Nuclear Details Research Articles

Fine-needle aspiration diagnosis of lymphomas with signet ring cell features: potential pitfalls and solutions.

Lymphoma with signet ring cell features (LSF) is a rare morphologic variant of non-Hodgkin lymphoma. Although it has been well documented in the surgical pathology literature, to the best of the authors's knowledge, the features of LSF in fine-needle aspiration (FNA) samples have rarely been reported. An accurate cytologic diagnosis of LSF is of important therapeutic significance. The authors retrospectively reviewed 7 FNA cases of LSF for cytologic features, ancillary studies, corresponding histologic findings, and the patients' clinical and radiologic information to illustrate the diagnostic clues and potential pitfalls. The final diagnoses, based on a multidisciplinary approach, were follicular lymphoma (5 patients), large B-cell lymphoma of follicular center cell origin (1 patient), and low-grade B-cell lymphoma with plasmacytoid features (1 patient). FNAs were obtained from both lymph node and extranodal sites. Common cytologic features included various percentages of signet ring cells in a background of nonvacuolated lymphomatous cells, lymphoglandular bodies, and cytoplasmic rings. The majority of signet ring cells contained a single, large, clear intracytoplasmic vacuole that pushed the nucleus laterally whereas fewer cells contained ≥ 2 vacuoles that indented the nucleus into a scalloped or stellate configuration. These cells resemble, to some degree, other lesions with signet ring cell features. One of the diagnostic clues of LSF was the similarity in nuclear details between signet ring cells and surrounding nonvacuolated lymphoid cells. Familiarity with cytologic features, correlation with clinical/radiologic information, and ancillary studies are important for an accurate diagnosis of LSF and for distinguishing it from other lesions with signet ring cell features in FNA samples.

Comparison of routine fixation of tissues with rapid tissue fixation.

Conventional formalin-fixed, paraffin-embedded tissue provides superior cellular morphology and long-term storage. Problems with formalin fixation comprise delay of fixation and variations in the duration of fixation. Microwave assisted tissue fixation removes the use of noxious and potentially toxic formalin that decreases the turnaround time and creates a personnel friendly workflow. The present study was conducted over a period of two years. One hundred and forty paired tissue sections were taken including both neoplastic and non-neoplastic tissues. One of the paired tissues was fixed in formalin and the other was fixed by using microwave irradiation in phosphate buffered saline. Both were then processed by conventional method. Each slide was examined and rated for the adequacy of fixation by two pathologists in a blinded fashion using 7 parameters: Cellular outline, cytoplasmic detail, nuclear detail, erythrocyte integrity, lymphocyte appearance, overall morphology and overall staining. Statistical analysis showed that sections obtained from microwave fixed tissues were comparable to that of routinely fixed tissue. The p-values of all parameters were not significant except for the overall morphology for which p-value was significant owing to loss of tissue in some cases. Microwave irradiation substantially shortened the time from specimen reception to diagnosis (turnaround time) and allowed same-day tissue processing and diagnosis of specimens without compromising the overall quality of the histologic section.

Comparison of conventional tissue processing with microwave processing using commercially available and domestic microwaves

Context: Microwave-accelerated tissue processing is believed to have brought a revolutionary improvement in the field of histopathology and histochemistry. This technique shortens the time for tissue processing from days to minutes. The technique fulfills the needs of the patient as well as those of the physician by improving the use of reagents while reducing or eliminating their toxicity, and places the laboratory in a better position to meet the demands of the surgeon. Aim: This study is being conducted to determine the efficacy of the microwave tissue processing method for orofacial biopsy specimens by comparing two different techniques of microwave histoprocessing with the conventional method, based on the clarity of nucleocytoplasmic differentiation and staining of tissues processed by each method . Materials and Methods: Twenty oral mucosal biopsy specimens were cut into three equal parts and each part was processed by three processing techniques. Hematoxylin and eosin staining was performed at the same time and grading was done by four oral pathologists . Statistical Analysis Used: The one-way ANOVA test using SPSS 17 was used in this study. Result: The parameters (color intensity, contrast, uniformity of staining, cytoplasmic and nuclear details) appeared slightly better in a commercial microwave, followed by the domestic microwave method and the routine method. The P values were statistically insignificant for all the parameters except color intensity . Conclusion: Microwaves can be used for tissue processing, which shortens the time for tissue processing without affecting the architecture of the cells and with increased intensity of staining.

Domestic Microwave Versus Conventional Tissue Processing: A Quantitative and Qualitative Analysis

Microwave irradiation has been tried as a replacement for the conventional tissue processing technique in histopathology laboratories for quite some time. Studies have shown that Domestic Microwave Tissue Processing (DMWTP) provides a faster delivery of the tissue sections with a morphology which is similar to that which is seen Conventional Tissue Processing (CTP). But many laboratories still confine the domestic microwave tissue processing method only to the handle selected specimens, for which urgent reports are needed. One of the probable reasons is that, understanding about the number of tissue sections which can be processed using a microwave oven at a time, with the appropriate quality, still remains unclear. The aim of this study was to quantitatively analyze the optimum number of samples that a domestic microwave could process at a time, as well as to qualitatively analyze the morphological outcome of those tissue sections with that of conventional processing. This study was approved by the research and ethical committee of Sree Balaji Medical College and Hospital. A total of 135 paired tissue sections were included in the study. Ten tissue sections (which are mentioned hereafter as A10) were processed in a domestic microwave and their paired 10 tissues were processed by a conventional method. Subsequently, the number of tissues which was to be processed was increased to B15, C20, D25, E30 and F35, after ascertaining that the morphological qualities of the previously processed tissue sections were satisfactory. Sections of 4 μm thickness were taken and they were stained by the Haematoxylin and Eosin method. The slides of the tissues which were processed by the microwave method and the conventional method were randomly numbered, for a blind study, which were independently evaluated by two observers. The qualities of slides were assessed, based on 4 parameters: the cytoplasmic details, the nuclear details, the tissue architecture and the staining characteristics. The statistical analysis was done by using SPSS 15.0. The morphological outcomes (quality) of the DMWTPs were comparable to that of the CTPs, when the sample load (quantity) in the microwave oven was up to 25 samples. Domestic microwave processing can be effectively used in laboratories with a maximum sample size of 25 samples per load. This has the advantage of being rapid, with its morphological quality being identical to that of conventional processing.

Cytopathologic diagnosis of spontaneous infarction of fibroadenoma of the breast

Infarction is an uncommon event in a fibroadenoma, which is the commonest benign tumor of the breast. Most often it occurs in pregnancy, lactation or is secondary to fine needle aspiration. Spontaneous infarction of a fibroadenoma in the absence of a predisposing condition is very rare. The cytopathologic features of infarction are necrosis and worrisome nuclear features, which are often misinterpreted as either inflammation or malignancy. We detail a report of accurate cytopathologic diagnosis of spontaneous infarction of fibroadenoma in a 17-year-old adolescent non pregnant girl. Careful attention to the cytopathologic clues like uniform thickness of the necrotic epithelial fragments, branching pattern reminiscent of the staghorn pattern despite atypical nuclear features and clinical details like young age of the patient and recent onset pain in a pre-existing lump helped arrive at the correct diagnosis and spared the patient of a radical excision. To the best of our knowledge, there are no earlier reports of correct cytopathologic diagnosis.

Phenomenologicalpn→ppπ−model ofA(p,π−)Breactions

A phenomenological $pn\ensuremath{\rightarrow}pp{\ensuremath{\pi}}^{\ensuremath{-}}$ model of $A(p,{\ensuremath{\pi}}^{\ensuremath{-}})B$ reactions is developed that incorporates the spin dependence of the former reaction. The model development follows closely that of the $pp\ensuremath{\rightarrow}d{\ensuremath{\pi}}^{+}$ model for $A(p,{\ensuremath{\pi}}^{+})C$ reactions. Predictions of the analyzing powers and differential cross sections for the $(p,{\ensuremath{\pi}}^{\ensuremath{-}})$ reaction on ${}^{13}$C at 200 MeV are presented and discussed. Calculated differential cross sections describe reasonably well the shape of the experimental data, although the predicted strengths vary considerably. Model predictions of the analyzing powers are in general agreement with the data. Nuclear structure amplitudes are obtained from a no-core multi-$\ensuremath{\hbar}\ensuremath{\omega}$ shell model. Sensitivities of the observables to the nuclear structure details and also the amplitudes of the primary $pn\ensuremath{\rightarrow}pp{\ensuremath{\pi}}^{\ensuremath{-}}$ reaction are investigated.

Comparison of diagnostic values of thyroid aspiration samples using liquid‐based preparation and conventional smear: one‐year experience in a single institution

Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy. Fine-needle aspiration (FNA) is the most useful tool in the diagnosis of thyroid nodules. Liquid-based preparation (LBP) in FNA of thyroid nodules is now widely used and is replacing the conventional smear (CS). We compared the diagnostic value of the LBP method with that of CS in thyroid lesions. A total of 1767 CS FNA samples and 2523 LBP FNA samples were included in this comparison of diagnostic values. We also assessed the differences in cytomorphologic features in 41 randomly selected cases of PTC. The measured sensitivities of CS and LBP were 78.9% and 76.3%, respectively, and the specificities were 64.2% and 54.9%, respectively, whereas the positive predictive values were 92.3% and 94.3%, respectively, and the negative predictive values were 89.5% and 81.8%, respectively. The cytomorphological features of LBP showed better-preserved nuclear details, cleaner background and fewer large papillae than were evident in CS. The results indicate that LBP reduces the diagnosis of non-diagnostic or unsatisfactory/atypia of undetermined significance or follicular lesion of undetermined significance, although the diagnostic values for CS and LBP are not appreciably different.

Rapid screening of cancer margins in tissue with multimodal confocal microscopy

BackgroundComplete and accurate excision of cancer is guided by the examination of histopathology. However, preparation of histopathology is labor intensive and slow, leading to insufficient sampling of tissue and incomplete and/or inaccurate excision of margins. We demonstrate the potential utility of multimodal confocal mosaicing microscopy for rapid screening of cancer margins, directly in fresh surgical excisions, without the need for conventional embedding, sectioning, or processing. Materials and methodsA multimodal confocal mosaicing microscope was developed to image basal cell carcinoma margins in surgical skin excisions, with the resolution that shows nuclear detail. Multimodal contrast is with fluorescence for imaging nuclei and reflectance for cellular cytoplasm and dermal collagen. Thirty-five excisions of basal cell carcinomas from Mohs surgery were imaged, and the mosaics analyzed by comparison with the corresponding frozen pathology. ResultsConfocal mosaics are produced in about 9min, displaying tissue in fields of view of 12mm with ×2 magnification. A digital staining algorithm transforms black and white contrast to purple and pink, which simulates the appearance of standard histopathology. Mosaicing enables rapid digital screening, which mimics the examination of histopathology. ConclusionsMultimodal confocal mosaicing microscopy offers a technology platform to potentially enable real-time pathology at the bedside. The imaging may serve as an adjunct to conventional histopathology to expedite screening of margins and guide surgery toward more complete and accurate excision of cancer.

Highlighting nuclear membrane staining in thyroid neoplasms with emerin: Review and diagnostic utility

Immunohistochemical staining (IHC) with emerin, an integral inner nuclear membrane protein, highlights nuclear membrane details in papillary thyroid carcinoma (PTC). We evaluated emerin for highlighting nuclear shape, grooves, inclusions, circumferential nuclear membrane irregularities ("garlands"), deep "stellate" membrane invaginations, and crescents in 34 fine-needle aspiration (FNA) cell blocks, PTC (n = 24) and follicular neoplasms (FN) (n = 10). Tissue microarrays were also examined for 182 cases, PTC (n = 95) and non-PTC (n = 87). Emerin IHC of PTC revealed a predominantly oval nuclear shape in the majority of cases, with FN demonstrating round nuclei and FV of PTC showing a roughly equal distribution of round and oval shapes. In addition to oval nuclear shape, the presence of emerin-positive nuclear grooves, circumferential emerin nuclear "garlands," nuclear crescent shapes, and chromatin clearing on cell block H&E staining were significant predictors of PTC by regression analysis. Emerin IHC of thyroid FNA and surgical specimens serves as a useful adjunct to conventional H&E staining in the diagnosis of PTC and its distinction from FN by delineating diagnostic nuclear membrane irregularities ("garlands" and crescents), nuclear grooves, and a characteristic oval nuclear shape. In diagnostically challenging cases with limited cellularity, emerin staining can help to provide a more definitive diagnosis of PTC.

Concordance between whole-slide imaging and light microscopy for routine surgical pathology

The use of high-resolution digital images of histopathology slides as a routine diagnostic tool for surgical pathology was investigated. The study purpose was to determine the diagnostic concordance between pathologic interpretations using whole-slide imaging and standard light microscopy. Two hundred fifty-one consecutive surgical pathology cases (312 parts, 1085 slides) from a single pathology service were included in the study after cases had been signed out and reports generated. A broad array of diagnostic challenges and tissue sources were represented, including 52 neoplastic cases. All cases were digitized at ×20 and presented to 2 pathologists for diagnosis using whole-slide imaging as the sole diagnostic tool. Diagnoses rendered by the whole-slide imaging pathologists were compared with the original light microscopy diagnoses. Overall concordance between whole-slide imaging and light microscopy as determined by a third pathologist and jury panel was 96.5% (95% confidence interval, 94.8%-98.3%). Concordance between whole-slide imaging pathologists was 97.7% (95% confidence interval, 94.7%-99.2%). Five cases were discordant between the whole-slide imaging diagnosis and the original light microscopy diagnosis, of which 2 were clinically significant. Discordance resulted from interpretive criteria or diagnostic error. The whole-slide imaging modality did not contribute to diagnostic differences. Problems encountered by the whole-slide imaging pathologists primarily involved the inability to clearly visualize nuclear detail or microscopic organisms. Technical difficulties associated with image scanning required at least 1 slide be rescanned in 13% of the cases. Technical and operational issues associated with whole-slide imaging scanning devices used in this study were found to be the most significant obstacle to the use of whole-slide imaging in general surgical pathology.

Application of liquid-based cytology to fine-needle aspiration biopsies of the thyroid gland.

Fine-needle aspiration biopsy is regarded as an important tool for diagnosing thyroid lesions because of its simplicity, safety, and cost–effectiveness. Its role in correctly characterizing the group of indeterminate lesions or follicular-patterned neoplasms (FN) might be more decisive. Liquid-based cytology (LBC) is a technique based on the use of a semi-automated device that has gained popularity as a method of collecting and processing both gynecologic and non-gynecologic cytologic specimens. It achieves a diagnostic sensitivity as accurate as conventional preparations especially for its excellent cell preservation and for the lack of background which decrease the amount of inadequate diagnoses. Moreover, the cellular material which has been stored in the preservative solution could be effectively used for the application of immunocytochemical and molecular techniques especially for the Follicular proliferations. In many cases the cytologic features are similar in both methods but the colloid film and the lymphocytic component are more easily evaluated on direct smears whereas nuclear details and colloid globules are better evaluated in LBC slides. The LBC-processed biopsies represent a valid alternative to conventional cytology. The possibility of applying special techniques enhance the efficacy of the cytological diagnosis of thyroid lesions.

Arias-Stella reaction of the cervix: The enduring diagnostic challenge

SummaryBackground:The Arias-Stella reaction is a hormone-related atypical endometrial change characterized by hypertrophy and vacuolization of glandular epithelial cells, associated with marked nuclear pleomorphism, enlargement, and hyperchromasia. When presenting in extra-uterine sites, the differentiation of Arias-Stella changes from other more ominous clear cell lesions may pose significant difficulties.Case Report:We report a case of an endocervical clear cell lesion incidentally discovered during a prenatal visit of a young pregnant woman. Interpretation of the pathological findings was complicated by the small size and fragmentation of the specimen.Conclusions:Recognition of Arias-Stella reaction in extra-uterine sites, especially in young women, is critical to avoid misdiagnosis of this innocuous lesion as clear cell adenocarcinoma. Attention to cellular and nuclear detail, as well as careful consideration of the clinical scenario, is crucial for establishment of the correct diagnosis.

Histological Fixation with Formalin under Microwave Irradiation

The use of microwaves can be a solution for rapid microwave-assisted tissue processing, thus in present days it is successfully applied in histopathologic diagnosis as well in electron microscopy, in certain histochemistry reactions and especially in immunocytochemistry. In this article, using a domestic microwave oven we limited our researches only to study the chemical fixation of samples immersed in formalin. There were collected samples from compacted organs (liver, kidneys, testicle, and tongue) of three guinea pigs and were fixed using two distinct methods. One samples’ lot was fixed by immersion in 10% neutral formalin during 24 hours at laboratory temperature, and the other samples’ lot was fixed in same fixative solution but using the microwave irradiation for only 20 minutes. After usual inclusion in paraffin and division into sections, was practiced the Hematoxyline-Eosine staining. We had not observed qualitative differences between the two preparations’ lots, neither as concerns the nuclear structural details nor the cytoplasm aspects, but the processing time was significant different.

Liquid-Based Cytology in Fine-Needle Aspiration Biopsies of the Thyroid Gland

Objectives: Fine-needle aspiration biopsy is regarded as the most important diagnostic tool for thyroid lesions because of its simplicity, safety, and cost-effectiveness. However, its pivotal role in the correct characterization of the majority of nodules is impaired by the difficulties in discriminating benign from malignant follicular-patterned lesions. Study Design: Liquid-based cytology (LBC) is a semiautomated device that has recently become widely available and has gained popularity as a method of collecting and processing both gynecologic and nongynecologic cytologic specimens. It achieves a diagnostic sensitivity as accurate as conventional preparations, especially for its excellent cell preservation and lack of background which decrease the amount of inadequate diagnoses. Results: In many cases the cytologic features are similar in both methods, but the colloid film and the lymphocytic component are more easily evaluated on direct smears whereas nuclear details and colloid globules are better evaluated in LBC slides. The material stored in the preservative solution could be effectively used for the application of immunocytochemical and molecular techniques. Conclusions: LBC-processed biopsies represent a valid alternative to conventional cytology. The possibility of applying additional techniques enhances the efficacy of the cytologic diagnosis of thyroid lesions.

Optimization of pupil design for point-scanning and line-scanning confocal microscopy

Both point-scanning and line-scanning confocal microscopes provide resolution and optical sectioning to observe nuclear and cellular detail in human tissues, and are being translated for clinical applications. While traditional point-scanning is truly confocal and offers the best possible optical sectioning and resolution, line-scanning is partially confocal but may offer a relatively simpler and lower-cost alternative for more widespread dissemination into clinical settings. The loss of sectioning and loss of contrast due to scattering in tissue is more rapid and more severe with a line-scan than with a point-scan. However, the sectioning and contrast may be recovered with the use of a divided-pupil. Thus, as part of our efforts to translate confocal microscopy for detection of skin cancer, and to determine the best possible approach for clinical applications, we are now developing a quantitative understanding of imaging performance for a set of scanning and pupil conditions. We report a Fourier-analysis-based computational model of confocal microscopy for six configurations. The six configurations are point-scanning and line-scanning, with full-pupil, half-pupil and divided-pupils. The performance, in terms of on-axis irradiance (signal), resolution and sectioning capabilities, is quantified and compared among these six configurations.

Morphology of rat testis preserved in three different fixatives

Histopathological examination of testes is important in assessing spermatogenesis and testicular function. Modified Davidson's fluid (mDF) has been proposed as a superior substitute for Bouin's fluid (BF) for fixation of adult animal testes. Besides, 4% paraformaldehyde (PFA) has been commonly used to fix testes with convenience. We compared the morphology of the rat testis fixed in 4% PFA, mDF, or BF using hematoxylin and eosin (HE)-stained sections. Fixation in 4% PFA resulted in obvious tissue shrinkage artifacts, especially between seminiferous epithelium cells. Shrinkage artifacts were also observed in the central area of the testes fixed in BF. Use of mDF did not cause shrinkage artifacts between seminiferous tubules, though a small amount can be observed in seminiferous tubules between germ cells. Clarity of nuclear detail in testes fixed in mDF and BF is better compared to 4% PFA. Our study demonstrated that fixation in mDF provided better morphologic details in the rat testis as compared with 4% PFA and BF.

Kitchen Microwave-Assisted Accelerated Method for Fixation and Processing of Oral Mucosal Biopsies: A Pilot Study

ABSTRACT Background and aim Conventional protocols of formalin fixation and processing add time and cost to histoprocessing. This study aims to evaluate an indigenously developed protocol for microwave histoprocessing and compare it with the routine protocol for histoprocessing. Materials and methods A total of 40 human buccal mucosal specimens were processed according to the microwave and the routine protocol. Sections obtained were coded and evaluated for the following: Section quality, staining quality, artifacts, cellular details in epithelium, nuclear detail and diagnostic ability. Parameters were graded as suboptimal, optimal and good, and results were statistically analyzed. Results The number of good and optimal specimens in both the protocols were almost similar for all parameters assessed with better results for cellular detail, nuclear detail, staining and sectioning quality seen in microwave-fixed samples. Artefacts were lesser in routine processing protocol. Conclusions Microwave alcohol fixation protocol provides good and faster fixation compared to routine fixation.

A comparative study on microwave and routine tissue processing

The present study was aimed at assessing the rapid microwave-assisted tissue processing and staining to determine if it can replace standard formalin-fixed and paraffin-embedded processing and staining technique. The study group consisted of 15 oral mucosal biopsies. The specimens were fixed in 10% formalin for 24 hours to ensure adequate fixation and their gross features were recorded, photographed, and then the specimens were cut into equal halves to be processed by both conventional and microwave histoprocessing methods and then subsequently stained with H and E by microwave and conventional methods. The stained slides in each group processed by both microwave and routine methods were randomly numbered for a blind study and circulated among six observers. All the observers were asked to grade each parameter into Excellent/Good/Average/Poor in a data sheet comprising a total of six parameters. These gradings were given a numerical value of 4, 3, 2 and 1, respectively. The parameters included in the data sheet were cellular clarity, cytoplasmic details, nuclear details, color intensity, interface of epithelium and connective tissue. Wilcoxon-matched pairs signed rank test (non-parametric) was used to calculate the test of significance (P value). The total processing time involved in microwave was 42 minutes and 270 minutes for the conventional method. H and E staining in microwave took 33 minutes and 40 minutes for conventional method. The individual scores by different observers regarding the various parameters included in the study were statistically insignificant, the overall quality of microwave-processed and microwave-stained slides appeared slightly better than conventionally processed and stained slides.

Evaluation and optimization for liquid-based preparation cytology in whole slide imaging

Background: Cytology poses different obstacles in whole slide imaging compared to surgical pathology slides. A single focal plane suffices for most of the latter, but cytology slides are thicker, potentially requiring multiple focal planes for adequate diagnostic information. Multiple focal planes adversely impact scanning time per slide, evaluation times, and file sizes. In this pilot study, we evaluated and compared the multilayer stack method to the extended focus algorithm as an alternative which collapses multiple focal planes into a single image, retaining only focused areas from each plane. Materials and Methods: 10 SurePath® cervical cytology slides were scanned at three thickness settings: 18, 24, and 30 μm. Three scanners were used: (1) Hamamatsu Nanozoomer 2.0-HT, (2) 3DHISTECH Mirax scan, and (3) Bioimagene iScan Coreo Au. The Nanozoomer and iScan utilized multilayer stacking, while the Mirax files were composited by extended focus. Scan times and file sizes were recorded, and image quality compared. Results: The Nanozoomer stacks averaged 1.58 gb and around 25 min for each slide, while the iScan stacks ranged from 6.23 to 9.3 gb and took 34-50 min to scan. The Mirax images averaged 210 mb and took 13-20 min to scan. Multilayer stack image quality from both Nanozoomer and iScan was fairly comparable. The iScan revealed significant mechanical issues that did not correspond to user settings. The Mirax images showed worrisome loss of crisp focus detail, worsening with increasing focal planes and impacting assessment of nuclear contours and chromatin detail. Conclusions: The optimal number of focal planes remains unknown for cytology. Multilayer stacks require excessive scanning time, network bandwidth, and file storage. Extended focus was evaluated as an alternative, but significant image quality issues were revealed. Further large-scale studies are needed to assess their clinical impact.

Use of pooled sodium acetate acetic acid formalin-preserved fecal specimens for the detection of intestinal parasites.

This study aimed at comparing detection of intestinal parasites from single unpreserved stool sample vs. sodium acetate acetic acid formalin (SAF)-preserved pooled samples, and stained with chlorazol black dye in routine practice. Unpreserved samples were collected from 120 patients and represented as Group I. Other three SAF-preserved samples were collected from the same patients over a 6-day period and represented as Groups IIa, IIb, and IIc. The latter groups were equally subdivided into two subgroups. The first subgroup of each of the three samples was examined individually, whereas the second subgroup of each were pooled and examined as a single specimen. All groups were examined by the routine diagnostic techniques; however, in group II when the diagnosis was uncertain, the chlorazol black dye staining procedure was carried out. Results demonstrated that out of 74 patients who continued the study, 12 cases (16%) were positive in group I, compared with 29 (39%) in the subgroups examined individually, and 27 (36%) in the pooled subgroups. Therefore, pooling of preserved fecal samples is an efficient and economical procedure for the detection of parasites. Furthermore, the chlorazol black dye was simple and effective in detecting the nuclear details of different parasites.