Prospective evaluation of malignant cell seeding after percutaneous endoscopic gastrostomy in patients with oropharyngeal/esophageal cancers.

BACKGROUND AND STUDY AIMS: Insertion of a percutaneous endoscopic gastrostomy (PEG) is standard care for many patients with oropharyngeal (ENT) and esophageal malignancies in order to ensure enteral feeding. The current pull-through insertion technique involves direct contact with the tumor and case reports have demonstrated the presence of metastases at insertion sites. The aim of the current study was to prospectively evaluate the risk of malignant cell seeding and the development of abdominal wall metastases after PEG placement. PATIENTS AND METHODS: A total of 50 consecutive patients with ENT/esophageal tumors were included. After PEG placement (40 pull-through technique, 10 direct insertion), brush cytology was taken from the PEG tubing and the transcutaneous incision site. A second cytological assessment was performed after a follow-up period of 3 - 6 months. RESULTS: In total, 26 patients with ENT cancer, 13 with esophageal cancer, and one with esophageal infiltration of lung cancer underwent pull-through PEG placement with no immediate complications. Cytology following brushing of tubing and incision sites demonstrated malignant cells in 9 /40 cases (22.5 %). Correlation analyses revealed a higher rate of malignant seeding in older patients and in those with higher tumor stages. At follow-up, cytology was undertaken in 32 /40 patients who had undergone pull-through PEG placement. Malignant cells were present in three on cytology, resulting in a metastatic seeding rate of 9.4 %. CONCLUSION: This study showed that malignant cells were present in 22.5 % of patients immediately after pull-through PEG placement; local metastases were verified at follow-up in 9.4 %, all of which were from esophageal squamous cell carcinoma. This risk is particularly high in the older age group and in patients with higher tumor stages. Therefore, pull-through PEG placement should be avoided in these patients and direct access PEG favored instead.

Back to the roots - should gastroenterologists perform their own cytology?

BACKGROUND: Onsite cytology is widely recommended to improve cytological results in endoscopic ultrasound fine-needle punctures. The question is how well a gastroenterologist can be trained to perform an immediate bedside cytology. PATIENTS AND METHODS: From September 2008 to May 2011 157 endosonographic fine-needle punctures with a 22 G needle where performed in a municipal hospital. A medium amount of 26 loaded slides resulted from every puncture and air drying was used to preserve the specimen. 2 promising slides were kept whereas the remaining slides were sent on to a professional laboratory for final examination. The preliminary diagnosis was compared with the results from the professional cytologist for final evaluation. RESULTS: 152/157 fine-needle punctures were evaluable. In 6 cases the final result was regarded as uncertain. 73 malignant specimens and 73 benign specimens could be used for comparison. The gastroenterologist's evaluation achieved a sensitivity of 87.7 % and specificity of 90.4 % when the decision between a benign and malignant specimen was made. The specification of the tumour could not be done reliably by the gastroenterologist. CONCLUSION: Doing a delayed onsite diagnosis of the specimen by a gastroenterologist can help to speed up the diagnostic process with reasonable certainty. However, it cannot replace a professional cytological diagnosis because of the possible misevaluation and the uncertainty in tumour specification. Additionally, advanced methods like immunocytology cannot be performed in an onsite hospital setting.

Granulomatous mediastinal adenopathy: can endoscopic ultrasound-guided fine-needle aspiration differentiate between tuberculosis and sarcoidosis?

BACKGROUND AND STUDY AIMS: Mediastinal lymphadenopathy may indicate diseases such as tuberculosis or sarcoidosis, and it is often difficult to establish a diagnosis when standard medical work-up is inconclusive. In this study we investigated the diagnostic yield of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) in the differentiation between tuberculosis and sarcoidosis. PATIENTS AND METHODS: In this prospective study, 72 consecutive patients with mediastinal lymphadenopathy, negative endoscopic investigations including bronchoscopic procedures, and no radiological evidence of lung cancer or other malignancies on computed tomography were enrolled. EUS-FNA and subsequent cytology, microscopy for acid-fast bacilli, and culture were performed. At least 12 months' follow-up including further investigations was included to exclude tuberculosis. RESULTS: Adequate samples were obtained from 71/72 patients (36 male; mean age 50.2 years). No complications occurred. The final diagnosis included 30 cases of sarcoidosis, 28 of tuberculosis, four malignancies, one abscess, and nine benign lymphadenopathies. The size of lymph nodes on EUS varied from 0.5 cm to 4.2 cm. Tuberculosis nodes were significantly smaller than those in sarcoidosis. Unrelated nodes were significantly smaller than in either tuberculosis or sarcoidosis. The sensitivity, specificity, and positive and negative predictive values of EUS - FNA for tuberculosis were 86 %, 100 %, 100 %, and 91 %, respectively; those for sarcoidosis were 100 %, 93 %, 91 %, and 100 %, respectively. For culture of tuberculosis, they were 71 %, 100 %, 100 %, and 84 %, respectively. EUS - FNA led to a definite diagnosis in 64/72 cases (89 %) that had not been previously diagnosed by routine methods. CONCLUSION: EUS - FNA offers a high diagnostic yield for the differential diagnosis of tuberculosis and sarcoidosis that have not been diagnosed by conventional methods.

Breast duct endoscopy: ductoscopy from a diagnostic to an interventional procedure and its future perspective.

OBJECTIVE: Breast duct endoscopy is increasingly used for evaluation of intraductal disease. We present a new rigid instrument for ductoscopy that allows intraductal biopsy and the removal of small lesions. METHODS: Overall, 102 women with breast cancer or pathologic nipple discharge were included in the analysis. All ductoscopies were performed with a rigid gradient index micro-endoscope (phi 0.7 mm) in combination with a special device for intraductal vacuum assisted biopsy. Ductoscopy, ductal lavage and intraductal biopsy were correlated with ductal cytology and histopathology of the resection specimen. RESULTS: Gradient index ductoscopy provided high resolution images of the breast ducts and identified additional intraductal lesions in 45% of the patients with breast cancer. The accuracy of ductal lavage, ductoscopy and mammography in the detection of an extensive intraductal component was 14%, 65% and 50%, respectively. Intraductal vacuum assisted biopsy yielded diagnostic material in 92% of 38 patients with nipple discharge and papillomatous lesions. Histology of the resection specimen confirmed the diagnosis in all cases including 2 in situ carcinoma and 2 invasive ductal carcinoma. CONCLUSIONS: Ductoscopy is a useful supplement for the standard radiological workup of breast cancer especially in patients with extensive intraductal carcinoma. Ductoscopic vacuum assisted biopsy is an effective technique for intraductal tissue sampling and allows ablation of small lesions. This technique provides new perspectives for interventional therapy of intraductal tumours.

Gastrointestinal stromal tumor in a patient with undiagnosed neurofibromatosis type I: an uncommon cause of extrahepatic cholestasis.

In this case report we present a 61-year-old patient with obstructive jaundice. Bile duct obstruction was caused by a tumor at the duodenal papilla and bile flow was restored by a plastic stent. Using endoscopic ultrasound and computed tomography imaging two additional tumors of the same morphology were found in the stomach wall and the pelvic region suggesting a multilocular gastrointestinal stroma tumor (GIST). Diagnosis of GIST was confirmed cytologically from the gastric lesion. Based on typical cutaneous manifestations (café-au-lait spots, several tiny dermal neurofibromata and Lisch nodules in the iris), a thus far unidentified neurofibromatosis type I was diagnosed which is known to promote multilocular GIST formation. Tumor resection failed because of cardiac decompensation due to a Takotsubo cardiomyopathy during induction of anesthesia. The patient has been started on imatinib instead and shows so far a stable disease over 6 months.

Value of endoscopic ultrasonography-guided fine needle aspiration (FNA) in the diagnosis of neoplastic tumor(-like) pancreatic lesions in daily clinical practice.

PURPOSE: To investigate the value of EUS-guided FNA in the diagnosis of solid and cystic pancreatic tumor(-like) lesions as well as metastatic tumor growth within peripancreatic lymph nodes and its impact on therapeutic decision-making. The results of the cytologic and pathohistological investigation were compared with i) each other and ii) the detection rates of various imaging procedures. PATIENTS AND METHODS: Overall, 153 patients (mean age, 56.9 years) underwent EUS-guided FNA from I/ 2000 - III/ 2003. RESULTS: Comparing various imaging procedures such as CT scan (80 %), MRI (57.1 %) and abdominal US (88.8 %), EUS achieved the highest diagnostic accuracy: 100 %. For EUS-based T-staging in 26 patients with malignant tumor lesions undergoing surgical intervention, there was a sensitivity of 73.3 % (specificity, 85.9 %; PPV, 69.2 %; NPV, 84.4 %), while the parameters for N-staging (n = 25) were: sensitivity, 61.5 %; specificity, 75 %; NPV, 64.3 %; PPV, 72.7 %. While the sensitivity of EUS-guided FNA in the group of patients who underwent surgical intervention (n = 55) was 81.4 % (specificity, 75 %; PPV, 92.1 %; NPV, 52.9 %), the parameters were as follows in the subgroup of individuals with chronic pancreatitis (n = 30): sensitivity in detecting a malignant pancreatic tumor lesion, 50 %; specificity, 91.7 %; PPV, 60 %; NPV, 88 %. Based on preoperative characteristics such as suspected diagnosis, TNM stage and tumor entity, a surgical intervention could be avoided in 29 / 153 patients (19 %). CONCLUSION: EUS-guided FNA allows more precise diagnosis clarification (malignant tumor growth and tumor entity) in solid and cystic pancreatic tumor(-like) lesions, which may assist in early and sufficient therapeutic decision-making.

Diagnosing sarcoidosis using endosonography-guided fine-needle aspiration.

STUDY OBJECTIVES: The ability to diagnose sarcoidosis cytologically has been reported previously, but the method is rarely used. Endoscopic ultrasonography (EUS) is a sensitive technique for detecting mediastinal lymph nodes, which in addition provides an opportunity to carry out guided fine-needle aspiration (FNA) cytology. We report herein on the use of EUS-FNA in the diagnosis of sarcoidosis. PATIENTS AND METHODS: Nineteen patients with suspected sarcoidosis were investigated using EUS-FNA with a linear echoendoscope and a 22-gauge Hancke-Vilman needle. MEASUREMENTS AND RESULTS: In all 19 patients, EUS revealed enlarged mediastinal lymph nodes (mean size, 2.4 cm), located subcarinally (n = 15), in the aortopulmonary window (n = 12), or in the lower posterior mediastinum (n = 5). The nodes had an isoechoic or hypoechoic appearance, with atypical vessels in five cases. The amount of aspirate obtained using EUS-FNA was adequate in all patients, and contained blood in excess of normal in some, indicating a high degree of vascularity. Cytology demonstrated epithelioid cell granuloma formation, suggesting sarcoidosis. Mycobacterial cultures were negative in all of the patients except one, in whom the final diagnosis was tuberculosis. The specificity and sensitivity of EUS-FNA in the diagnosis of sarcoidosis were 94% and 100%, respectively. CONCLUSIONS: EUS of mediastinal lymph nodes in sarcoidosis reveals certain characteristic features. However, it is not capable of differentiating the lesions from tuberculosis or malignancy. EUS-FNA is a safe and sensitive method of aspirating material for cytology and mycobacterial cultures. We believe it will provide a useful alternative in the diagnosis of sarcoidosis.

Cell preparation methods and criteria for sample adequacy. International Academy of Cytology Task Force summary. Diagnostic Cytology Towards the 21st Century: An International Expert Conference and Tutorial.

ISSUES: Cell Preparation Methods Standardized fixation and optimal staining Sampling of cervix, sampling error, homogenization of sample, subsampling Assessment of liquid-based preparations: efficacy and economic impact Training and transitional procedures before full implementation of new technologies Criteria for Sample Adequacy Clinician responsibility for collecting and providing representative sample to laboratory Collection instruments, number of slides Cellular content of samples: evidence of transformation zone (TZ) sampling, number of squamous cells present, obscuring factors Screening issues CONSENSUS POSITION The conventional cervical smear remains the standard method of cervical cancer screening but has limitations in individual test sensitivity and specificity. Sample takers should: (1) receive appropriate training in sample collection, (2) be held responsible for providing the laboratory with appropriate samples, and (3) have their performance monitored. The instruments used for sampling should collect cells from both the ectocervix and endocervix; optimally, TZ sampling, represented by the presence of endocervical or squamous metaplastic cells, should be identifiable in samples other than atrophic specimens. The adequacy of a specimen (as judged microscopically) does not guarantee that it is representative of the cervix. Each cytology report should include a comment on cellular content/adequacy of the specimen. Liquid-based preparations may overcome many of the inherent problems with the conventional cervical smear. ONGOING ISSUES: We need further data on the cost-effectiveness of making two slides from cervical specimens and/or using two samplers rather than a single one. Do we have enough information to make recommendations as to the appropriate type of sampler to be used in particular situations, such as routine screening? What is the best method of screening for/detecting endocervical glandular neoplasia? How are such terms as unsatisfactory and inadequate defined in cervical cytology classifications other than the Bethesda System? What number and types of epithelial cells should be present (visualized) in a cervical smear or liquid-based preparation for it to be considered adequate? Do we need to have evidence of TZ sampling in specimens taken during the follow-up period after treatment of squamous intraepithelial lesion or after detection of endocervical glandular neoplasia? What criteria for obscuring factors, such as blood and inflammation, should be used in assessing adequacy? Cost-benefit analyses of utilizing liquid-based preparations are needed. Should we inform women about the technical details of the test methods available or chosen by the laboratory? Are women in a position to decide which method is the most appropriate to assess their cervical scrape sample? We need to obtain more information about the properties of proprietary liquid fixative/transport media with respect to inactivation of viral pathogens, tuberculosis and other bacterial pathogens and suitability for immunobiologic and molecular tests, etc. We need to obtain more information on the use of stoichiometric stains and the limitations of Papanicolaou stain for image analysis systems. The use of liquid-based preparations for nongynecologic cytopathology and ancillary tests must be considered, including criteria for adequacy. We need to obtain more information on the time required for and best methods of training experienced cytotechnologists to become competent at assessing liquid-based cervical preparations.

Computerized screening devices and performance assessment: development of a policy towards automation. International Academy of Cytology Task Force summary. Diagnostic Cytology Towards the 21st Century: An International Expert Conference and Tutorial.

ISSUES: The extension of automation to the diagnostic assessment of clinical materials raises issues of professional responsibility, on the part of both the medical professional and designer of the device. The International Academy of Cytology (IAC) and other professional cytology societies should develop a policy towards automation in the diagnostic assessment of clinical cytologic materials. CONSENSUS POSITION: The following summarizes the discussion of the initial position statement at the International Expert Conference on Diagnostic Cytology Towards the 21st Century, Hawaii, June 1997. 1. The professional in charge of a clinical cytopathology laboratory continues to bear the ultimate medical responsibility for diagnostic decisions made at the facility, whether automated devices are involved or not. 2. The introduction of automated procedures into clinical cytology should under no circumstances lead to a lowering of standards of performance. A prime objective of any guidelines should be to ensure that an automated procedure, in principle, does not expose any patient to new risks, nor should it increase already-existing, inherent risks. 3. Automated devices should provide capabilities for the medical professional to conduct periodic tests of the appropriate performance of the device. 4. Supervisory personnel should continue visual quality control screening of a certain percentage of slides dismissed at primary screening as within normal limits (WNL), even when automated procedures are employed in the laboratory. 5. Specifications for the design of primary screening devices for the detection of cervical cancer issued by the IAC in 1984 were reaffirmed. 6. The setting of numeric performance criteria is the proper charge of regulatory agencies, which also have the power of enforcement. 7. Human expert verification of results represents the "gold standard" at this time. Performance characteristics of computerized cytology devices should be determined by adherence to defined and well-considered protocols. Manufacturers should not claim a new standard of care; this is the responsibility of the medical community and professional groups. 8. Cytology professionals should support the development of procedures that bring about an improvement in diagnostic decision making. Advances in technology should be adopted if they can help solve problems in clinical cytology. The introduction of automated procedures into diagnostic decision making should take place strictly under the supervision and with the active participation and critical evaluation by the professional cytology community. ONGOING ISSUES: Guidelines should be developed for the communication of technical information about the performance of automated screening devices by the IAC to governmental agencies and national societies. Also, guidelines are necessary for the official communication of IAC concerns to industry, medicolegal entities and the media. Procedures and guidelines for the evaluation of studies pertaining to the performance of automated devices, performance metrics and definitions for evaluation criteria should be established.

Quality assurance/control issues. International Academy of Cytology Task Force summary. Diagnostic Cytology Towards the 21st Century: An International Expert Conference and Tutorial.

ISSUES: General definitions of quality assurance and quality control (QA/C) have existed in many forms for decades, and a new discipline guides their application to diverse industrial and recently medical processes without much fanfare. However, in the field of cervical cytology screening, the range of QA/C options has recently broadened and become controversial. With the advent of new systems of terminology, larger-scale laboratories and new technologies--plus strong governmental and legal pressures in some nations--the range of extremely difficult and sometimes expensive QA/C choices our community faces is greater than ever. CONSENSUS POSITION: At our conference, the basic definitions of QA/C posed little difficulty. Presentation of the range of methods in use today and of those based on new technologies where use is proposed or has just begun also was achieved with little or no dispute. However, there was lack of consensus on exactly how QA/C methods are to be assessed. Indeed, there was little consistency in the use of different outcome measures with which we can judge success or failure of specific QA/C options. In addition, the tension between pressure to adopt sometimes uncertain or expensive method enhancements and pressure to maintain affordability and the widest possible access for populations that most need cervical cytology screening is greater than ever. ONGOING ISSUES: More data are required that would enable assessment of QA/C options with the clearest possible understanding of cost/benefits and current or new assumptions of risk. Other task forces, such as medicolegal, cost/benefit and those devoted to new technologies, are our essential partners in meeting the challenges described above.

[Contrast enhanced endosonography for improving differential diagnosis between chronic pancreatitis and pancreatic cancer]. / Kontrastverstärkte Endosonographie zur Verbesserung der Differenzialdiagnose zwischen chronischer Pankreatitis und Pankreaskarzinom.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate contrast-enhanced endosonography as a method for improving the differentiation between chronic focal pancreatitis and ductal pancreatic cancer, based on perfusion characteristics of the microcirculation. PATIENTS AND METHOD: In 194 patients [75 women, 119 men; age: 64 +/- 11 years] with chronic pancreatitis (n = 73) or pancreatic cancer (n = 121), pancreatic lesions were examined by conventional endoscopic B-mode ultrasound, power Doppler ultrasound and contrast-enhanced power mode, using the following criteria for malignant lesions: no detectable vascularization with conventional power Doppler scanning, irregular appearance of arterial vessels over a short distance using contrast-enhanced technique and no detection of venous vessels inside the lesion. A malignant lesion was assumed to be present if all criteria were detectable. The criteria of chronic pancreatitis without neoplasia were defined as no detectable vascularization before injection, regular appearance of vessels over a distance of at least 20 mm after injection of the contrast medium and detection of arterial and venous vessels. The gold standard was the histological diagnosis by endoscopic ultrasound (EUS)-guided fine needle aspiration cytology or operation. RESULTS: Using conventional EUS the diagnosis was correct in 96 of 121 patients with pancreatic cancer (sensitivity 79.3 %; 95% confindence interval 71 - 85.2%) and in 60 of 73 patients with chronic pancreatitis (specificity 82.2 % [71.5 - 90.2%]). Using contrast-enhanced EUS malignant pancreatic lesions were correctly diagnosed in 111 of 121 patients, thus increasing sensitivity to 91.7 % (85.3 - 96%). In 70 of 73 patients chronic inflammatory pancreatitis was correctly diagnosed (specificity 95.9 % [88.5 - 99,1%]) . CONCLUSION: Contrast-enhanced endoscopic ultrasound improves the differentiation between chronic pancreatitis and pancreatic carcinoma.

[Autoimmune pancreatitis with normal IgG4-Levels: 4 case reports and review of the literature]. / Autoimmune Pankreatitis bei normalem IgG4: 4 Fallberichte und Literaturübersicht.

We report four cases of autoimmune pancreatitis in an 18-, a 22- and a 26-year-old male patient and a 20-year-old female patient. The 20-year-old female patient was admitted to the hospital with upper abdominal pain and jaundice, the 18-year-old patient with recurrent acute pancreatitis and cholestasis, the 26-year-old patient with right upper abdominal pain for four weeks and laboratory findings suggesting an acute pancreatitis. The 22-year-old patient presented with painless jaundice. EUS-guided fine needle aspiration was performed in all patients. The cytological findings and the EUS were decisive for the diagnosis of autoimmune pancreatitis in all four cases. In contrast, no patient showed elevated IgG4, or antibodies for carboanhydrase-II, for lactoferrin, or rheumatoid factor, serum markers reported to be positive in autoimmune pancreatitis. All patients were treated successfully with steroids, one patient relapsed after discontinuing the steroid medication and required renewed therapy. These case reports demonstrate that autoimmune pancreatitis should be considered in the differential diagnosis in cases of pancreatitis and/or jaundice also in western countries. As demonstrated, the diagnosis should not be based solely on the elevation of IgG4 or autoantibodies.

Endoscopic ultrasonography (EUS) and fine-needle aspiration (FNA) cytology for diagnosis of chronic pancreatitis.

BACKGROUND AND STUDY AIMS: Previous studies have shown that endoscopic ultrasonography (EUS) sensitively detects morphologic abnormalities due to chronic pancreatitis. However, morphologic EUS findings have limited specificity, particularly at the early stages of chronic pancreatitis. Our aims were to study pancreatic morphology and inflammation in patients with chronic pancreatitis, using EUS and fine-needle aspiration cytology (EUS-FNA), and to compare the results with those of endoscopic retrograde cholangiopancreatography (ERCP) and pancreatic function tests. PATIENTS AND METHODS: 37 patients (48 +/- 13 years) with clinical symptoms and laboratory test findings suggestive of chronic pancreatitis were prospectively studied. Patients with malignancy or major concomitant disorders were excluded. Clinical evaluation included indirect pancreatic function tests. Morphologic criteria for chronic pancreatitis included echo-intense septae/echo-reduced foci (i. e. pseudolobularity), ductal irregularities, and calcifications. EUS-FNA was performed in 27/37 patients, by means of the Hitachi FG34-UX echo endoscope and a 22-gauge needle, and tissue specimens were submitted for standard cytological evaluation. ERCP served as reference in all patients, using the Cambridge classification. RESULTS: 31 patients had chronic pancreatitis while six had normal findings at ERCP. EUS showed morphologic abnormalities of the pancreas in 33 patients. Morphologic abnormalities alone reached a sensitivity of 97 % for chronic pancreatitis with a specificity of only 60 %, while the positive predictive value (PPV) was 94 %, and the negative predictive value (NPV) was 75 %. EUS-FNA increased the negative predictive value to 100 % and the specificity to 67 %. On average, 2.3 needle passes were necessary to obtain sufficient amounts of tissue. The correlation of EUS findings with pancreatic function tests was poor. EUS results were in agreement with regard to the severity of chronic pancreatitis in 5/8 patients with grade I disease, in 11/13 patients with grade II, and in 10/10 patients with grade III disease. Minor complications occurred in two patients (7 %). CONCLUSIONS: EUS is as sensitive and effective as ERCP in the detection of chronic pancreatitis, particularly when only mild disease is present. However, EUS findings have limited specificity, particularly in patients with mild disease. EUS-FNA with cytology is safe and improves the negative predictive value. Negative EUS-FNA findings rule out chronic pancreatitis, but cytological investigation alone does not improve the specificity of EUS findings, suggesting that further improvements in tissue sampling and analysis are necessary to support routine use of FNA in patients with chronic pancreatitis.

[Cytochemical studies for determining the histogenesis of anaplastic and poorly differentiated lung tumors]. / Zytochemische Untersuchungen zur Bestimmung der Histogenese bei anaplastischen bzw. schlecht differenzierten Lungentumoren.

Proper histogenetic classification of pulmonary tumours is most important in choosing the best possible treatment. Since this is very difficult especially in case of anaplastic or poorly differentiated tumours, additional pointers on histogenesis, supplied by complementary histochemical examinations, are very helpful. 105 bronchial carcinomas were examined cytochemically by means of air-dried smear preparations (imprint, brush an puncture smears). Cytochemical examinations were performed in respect of alkaline phosphatase, acid phosphatase, PAS reaction and unspecific esterase. It was found that the dedifferentiated squamous cell carcinomas were alkaline phosphatase-negative and weakly positive to acid phosphatase and unspecific esterase, whereas dedifferentiated adenocarcinomas were strongly positive to acid phosphatase and unspecific esterase. The PAS reaction was always slightly to moderately positive. Small-cell bronchial carcinomas were negative in all cytochemical examinations.