Sample Turnaround Times for Three Histoprocessing Methods

Sample Turnaround Times for Three Histo treat MethodsIntroductionTurnaround time for whatever pathological laboratory is very important which depends upon the preparation and diagnosing of the histopathologic material. The fastity advantages the clinician to treat acutely carsick patients and influence the process practice of the pathologist. With the advent of modernization weave report card bear upon is change from the point of meander removal to embedding for instant histopathological diagnosis by various techniques or methods.After the surgical removal, the tissue undergoes preceding(prenominal) protocol for preparation of sections which usually involves impregnation with a suitable supporting medium. The stages of tissue bear upon implicate fastening, dehydration, clea pack, impregnation and embedding for designated durations of time to ensure completion of the procedure. Culling 1974, Bancroft Gamble 2002The reproducibility and relatively low expense abandoned to the most commonly employed method continues to recommend it as a valuable tool afterward nearly 100 years of existence. But with the demand of faster or early reporting, newer techniques like rapid manual(a) of arms and zap touch atomic number 18 getting introduced. Each of them is unique with their own set of advantages and disadvantages.The naturalized tissue bear on is reliable and address effective notwithstanding time consumption, reagent toxicity and delay in providing diagnosis are the major disadvantages. The rapid manual tissue processing has major disadvantages like the utilise of noxious chemicals, greater degree of tissue distortion and shrinkage which led to exploration for new before long processing schedules. The atom-bomb tissue processing eliminates the use of noxious chemicals, causes lesser distortion of tissue and has shorter processing time but the cost involved in instrumentation is very high. (Panja et al. 2007)Microwaves were invented by Perc y Spencer in 1945 which work on the principle of producing heat by oscillating or exciting polar molecules. The cook radiation syndrome forces dipolar molecules of proteins to rotate through 180 at the rate of 2.45 billion cycles per second (Srinivasan, Sedmak Jewell 2002, Bancroft Gamble 2002). These harebrained molecules referable to kinetics cause collision with adjacent molecules resulting in transfer of rotational energy. This friction causes drudgery of heat within the material itself leading to accelerated diffusion of processing fluids hence faster processing is possible.The advantages associated with atom-bomb processing led to the production of commercially available microwaves specifically designed for tissue processing, however, the cost involved in these is very high (Leong 2004, Rohr et al. 2001). Domestic microwaves are readily available, affordable and had been employ for tissue processing with good results earlier by some authors.Thus, the aim of the presen t study was to compare and psychoanalyze the efficacy of iii histoprocessing methods and to determine the meeting on verso times of tissue processing by these three methods.Materials and methodIn the present study, 60 specimens were selected randomly. The soft tissue specimen laid in 10% NBF for 24h were included in the study and hard tissues like cartilage, bone and tooth were not included in the study. The gross features of the specimen were recorded and tissues were cut into three pieces of approximately same size to be svelte by three methods. The sections obtained after processing were subsequently stained with HE by routine and microwave staining method.The stained slides in from individually one group bear upon by three techniques were randomly numbered for a blind study and circulated among four observers referred as O1-O4. The observers graded each parameter on the format prone in accessory D by following specific criteria as given in appendix C.MethodologyMicro wave tissue processingThe microwave oven was calibrated as the microwave energy is non changeless within the chamber. Thus, thermal and rimy slurs were detected in the chamber with the use of thermal paper sheet instead of the use of extra water load as suggested by various authors. The common frigidness spot provided the most consistent results every time.The technique was self-standardized by trial and error method in the LG domestic Microwave (Model no. MS-285SD). The microwave was operated at the maximum output power of 40% (approximately 360 W) with rotating tray and ring removed. The cut piece from a unyielding tissue sample was placed in a credit card tissue cassette and water rinse in running tap water so that tissue was excess of formalin. The tissue was irradiated in 200ml of 100% methanol and 200ml of 100% isopropyl alcohol for dehydration at cold spot for 2 cycles of 10 minutes each respectively in the microwave. After dehydration tissue was impregnated in 200 ml of melt down paraffin series wax for 2 cycles at cold spot of 10 min each and was embedded in paraffin wax.Conventional Tissue ProcessingThe cut piece from a fixed tissue sample was placed in a metal tissue cassette and water washed in running tap water so that tissue should be free of formalin. The tissue was dehydrated in 70% alcohol (one change), 90% alcohol (one change) and 100% alcohol three changes of 1 h each respectively. After dehydration tissue was cleared in two changes of xylene of 1 h each. Finally, tissue was impregnated in 2 changes of molten paraffin wax for 1 h each and was embedded.Rapid manual Tissue ProcessingThe cut piece from a fixed tissue sample was water washed in running tap water so that tissue should be free of formalin, after that tissue was wrapped in filter paper and dehydrated in 95% alcohol, 100% alcohol for 20 min on a call down plate. The dehydrated tissue was cleared in xylene for 20 min on a stir plate 20 min on a stir plate. Tissue was imp regnated in 2 changes of molten paraffin wax of 1h each and was then embedded.The microwave processed tissue were stained as given in table no. ____. Conventional and rapid manual processed tissues were stained as given in table no.___________.Statistical AnalysisThe values obtained from diametrical observers after assessment of sections processed by the three techniques were subjected to statistical analysis by Kruskal Wallis Test. One fashion ANOVA (Analysis of variance) was used for comparability mean shrinkage in tissues processed by the three histoprocessing methods. The P value Results tout ensemble observers were assumed to be reliable as the Cronbachs reliability test was statistically profound. Complete concordance was found amongst all pathologists in most of the cases. Hence, observer 1 was randomly selected for foster analysis.The histopathological evaluation of the epithelium, fibrous tissues and glandular tissue revealed that the nuclear cytoplasmic lineage was g ood and cellular draft was distinct in tissues processed by microwave assisted technique followed by conventional processing and rapid manual processing techniques. The stroma was good with distinct cellular outline. The secretory products can be slow appreciated and the RBCs, inflammatory cells were intact. The results were statistically non-significant as ascertained by Kruskal Wallis test.The colour intensity of the tissues graded by four observers revealed that the microwave sections were crisper and there was a good contrast between the hematoxyphilic and eosinophillic areas. Though some slides were not visualised up to the mark, all the three histoprocessing methods were comparable to each other.One way Analysis of partition (ANOVA) revealed a significant value on comparison of the tissue shrinkage processed by the three techniques. The dimensions of the tissues were recorded before dehydration and paraffin embedding. The mean percentage of shrinkage in rapid manual techniq ue was significantly higher as compared to the other two techniques whereas statistically non-significant value was obtained on comparing conventional and microwave method of tissue processing.DiscussionMicrowaves are electromagnetic waves(Microwave Processing Techniques for Microscopy) which causes oscillation and inflaming of polar molecules which are usually dipolar molecules of proteins in tissues. The excited molecules cause collision with adjacent molecules due to kinetics producing friction and causes production of heat within the material itself. The heat produced enhances the rate of diffusion of fluids to trickle into the tissues. The rise in temperature decreases the viscosity of processing fluids that facilitates diffusion. Therefore it is theoretically possible to fasten the tissue fixation and processing. This has resulted in a substantial reduction in the basic steps of histoprocessing, thereby reducing turnaround time and providing same day diagnosis.The applicatio ns of microwaves are extensive which includes tissue fixation, stabilization of large specimens, tissue processing for light and electron microscopy, histochemical and immunohistochemical staining.Microwave tissue processing technique was introduced by Boon and Kok in 1985 (Leong 2004) but the potential application of microwave energy was first recognized by Mayers in 1970 who successfully fixed tissue with a microwave generator (Kok, Visser Boon 1988). Boon et al. (1986) inform that it was possible to produce significant acceleration of tissue processing by using microwave radiation. Visinoni et al. (1998) first described the tissue central processor that completed the processing in 30-120 min, thus reducing the processing time from 24 h to on the dot 1-2 h providing early reporting and easy patient management.Thus, the aim of the present study was to compare the cytoplasmic and nuclear details as well as staining characteristics of tissue sections processed by conventional, ra pid manual and microwave techniques.The noxious chemicals used in conventional tissue processing were replaced in microwave tissue processing. In the microwave processing in contrast to conventional tissue processing, isopropyl alcohol was replaced by methanol as dehydrating agent and xylene by isopropyl alcohol as intermediate agent. Molten paraffin wax remained the impregnating and embedding medium for both the techniques. The reagent selection was in consonance with Babu, Malathi and Mangesh (2011) who also used methanol, isopropyl alcohol and molten paraffin wax for microwave tissue processing.Microwave radiation produced when enter the chamber it is reflected by the chamber walls until these gets absorbed by the material placed inside the chamber (Wong 2011). However, the spreading is not even throughout the chamber leading to formation of hot and cold spot zones. (Kok, Boon Smid 1993, Thostenson Chow 1999, Rutgers 2013).Hence, hot cold spots should be detected to achieve co nsistent results. Various authors have described methods for detection of hot and cold spots.Microwave processing was self-standardized by trial and error method in which the hot and cold spots were detected by using a damp thermal paper Kok, Boon and Smid (1993). All the procedures in the microwave were carried out in the cold spot zones as suggested by Sharp and Paperiello (1971), Benard (1974), Rangell and Keller (2000) in their respective studies.Microwavable plastic tissue cassettes were used for microwave tissue processing which are cheap and reclaimable as metallic utensils are contraindicated in the microwave because the electric fields of the waves produced by microwave magnetron are completely reflected at the same frequency by metals which can lead to sparking. (Vollmer 2004).In the present study, the staining protocol for microwave was followed as given by Babu, Malathi and Mangesh (2011) which included the stains used to be accelerated in the microwave. Kayser and Buben zer (1990) used domestic microwave oven for acceleration of the various stains which also included HE stain. Valle (1986), Moorlag, Boon and Kok (1987) and Mathai et al. (2008) modified various special stain protocols for microwave and concluded that microwave did not produce any detrimental effectuate on staining.In our study, the three pieces of tissue processed by three techniques sectioned by a soft tissue microtome and stained as per their respective protocols were evaluated. We adopted the meter for evaluation of tissue sections given by Kango and Deshmukh (2011).The overall quality of the tissue sections processed by microwave and manual methods was comparable. The microwave processed sections had same or resembling cytoplasmic and nuclear details with good erythrocyte legality and lymphocytic appearance than the manual methods. Similar results are given by Mathai et al. (2008), Morales et al. (2002), Bhuvanamha et al. (2013), Panja et al. (2007), Boon et al. (1986), Kok et al. (1988).We also observed that the stroma in some cases was slightly more condensed focally in microwave processed tissue sections which is similar to the findings reported by Boon, Kok and Ouwerkerk-Noordam (1986) This lead to the erroneous categorization of these cases as indistinct in studies by Kango and Deshmukh (2011). Since our criterion was adopted from the above mentioned study we also placed focal condensation of stroma as indistinct. In contrast Kok, Visser and Boon (1988) refuted the importance of focal condensation of stroma in diagnostic pathology.The colour intensity of the tissues graded by four observers revealed that the microwave sections were crisper and there was a good contrast between the hematoxyphilic and eosinophillic areas. The microwave processed tissues showed an change magnitude reaction to HE. The sections stained were slightly more eosinophilic as compared to the manual techniques. Similar findings are reported by Hopwood et al. (1984), Boon et al. (1986), Chaudhari, Chattopadhyay and Dutta (2000), Leong and Price (2004), Panja et al. (2007), Mathai et al. (2008), Babu, Malathi and Mangesh (2011). Hopwood et al. (1984) suggested that this eosinophilia could be easily corrected by altering the stain writing or staining time in eosin. In contrast Leong Price (2004) observed that eosinophilia of the cytoplasm was good as it produced good nuclear cytoplasmic contrast and enhancement of the cellular features.The dysplastic features i.e. hyperchromatism, pleomorphism of neoplasm cells and mitotic figures were easily appreciable in the microwave processed tissue sections of malignancy. There was also an easy clasp of the giant cells in the tissues of Central Giant cell Granuloma and tubercular lymphadenitis processed by microwave processing technique.Rapid processing of histopathologic material is becoming increasingly desirable for intraoperative consultations and timely diagnosis. We found positive impact on turnaround tim e in microwave method as the time taken for avoid preparation from fixed tissue was 1h as compared to conventional method (9h) and rapid manual method (3h).In some cases, proper diagnosis could not be reached as the size of the tissue was small and the sample was not representative of the site. Similar difficulties were also encountered by Suri et al. (2006), Kango and Deshmukh (2011) in their respective studies.As assessed in our study, the effects of the three methods of histoprocessing on cytoplasmic and nuclear details of epithelial, fibrous and glandular tissue showed no statistically significant variation. The microwave technique was comparable or slightly better than the manual methods.ConclusionThe applications and versatility of microwave processing methods are unattainable with conventional procedures. The method reported herein reproducibly yields similar histological quality to that provided by conventional processing. It has many advantages including feasibility, safet y and elimination of noxious chemicals that might be used for improvement in the practice of the histopathology laboratory, permitting the preparation of diagnostic material within a day. Domestic microwaves are easily available and cost effective but have certain notable disadvantages like spotted heating and inability to record and maintain temperature within the chamber. Further exploration in the field is need for development of cost effective microwave histoprocessors for histopathology which provide similar histologic material for rapid diagnosis and reporting.References