Rapid tissue processing using a temperature-controlled collection device to preserve tumor biomarkers.

Precision tissue diagnostics rely on high quality input specimens so that assay results are not affected by artifact, but advances in collection and processing of tissue specimens have lagged behind innovations in diagnostic assay development. Therefore, we have designed and evaluated a novel surgical tissue collection device that maintains and monitors sample temperature and motion throughout transport so that the major preanalytical variable of tissue temperature can be controlled and measured. This device, in combination with an improved cold-hot tissue fixation protocol affords optimal biomarker preservation in less overall time, thereby simultaneously improving diagnostic quality and turnaround time. We collected 50 primary and metastatic liver tumors using a novel transport device. Tissue was fixed using a rapid cold-hot fixation protocol and immunohistochemical assays were used to assess the performance of the device, in comparison to control tissue preserved using standard clinical fixation protocol. Two pathologists evaluated the IHC studies in a blinded fashion to determine the immunophenotype of each tumor. The observed IHC staining intensities and the clinical impressions of the immunophenotypes did not differ between tissue collected with the novel device and control tissue, while improvements in processing time were achieved. The novel cold transport device and rapid fixation protocol can be successfully and safely combined and used to monitor specimen conditions, thus preserving the diagnostic utility of specimens and improving the overall turn-around time of the diagnostic process.

Efficient and cost-effective extraction of genomic DNA from formalin-fixed and paraffin-embedded tissues.

Diagnostic and investigative molecular pathology frequently has to resort to extraction of DNA from formalin-fixed and paraffin-embedded tissue samples. Although many different protocols are reported for this type of material, extraction of sufficient amounts of intact DNA is still challenging. Here, the authors report a reproducible, simple, cost-effective, and efficient protocol that yields up to 140 µg of DNA from approximately 10 to 15 mg of formalin-fixed and paraffin-embedded tissue samples and compare it to available protocols. The protocol allows stable amplification of DNA fragments up to 600 bp in length in a wide variety of tissues.

Cell block alone as an ideal preparatory method for hemorrhagic thyroid nodule aspirates procured without onsite cytologists.

OBJECTIVE: To study diagnostic efficacy of direct smears (DS) vs. cell block (CB) alone in hemorrhagic thyroid fine needle aspirations (FNAs) performed without a cytotechnologist or cytopathologist. STUDY DESIGN: Ultrasound-guided thyroid FNAs from an offsite location were retrospectively searched during a 53-month period. Aspirates in the initial 13 months were submitted as air-dried DSs. Subsequent specimens were submitted as CBs. Each case was classified into 1 of 4 categories: (1) nondiagnostic, (2) nonneoplastic, (3) follicular lesions and (4) papillary thyroid carcinoma (PTC). RESULTS: There were 77 aspirates: DS = 20 (26%) and CB = 57 (74%). Two cases had both DSs and CBs. Diagnoses of DS: nondiagnostic = 12 (60%); nonneoplastic = 7 (35%); follicular lesion = 1 (5%). Diagnoses of CB cases: nondiagnostic = 4 (7.0%); nonneoplastic = 43 (75.4%); follicular lesion, including 1 Hürthle cell neoplasm = 7 (12.3%), PTC = 3 (5.3%). Repeat FNAs on 4 nondiagnostic cases (3 DSs, 1 CB) utilizing the CB-only technique were diagnostic and included nodular goiter, follicular neoplasm, PTC, and reactive lymph node. CONCLUSION: Without onsite assessment, CB alone is superior to DSs for hemorrhagic thyroid FNAs. It shows increased diagnostic efficacy and slide reduction and obviates repeat FNAs.

Validation of a New Low-Cost, Methanol-Based Fixative for Cervical Cytology and Human Papillomavirus Detection.

OBJECTIVE: To test the performance of a new fixative for pap smear collection for liquid-based cervical cytology, CellPreserv® and compare it with the commercially available, PreservCyt® used in the diagnosis and detection of human papillomavirus (HPV). METHODS: Seven hundred twenty five women participated in this study after signing an informed consent. The specimens were collected using a traditional device, agitated in PBS, and equally divided in both fixatives. The slides were prepared routinely, stained by Papanicolaou, examined blindly by 2 cytologists, and reviewed by one cytopathologist. To search for HPV, 1,000 µL from each fixative was taken and processed by polymerase chain reaction. RESULTS: Considering the adequacy of samples, both fixatives had similar results - 0.33 and 0.32% of the cases unsatisfactory for PreservCyt® and CellPreserv®, respectively. Considering the 701 satisfactory cases and comparing the new fixative to the traditional fixative, there was 99.3% concordance between both. The results regarding the HPV detection was 100% concordant between the 2 fixatives. CONCLUSION: The new methanol-based fixative, CellPreserv®, is cheaper and equally efficient for treating cervical cancer screening and for HPV detection, and can be safely used by the health system prevailing in low-income countries.

Application of domestic microwave for urgent histopathology reporting: an evaluation.

Rapid diagnosis of histopathological material is becoming increasingly desirable. In neuropathology, crush smear preparation and frozen section diagnosis of tissues removed during operative procedures, have remained as essential tools for rapid diagnosis. Microwave technology has been introduced into the field of tissue processing and staining in past decade. Now-a-days even automated microwave assisted rapid tissue processors are available. In our study we have analysed the use of a domestic microwave (cost approximately Rs.5000) for urgent histoprocessing (30 minutes). This could be useful in small laboratories or the ones which are in the phase of establishing the department as the procedure is much more economical than obtaining a frozen section (which requires a cryostat worth 3-6 lakhs) and the interpretation of the section obtained does not require any extra experience as these resemble the routinely processed tissue sections. The advantages and limitations of the procedure have been discussed.

Comparison of modified Thiel embalming and ethanol-glycerin fixation in an anatomy environment: Potentials and limitations of two complementary techniques.

Thiel-fixed specimens have outstandingly lifelike visual and haptic properties. However, the original Thiel method is expensive and requires an elaborate setup. It is therefore of principal interest to modify the Thiel method in order to make it available to a broader user group. A modified Thiel embalming method will be described in detail and compared to ethanol-glycerin fixation with the help of illustrative examples. The visual properties, haptic properties, the usability for performing histological investigations, costs and potential health aspects will be considered. Tissues fixed with the modified Thiel technique gave results similar to the original method, providing more realistic visual and haptic properties than ethanol-glycerin embalming. However, Thiel fixation is significantly more expensive and requires more precautions to minimize potential health hazards than ethanol-glycerin-fixed tissues. In contrast to ethanol-glycerin-fixed specimens, the Thiel-fixed specimens are not suitable for histological investigations. Both modes of fixation are inappropriate for biomechanical testing. Modified Thiel embalming simplifies the availability of body donors with lifelike properties and has cost-saving advantages to the original technique. Thiel-embalmed body donors are ideally suited for clinical workshops but have restrictions for student dissection courses in facilities with limited storage space, air circulation or technical staff. Vice versa, ethanol-glycerin-fixed body donors are well suited for student dissection courses in such an environment but are limited in their use for clinical workshops. Modified Thiel embalming therefore ideally complements ethanol-glycerin fixation in order to provide customized solutions for clinical workshops and student dissection courses in a wide range of applications.

GEWF solution.

BACKGROUND: Lymph node status is an important prognostic factor in the staging of colorectal carcinoma. Several adjunctive solutions have been used to increase the yield of pericolic lymph nodes from colorectal cancer resection specimens. METHODS: During 1998 at the Grey Bruce Regional Health Centre (Owen Sound, Ontario), 67 colonic resections were performed for colorectal cancer. Lymph nodes were identified using GEWF solution (glacial acetic acid, ethanol, distilled water, and formaldehyde) in 35 cases, and by the conventional method of sectioning, inspection, and palpation in 32 cases. RESULTS: There were no significant differences between GEWF and non-GEWF cases with respect to patient age, length of resection, size of tumor, tumor histologic type, tumor differentiation, or depth of tumor penetration into the bowel wall. Use of GEWF led to a significant increase in the number of lymph nodes found (10.2 +/- 4.9 per case) compared with non-GEWF cases (6.8 +/- 3.9 per case) (P =.002). In GEWF cases 358 lymph nodes were identified, 82 with metastases, whereas in the non-GEWF cases 218 lymph nodes were found, 41 with metastases. The size of positive lymph nodes in the GEWF group (0.5 +/- 0.2 cm) was significantly smaller than in the non-GEWF group (0.7 +/- 0.4 cm) (P =.046). A greater percentage of positive lymph nodes in the GEWF cases (49/82, 60%) were 0.5 cm or smaller compared with the non-GEWF cases (17/41, 41%). CONCLUSIONS: GEWF increases the yield of lymph nodes recovered from colorectal cancer specimens and may lead to improved staging of this cancer; it is inexpensive and simple to use.

DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells.

The objective of the present study was to develop a simplified low cost method for the collection and fixation of pediatric autopsy cells and to determine the quantitative and qualitative adequacy of extracted DNA. Touch and scrape preparations of pediatric liver cells were obtained from 15 cadavers at autopsy and fixed in 95% ethanol or 3:1 methanol:acetic acid. Material prepared by each fixation procedure was submitted to DNA extraction with the Wizard genomic DNA purification kit for DNA quantification and five of the preparations were amplified by multiplex PCR (azoospermia factor genes). The amount of DNA extracted varied from 20 to 8,640 microg, with significant differences between fixation methods. Scrape preparation fixed in 95% ethanol provided larger amount of extracted DNA. However, the mean for all groups was higher than the quantity needed for PCR (50 ng) or Southern blot (500 ng). There were no qualitative differences among the different material and fixatives. The same results were also obtained for glass slides stored at room temperature for 6, 12, 18 and 24 months. We conclude that touch and scrape preparations fixed in 95% ethanol are a good source of DNA and present fewer limitations than cell culture, tissue paraffin embedding or freezing that require sterile material, culture medium, laboratory equipment and trained technicians. In addition, they are more practical and less labor intensive and can be obtained and stored for a long time at low cost.

Fixation techniques for fine needle aspiration biopsy smears prepared off site.

OBJECTIVE: To identify a simple, cost-effective, reliable fixation method for fine needle aspiration biopsy (FNAB) yielding a specimen suitable for mail transport. STUDY DESIGN: Smears prepared from 59 FNABs of surgical specimens were fixed by continuous fixation in 95% ethanol, spray fixation, air drying, ethanol fixation for either 5 minutes or 4 hours followed by spray fixation, or fixation in 95% ethanol for either 30 minutes or 4 hours followed by air drying. Fixation was graded as unsatisfactory, suboptimal, average, good or excellent. RESULTS: Of smears continuously fixed in ethanol, 96.6% were graded as excellent. Of smears fixed in ethanol followed by spray fixation, 93.2% were excellent irrespective of fixation time; 64.4% of spray-fixed smears were excellent and 27.1% good. Of air dried smears, 93.2% were unsatisfactory or suboptimal; 83.0% of smears fixed in ethanol for 30 minutes and 74.6% of smears fixed for 4 hours prior to air drying were unsatisfactory or suboptimal. CONCLUSION: Fixation of smears in 95% ethanol followed by spray fixation produces excellent results, comparable to those with continuous fixation in ethanol. Spray fixation is generally good but not consistently excellent. Air drying or fixation in ethanol followed by air drying yields unsatisfactory or suboptimal results in most cases.

Chromogen detection of microRNA in frozen clinical tissue samples using LNA™ probe technology.

Specific chromogen- and fluorescence-based detection of microRNA by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded (FFPE) tissue sections has been facilitated by locked nucleic acid (LNA)-based probe technology and can be performed within a single working day. In the current method paper, we present a similar simple 1-day ISH method developed for cryostat sections obtained from clinical cryo-embedded tissue samples. The presented chromogen-based ISH method does not involve proteolytic pretreatment, which is mandatory for FFPE sections, but still retains a sensitivity level similar to that obtained in FFPE sections. The LNA-based ISH method is not only applicable in situations where only access to cryo-embedded material is possible, but it also has a potential use if combining microRNA ISH with immunohistochemistry in double fluorescence staining with antibodies not being compatible with proteolytic predigestion.

An examination of non-formalin-based fixation methods for Xenopus embryos.

Despite the growing availability of non-formalin-based fixatives, the vast majority of researchers in developmental biology continue to fix embryos and tissue in 4% paraformaldehyde. This fixation method has proven useful for both immunohistochemistry and in situ hybridization, yet working with paraformaldehyde has distinct disadvantages in its toxicity and the short shelf life of prepared solutions. In a search for viable alternative fixatives, we have evaluated two non-formalin-based commercial products, FineFIX (Milestone Microwave Laboratory System) and NOTOXhisto (Scientific Device Laboratory). These products were tested side-by-side with a commonly used 4% paraformaldehyde solution (MEMPFA) on Xenopus laevis embryos and assayed using whole mount immunohistochemistry and whole mount in situ hybridization. The results indicate that NOTOXhisto can be used as a substitute for MEMPFA in both tested Xenopus protocols with no loss of sensitivity or tissue morphology.