blood smears without the use of solvents such as methanol, the effect of heat fixation on human and rat blood smears was studied. Slides were placed in a fused silica dish, covered by a Pyrex petri dish lid, and heated for 5 min in an oven at 150° C. After such fixation, smears stained by Giemsa' s solution showed that the blood elements retained their characteristic morphology. Thus, even though volatile substances may be lost and heat-labile constituents may be modified, this simple method avoids contact of the cells with solvents.     

A quantitative method for assessing stages of the rat estrous cycle

The impact of gender and/or hormone variations on a wide variety of neural functions makes the choice between studying males or females (or both) of a given species difficult. Although female rats are widely used experimentally, few studies control for the stage of estrus. More detailed information about how to distinguish the various stages of the estrous cycle is needed. For the present study, vaginal smears were obtained once a day and stained using an adaptation of the Papanicolaou (PAP) procedure. Images are provided of unstained “wet” samples and the corresponding PAP stained smears illustrating the cellular profile for each stage of the cycle as well as post-ovariectomy. The different cell populations across the cycle were quantified and ratios determined to show trends between the predominant and other cell types in each stage of the estrous cycle. Both stained and unstained images and cell quantification data provide valuable guidelines for distinguishing the stages of the estrous cycle.     

Understanding microwave-stimulated Romanowsky-Giemsa staining of plastic embedded bone marrow

Summary Bone marrow smears were made and fixed in methanol or formaldehyde. Marrow sections of various thicknesses were also prepared from formaldehyde fixed marrows embedded in paraffin or plastic (glycol methacrylate). The different smears and sections were then stained by a Romanowsky-Giemsa procedure. Some specimens were stained using a standard microwave-stimulated method previously used diagnostically. The effects of technical variations were studied, including degree of microwave irradiation and the staining time. Comparisons of the resulting staining outcomes showed that microwave stimulated Romanowsky-Giemsa staining of plastic sections is a rate controlled process. Unusual aspects of the staining pattern of plastic sections (namely the purple basophilic cytoplasms and nucleoli, and blue chromatin) are due to microwave stimulation and formaldehyde fixation respectively.     

Microscopic diagnosis of vulvovaginal candidiasis in stained vaginal smears by Dutch general practitioners

OBJECTIVE: To determine the accuracy of the microscopic diagnosis of vulvovaginal candidiasis (presence of [pseudo] hyphae and blastospores) in stained vaginal smears in clinical practice. STUDY DESIGN: General practitioners trained in diagnosing vulvovaginal candidiasis performed microscopy of 324 stained vaginal smears. These smears were sent to the pathologist for confirmation of the microscopic diagnosis of the clinician; cytologic diagnosis by the pathologist was considered the gold standard. RESULTS: In 104 of the 342 cases Candida was established by the pathologist. The clinicians made 24 false positive and 50 false negative diagnoses of Candida. Sensitivity and specificity of the microscopic diagnoses of the clinicians were 52% and 89%, respectively. The most frequent reason for a false positive diagnosis was presence of hairs, whereas the most frequent reason for a false negative diagnosis was understaining of the smear. CONCLUSION: This study shows that even in stained smears it is difficult for clinicians to recognize blastospores and (pseudo)hyphae. Efforts are clearly needed to improve the quality of the clinical diagnosis of vulvovaginal candidiasis.     

Maturation value as an indicator of the serum estrogen concentration during treatment with gonadotropins

Smears were taken each day from the middle third of the lateral vaginal wall of 14 infertile women being treated with daily intramuscular injections of human menopausal gonadotropins followed by human chorionic gonadotropins to induce ovulation. Each smear was stained with modified Papanicolaou stain and screened for the maturation index (MI), which was then converted to Meisel's maturation value (MV). In the 23 treatment cycles, the MV of the vaginal smears was compared to daily serum estrogen concentrations. In 7 of the 23 cycles the MV predicted a rise in serum estrogens; in 16, however, the MV did not predict the increasing serum estrogen concentrations. Although determination of the MV is inexpensive, easily obtained and rapidly performed, the cytologic examination of the vaginal epithelium cannot be used to predict the dynamic changes in serum estrogen concentrations during gonadotropin treatment.     

A quantitative method for assessing stages of the rat estrous cycle

The impact of gender and/or hormone variations on a wide variety of neural functions makes the choice between studying males or females (or both) of a given species difficult. Although female rats are widely used experimentally, few studies control for the stage of estrus. More detailed information about how to distinguish the various stages of the estrous cycle is needed. For the present study, vaginal smears were obtained once a day and stained using an adaptation of the Papanicolaou (PAP) procedure. Images are provided of unstained “wet” samples and the corresponding PAP stained smears illustrating the cellular profile for each stage of the cycle as well as post-ovariectomy. The different cell populations across the cycle were quantified and ratios determined to show trends between the predominant and other cell types in each stage of the estrous cycle. Both stained and unstained images and cell quantification data provide valuable guidelines for distinguishing the stages of the estrous cycle.     

Sudanophilia and lipids in the blood and marrow cells

Summary The method of Sheehan and Storey, used for demonstrating stable Sudanophilia of neutrophils in smears, was modified by substituting other alcohols (from methanol to amyl alcohol) for ethanol, and the effect of this on the resulting staining reactions was investigated. The intensity of the staining of the neutrophil granules decreases with increasing number of carbon atoms in the alcohol, with a gradation of staining from black to yellow, while, at the same time the staining of the erythrocytes increases. The best solvent for Sudan black B for haematological purposes is methanol. This solvent was further used for investigations on various procedures for detecting so-called masked sudanophilia. The most constant and interesting results were obtained after the action of mercuric ions on smears fixed in formalin vapour. Under certain conditions, a characteristic binding of different fractions of Sudan black B on different blood cells can be attained, so that neutrophil granules are stained brown, monocytes mostly grey, lymphocytes, platelets, megakaryocytes, plasmocytes and perinuclear areas of normoblasts blue. The present experiments with various substrates indicate that demasked blue staining in the cytoplasm may be conditioned by phospholipids, although a reaction with other components of the cytoplasm cannot be excluded. The method described may be useful in haematology for studying cytogenetic relations in the monocytic and granulocytic series.Technical Assistance: V. LodrovÁ.     

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.     

A simple and rapid microwave-assisted hematoxylin and eosin staining method using 1,1,1 trichloroethane as a dewaxing and a clearing agent.

The use and practicability of microwave-assisted staining procedures in routine histopathology has been well established for more than 17 years. In the study reported here, we aimed to examine an alternative approach that would shorten the duration of dewaxing and clearing steps of hematoxylin and eosin (H & E) staining of paraffin sections by using a microwave oven. Although xylene is one of the most popular dewaxing and clearing agents, its flammability restricts its use in a microwave oven; thus we preferred 1,1,1 trichloroethane, which is not flammable, as the dewaxing and clearing agent in the present study. In Group I and Group II (control groups), intestine was processed with xylene and 1,1,1 trichloroethane, respectively. The sections were then stained with H & E according to the conventional staining protocol at room temperature and subdivided into two groups according to the duration of dewaxing and clearing in xylene. In Groups III and IV (experimental groups) similar tissues were processed with xylene and 1,1,1 trichloroethane, respectively; however, sections from these groups were divided into four subgroups to study the period required for dewaxing and clearing in 1,1,1 trichloroethane, then stained with H & E in the microwave oven at 360 W for 30 sec. Our conventional H & E staining procedure, which includes dewaxing, staining and clearing of sections, requires approximately 90 min, while our method using 1,1,1 trichloroethane and microwave heating required only 2 min. Our alternative method for H & E staining not only reduced the procedure time significantly, but also yielded staining quality equal or superior to those stained the conventional way. Our results suggest that 1,1,1 trichloroethane can be used effectively and safely as a dewaxing and clearing agent for H & E staining in a microwave oven.     

An alternative sputum processing method using chitin for the isolation of Mycobacterium tuberculosis

An alternative bio-friendly sputum processing method is the need of the hour to augment the rate of detection of TB cases and to improve the sensitivity of rapid growth based diagnostic methods. Chitin, mucolytic in nature and present ubiquitously in animal kingdom, was found to have decontaminating activity when used for processing sputum specimens. The aim of the present study is to develop an alternative bio friendly sputum processing method using chitin. Smear microscopy was done on direct sputum samples and on the deposits obtained after processing with modified Petroff’s method as well as Chitin method. Two direct smears were made from each of the sputum samples and stained by Ziehl Neelsen and Auramine phenol (AP) method. The samples were divided in to two aliquots and processed by chitin and modified Petroff’s method. Smears were made from each of the deposits and stained by both methods. The deposits were inoculated on to two Lowenstein Jensen slopes. AP method showed a sensitivity of 95% in direct smear. Samples processed by chitin and the deposit smears stained by AP method showed a sensitivity of 80% and a specificity of 89% compared to that of modified Petroff’s method. The sensitivity of chitin culture is 87% and the specificity is 85%. Chitin–H₂So₄ solution took less time compared to 4% NaOH to homogenize the mucopurulent sputum specimens. Chitin–H₂So₄ can be used as an alternative method of sputum processing for the detection of M. tuberculosis.     

A New Stage in the Performance of a Human Immunogram: A Visual Streptavidin-Biotin Approach

The production of a reagent kit has been recently organized by DAKO, Immunotekh and other companies, for phenotyping of lymphocytes by the streptavidin–biotin method. The method needs no sophisticated equipment, is highly sensitive, and allows rapid staining of different lymphocyte subpopulations in capillary blood smears and subsequent observation of them under a light microscope. We have modified this method for staining leukocytes in the monolayer prepared with a plate cytorotor. Not decreasing the above-mentioned advantages of the method, this modification significantly cheapens and simplifies the staining procedure; the blood cells of 16 subjects can be stained concurrently, and the staining can be performed by a technician. The streptavidin–biotin method of lymphocyte phenotyping can be mastered in every immunological laboratory, thus improving its technical level.     

Sequential silver staining and hybridization in situ on nucleolus organizing regions in human cells.

Chromosome preparations from eight individuals were first stained with silver nitrate to reveal the nucleolus organizing regions (NORs) and then hybridized in situ with ribosomal RNA. In six individuals the size of the silver-staining regions was positively correlated with the amount of label present after hybridization in situ. Thus the variation in silver-staining intensity among chromosomes was largely explained by variation in the number of rDNA gene copies per NOR. However, in two individuals this correlation was absent, suggesting that other factors can also influence the size of the silver-staining region.     

Fish health assessment: a preliminary report on the use of impression smears of skin mucus

Preliminary results from this study suggest that impression smears of the skin mucus of fish can be used to aid in the evaluation of the fish's health status. Nonstressed Lagodon rhomboides and Carassius auratus have numerous superficial epithelial cells, occasional lymphocytes and other mononuclear cells in their cutaneous mucus. Smears of these fish 24 h post-stress had more cellular debris, micro-organisms and erythrocytes when compared to those made pre-stress. Components in the mucus of fish which recovered gradually became normal. Smears of fish, which died, had increasing amounts of debris and micro-organisms. The use of this relatively nontraumatic technique may be a useful adjunct in monitoring fish health.     

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

 We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. They were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P=0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients. Accepted: 29 April 1997     

THE PAPANICOLAOU-TRAUT METHOD OF CANCER DIAGNOSIS,Its Use as a Routine Pathologic Laboratory Procedure

In a series of 450 examinations by the smear technique in the pathologic laboratory of a small private hospital, the total diagnostic error on all smears was 6.6 per cent. The error on vaginal smears was 4.7 per cent. These figures include false positives, false negatives, and all those smears classified as suspicious even though positive or negative diagnoses were made on subsequent examinations.The Papanicolaou-Traut method of cancer diagnosis can easily be made one of the routine pathologic procedures in the small hospital laboratory. Technicians with little previous cytologic experience can be trained to screen vaginal and other smears accurately after a short training period. This cytologic method is proving to be of value in the early detection of some neoplasms, and if its application is to be extended, the practicing pathologist should add the test to his diagnostic routine.     

Alternative, cost-effective fungus-staining method for control slides in cytology and histopathology

OBJECTIVE: To develop a cost-effective, reliable and safe method of providing fungal control slides for routine use in pathology laboratories. STUDY DESIGN: A set of easily available, low-cost material was tested to obtain fungal colonies on substrate adequate for paraffin-embedded sections or smears. RESULTS: Such material as cheese is a simple, inexpensive and practical culture medium for silver-positive fungi. A batch of paraffin blocks can be prepared to maintain a stock of control material in the laboratory. CONCLUSION: It is useful to maintain fungal colonies to produce staining control specimens using small pieces of refrigerated cheese to easily produce silver-staining control specimens or smears embedded in paraffin, reducing the risk of accidental exposure to potentially infective pathogens in the laboratory. This method might also be a good alternative for conserving routine surgical specimens, considering the currently decreasing numbers of necropsy and large specimens, particularly from immunosuppressed and infected patients.