Diastase-resistant Periodic acid-Schiff Research Articles

The Value of DPAS in the Detection of Breast Malignant Cells

Background: Fine-needle aspiration (FNA) has become an essential, critical test for breast masses. This study aimed to determine the value of diastase-resistant periodic acid-Schiff (DPAS) staining in the detection of malignant breast cells. Methods: This prospective cross-sectional study was conducted in Khartoum state (Sudan) among Sudanese women who suffered from breast lumps. FNA samples were collected from each patient, and the material was simultaneously smeared onto two labeled glass slides. The DPAS score and aspiration cytology (AC) grade are expressed as mean +/- SD, and the 95% confidence intervals of the means were calculated. Results: The findings revealed the following DPAS score frequencies among the studied women: negative (+/-) (28, 13.9%), one plus (+) (114, 56.7%), two plus (++) (27, 13.4%), and three-plus (+++) (32, 15.9%). Comparison of DPAS scores with the cytological categories (cytology results) revealed that DPAS positivity (++, +++) correlated best with malignancy. Of the 201 patients, the AC grades according to the International Academy of Cytology (IAC) system were: AC2 (30, 14.9%), AC3 (112, 55.7%), AC4 (27, 13.4%), and AC5 (32, 15.9%). Conclusions: DPAS positivity in atypical cells in FNA aspirates may assist in upgrading from a suspicious to a malignant diagnosis in women with breast lumps.

Pediatric granular cell tumor in the posterior wall of the larynx extending to the trachea.

Granular cell tumor (GCT) is a slow-growing benign neoplasm that can be found in any organ. Pediatric laryngotracheal GCT is rare. We experienced a 6-year-old boy suffering from a barking cough and symptoms of stridor and croup for one month. Head and neck computed tomography revealed a protruding mass that occluded 60% of the airway lumen. Under the impression of hemangioma or papilloma, excision revealed a submucosal non-encapsulated mass. Histologically, the mass was composed of sheets of large polyhedralshaped tumor cells containing plump eosinophilic granular cytoplasm and centrally placed, small, bland-appearing nuclei. The tumor cells were positive for S-100 protein, and voluminous eosinophilic cytoplasm was stained by diastase-resistant periodic acid-Schiff. The present report describes a unique case of a huge pediatric laryngeal GCT extending to the subglottic trachea. We also review the clinical course of pediatric laryngotracheal GCT and emphasize the importance of diagnosing GCT in children.

Alveolar soft-part sarcoma in paranasal sinuses

Objective To investigate clinicopathological features, immune phenotype, diagnosis and differential diagnosis of alveolar soft-part sarcoma (ASPS) in paranasal sinuses. Methods Retrospective study of clinical manifestations, histopathological features and immunohistochemical features was conducted in one case of ASPS in paranasal sinuses. Results A 28-year-old female presented with bulging forehead for 2 months. MRI revealed a well-circumscribed lesion in left frontal and ethmoid sinuses extending to anterior skull base that showed slightly hyperintense signal on T 1 WI and hypointense signal on T 2 WI without obvious enhancement after contrast administration. The patient subsequently underwent endoscopic open surgery on left ethmoid and bilateral frontal sinuses and performed partial resection of the lesion. Three months after the initial surgery, the patient received reoperation for total removal of residual lesion and reconstructive surgery of anterior skull base. Adjuvant chemotherapy and radiotherapy were not administered. Histologically, the tumor was composed of epithelioid cells arranged in organoid nests and/or alveolar structures varying in size and shape, which were separated by connective tissue richly containing sinusoidal vascular channels. The tumor cells were generally large-sized, round, oval or polygonal with abundant eosinophilic granular or translucent vacuolated cytoplasm. The nuclei showed round or oval shape containing centrally placed and obvious nucleoli. The presence a lot of mono- or multi-nuclear giant cells served as another striking feature. Mitotic activities were rare. Reticular fiber staining indicated that reticular fibers surrounded the nest of tumor cells, and diastase-resistant periodic acid-Schiff (PAS)-positive crystalline inclusions were identified within the cytoplasm of tumor cells. Immunohistochemically, the tumor cells were reactive for TFE3, while were negative for glial fibrillary acidic protein (GFAP), melanophore markers [HMB45, Melan-A and S-100 protein (S-100)], and epithelial markers [cytokeratin (CK) and epithelial membrane antigen (EMA)]. Ki-67 labeling index was low (2%). The patient remained well without recurrence 15 months after the second surgery. Conclusions ASPS is a rare malignant tumor that tends to occur in adolescents. The tumor is predominantly located in deep soft tissues, such as legs and buttocks. ASPS of paranasal sinus and/or extending to skull base is extremely rare. The accurate diagnosis of ASPS mainly depends on histological and immunohistochemical features, and should be considered in differential diagnosis including the similar morphological pattern of primary or metastatic neoplasms. ASPS is characterized by ASPL-TFE3 gene fusions, and TFE3 protein is a specific marker for ASPS which displays nuclear labeling with TFE3 by immunohistochemistry. DOI: 10.3969/j.issn.1672-6731.2015.07.012

The impact of intracytoplasmic mucin in lung adenocarcinoma with pneumonic radiological presentation

ObjectivesThis study investigated the clinical and prognostic impact of intracytoplasmic mucin in lung adenocarcinoma with “pneumonic” radiological presentation, formerly known as bronchioloalveolar carcinoma (BAC). Patients and methodsBetween 1986 and 2011, clinical and pathological data from 120 consecutive patients with lung adenocarcinoma with “pneumonic” radiological presentation were reviewed. Intracytoplasmic mucin was assessed using a diastase-resistant periodic acid-Schiff staining. The presence of EGFR or K-Ras mutations and ALK rearrangement were determined in surgical samples. ResultsThe two predominant histological patterns were invasive mucinous adenocarcinoma (40%) and lepidic predominant adenocarcinoma (32%). Intracytoplasmic mucin was detected in 71 patients (59.2%) who were more likely to be non-smokers (p=0.04) and have bronchorrhea (p=0.006), crepitant rales (p=0.02), or neutrophil alveolitis (p=0.0004). In mucin-producing tumors, EGFR mutation was not detected, K-Ras mutations and ALK rearrangement were present in 32% and 3% of cases, respectively. In non-mucin-producing tumors, EGFR and K-Ras mutations were detected in 17% and 10% of cases, respectively, no ALK rearrangement was detected. In univariate analysis, performance status>0, crepitant rales, bronchorrhea, neutrophil alveolitis, bilateral extension, intracytoplasmic mucin and no surgery were associated with worse survival. In multivariate analyses, intracytoplasmic mucin, neutrophil alveolitis, and no surgery were independent factors for worse survival. ConclusionIntracytoplasmic mucin is associated with specific clinical characteristics and is an independent factor for worse survival in lung adenocarcinoma formerly known as BAC.

Actinomycosis of the middle ear

To present the first case of middle-ear actinomycosis in the UK in the last 60 years. The diagnosis and management of actinomycosis of the middle ear is also presented, as well as a review of the recent literature. This paper reports the case of a nine-year-old girl who presented with recurrent otorrhoea caused by actinomycosis. Mastoid exploration with clearance of the infected tissue, in conjunction with six months amoxicillin, resulted in long-term disease remission. Histology revealed Gram-positive, silver avid (on Grocott staining) and diastase-resistant periodic acid-Schiff negative staining of colonies; this profile was in keeping with the characteristic 'sulphur granules' of actinomycosis. Actinomycosis of the middle ear and mastoid is rare, with less than 45 cases presented in the worldwide literature. This case confirms that the disease should be treated with full surgical clearance and long-term antibiotics.

Histochemical and Immunohistochemical Study in Melasma: Evidence of Damage in the Basal Membrane

The pathogenesis of melasma has not been clearly elucidated. Using Fontana Masson; diastase-resistant periodic acid-Schiff stains; and immunohistochemistry to stem cell factor (SCF), its receptor c-kit, anti-mast cell tryptase, and anti-collagen type IV antibody, we evaluated melasma lesions and compared them with perilesional skin and photoprotected skin. Samples were taken from lesional and photoprotected nonlesional skin in 24 patients. In other 24 patients, we took biopsies of lesional and perilesional skin. With Fontana Masson, we observed many pigmented basal cells protruding into the dermis of the melasma skin. Periodic acid-Schiff stain and anti-collagen type IV showed damage on the basal membrane in 95.5% and 83%, respectively, in melasma lesion. The immunoreactivity of SCF and the prevalence of mast cells were increased in the dermis of melasma compared with perilesional dermis. The expression of c-kit was significantly increased at lesional epidermis; a frequent protrusion of c-kit-positive basal cells into the dermis was evident in 70% versus that in 29% of perilesional skin. The expression of c-kit was increased at lesional dermis of melasma compared with perilesional skin. We found a low correlation between c-kit expression and prevalence of mast cells; these were increased in melasma skin. The results may suggest a role of SCF, c-kit, and mast cells in the pathogenesis of melasma. We were surprised by the unexpected evidence of damage to basal membrane (BM), which could facilitate the fall or the migration of active melanocytes and melanin into the dermis allowing the constant hyperpigmentation in melasma.

Meningeal alveolar soft part sarcoma confirmed by characteristic ASPCR1‐TFE3 fusion

Sarcoma metastatic to the brain is uncommon and rarely occurs as the initial manifestation of tumor. Alveolar soft part sarcoma (ASPS) is a rare but well-studied subtype of sarcoma. A 39-year-old man presented with seizures due to a left temporal meningeal-enhancing lesion with striking brain edema on MRI. The patient underwent neurosurgical resection for suspected meningioma. Histology showed large tumor cells clustering and forming small nests, in places with pseudoalveolar pattern. Diastase-resistant periodic acid-Schiff revealed very rare granular and rod-like cytoplasmic inclusions. Immunohistochemistry showed convincing positivity only with vimentin and smooth muscle actin. The histological features were strongly suggestive of ASPS. At the molecular level RT-PCR and sequencing analysis demonstrated ASPCR1-TFE3 fusion confirming the histological diagnosis of ASPS. There was no evidence of primary extracranial tumor by physical examination and on chest and abdominal CT scan 11 months after presentation. ASPS typically arise from the soft tissues of the extremities and develop multiple metastatic deposits usually with a long clinical course. This case may represent primary meningeal ASPS although metastatic deposit from an undiscovered primary site cannot be entirely excluded.

Solitary cutaneous histiocytosis with granular cell changes: a morphological variant of reticulohistiocytoma?

We first report a case of granular cell histiocytosis occurring as a solitary polypoid lesion of the nipple in a 15-year-old girl. Histologically, the lesion was composed of a dermal population of medium- to large-sized, short spindle- to round- to epithelioid-shaped cells with eosinophilic cytoplasm containing numerous and small diastase-resistant periodic acid-Schiff (PAS) positive granules. No associated inflammatory cells were observed. Immunohistochemical studies, revealing immunoreactivity exclusively to vimentin and CD68, were consistent with their histiocytic profile. Based on clinical, morphological and immunohistochemical features, the diagnosis of 'solitary cutaneous histiocytosis with granular cell changes' was proposed. The absence of an inflammatory cell component, such as lymphocytes and leucocytes, along with no history of a previous trauma or medical treatment, suggest that the present lesion could fit into the morphological spectrum of the so-called solitary epithelioid histiocytoma, also known as reticulohistiocytoma. Alternatively, the possibility of a histiocytic reaction to unknown stimuli cannot be completely ruled out. Nevertheless, awareness of solitary cutaneous histiocytosis with granular cell changes is useful to avoid confusion with other dermal tumors, especially 'granular cell tumor' and 'dermal non-neural granular cell tumor'.

Breast secretory carcinoma: a clinicopathologic study of four cases

Objective: To explore the clinicopathologic features of secretory carcinoma of breast (SCB). Methods: Four cases of SCB were analyzed by light microscopy, histochemistry, immunohistochemistry and electron microscopy. The clinical data were also analyzed. Special staining of periodic acid-Schiff reaction with diastase pretreatment, immunohistochemical staining of ER, PR, HER-2, p53, MIB-1, S-100, p63, CK8/18 and EMA by En Vision method were performed. Results: Solid, cribriform, tubular, or papillary architecture may be seen. Tumor forms cystic spaces filled with abundant pale pink secretory material, positive with diastase resistant periodic acid-Schiff (PAS-DR) stains. Tumor cells were small with bland nuclei and abundant pale, eosinophilic cytoplasm, rare mitotic activity and necrosis. Immunohistochemically, tumor cells were positive for CK8/18, EMA, negative for S-100, p63 and variable partially positive for p53, ER, PR, HER-2 and MIB-1. Under electron microscopy, the tumor cells possessed numerous membrane-bound secretory vacuoles in cytoplasm lined by microvilli. Con- clusion: SCB is a rare, low-malignant neoplasm. SCB have pathology, clinical picture, treatment, follow-up, immunohisto- chemical and genetic features that distinguish them from invasive ductal carcinomas of the usual type.

Sister mary joseph′s nodule: A case of umbilical cutaneous metastasis with signet ring cell histology

The metastasis of a visceral malignancy to the umbilicus is known as "Sister Mary Joseph's nodule". It is a rare clinical sign indicating advanced, metastasizing intraabdominal cancer. We report a 50-year-old man who developed metastatic skin cancer in the form of semicircular indurated plaque on top of which was a firm mobile rounded nodule at the umbilical area. Histopathological examination demonstrated diastase-resistant periodic acid-Schiff and mucicarmine positive signet ring cells, suggesting gastric carcinoma. Immunohistochemical staining showed that these cells were positive for cytokeratins and epithelial membrane antigen, suggesting epithelial origin.

Recombinant basic fibroblast growth factor inhibits the airway hyperresponsiveness, mucus production, and lung inflammation induced by an allergen challenge

IL-13 is believed to be a central mediator of asthma, and TGF-beta1 is a key downstream mediator in the development of IL-13-mediated asthma phenotypes. To evaluate the biological roles of basic fibroblast growth factor (FGF2) in phenotype expression in transgenic (TG) mice overexpressing lung-specific TGF-beta1, and the therapeutic effects of recombinant FGF2 in the development of asthma phenotypes. To evaluate the roles of FGF2 in airway hyperresponsiveness (AHR) expression induced by high levels of TGF-beta1, TGF-beta1 TG (+) mice were bred with FGF2-deficient mice. To evaluate the therapeutic effects of recombinant FGF2 (rFGF2) in the development of asthma, mice were given 10 mug of rFGF2 subcutaneously once a day, 1 hour before the allergen challenge in an asthma mouse model. AHR was evaluated using noninvasive whole-body plethysmography, mucus production by diastase-resistant periodic acid Schiff (DPAS) staining, and lung inflammation using bronchoalveolar lavage (BAL) cellularity and lung histology. AHR decreased in TGF-beta1 TG (+) mice and was accompanied by the upregulation of FGF2 mRNA expression in lung tissues, when compared with littermate wild-type control mice. Interestingly, AHR was enhanced markedly in TGF-beta1 (+) mice with homozygous FGF2 gene disruption. In an asthma mouse model, AHR, mucus production, and lung inflammation were inhibited markedly by rFGF2 treatment. This inhibition was accompanied by downregulation of the allergen-induced proliferation of T cells from regional lymph nodes. FGF2 seems to be a key inhibitor in the development of AHR, and rFGF2 treatment constrains the development of asthma phenotypes.

Melasma: histopathological characteristics in 56 Korean patients.

Melasma is a common acquired symmetrical hypermelanosis characterized by irregular light to dark brown macules and patches on sun-exposed areas of the skin. Its histopathological characteristics are not fully understood. To characterize the histopathological features of facial melasma skin in comparison with adjacent normal skin. Biopsies were taken from both melasma lesional skin and adjacent perilesional normal skin in 56 Korean women with melasma. The sections were stained using haematoxylin and eosin, Fontana-Masson, diastase-resistant periodic acid-Schiff, Masson trichrome and Verhoeff-van Gieson stains, and immunostaining for melanocytes. Data on the changes in number of melanocytes and melanin contents of the epidermis were analysed by a computer-assisted image analysis program. The ultrastructure of the skin was also examined. The amount of melanin was significantly increased in all epidermal layers in melasma skin. The staining intensity and number of epidermal melanocytes increased in melasma lesions. Lesional skin showed more prominent solar elastosis compared with normal skin. Melanosomes increased in number and were more widely dispersed in the keratinocytes of the lesional skin. Lesional melanocytes had many more mitochondria, Golgi apparatus, rough endoplasmic reticulum and ribosomes in their cytoplasm. A dihydroxyphenylalanine reaction was apparent in the cisternae and vesicles of the trans-Golgi network in melanocytes from lesional skin. Melasma is characterized by epidermal hyperpigmentation, possibly caused both by an increased number of melanocytes and by an increased activity of melanogenic enzymes overlying dermal changes caused by solar radiation.

Microcalcifications in the anterior pituitary gland of the fetus and the newborn: A histochemical and immunohistochemical study

Calcified concretions are a normal and constant finding in the anterior pituitary gland of fetuses and newborns. Their light and electron microscopic characteristics have been recently reported by the authors. In this study, undecalcified and decalcified sections from 20 neonatal and 60 fetal anterior pituitary glands were studied by histochemical and immunohistochemical methods to further clarify their nature and mechanism of formation. All the glands revealed homogeneous and/or laminar calcifications located either within the interstitium or follicular structures. They were composed of a diastase-resistant periodic acid-Schiff—positive carbohydrate-rich matrix. The Feulgen method for DNA was negative. Their core frequently reacted to Alcian blue and epithelial membrane antigen (EMA). EMA also stained the apical membranes of adjacent epithelial cells. Other immunostains (vimentin, keratin, and pituitary hormones) were negative. The positive staining for Alcian blue and EMA and the negative staining with the Feulgen method for DNA suggest that the core of the calcifications consists of acidic mucosubstances and EMA-positive proteinaceous material previously secreted by viable pituitary cells. The EMA-negative periphery of the concretions probably develops from further extracellular peripheral mineralization that leads to larger, sometimes laminated psammoma bodies. The occurrence of pituitary calcifications in states of adult physiological and pathological hyperprolactinemia suggests that the marked proliferation of lactotrophs occurring during the fetal life play an important role in the pathogenesis of the fetal and neonatal concretions.

Pulmonary giant cell carcinoma: pathological entity or morphological phenotype?

To evaluate the morphological spectrum and clinical significance of giant cell carcinoma and to assess the frequency of tumour giant cell production in a consecutive series of primary (non-giant cell) lung tumours. Forty-six cases of giant cell carcinoma of the lung were collated from two centres over a 12-year period. Giant cell carcinoma was found to be associated with areas of clear cell carcinoma, spindle cell carcinoma and showed trophoblastic differentiation (syncytiotrophoblastic giant cells and beta-human chorionic gonadotrophin immunopositivity) in 57%, 34% and 26% cases, respectively. 'Pure' giant cell carcinoma was identified in five (11%) cases. Eleven of the tumours contained diastase-resistant periodic acid-Schiff positive material and were separately designated as giant cell adenocarcinomas. Areas of squamous cell and neuroendocrine differentiation (as determined by chromogranin A and Leu-7 immunopositivity) were not found. The median survival for giant cell carcinoma (excluding the giant cell adenocarcinomas) was 18 months. Median survival was not adversely affected by the extent of tumour giant cell formation or by the presence of trophoblastic differentiation. Of 200 consecutive non-small cell lung carcinomas, tumour giant cells constituting < 10% of the tumour were identified in 32% of adenocarcinomas and 26% of squamous cell carcinomas. The presence of tumour giant cells in lung carcinoma does not, in itself, indicate a more aggressive tumour type, Giant cell carcinoma of the lung does not appear to be a distinct entity but a morphological phenotype expressed by a heterogenous group of tumours. We support and advocate the use of an encompassing term such as 'pleomorphic' or 'anaplastic' carcinoma for those tumours showing no specific differentiation pattern but which express diverse morphological features such as giant cell formation, clear or spindle cell change.

Cell renewal and functional morphology of human lactating breast: presentation of a new mode of cell death ('magentosis') characterized by nuclear periodic acid-Schiff reactivity.

Cell kinetics and functional morphology of the human lactating breast were analyzed using 21 paraffin-embedded specimens of the lactating breast obtained by biopsy or surgery and two of the 'resting' breast. Four types (I-IV) of lactating lobules were categorized histologically, and were well correlated with their functional status demonstrated by immunohistochemistry for epithelial membrane antigen, beta-casein, lipase, lactoferrin, secretory component and IgA. Type I corresponded to a pre-lactating state, type II to an actively lactating state, type III to an early stage of regression, and type IV to an advanced stage of regression. Cell proliferation monitored by Ki-67 (MIB-1) immunostaining was at the highest level in type I lobules followed by type IV, while the labeling indices were low in types II and III. Apoptosis demonstrated by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) method showed labeling profiles comparable with the proliferative activity. Holocrine-type desquamative cells with intact-appearing nuclei were frequently recognized in type II lobules. In type III, a peculiar form of cell death, designated 'magentosis', was demonstrated. These degenerative cells occasionally shedding into the acinar lumen possessed homogeneous pale, TUNEL-negative nuclei with diastase-resistant periodic acid-Schiff (PAS) reactivity. 'Magentosis' specifically identified in an early phase of involution of the human lactating breast might represent a unique mode of cell death distinguishable from apoptosis and necrosis.

A hepatic tumor associated with bilateral renal angiomyolipomas: A variant of angiomyolipoma?

Angiomyolipoma is usually derived from the kidney and composed of well developed vessels, smooth muscle and fat tissue. The liver is the only extra-renal site of angiomyolipoma. A peculiar type of hepatic tumor accompanied by bilateral renal angiomyolipomas is reported here. The tumor was mostly composed of large epithelioid cells and a small part of hyalinized large vessels and foam cell infiltration. Mature adipose tissue was absolutely absent. Epithelioid tumor cells arranged in an alveolar pattern had abundant glycogen and some diastase-resistant periodic acid-Schiff granules without obvious crystals. Immunohistochemical studies revealed that the epithelioid cells were positive for melanoma specific antibody (HMB-45), S-100 protein, aromatic L-amino acid decarboxylase and focally alpha-smooth muscle actin. Many melanosome- or premelanosome-like, electron-dense granules were observed in these cells. Thus, these cells were characterized by differentiation to both immature melanocytes and smooth muscle cells. These epithelioid cells were similar to some cells in the renal angiomyolipomas of the same patient. The hepatic tumor was considered to be a result of monotonous proliferation of the epithelioid cells seen in renal angiomyolipoma. Differential diagnosis of this tumor was discussed.

Aipha1-antitrypsin, Protein Marker in Oral Contraceptive-associated Hepatic Tumors

Tissue specimens from a series of 46 hepatic tumors occurring in young female oral contraceptive users were tested for alpha1-antitrypsin deposition, utilizing immunocytochemical and histochemical methods. In two instances serum alpha1-antitrypsin phenotyping was also performed. Immunoreactive alpha1-antitrypsin deposits were demonstrated in benign lesions, including 56% of cases of focal nodular hyperplasia and 68% of cases of liver-cell adenoma, and in 89% of cases of malignant hepatoma. There was good correlation between alpha1-antitrypsin deposits and variable amounts of finely granular, or globular, diastase-resistant periodic acid-Schiff positivity within tumor cells. While quantitative differences in alpha1-antitrypsin deposits between benign and malignant cell proliferations were not observed, a qualitative continuum that linked all tumors in the study group was found. The findings suggest that alpha1-antitrypsin deficiency is not related to the hepatic tumors developing in oral contraceptive users. The tumor tissue deposits of alpha1-antitrypsin observed represent a marker protein, the significance of which is undefined.

Adenoid cystic carcinoma of minor salivary gland: Histochemical and electron microscopic studies of cystlike spaces

Five cases of adenoid cystic carcinoma of minor salivary glands were studied. The mucoid material in the characteristic cystlike spaces of this neoplasm was diastase-resistant periodic acid-Schiff (PAS)—positive, alcian blue-positive, toluidine blue-positive, and mucicarmine-positive. Verhoeff-Van Gieson's method and Weigert's method did not reveal elastic tissue in the cystlike spaces. Mallory's method revealed that a central core in cystlike spaces was similar in stainability to collagen. Wilder's method did not reveal reticular fibers in these spaces. Electron microscopy revealed three readily recognizable zones: a juxtacellular zone of a network of replicated basal lamina, an intermediate zone of stellate granules of mucoid material, and a central core of densely packed aperiodic filaments or collagen fibrils. The histogenesis of cystlike spaces and their relationship with biologic behaviors of the neoplasm were discussed.

Isolation and characterization of an aetiological agent in Whipple's disease.

A cell wall deficient form of an alpha-haemolytic streptococcus was grown from a prolonged monolayer cell culture of a lymph node taken from a patient with Whipple's disease. Serological cross reactivity was shown between the organism and the material within Whipple's disease macrophages positive for diastase-resistant periodic acid-Schiff (D./P.A.S.). In vitro studies characterized the organism as a facultative intracellular parasite which caused the accumulation within cells of D./P.A.S.-positive material. These results suggest that a pathogenic bacterium is the essential aetiological agent and that the culture of Whipple's disease tissues in hypertonic media may have practical value.