May-Grunwald Giemsa Research Articles

Comparative cytomorphology of skin, lymph node, liver and bone marrow in patients with lepromatous leprosy1

The aim of this study was to compare the cytological changes in skin, lymph nodes, liver and bone marrow in patients with lepromatous leprosy. Skin lesion, lymph node, liver and bone marrow aspirates were analysed. May-Grunwald-Giemsa (MGG) and Ziehl-Neelsen (Z-N) stains were employed. Comparative cytomorphology was studied. Twenty patients with lepromatous leprosy were studied. Lepra cells (LC) predominated in the skin aspirates of 12 patients with lepromatous leprosy (LL), lymphocytes accompanied LC in eight patients with borderline-lepromatous (BL) leprosy. Three patients of LL leprosy and two of BL leprosy in type 2 reaction additionally had numerous neutrophils. Two patterns of lymph node aspirates were seen: partial replacement with few LC in a reactive lymphoid background (10), complete replacement with either only LC or LC in a background of degenerating neutrophils (10), the latter a feature of type 2 reaction. Liver aspiration was performed in seven patients and of bone marrow in eight patients. Occasional LC were present in five liver-aspirated patients, steatosis and Kupffer cell hyperplasia in four patients, and myelopoiesis in two patients. Bone marrow smears invariably had occasional LC and a relative increase in mature plasma cells; sea-blue histiocytes were seen in six patients. Lepra cells predominated in skin and lymph node aspirates with complete replacement. In comparison, liver, bone marrow and lymph node aspirates with partial replacement were dominated by a preponderance of cells native to these organs with only few or occasional LC.

Leishman-Giemsa Cocktail

To develop an economical, quick, readily available and simple alternative to May-Grünwald-Giemsa (MGG) stain and so to explore the combination of Leishman and Giemsa stain (LG cocktail).One wet-fixed and 2 air-dried smears were prepared from 720 cases during the period January 2003-November 2004. The LG cocktail and MGG stain were used on the air-dried smears and Papanicolaou stain in wet-fixed smears. Diagnoses of the cases were made using the LG cocktail-stained smears, and then its diagnostic efficacy was cross-checked with the MGG- and Papanicolaou-stained smears by the same cytopathologist. A comparative study of the LG cocktail and MGG-stained smears was done.The results achieved with the LG cocktail-stained smears were comparable to or even better than those with the MGG-stained smears, with excellent diagnostic efficacy.As compared to MGG stain, the 1-step LG cocktail is cheaper, easier to standardize and quicker.

Evaluation of Papanicolaou Stain for Studying Micronuclei in Buccal Cells Under Field Conditions

To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies.Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN.Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm.Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.

Application of modified ultrafast Papanicolaou stain in cytology of various organs

This prospective study was conducted to assess the use of modified ultrafast Papanicolaou (MUFP) stain for fine-needle aspiration cytology (FNAC) of various organs. In India, many reagents are not easily available. In this modification, Gill's haematoxylin, EA-36, and isopropanol substitute Richard-Allan haematoxylin, cytostain, and ethanol, respectively. Group I included 40 FNAC smears of various organs. In each case, three smears were prepared and stained by MUFP, Papanicolaou, and May-Grunwald Giemsa (MGG) stains. In group II, 10 intraoperative cytology smears of different organs were stained with MUFP and rapid haematoxylin-eosin (H&E). For assessment of MUFP stain, scores were given on four parameters: background of smears, overall staining pattern, cell morphology, and nuclear staining. Quality index was calculated from ratio of score achieved to maximum possible score. Diagnosis made by MUFP stain was compared with standard stains. It was observed that MUFP-stained smears had clear RBC free background, crisp nuclear chromatin, well-stained nucleoli, and transparent cytoplasm. The diagnosis made was correct except in three cases of metastatic squamous-cell carcinoma. Hence, it was concluded that MUFP stain is useful for rapid diagnosis by FNAC, but is not useful for squamous-cell lesions.

Assessment of Genetic Aberrations in Plasma Cells Using a Sequential Morphology (Phenotype)/FISH (Genotype) Approach Significantly Improves the Detection of Residual Disease in Post Treatment Multiple Myeloma.

Multiple myeloma (MM) is a B cell malignancy characterized by clonal expansion of plasma cells. Many MM patients achieve a complete remission by conventional criteria; yet most patients relapse as a consequence of residual disease. Current approaches for the measurement of minimal residual disease (MRD) in bone marrow (BM) are based on morphologic assessment of BM aspirate and biopsy, flow cytometry, immunohistochemistry, molecular (PCR) studies, conventional cytogenetics (CC) and fluorescence in situ hybridization (FISH) analyses. Morphologic assessment of MRD is often difficult due to the fact that normal plasma cells may also be present in the BM. Genetic factors have emerged as significant prognostic factors in MM; however, CC studies are hampered by the low proliferative nature of the malignant cells. FISH analyses have detected clonal abnormalities of -13/del(13q), 14q32/IGH, del(17p), and hyperdiploidy (+5,+9,+15) in >80% of newly diagnosed MM cases; yet, detection of these abnormalities post treatment by the standard FISH approach has proven to be very difficult in samples with less than 20% BM involvement. PCR-based approaches are sensitive but suffer a critically high false-negative rate. In this study, we investigated 137 post treatment samples collected from 101 MM patients (31 patients with multiple studies), all showing < 20% BM involvement, using a sequential May-Grünwald Giemsa (MGG) (morphology)/FISH approach to determine the plasma cell genotype (target or T-FISH). Cytospin slides were made using 200 μl of BM and stained with MGG for morphologic classification on a Duet™ Image Analyzer (Bioview Ltd., Rehovot, Israel). After identifying and mapping the plasma cells, the slides were destained and hybridized with one of four FISH probe sets corresponding to the chromosome aberrations listed above. The T-FISH results were correlated with CC, BM pathology, which quantified the percentage of plasma cells in the BM aspirate, and BIOMED-2 PCR analysis for IGH (FR1, 2 and 3) and IGK gene rearrangements (InVivoScribe Technologies, San Diego, CA). T-FISH identified MM aberrations in 123 of 137 (89.8%) samples, a finding significantly higher than the 50/83 (60.2%) positive cases detected by combined molecular IGH and IGK PCR studies (two-sided Fisher's Exact p < 0.0001). T-FISH aberrations observed were IGH in 77 samples, del(13q)/-13 in 48, hyperdiploidy in 37, hypodiploidy in 6, and del(17p) in 4, with 42 samples showing more than one abnormality. Only 10 samples showed clonal karyotypic aberrations by CC; an additional 3 samples showed a “presumed” stemline with only one abnormal cell (9.5%). A comparison with the percentage of plasma cells in the BM smears showed T-FISH detected residual disease in all 48 samples with ≥6 % plasma cells, 14 of 15 hemodilute samples, one smoldering MM sample and 82.2% (60/73) of the samples with 1–5% plasma cells. Our data indicate T-FISH is a quick, universally applicable, and robust assay to quantitate neoplastic plasma cells regardless of treatment status, making it the most sensitive molecular assay currently available to monitor a patient's clinical course. Furthermore, the T-FISH molecular cytogenetics strategy provides a novel approach to monitor both traditional and targeted therapies in low proliferative malignancies by their underlying genetic abnormalities.

Circulating B Lymphocytes from Patients with Multiple Myeloma Harbour T(4;14) Translocations and Chromosome 13 Deletions.

Multiple Myeloma (MM) is clinically characterized by accumulations of plasma cells in the bone marrow (BM). We have identified drug resistant clonotypic B lymphocytes in the peripheral blood of MM patients that have malignant characteristics and the ability to xenograft MM to immunodeficient mice. Using an automated scanning system (Bioview Duet), for blood samples from 72 MM patients, we scanned cytospin slides stained with May-Grunwald Giemsa to identify lymphocytes and determine whether they have the same chromosomal abnormalities that characterize autologous plasma cells. The location on the slide of each morphologically identified cell is recorded during an initial scan, followed by FISH, and examination of the same cells for genetic abnormalities. For this study, we chose probes to detect 1) deletion of chromosome 13 using D13S319 (Vysis), and 2) the t(4;14)(p16;q32) translocation using a dual fusion probe (Vysis). Both abnormalities correlate with adverse prognosis. Lymphocytes from PBMC of healthy donors show 2.8+/−1% of lymphocytes with Ch13 deletion, and less than 1% with t(4;14). An MM PBMC sample was considered to have lymphocyte abnormalities if it scored above a cut off value of 10% for Ch13 deletions and 2% for t(4;14), this is likely to underestimate the extent of chromosomal abnormalities in MM B cells. The number of abnormal lymphocytes in MM PBMC is sufficiently large, and the number of abnormal cells in comparable populations from healthy donors is sufficiently small, that we are readily able to detect significantly increased numbers of abnormal lymphocytes in MM PBMC. We found that peripheral lymphocytes from 19/60 (32%) MM patients have Ch13 deletion (27%) and/or t(4;14) (26%); for those patients with an available BM sample, these same abnormalities were found in their BM plasma cells. Interestingly, intraclonal heterogeneity is apparent in malignant cells from a t(4;14) patient; these plasma cells include a minority population with apparent Ch14 monosomy (23%) and a major population having two copies of Ch14 (77%). This was internally controlled in that for both populations, the plasma cells have two copies of the fused t(4;14) chromosome, and the polymorphonuclear cells on the same slide were normal. One MM patient had Ch13 deletion in 60% of lymphocytes from PBMC, consistent with the 63% of BM plasma cells from this same patient showing Ch13 deletion. For another MM patient known to be t(4;14)+, sorted sIgM+ B cells were analyzed and found to include a small subset with t(4;14) translocations. For 2/6 MM PBMC, abnormalities in Ch19 were detectable in 25–37% of lymphocytes. For mobilized blood autografts, preliminary data indicates the presence of lymphocytic cells with detectable Ch13 deletion. By performing FISH after immunostaining with anti-CD20 of PBMC from MM patients whose bone marrow plasma cells had Ch13 deletion/monosomy, we detected chromosomal abnormalities in CD20+ B cells from MM blood. No abnormalities were detected for CD20+ B cells from healthy donors. In the context of our previous work, this analysis demonstrates that two molecular signatures of MM, clonotypic IgH gene rearrangements and, as reported here, prognostically important chromosomal abnormalities are found among circulating CD20+ B cells from the blood of patients with MM. This work confirms that the MM clone includes circulating B lymphocytes harbouring chromosomal deletions or IgH switch region translocations known to be clinically significant in MM, further implicating them as a source of relapse.

HER-2/neu Evaluation by Fluorescence in Situ Hybridization on Destained Cytologic Smears from Primary and Metastatic Breast Cancer

To evaluate HER-2/neu amplification by fluorescence in situ hybridization (FISH) (HER-2/neu by FISH) on archival cytologic smears stained with May-Grünwald-Giemsa (MGG) stain.Cytologic specimens from 69 breast cancer lesions (48 primary and 21 metastatic), stained with MGG stain for routine diagnostic cytology, were destained and subjected to HER-2/neu by FISH. Fifteen of the 69 samples were also evaluated by FISH on paired fresh smears.HER-2/neu by FISH was successfully assayed in 25 of the 48 primary tumors and in 15 of the 21 metastatic lesions, corresponding to an overall feasibility of 58%. These cases had been archived between 1 month and 10 years prior to FISH analysis. Eight of the 25 primary and 5 of the 15 metastatic tumors were amplified. In 15 of the 40 evaluable cases, HER-2/neu was also assessed on the corresponding fresh smears: 8 tumors were amplified and 7 unamplified on both destained MGG and fresh smears.HER-2/neu can be detected by FISH on routinely MGG-stained cytologic slides. This approach allows HER-2/neu evaluation whenever histologic sections or fresh cytologic material are not available. In these cases, HER-2/neu assessment on destained cytologic smears plays a role in the selection of targeted therapy.

Increased radiation sensitivity of an eosinophilic cell line following treatment with epigallocatechin-gallate, resveratrol and curcuma

Ionizing radiation is widely used in radiotherapy, in order to promote an apoptotic response in cancerous cells. Since the need to find new substances that would enhance the radiation-induced apoptosis in cancerous cells is great, we studied the effect of epigallocatechin-gallate (EGCG, a tea component), resveratrol (a wine component) and curcuma on cell proliferation and radiation-induced apoptosis in the human leukaemic cell line, EOL-1, derived from a patient with eosinophilic leukaemia. Cells were X-irradiated with 0, 2, 4, 6 or 8 Gy and cultured in the presence of EGCG, resveratrol or curcuma (concentrations ranging from 0 to 200 microM) for 1, 2 or 3 days of culture. Cell proliferation was measured using trypan blue exclusion. Apoptosis was evaluated using light microscopy (morphology study after May-Grunwald Giemsa staining) and flow cytometry (annexin-V staining). Irradiation alone induced a dose-related reduction in cell proliferation and the appearance of polyploid cells in EOL-1 cells. Additionally, EOL-1 cells underwent a dose-related increase of apoptosis which, from the second day on, was accompanied by a dose-related increase of necrosis. When cells were exposed to EGCG, resveratrol or curcuma alone, a decrease in cell proliferation was observed, beginning from 25 microM EGCG and 50 microM resveratrol and curcuma, while an increase in the percentage of apoptotic cells was noted from 50 microM EGCG, 100 microM resveratrol and curcuma in EOL-1 cells, after only one day of culture. Simultaneous exposure to X-irradiation and, EGCG, resveratrol or curcuma resulted in a synergistic decrease of cell proliferation as well as in a synergistic increase of apoptosis and necrosis. These results suggest that, depending on the concentration, EGCG, resveratrol and curcuma enhance radiation-induced apoptosis in the leukaemic cell line, EOL-1 (EGCG >resveratrol >curcuma). In order to further characterise the radiation-induced apoptosis of this leukaemic cell line, other flow cytometrical analyses are in progress.

Cytopathology of uncommon malignant renal neoplasms in the pediatric age group

Malignant renal neoplasms are common solid tumors in pediatric oncology practice. These include the common Wilms' tumor/nephroblastoma and the uncommon neoplasms such as clear-cell sarcoma of the kidney (CCSK), rhabdoid tumor, renal-cell carcinoma, and others. The aim of this study was to describe in detail the cytopathological features of the histopathologically proven uncommon pediatric renal tumors. Aspirates from Wilms' tumor, which are mesenchyme predominant, show clusters of spindle cells associated with the matrix material. Evidence of rhabdomyoblastic differentiation may be present. CCSK, classic subtype, is characterized by round to oval cells arranged perivascularly and also in sheets and clusters intimately associated with a metachromatic matrix mucopolysaccharide material better appreciated in May-Grunwald-Giemsa (MGG)-stained smears. The cells also have more abundant cytoplasm and may show nuclear grooves. Spindle-cell pattern of CCSK is difficult to diagnose on aspiration cytology. Renal-cell carcinoma of childhood shows similar cytological features as its adult counterpart. Rhabdoid tumor of the kidney is characterized by a monomorphic population of cells with abundant cytoplasm, eccentric nuclei with prominent nucleoli. Intrarenal yolk sac tumor is a rare neoplasm and shows severely pleomorphic cells on aspiration. Awareness of these entities is important for the practicing cytopathologist. Further, non-Wilms' renal malignant neoplasms must be distinguished from the common Wilms' tumor so that appropriate chemotherapy protocols may be instituted in cases where the tumor is in an advanced stage of malignancy.

Assessing HER‐2 status in fresh frozen and archival cytological samples obtained by fine needle aspiration cytology

Determination of HER-2 status is an essential prerequisite in considering a patient's eligibility for anti-HER-2 therapy; few studies have focused on its evaluation on cytological material. We present a new method of assessing HER-2 status on cytological samples, by fluorescence in situ hybridization (FISH) with an automated detection system, using fresh frozen (FF) and May-Grunwald-Giemsa (MGG) stained smears, and we evaluate the reliability of HER-2 determination on fine needle aspiration cytology (FNAC). The pre-treatment protocol for FF smears is easier and faster than for MGG stained slides. However, with the described procedure, FISH is also feasible on archival MGG stained slides. We conclude that with this method, cytological samples obtained by FNAC, either FF or MGG, are a reliable option for assessing HER-2 status.

Fluorescence In Situ Hybridization (FISH) for the Detection of the (8;14) Translocation in Archival Fine Needle Aspirates of Suspected Burkitt Lymphomas (BL).

The detection of the chromosomal translocations involving the myc gene, characteristic of BL, is important in the diagnostic workup of agressive lymphomas given its impact on treatment strategies and prognosis. Recently this has been accomplished using FISH techniques either in paraffin-embedded tissue sections or fresh tumor aspirates. BL has an elevated cellular turn-over and a rapid diagnosis is highly desirable; this, and its high incidence in pediatric populations in whom organ biopsy needs general anesthesia, makes fine needle aspiration (FNA) samples attractive for diagnosis. In some cases, confirmation of the diagnosis may require the analysis of archival pre-stained samples. However, only recent samples have been reported as suitable for FISH analysis. We analysed 18 cytological samples (8 cytospins and 10 smears) obtained by FNA from 16 male and two female patients, median age 7.5 (3 to 48) years old, diagnosed with BL (15), atypical BL (1) or high grade lymphoma with a difficult differential diagnosis with BL (3) between February 2000 and July 2004 on the basis of morphology and phenotype of cytological and/or histological samples. In all cases the presentation and further clinical course suggested the possibility of BL and treatment was administered accordingly. Sixteen smears were May-Grunwald-Giemsa (MGG) stained whereas two were fresh-frozen; the samples had been stored for variable periods (1 day to 53 months). Cytology specimens (6 MGG-stained and 6 frozen) obtained from 12 reactive lymph nodes were used as controls, to determine the cut-off value for positive hybridization signals. After removal of the coverslips with xylene, the slides were destained in a series of water and ethanol washes. Pretreatment with pepsin was followed by the hybridization procedure. The dual fusion t(8;14) fluorescent probe was purchased from Vysis. Eleven out of 18 (61%) samples could be hybridized using this technique. The 7 cases without an interpretable signal were between 9 and 53 months old. The cut-off value for t(8;14) was 0%, meaning that in 12 negative controls the pattern of two fusion signals with or without a remaining signal from der(8) chromosome was never observed. In aggreament to previous publications 8 out of 11 specimens (72%) were positive for the (8;14) translocation, including 3 cases in which a definitive BL diagnosis could not be made by cytology/histology alone. The median percentage of positive cells was 80% (46 and 92%). Although the quality and age of the smears influenced the results, and better signals were observed with frozen specimens, FISH analysis of archival cytopathological material stored for prolonged periods was frequently possible and allowed the diagnosis of 3 difficult cases, whereas it confirmed the morphological results in another 5 patients.

Magnetic Cell Separation Using Antibody Binding with Protein A Expressed on Bacterial Magnetic Particles

Bacterial magnetic particles (BacMPs) are efficient platforms of proteins for surface display systems. In this study, mononuclear cells from peripheral blood were separated using BacMPs expressing protein A on the BacMP membrane surface (protein A-BacMPs), which were complexed with the Fc fragment of anti-mouse IgG antibody. The procedure of positive selection involves incubation of mononuclear cells and mouse monoclonal antibodies against different cell surface antigens (CD8, CD14, CD19, CD20) prior to treatment with protein A-BacMP binding with rabbit anti-mouse IgG secondary antibodies. Flow cytometric analysis showed that approximately 97.5 +/- 1.7% of CD19(+) and CD20(+) cells were involved in the positive fraction after magnetic separation. The ratio of the negative cells in the negative fraction was approximately 97.6 +/-1.4%. This indicates that CD19(+) and CD20(+) cells can be efficiently separated from mononuclear cells. Stem cell marker (CD34) positive cells were also separated using protein A-BacMP binding with antibody. May-Grunwald Giemsa stain showed a high nuclear/cytoplasm ratio, which indicates a typical staining pattern of stem cells. The separated cells had the capability of colony formation as hematopoietic stem cells. Furthermore, the inhibitory effect of magnetic cell separation on CD14(+) cells was evaluated by measurement of cytokine in the culture supernatant by ELISA when the cells were cultured with or without lipopolysaccharide (LPS). The induction of IL1-beta, TNFalpha, and IL6 was observed in the presence of 1 ng/mL LPS in all fractions. On the other hand, in the absence of LPS, BacMPs had little immunopotentiation to CD14(+) cells as well as that of artificial magnetic particles, although TNFalpha and IL6 were slightly induced in the absence of LPS in the positive fraction.

Morphology, cytochemical staining, and ultrastructural characteristics of the blood cells of the giant lizard of El Hierro ( Gallotia simonyi)

The object of this study was to examine the erythrocytes, leukocytes and thrombocytes of the giant lizard of El Hierro ( Gallotia simonyi) by light and electron (TEM) microscopy, and cytochemical staining. Smears were prepared from blood from the ventral coccygeal vein of 10 healthy adult lizards (five males and five females) from the Giant Lizard of El Hierro Reproduction and Research Centre, Canary Islands, Spain. The cytochemical stains used were: benzidine peroxidase (BP), chloroacetate esterase (CAE), alpha-naphthyl acetate esterase (ANAE), acid phosphatase (AP), periodic acid-Schiff (PAS), toluidine blue (TB) and May–Grünwald–Giemsa (MGG). Electron microscopy was also performed on all samples. Heterophils had granules that were heterogeneous in both size and electron density, and stained with BP, PAS and ANAE. Eosinophil granules were homogeneously electron-dense and stained for AP, CAE and ANAE. Basophils had both highly and moderately electron-dense granules, and stained with TB and ANAE. Azurophil granules were of low electron-density and stained for AP, CAE and ANAE. Azurophil cytoplasm was vacuolated on TEM. The cytoplasm of lymphocytes contained many ribosomes and was positive for AP. Monocytes had a large nucleus and a vacuolated cytoplasm but did not stain by any of the cytochemical methods used. Thrombocytes had a relatively large nucleus but little cytoplasm; they did not stain cytochemically. The blood cells of the giant lizards of El Hierro differ from those of other members of the Order Squamata both morphologically and cytochemically. The variation in cytochemical responses in the blood of reptiles makes it necessary to study species individually if meaningful clinical decisions are to be made.

Enhanced radiation-induced apoptosis of cancer cell lines after treatment with resveratrol

A search for new agents that can sensitise cancer cells to ionising radiation is of continual interest and mainly due to the use of radiation in cancer therapy. Resveratrol, a powerful antioxidant has been shown to inhibit carcinogenesis in animal models. The purpose of this study was to examine whether resveratrol can sensitise cancer cells to X-irradiation. The human cancer cell lines examined were HELA (cervix carcinoma), K-562 (chronic myeloid leukemia) and IM-9 (multiple myeloma). The assays that were performed following X-irradiation (doses from 0 to 8 Gy) and/or incubation in the presence of resveratrol (concentrations ranging from 0 to 200 microM), were the following: trypan blue exclusion test to determine cell viability, cell morphology after May-Grunwald Giemsa staining, DNA profile analysis by flow cytometry to assess cell cycle distribution and the presence of the sub-G1 peak. The cell lines showed different radiation sensitivity (IM-9, high radiation sensitivity, K-562, intermediate radiation sensitivity and HELA, low radiation sensitivity) as seen by the X-irradiation dose related inhibition of cell growth and induction of apoptosis. The addition of resveratrol alone to the cell cultures induced apoptosis and inhibited cell growth from 50 (IM-9), 100 (EOL-1) or 200 microM (HELA) resveratrol concentrations. Concomitant treatment of the cells with either resveratrol and X-irradiation induced a synergical effect at the highest dose of 200 microM. These results show that resveratrol can act as a potential radiation sensitiser at high concentrations. Further studies need to address the toxicity of resveratrol on normal cells.

Field's stain a quick alternative to MGG (May Grunwald Giemsa)

Field's stain a quick alternative to MGG (May Grunwald Giemsa)

Cytoplasmic vacuolation, intracytoplasmic lumina, and DPAS staining in ductal carcinoma of the breast.

A retrospective study was carried out on 30 cases of histologically proven invasive ductal carcinoma of the breast with a prior fine-needle aspiration (FNA) cytology. On evaluating the May-Grünwald-Giemsa (MGG) FNA smears, cytoplasmic vacuolation was observed in 70% cases. Positivity with periodic acid Schiff-positive, diastase-resistant (DPAS) staining was observed in 90% of cases. The chi(2) value on a McNemar test was 4.16. Thus, DPAS staining was significantly superior to MGG staining for picking up cytoplasmic vacuoles (P < 0.05). In 56.67% cases, DPAS staining showed an improvement in score as compared to MGG smears. This was highly significant (P < 0.001) on Wilcoxon matched-pairs signed-ranks test. Applying the strict criteria of thick-walled cytoplasmic vacuoles with a central darkly stained dot, none of our cases revealed true intracytoplasmic lumina. Larger studies are required to establish a role for DPAS staining in separating borderline, in situ, and invasive breast lesions, and to see if such positvity can be incorporated into the grading systems for breast carcinoma.

Fossomatic Cell-Counting on Ewe Milk: Comparison with Direct Microscopy and Study of Variation Factors

Using the Fossomatic method (FSCC) a total of 23,003 analytical SCC observations were carried out on 6400 aliquots taken from 80 individual ewe milk samples with the objective of studying the influence of 4 preservation procedures (without preservation, potassium dichromate, azidiol, and bronopol), 2 storage temperatures (ambient and refrigeration), 10 milk ages (3, 6, 12, and 24h, and 2, 3, 4, 5, 7, and 9d postcollection), and two analytical temperatures (40 and 60°C). In addition, each sample was analyzed with direct microscopic method (DMSCC), using 3 different stainings for each sample: methylene blue (MB), May-Grünwald-Giemsa (MGG) and Pyronin Y-methyl green (PGM). This allowed DMSCC and FSCC (at 24h of age) to be compared.The reference DMSCC from MB staining was a reliable method in ewe milk, though more specific stainings such as MGG and PMG slightly improve the residual standard deviation for repeated SCC. Between DMSCC and FSCC, the highest coefficients of correlation (0.972 to 0.996) corresponded to preserved and refrigerated milk, and the lowest (0.708 to 0.919) to unpreserved and ambient stored aliquots. Except for the unpreserved and ambient stored aliquots, SCC values were similar in all aliquots.Under FSCC, preservation, storage and analytical temperature, milk age, and most of the interactions showed a significant effect on SCC variation. In preserved samples, logSCC values ranged between 5.67 (bronopol) and 5.62 (azidiol). The higest values (5.72) were for unpreserved milk, which showed false overestimation of SCC due to bacterial proliferation. LogSCC was higher at 60°C (5.68) than at 40°C (5.65). The interaction between age, preservation and storage temperature showed no cell degeneration in properly handled samples over the 9 d of study.

The contribution of rapid intraoperative cytology to the improvement of ovarian cancer staging.

The goal of this work was to evaluate the use of intraoperative cytology in the improvement of ovarian cancer staging. Fifty-two patients with clinical stage IA-IIB ovarian cancer underwent peritoneal washing (PW) cytology and imprint cytology performed on retroperitoneal lymph node samples, during primary surgical treatment. Cytologic specimens were stained by the May-Grünwald-Giemsa (MGG) and hematoxylin-eosin (HE) techniques. Pertinent histologic sections of the ovarian lesions, cell blocks prepared from PW sediments, and lymph node samples were studied and compared with the cytologic findings. Our study reveals that, when malignant cells are present in the peritoneal cavity, PW cytology has 84.6% sensitivity and 94.7% specificity in detecting them. Imprint cytology performed on lymph node samples presented 94.1% sensitivity and 94.1% specificity in the diagnosis of retroperitoneal metastasis of ovarian cancer. Only 7 patients (13.4%) were upstaged with either cytologic method. PW cytology alone upstaged 6 more patients, while imprint cytology alone upstaged 11 more patients. This corresponds to a total of 17 patients (32.6%) who benefit from the combined performance of both cytologic methods. HE stain presents lower values of sensitivity and specificity compared with MGG. Cytologic evaluation of intraperitoneal and retroperitoneal spread of ovarian cancer by use of PW cytology and imprint cytology performed on lymph node samples contributes to the improvement of ovarian cancer staging.

The Significance of Rapid Intraoperative Cytology in the Evaluation of Intraperitoneal and Retroperitoneal Spread of Cervical Cancer

Objective. To evaluate the contribution of the rapid intraoperative cytology (peritoneal washing [PW] cytology together with imprint cytology performed on lymph node samples) in the assessment of peritoneal and retroperitoneal dissemination of cervical cancer.Methods. Seventy-nine patients with clinical Stage IA2–IIA cervical cancer underwent PW cytology and imprint cytology performed on retroperitoneal lymph node samples during primary surgical treatment. Cytologic specimens were stained with the May-Grünwald-Giemsa (MGG) and hematoxylin–eosin (HE) techniques. Pertinent histologic sections of the cervical lesions, of the cell blocks prepared from PW sediments, and of the lymph node sampling were studied and compared with the cytologic findings.Results. PW cytology was positive in 2.5% of the patients, and lymph node imprints revealed metastases in 19% of the patients. Cervical adenocarcinomas presented increased rates of metastatic disease in both the peritoneal cavity and retroperitoneum. HE presented higher sensitivity in detecting malignant cells in the peritoneal fluids, while MGG gave more accurate results in the diagnosis of lymph node metastases.Conclusions. Cytologic evaluation of intraperitoneal and retroperitoneal spread of cervical cancer by use of PW cytology and imprint cytology performed on lymph node samples contributes to the assessment of the extent of disease and therefore could be useful in further treatment of the patient.

Estudos Sobre Leishmaniose: I. Primeiros Casos de Leishmaniose Espontânea Observados em Cobáios

The author describes the first cases of spontaneous leishmaniasis in guinea-pigs, as verified for the first time, in Curitiba, at the Institute of Biology and Technological Research. Studying the inoculability of Paracoccidioide brasiliensis in guinea-pigs, he found animals which presented tumoral lesions in the ears and peritesticular skin. The examination of these tumors, mixed with a small drop of Giemsa's original solution, plus the exam of the rubbings of the same material, coloured by the May-Grunwald Giemsa after fixation by Heidenhein's solution and the histological exams of the cuts in the tumoral lesions revealed the presence of elements which possessed the same morphology and aspect of the LEISHMAN-DONOVANI corpuscles. In the meanwhile, a parasitosis by protozoarians of the Leishmania variety might have been suspected, because large macrophagi could be seen in the histological cuts, all parazitized, identical with those which are found in other cutaneous leishmaniosis. The author then proceeds to the experimental study of the sickness, first by endeavouring to obtain material for the inoculation and cultures, by means of punctures in the tumors. Part of the product of the punctures was sown in N. N. N. medium and part inoculated in other guinea-pigs. After the third day the cultures presented the flagellated forms of the parasite, free or grouped in rosettes in the culture liquid. All the guinea-pigs inoculated with part of the material obtained by puncturing and with the liquids of the cultures, reproduced the tumors found in the animals spontaneously infected. With a view to reproducing the same features of generalization which were observed in other experimental animals, with other strains of leishmania, new animals were inoculated after having been tarred and blocked in order to attempt the breaking down of possible natural resistences, which proved unnecessary, however, for the generalization went on of itself naturally and without artifice. Inoculations were made by the subcutaneous, intraperitonial, intrasplenic, intramedular, intracerebral and endovenous channels and by instillation in the conjunctive, in those guinea-pigs which received subcutaneous inoculations, the incubation period varied between 11 and 15 days, this period being longer when the internal channels were used, when it was generally one or two months before the lesions appeared. When the lesions are on the skin or when the inoculation is done there, the first manifestations of lesions are characterized by a slight thickening of the subcutaneous tissue, which, little by little, becomes more consistent, giving place to a tumoral growth which attains great volume and characterizes the most developed phase. The lesions, when metastatic, are localized mostly in the tegumentary extremities and periarticular zones, having the same appearance as the lesions observed by other researchers who have studied the infection of hamsters, mice and dogs infected by L. donovani, L. infantum and L. tropica. The experiments carried out in all the tumoral lesions in guinea-pigs, were positive for leishmania, where they were found free in the tissues or parasitizing large macrophagi. At times the tumors ulcerate and become covered with a crusty layer, easily detachable, covering a rosy surface of granulomatosis aspect. The spleen and lymphatic ganglia are the internal organs which most react, hypertrophy always being present, and it has been possible to re-isolate the parasite of these organs as well as those of the liver, suprarenal, testicle, medulla of the bones and cardiac blood. A series of sixty guinea-pigs were inoculated in the skin of the nose and sacrificed within anything from a few minutes' up to twenty-four hours' interval. The material obtained from these animals was fixed in Bouin-Hollande, included in paraffin and coloured by hematoxilin-eosine and Maxinow's hematoxilin-azur II-eosine. The study of the histological cuts in the different periods of evolution showed that the inflammatory process passes through a series of modifications which start by an acute inflammatory phase followed by a simple granulomatosis phase, finally followed by another characterized exclusively by large parasitized macrophagi - the macrophagi phase. During the period of evolution of the inflammatory lesion, the infiltrative phenomena are constant and are at first constituted of granulocited neutrophils, eosinophils and mononuclear cells, followed by a preponderance of monocytes and lastly by the sole presence of large parasitized macrophagi. The author compares his findings in the guinea-pigs with those in other animals inoculated with different strain of Leishmania, calling attention to a real dermatropism of the parasite under study to its dissemination through the lymphatic channels and to the histologycal lesions identical to those other leishmaniosis, principally by those produced by L. tropica.