Field histology: point-of-care microscopic technique.
In this study, a point-of-care protocol for rapid tissue processing and field microtome sectioning was developed and tested using portable lightweight equipment.
MATERIALS AND METHODS
Twenty-one tissue samples were evaluated. Tissues from various sites with a range of diagnoses were procured from specimens left after completion of pathologic evaluation. Hospital institutional review board approval was obtained for use of clinical material.
Tissue samples less than 0.5 cm in thickness were taken from specimens previously fixed in 10% buffered formalin. They were serially placed 15 minutes each in capped tubes containing 30 mL of reagent alcohol, isopropyl alcohol, xylene, and paraffin. The tubes were heated in a portable electric hot pot at 95[degrees]C to 99[degrees]C (Figure 1). The processed samples were embedded in a mold consisting of a 2-inch length of polyvinyl chloride pipe 1 inch in diameter with an inner plastic film divider (Figure 2). The pipe was covered on one end with a 3 X 3-inch plastic sheet held in place with a rubber band and filled with melted paraffin (Paraplast X-TRA, Thermo Scientific, Waltham, Massachusetts). The tissue samples were centrally placed in the crescent-shaped block to fit the microtome. The paraffin blocks were allowed to harden on ice for 10 minutes. The blocks were removed from the mold and trimmed slightly to fit the holder and sectioned with a portable microtome (Uchida DK-10 slide microtome, Edmund Scientifics, Tonawanda, New York), which has a blade fixed at 0 degrees relative to the surface of the block (Figure 3). The blade is reinforced with 2 metal washers measuring up to 2 inches in diameter to improve stability. The sections were cut at 10 to 20 mm, floated in a heated water bath (35[degrees]C-0[degrees]C), mounted on glass slides, allowed to dry for 10 minutes, and stained with standard hematoxylin-eosin stain (AutoStainer XL, Leica, Wetzlar, Germany) or hand stained with frozen section hematoxylineosin protocol (xylene 3 minutes X 2; 100% alcohol (reagent) 10 dips; 70% alcohol 10 dips; tap water 10 dips; Gill hematoxylin 1 minute; tap water to clear; ammonium hydroxide bluing 5 dips, tap water to clear, 80% alcohol 5 dips; 95% alcohol 10 dips, 100% alcohol 10 dips X 2; xylene 10 dips X 2; coverslip with mounting medium).
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The slides were evaluated independently by 3 of the authors (P.B.-G., C.L., T.N.) for preparation traits and overall diagnostic quality. The preparation traits were scored on completeness and uniformity of the sections, integrity of architectural features, cytologic detail, and stain quality. An arbitrary score was assigned as follows: 3 (good), sections showed greater than 90% diagnostic quality features; 2 (fair), 50% to 90% of the section showed acceptable quality for the trait; and 1 (poor), less than 50% of the section with acceptable features. The mean response of the 3 observers was used as the final score.
For overall diagnostic quality score, a similar system was used: 3, preparation was felt to be of diagnostic quality with minimal limitations compared with conventionally processed and prepared histologic slides; 2, sufficient quality for diagnostic purpose but significant limitation was present as seen typically in frozen section slides; and 1, nondiagnostic preparation. The mean responses were reported. To test the diagnostic reproducibility with the preparations, 2 of the observers (P.B.-G., C.L.) rendered a histologic diagnosis for each case after being provided with patient demographic information and gross specimen findings. Their diagnoses were compared with one another and against the file diagnosis based on conventionally processed tissue.
The scores of the slide quality are summarized in the Table. Sample photomicrographs are shown in Figures 4 to 7. All 21 cases yielded slides with adequate diagnostic quality, although in 16 of the cases at least 1 of the 3 observers felt there was some compromise in overall quality. None were considered nondiagnostic. The most common feature associated with at least 1 suboptimal trait was tissue containing densely cellular spindle cell proliferation (eg, myoma, cellular scar tissue) that yielded sections with variable thickness and diminished cytologic details in areas where the sections were thicker. In 1 case, extensive tumor necrosis required a slightly thicker section and diminished visualization of nuclear detail. The hematoxylin-eosin stain quality was uniformly good. Fatty tissue processed well and yielded sections similar in quality to routine processing. Moderate retraction artifact of tumor cells was present in 1 case.
The 2 observers that were blinded to the file diagnosis rendered completely concordant diagnosis except discrepant tumor type or grade in the 2 cases of uterine carcinomas. The undifferentiated carcinoma was designated as grade III endometrioid carcinoma and poorly differentiated carcinoma, not otherwise specified. The case of grade I endometrioid carcinoma was interpreted as grade III carcinoma by one observer.
Field histology has been a technique described for preparation of nonclinical material (eg, plant tissue) for microscopy. The microtome used for this procedure is designed for such a purpose. To our knowledge, there are no descriptions or established procedures for rapid processing and microscopic slide preparation of clinical tissue samples outside of dedicated histology laboratories. The aim of this study was to establish and test a point-of-care procedure in the hope that it can be used for treatment decisions on medical missions to underserved and geographically isolated locations.
Standard histologic preparation involves use of heavy commercial equipment not practical for transport to remote areas. The tissue processor is one of the key pieces of equipment we felt was problematic for point-of-care histology. Manual processing is an alternative that may take up to 24 hours. (5) Microwave-assisted devices can cut the time to 1 hour. (6) Our method is a modification of the microwave technique that also reduces the processing time to 1 hour. When fresh tissue is received for processing, fixation can also be expedited by placing the tissue in heated formalin for 10 minutes. The second key equipment is the microtome, which typically weighs 25 kg. We previously used a number of hand-held portable microtome kits from vendors of science class supplies designed for introductory biology and histology work. These microtomes have circular stages and central advancing wells for the specimen. The devices were small and convenient for travel, but we found the sectioning inconsistent and generally unsatisfactory for diagnosis.
We found our technique to have limitations. The small size of the block limits the size of the tissue to 1.0 cm or less. Despite reinforcement of the blade, the thickness of the sections varied considerably and required several attempts in some cases to yield an optimal section. Sectioning the one case of extensively necrotic material was more difficult as is the case with using usual methods. Densely cellular spindle cell tissue seemed to yield thicker sections in some specimens and made it more difficult to see the cytologic details. Previous attempts to skip the xylene step resulted in incomplete penetration of the paraffin into the tissue, resulting in incomplete sections. Unlike the alcohol and paraffin steps, xylene requires a glass container. In the embedding step, pouring in the melted paraffin before the tissue becomes cold and hardened is important to prevent separation of the tissue from the block during microtome sectioning.
The prepared sections were generally of adequate quality for use in medical missions where the need for detailed pathologic assessment is limited. Treatment options available for pathologic conditions in these settings include surgical excision of tumors, obtaining clear margins if the tumor is identified to be malignant, antibiotic therapy for infectious diseases, palliation or counseling for incurable conditions, and referral to a tertiary center once a diagnosis has been established. The processing was sufficiently simple that a person with minimal training could perform it. Using the microtome posed some challenges but is not significantly different from cutting frozen section slides, a technique most histopathologists are familiar with. Overall, the preparation quality was between that of frozen section slides and routine permanent section slides. The limitation in tissue size and occasional suboptimal preparation quality were contributing factors to discrepant tumor grade assignment in 2 of the cases in our series. The manual steps also impose a quantitative limit and preclude processing more than 3 or 4 samples at a time. These limitations preclude use of the technique for routine histologic processing.
Point-of-care microscopy service requires a lightweight microscope. The Swift Field Microscope FM31-LWD (Swift Instruments, Scientific Instrument Division, San Jose, California) with x4, x10, and x40 objectives and a compact flashlight for light source has worked well for us. On medical missions, we will not have access to a hematoxylin-eosin autostainer and we intend to hand stain the slides using the frozen section staining protocol. Other useful supplies for pathology field work include fine-needle aspiration biopsy material, core biopsy needles, Diff-Quik stain, 10% potassium hydroxide solution, formalin containers, and culture/transport media for bacteria and fungi. A digital camera with macro capability is useful to document gross specimen and clinical findings.
The entire processing equipment with reagents, stains, forceps, 100 mL of formalin, forceps, a dozen scalpel blades, brushes, thermometer, a box of slides, microscope, and empty specimen containers has a combined weight of 3.6 kg and fits in a hand-held case. Inclusion of fine-needle aspiration and core needle biopsy supplies will not appreciably add to the weight. The field microscope ($700), portable microtome ($230), and disposable core biopsy needles ($45 each) are the most expensive components and the estimated cost of the entire kit is less than $1500. Portable ultrasound devices that one may have on a fine-needle aspiration cart are generally not necessary on medical missions due to the usually advanced mass lesions encountered. The minimal resources needed at the testing site include electricity for the hot pot, water, and ice for paraffin embedding.
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The sample size used for the study is small and additional experience in actual field conditions is needed to validate the methodology. We hope others will take an interest in our study and find improvements to the technique. Additional areas to be explored for point-of care microscopy include special stains of the rapidly processed slides for fungal and acid-fast bacilli organisms, a problem frequently encountered in previous missions.
The thoughtful advice that the histology staff gave was instrumental in developing the field histology technique.
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(5.) Prophet EB. Tissue processing: dehydration, clearing, and infiltration. In: Prophet EB, Mills B, Arrington JB, et al, eds. Laboratory Methods in Histotechnology. Washington, DC: American Registry of Pathology, 1992:29-31.
(6.) Rohr LR, Layfield LJ, Wallin D, Hardy D. A comparison of routine and rapid microwave tissue processing in a surgical pathology laboratory: quality of histologic sections and advantages of microwave processing. Am J Clin Pathol. 2001;115(5):703-708.
Kaumakaokalani Calhoun, BS; Alexander Lin, BS; Peter Bryant-Greenwood, MD; Christopher Lum, MD; Douglas Johnson, MD; Thomas Namiki, MD
Accepted for publication May 18, 2010.
From the Departments of Pathology (Drs Bryant-Greenwood, Lum, and Namiki and Ms Calhoun and Mr Lin) and Medicine (Dr Johnson), The Queen's Medical Center and University of Hawaii School of Medicine, Honolulu.
The authors have no relevant financial interest in the products or companies described in this article.
Reprints: Thomas Namiki, MD, The Queen's Medical Center, 1301 Punchbowl St, Honolulu, Hawaii 96813-2499 (e-mail: tnamiki@pol. net).
Summary of Specimens and Quality of Preparations Case Section Completeness No. Tissue Diagnosis and Uniformity (a) 1 Breast Benign fibrosis 2.7 2 Breast Phyllodes 2.0 tumor 3 Breast IDCA 3.0 4 Breast Fibroadenoma 3.0 5 Breast Benign fibrosis 2.7 6 Uterus Undiff Ca 2.0 7 Uterus Myoma 3.0 8 Uterus Endomet Ca 3.0 9 Uterus Endomet polyp 2.7 10 Uterus Myoma 2.7 11 Skin SCC 2.0 12 Skin BCC 2.7 13 Skin Keloid 2.3 14 Skin EIC 2.0 15 Skin Acrochordon 2.3 16 Soft tissue Fibromatosis 2.3 17 Colon Adenoca 2.7 18 Colon Adenoca 2.0 19 Colon Adenoca 2.3 20 Colon Adenoma 3.0 21 Colon Adenoca 2.7 Case Architectural Cytologic Stain Overall Diagnostic No. Features (a) Detail (a) Quality (a) Quality Scores (b) 1 3.0 2.0 3.0 2.3 2 3.0 2.0 3.0 2.3 3 3.0 2.7 3.0 2.7 4 3.0 3.0 3.0 3.0 5 3.0 3.0 3.0 3.0 6 2.7 2.7 3.0 2.7 7 3.0 2.0 3.0 2.0 8 3.0 3.0 3.0 3.0 9 3.0 3.0 3.0 3.0 10 3.0 3.0 3.0 3.0 11 2.7 2.7 3.0 2.7 12 3.0 2.7 3.0 2.7 13 3.0 2.3 3.0 2.3 14 3.0 2.3 3.0 2.3 15 3.0 2.7 3.0 2.7 16 3.0 3.0 3.0 2.7 17 3.0 2.7 3.0 2.7 18 2.7 2.7 3.0 2.3 19 3.0 2.3 3.0 2.3 20 3.0 2.7 3.0 2.7 21 2.3 2.3 3.0 2.3 Abbreviations: Adenoca, adenocarcinoma; BCC, basal cell carcinoma; Ca, carcinoma; EIC, epidermal inclusion cyst; Endomet, endometrial; IDCA, invasive ductal carcinoma; SCC, squamous cell carcinoma; Undiff, undifferentiated. (a) Preparation quality mean scores: 1, poor (<50% of tissue diagnostic quality); 2, fair (50%-90% diagnostic quality); 3, good (>90% diagnostic quality). (b) Overall diagnostic quality mean scores: 1, nondiagnostic quality due to poor preparation; 2, diagnostic quality but with moderate limitation similar to frozen section slide; 3, diagnostic quality without significant deficiencies.
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|Author:||Calhoun, Kaumakaokalani; Lin, Alexander; Bryant-Greenwood, Peter; Lum, Christopher; Johnson, Douglas|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Feb 1, 2011|
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