Development of an Ultrasound-emitting Device for

Volume 58(5): 421–428, 2010Journal of Histochemistry & Cytochemistry

http://www.jhc.org

ARTICLE

Development of an Ultrasound-emitting Device for PerformingRapid Immunostaining Procedures

Hideki Hatta, Koichi Tsuneyama, Takashi Kondo, and Yasuo Takano

Department of Diagnostic Pathology (HH,KT,YT) and Radiological Sciences (TK), Graduate School of Medicine andPharmaceutical Sciences, University of Toyama, Toyama, Japan

SUMMARY Although intraoperative rapid diagnosis is conventionally performed usinghematoxylin–eosin (HE)-stained specimens, the use of additional special staining, togetherwith immunostaining techniques, has been examined in recent years to improve diagnosticaccuracy. In intraoperative rapid diagnosis, immunostaining should be completed within7–10 min, because the pathologist is typically presented with an HE-stained specimen withinthe same time period. We hypothesized that ultrasound may enhance antigen–antibodyreactions and reduce the number of immunostaining steps. To clarify the ability of ultra-sound to support immunostaining, we first created an ultrasonic generator specifically forimmunostaining. Next, we explored the optimal conditions for immunostaining of formalin-fixed specimens to examine the utility of the ultrasonic generator. Finally, we tried immuno-staining with the ultrasonic generator using frozen specimens to simulate intraoperativerapid diagnosis. We report herein that ultrasound enables immunostaining of frozen speci-mens in ?10 min. (J Histochem Cytochem 58:421–428, 2010)

KEY WORDS

immunostaining

ultrasound

intraoperative rapid diagnosis

TISSUE SAMPLE PREPARATION in pathology involves multi-ple steps of fixation, paraffin-embedded block creation,cutting of sections, and various staining procedures, aprocess generally requiring several days for comple-tion. Because reducing the time required for this pro-cess will directly affect the time required to make acomplete diagnosis, improvement of this process with-out affecting the quality of each step has been investi-gated (Leong et al. 1985,2002; Leong and Duncis1986; Login et al. 1987; Kayser et al. 1988; Vinceket al. 2003; Hafajee and Leong 2004; Morales et al.2004; Nadji et al. 2005). In particular, the applicationof microwaves is being investigated worldwide. Micro-waves have been applied to fixation, a special stainingmethod, and immunohistochemistry with impressiveresults. (Leong et al. 1985,2002; Leong and Duncis1986; Login et al. 1987; Kayser et al. 1988; Kumadaet al. 2004; Morales et al. 2004; Hatta et al. 2006).

It has recently been reported that ultrasound can re-duce the reaction time of processes such as fixation,decalcification, and defatting in the production ofpathological specimens and can increase protein andnucleic acid stability (Chu et al. 2006; Kitayama andYamada 2006; Reineke et al. 2006). The utility ofmicrowaves in pathological specimen production andrapid immunostaining is believed to be the result ofthe greater agitation effect produced by high-frequencyvibrations (Leong et al. 1985; Kumada et al. 2004;Hatta et al. 2006). On the other hand, ultrasound isalso known to produce high churning/osmosis effectsresulting from repeated compression and depressionof the liquid level, and these are expected to have thesame effects as microwaves (Chu et al. 2006; Kitayamaand Yamada 2006; Reineke et al. 2006).

We hypothesized that immunostaining reactiontime would be reduced using an ultrasonic generator,

Correspondence to: Koichi Tsuneyama, MD, PhD, Departmentof Diagnostic Pathology, Graduate School of Medicine and Phar-maceutical Sciences, University of Toyama, 2630 Sugitani, Toyama930-0194, Japan. E-mail: [email protected]

Received for publication September 24, 2009; accepted January 12,2010 [DOI: 10.1369/jhc.2010.955096].

© 2010 Hatta et al. This article is distributed under the terms of aLicense to Publish Agreement (http://www.jhc.org/misc/ltopub.shtml). JHC deposits all of its published articles into the U.S.National Institutes of Health (http://www.nih.gov/) and PubMedCentral (http://www.pubmedcentral.nih.gov/) repositories for publicrelease twelve months after publication.

0022-1554/10/$3.30 421

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and thus for this purpose, we created an ultrasonicgenerator specifically for immunostaining. Next, weexplored the optimal conditions for immunostainingusing a formalin-fixed specimen to examine the utilityof immunostaining with the ultrasonic generator.Finally, we attempted to immunostain frozen speci-mens using the ultrasonic generator, as a simulationof its application in intraoperative rapid diagnosis, toverify the amount of time required, its staining preci-sion, and its potential for practical use.

Materials and Methods

Creation of an Ultrasound Generatorfor Immunostaining

In collaboration with the Kurokawa Corporation(Toyama, Japan) and Honda Electronics Corporation(Aichi, Japan), we experimentally produced an ultra-sound generator exclusively for immunostaining; thisgenerator has equal irradiation states in all vibrationplates and enables the simultaneous investigation oftwo or more glass slides (Figure 1). The driving fre-quency of 1 MHz was utilized to minimize the chemi-cal effects of ultrasound resulting from cavitation(Koda et al. 2003). This device has an independentvibration plate at 1 MHz (plate diameter: 3 cm) withsix channels and allows the simultaneous processingof six glass slides under identical conditions. All thechannels produce continuous waves at 1000 kHz. Be-tween the glass slides and the vibration plate, a gelsheet was inserted to ensure that no gaps were present.This gel sheet protects the attenuation of ultrasoundtransmission by less than 1 dB.

Immunostaining Study

Examination of Ultrasonic Intensity and TimeRequired for Primary Antibody Incubation UsingFormalin-fixed Paraffin-embedded Sections. The mosteffective combination of ultrasonic power [intensity 5ultrasonic power (W)/area of plate(cm2)] and irradia-tion time in each stained specimen was examined usingultrasonic power (intensity) to 0.4 W (0.057 W/cm2),1.9 W (0.269 W/cm2), 3.3 W (0.467 W/cm2), and

4.4 W (0.628 W/cm2), and ultrasound exposure timesof 1, 2, and 3 min. Five different mouse monoclonalantibodies (cytokeratin AE1/AE3, cytokeratin CAM.5.2,CD3, CD20, and Ki-67) frequently used in routinelaboratory tests were used as primary antibodies, andEnvision peroxidase for mouse monoclonal antibodies(DAKO; Carpinteria, CA) was used as the secondaryantibody. A much higher concentration of primary anti-bodies was used in this examination. Details of the anti-bodies are shown in Table 1. The irradiation time of thesecondary antibody was set at 2 min, and color devel-opment was performed using the DAB substrate kit(DAKO). The color development time was set at1 min. Formalin-fixed paraffin-embedded sections oflymph nodes including metastatic lesions of colorectalcancers were used as specimens. Each section wasused for experimentation after performing the typicalantigen retrieval process using a target retrieval solu-tion (DAKO).

Validation Study of the Ultrasound-supportedMethod Using Formalin-fixed Paraffin-embeddedSections. To evaluate the practical utility of the ultra-sonic method, we selected 10 formalin-fixed paraffin-embedded blocks of common malignant tumors andprepared 30 unstained specimens and a hematoxylin–eosin (HE)-stained specimen. Then, we performedimmunostaining with 10 primary antibodies, including5 common antibodies (cytokeratin AE1/AE3, cyto-keratin CAM5.2, CD3, CD20, and Ki-67) and 5 otherantibodies, using three different staining procedures(common method, microwave-supported method, andultrasound-supported method). In brief, the commonmethod is a standard immunostaining procedure with

Table 1 Five common antibodies

Name (clone) Supplier Dilution

Cytokeratin (AE1/AE3) DAKO 1:80Cytokeratin (CAM5.2) Becton-Dickinson; San Jose, CA NeatCD3 (PS1) Nichirei Co.; Tokyo, Japan NeatCD20 (L26) DAKO 1:200Ki-67 (MIB1) DAKO 1:40

Figure 1 Ultrasound generator devicefor immunostaining and its attach-ments. This device has six separatechannels. In the image, six channels(channels 1–6) were used for immu-nostaining (A). Five slides were seton vibrating plates (B). A gel sheetwas loaded between the vibratingplates and glass slides.

422 Hatta, Tsuneyama, Kondo, Takano

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30-min incubation of primary and secondary antibodiesat room temperature, and the microwave-supportedmethod is a procedure with 10-min incubation of pri-mary and secondary antibodies under intermittentmicrowave irradiation (Kumada et al. 2004). For theultrasound-supported method, we performed a 3-minincubation of primary and secondary antibodies under4.4Wultrasound using our aforementioned equipment.The 10 malignant tumors and selected antibodies aredescribed in Table 2. Each section was used for experi-mentation after performing the typical antigen retrievalprocess using a target retrieval solution (DAKO).

Examination of Irradiation Intensity and TimeRequired for Incubation of the Primary Antibodyin Cryosections. A protocol in which immunostainingof frozen specimens was completed within 10 min wasdeveloped, and staining quality was evaluated. Weused frozen specimens that were presented for rapiddiagnosis at Toyama University Hospital. These speci-mens also had sufficient slices to act as controls foractual diagnoses. After making 5-mm-thick sliced sec-tions, the frozen specimens were fixed for 2 min withacetone (4C), and endogenous peroxidase was re-moved by a 30-sec incubation with 3%H2O2 (DAKO).The ultrasonic power (intensity) was fixed at 4.4 W(0.628 W/cm2) during the 90-sec incubation with theprimary antibody, and the secondary antibody wasincubated for 90 sec after washing the specimens for15 sec with Tris-buffered saline. After DAB coloringfor 1 min, nuclear counterstaining was performed for15 sec using hematoxylin, and the specimens were thencovered with coverslips (Table 3).

Results

Staining Conditions Employing Formalin-fixedParaffin-embedded Sections

The best-suited staining conditions were examinedusing 5 different primary antibodies (cytokeratinAE1/AE3, cytokeratin CAM5.2, CD3, CD20, and

Ki-67) by adjusting the intensity and duration ofultrasound irradiation. With cytokeratin AE1/AE3,the staining intensity increased with irradiation in-tensity at all reaction times, and thus, the maximal

Table 2 Ten malignant tumors and selected antibodies

Organ Diagnosis Primary antibodies

Uterine cervix Squamous cell carcinoma CK5/6 CK14 p53 D2-40 FibronectinStomach Adenocarcinoma CK7 CK20 Muc1 Muc5AC Muc6Colon Adenocarcinoma Muc2 CDX2 CEA MLH1 EGFRLymph node Micrometastasis of adenocarcinoma CD31 CD34 CD68 CD5 CD21Breast Adenocarcinoma ER PgR HER2 E-cadherin b-CateninProstate Adenocarcinoma p63 CK34bE12 p504S PSA a-SMALiver Hepatocellular carcinoma AFP Hepatocyte CD10 CK19 Glypican-3Lung Small-cell carcinoma TTF-1 Chromogranin A CD56 Calretinin SynaptophysinBrain Glioma GFAP Olig-2 EMA MIC-2 Neu NBone marrow Involvement of lymphoma MPO Glycophorin A CD138 CD30 CD61

Immunostaining with five common primary antibodies (AE1/AE3, CAM5.2, CD3, CD20, Ki-67) was also performed in all organs.

Table 3 Immunostaining protocol for frozen specimens

Time (min)

Ultrasound-supported methodSpecimens fixed by acetone (4C) 2Endogenous peroxidase blocking by 3% H2O2 0.5Incubation with primary antibodies under ultrasound

irradiation (4.4 W)1.5

Washing of specimens with Tris-buffered saline 0.25Incubation with peroxidase-conjugated secondary

antibodies under ultrasound irradiation (4.4 W)1.5

Washing of specimens with Tris-buffered saline 0.25Reaction with substrate (DAB) for color imaging 1Counterstaining with hematoxylin (Meyer) 0.25Dehydration and placement of a coverslip with

mounting agentStandard immunostaining method

Specimens fixed by acetone(4C) 2Endogenous peroxidase blocking by 3% H2O2 0.5Incubation with primary antibodies 5–10Washing of specimens with Tris-buffered saline 1Incubation with peroxidase-conjugated

secondary antibodies5–10

Washing of specimens with Tris-buffered saline 1Reaction with substrate (DAB) for color imaging 1Counterstaining with hematoxylin (Meyer) 0.25Dehydration and placement of a coverslip with

mounting agentMicrowave-supported method

Specimens fixed by acetone (4C) 2Endogenous peroxidase blocking by 3% H2O2 0.5Incubation with primary antibodies under intermittant

microwave irradiation (4.4 W)5

Washing of specimens with Tris-buffered saline 0.25Incubation with peroxidase-conjugated

secondary antibodies5

Washing of specimens with Tris-buffered saline 0.25Reaction with substrate (DAB) for color imaging 1Counterstaining with hematoxylin (Meyer) 0.25Dehydration and placement of a coverslip with

mounting agent

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power (intensity) of 4.4 W (0.628 W/cm2) was con-sidered to be the optimal ultrasound irradiation in-tensity. Although staining intensity increased with areaction time of 2 min compared with 1 min, attenu-ation of staining intensity was occasionally observedwith a reaction time of 3 min (Figure 2). Long ultra-sound exposure times were assumed to have harmfuleffects (destruction, denaturing, etc.) on the antibody(Figure 2). Similar tendencies were recognized in otherantibodies examined. Taken together, the optimalconditions in formalin-fixed paraffin-embedded sec-tions were 4.4 W of ultrasound irradiation intensityand 2–3 min irradiation time for both primary andsecondary antibodies.

Validation of Ultrasound-supported Method

We fixed the staining condition of the ultrasoundmethod as 3 min of incubation for primary and second-ary antibodies with 4.4 W ultrasound irradiation and

compared its ability to the common staining procedureand the microwave-supported procedure for a valida-tion study. We then performed immunostaining of atotal of 55 primary antibodies (5 common antibodiesand 50 other antibodies) in 10 samples of malignanttumors using three different immunostaining proce-dures. Of the 10 primary antibodies, 5 were commonprimary antibodies (cytokeratin AE1/AE3, cytokeratinCAM5.2, CD3, CD20, and Ki-67) and 5 were other pri-mary antibodies that were chosen based on the sourceof the tumor. In total, 55 different primary antibodieswere examined in this validation study (Table 2). Onstaining with all 55 primary antibodies using the com-mon and microwave-supported methods, all 100 speci-mens showed reasonable immunostaining with clearbackground. All the samples stained by the commonand microwave-supported methods retained good mor-phology. The total staining time of the method usingmicrowave (60 min) was substantially shorter than thatof the common method (100 min). In the method using

Figure 2 Examination of the irradiation intensity and staining time required for the primary antibody cytokeratin AE1/AE3, using formalin-fixed paraffin-embedded sections of lymph nodes including metastatic lesions of colorectal cancers. The optimal ultrasound irradiationintensity was considered to be 4.4 W. Although staining intensity was reinforced at the reaction time of 2 min compared with 1 min, attenua-tion of staining intensity was occasionally observed in irradiation for 3 min. Bar 5 0.1 mm.


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ultrasound, of the 55 primary antibodies, 39 producedstrong immunostaining and 9 produced weak butreasonable immunostaining. Taken together, 48 of55 primary antibodies (87%) were effective using theultrasound-supported method. Figure 3 shows anexample of HE staining and 10 examples of immuno-staining of prostate adenocarcinoma. Although 7 pri-mary antibodies (CD5, ER, PgR, b-catenin, Olig-2,Neu N, and CD61) did not produce recognizableimmunostaining, 5 common primary antibodies (cyto-keratin AE1/AE3, cytokeratin CAM5.2, CD3, CD20,

and Ki-67) produced appropriate immunostaining inall cases examined. Almost all slides indicated a reason-able immunostaining pattern; however, cytokeratinCAM5.2 not only occasionally reacted with epithelialcells but also produced nonspecific staining on somefoamy macrophages (Figure 4). Of 10 samples,4 showed various degrees of morphological damage.Bone marrow samples tend to show morphologicaldamage (Figure 4). The ultrasonic-supported methodtook the shortest time for staining (45 min). Theseresults are described in Table 4.

Figure 3 Results of validation studyof formalin-fixed paraffin-embeddedsections. In prostate adenocarcinoma,all 10 primary antibodies showed rea-sonable immunostaining by the ultra-sound method. (A) HE; (B) AE1/AE3;(C) CAM5.2; (D) CD3; (E) CD20; (F) Ki-67;(G) CK34bE12; (H) p63; (I) p504S; (J)PSA; and (K) aSMA. Arrows in F indi-cate positive cells. Bar 5 0.1 mm.

Figure 4 Examples of the disadvan-tage of the ultrasound method. Mor-phological damage was observedoccasionally. (A) Bone marrow samplewith HE staining. (B) Bone marrowsample with CD20. Nonspecific reac-tion against foamy macrophages isobserved by CAM5.2 immunostainingin the lymph node. (C) Micrometasta-sis of lymph node with HE staining.(D) Micrometastasis of lymph nodewith CAM5.2. Insets in B,D show highermagnification. Bar 5 0.1 mm.


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Staining Conditions in Frozen Sections

We then examined 75 cases of frozen sections using4 different primary antibodies (cytokeratin AE1/AE3,cytokeratin CAM5.2, CD3, and CD20) with 10-minsonication. Although 10 cases showed slight morpho-logical damage at the edge of the specimens, they hadsufficient area without morphological damage to beincluded in this experiment. In all the tested specimens,reasonable immunostaining results were obtainedusing 10-min sonication. Representative immunostain-ing images are shown in Figure 5. The staining in-tensity of each specimen was sufficient, the stainingpattern was reasonable, and fewer nonspecific reac-tions were seen in most of the cases examined.

DiscussionAlthough intraoperative rapid diagnosis is convention-ally performed using HE-stained specimens, the use ofadditional special staining, together with immunostain-ing techniques, has been examined in recent yearsto improve diagnostic accuracy (Tsutsumi et al. 1995;Viberti et al. 2001; Matsumoto et al. 2003; Salem et al.2003; Hatta et al. 2006; Monig et al. 2006; Jyllinget al. 2008). However, a certain amount of reactiontime is required for an antigen–antibody reaction, andthe time required for the common immunostaining pro-

cedure (dextran polymer method) can only be shortenedto around 13–20 min (Ichihara et al. 2001). In intra-operative rapid diagnosis, immunostaining should becompleted within 7–10 min, because the pathologist istypically presented with an HE-stained specimen withinthe same time period.

In this study, we experimentally produced an ultra-sonic device specifically for immunostaining using 6 dif-ferent antibodies simultaneously and conducted apreliminary examination of the optimal immunostain-ing conditions for this device (data not shown). As aresult, when a particular intensity of sonication wasexceeded, the reaction time for immunostaining wassignificantly shortened, but the quality of the immuno-staining was reduced and the specimen was damagedby long-term ultrasonic irradiation. These facts indi-cate that regulation of output and reaction time is es-sential for immunostaining with ultrasonic irradiationequipment. Using 5 different commonly used anti-bodies, we observed that the optimal conditions forour equipment were 4.4 W of irradiation and 2–3 minof antigen–antibody reaction time. Then we performeda validation study using a total of 55 primary anti-bodies (5 common antibodies and 50 other antibodies),and compared the quality of staining using three differ-ent immunostaining procedures (common method,microwave-supported method, and ultrasound-supported

Table 4 Summary of validation study for three different immunostaining procedures

Antibodies showing recognizablestaining/total 55 antibodies

Samples with nonspecificreaction/total 100 samples

Damaged samples/total100 samples Total time (min)

Standard immunostaining method 55/55 (100%) 0/100 (0%) 0/100 (0%) 100Microwave-supported method 55/55 (100%) 0/100 (0%) 0/100 (0%) 60Ultrasound-supported method 48/55 (87%) 1/100 (1%) 4/100 (4%) 45

Figure 5 Immunostaining images ofcryosections according to the protocolshown in Table 3. (A) AE1/AE3 in fol-licular epithelial cells of the thyroidgland. (B) CAM5.2 in metastatic adeno-carcinoma of a lymph node. (C) CD3 ina lymph node. (D) CD20 in a lymphnode. Bar 5 0.1 mm.


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method). Of the 55 primary antibodies examined, only7 did not produce recognizable immunostaining usingultrasound. However, 5 commonly used primary anti-bodies (cytokeratin AE1/AE3, cytokeratin CAM 5.2,CD3, CD20, and Ki-67) produced recognizable immu-nostaining. Four of 100 cases showed morphologicaldamage by ultrasound, but most of them were soft,easily damaged organs such as brain and bone marrow.In one case, nonspecific reaction of CAM5.2 to foamymacrophages was observed. Care should be taken whileusing this antibody for micrometastasis detection. Theultrasound-supported method is beneficial becauseit reduces the staining time to 45 min.

Next, we attempted to establish a staining protocol inwhich the immunostaining of frozen specimens could becompleted in 10 min. Although various steps areneeded for immunostaining in addition to the antigen–antibody reaction, the antigen–antibody reaction time isthe rate-determining step of the overall reaction time.Although frozen specimens tend to suffer mechanicaldamage, their antigenicity is more strongly retainedcompared with formalin-fixed paraffin-embeddedspecimens. Considering this fact, the antigen–antibodyreaction time of the primary and secondary antibodieswas reduced by 90 sec compared with the conditionsfor formalin-fixed paraffin-embedded specimens, thusproducing a protocol with an overall reaction time of10 min. When immunostaining was performed in75 cases using this protocol, reasonable and recogniz-able immunostaining was observed in all cases exam-ined, although slight morphological damage wasapparent in some cases.

The mechanism by which antigen–antibody reac-tion time is shortened by ultrasound or microwaveirradiation has not been fully clarified. We assumethat when ultrasound waves emitted from a vibrationplate repeatedly compress and depress the fluid levelin a preparation, a high agitation effect is inducedand the opportunity for antigens and antibodies to in-teract significantly improves (Leong et al. 1985,2002;Kumada et al. 2004; Hatta et al. 2006). Because thesame effect can also be induced by raising the dilutionconcentration of antibodies, an antibody concentra-tion two to four times greater than normal is oftenused for rapid immunostaining. However, increasedconcentration of antibodies results in cost increase,introduction of nonspecific reactions, and reductionin immunostaining quality. Using our ultrasonic de-vice resulted in a decrease in nonspecific reactionswithout producing morphological damage, suggestingthat ultrasound can produce sufficient vibration andagitation to enhance specific reactions and inhibit non-specific reactions.

Using the ultrasonic device developed in this study,immunostaining in frozen specimens can be performedin ?10 min. It is believed that routine immunostaining

under the abovementioned conditions is now a viablemethod for rapid diagnosis.

Acknowledgments

This study was supported by Toyama New Industry Or-ganization (2007).

We thank Tokimasa Kumada (University of Toyama),Shinichi Kurokawa (Kurokawa Co.), and Kazuto Kobayashi(Honda Electronics Co.) for their help and technical assistance.

Literature Cited

Chu WS, Liang Q, Tang Y, King R, Wong K, Gong M, Wei M, et al.(2006) Ultrasound-accelerated tissue fixation/processing achievessuperior morphology and macromolecule integrity with storagestability. J Histochem Cytochem 54:503–513

Hafajee ZA, Leong AS (2004) Ultra-rapid microwave-stimulated tis-sue processing with a modified protocol incorporating microwavefixation. Pathology 36:325–329

Hatta H, Tsuneyama K, Kumada T, Zheng H, Cheng C, Cui Z,Takahashi H, et al. (2006) Freshly prepared immune complexeswith intermittent microwave irradiation result in rapid andhigh-quality immunostaining. Pathol Res Pract 202:439–445

Ichihara T, Nomoto S, Takeda S, Nagura H, Sakamoto J, Kondo K,Horisawa M, et al. (2001) Clinical usefulness of the immuno-staining of the tumor markers in pancreatic cancer. Hepatogastro-enterology 48:939–943

Jylling AM, Lindebjerg J, Nielsen L, Jensen J (2008) Immuno-histochemistry on frozen section of sentinel lymph nodes in breastcancer with improved morphology and blocking of endoge-nous peroxidase. Appl Immunohistochem Mol Morphol 16:482–484

Kayser K, Stute H, Lubcke J, Wazinski U (1988) Rapid microwavefixation: a comparative morphometric study. Histochem J 20:347–352

Kitayama Y, Yamada K (2006) [Ultrasound-mediated treatment ofpathology archival samples]. Rinsho Byori 54:1215–1222

Koda S, Kimura T, Kondo T, Mitome H (2003) A standard methodto calibrate sonochemical efficiency of an individual reaction sys-tem. Ultrason Sonochem 10:149–156

Kumada T, Tsuneyama K, Hatta H, Ishizawa S, Takano Y (2004)Improved 1-h rapid immunostaining method using intermittentmicrowave irradiation: practicability based on 5 years applicationin Toyama Medical and Pharmaceutical University Hospital. ModPathol 17:1141–1149

Leong AS, Daymon ME, Milios J (1985) Microwave irradiation asa form of fixation for light and electron microscopy. J Pathol146:313–321

Leong AS, Duncis CG (1986) A method of rapid fixation of largebiopsy specimens using microwave irradiation. Pathology 18:222–225

Leong AS, Lee ES, Yin H, Kear M, Haffajee Z, Pepperall D (2002)Superheating antigen retrieval. Appl Immunohistochem MolMorphol 10:263–268

Login GR, Schnitt SJ, Dvorak AM (1987) Rapid microwave fixa-tion of human tissues for light microscopic immunoperoxidaseidentification of diagnostically useful antigens. Lab Invest 57:585–591

Matsumoto M, Natsugoe S, Ishigami S, Uenosono Y, Takao S, AikouT (2003) Rapid immunohistochemical detection of lymph nodemicrometastasis during operation for upper gastrointestinal carci-noma. Br J Surg 90:563–566

Monig SP, Luebke T, Soheili A, Landsberg S, Dienes HP,Holscher AH, Baldus SE (2006) Rapid immunohistochemicaldetection of tumor cells in gastric carcinoma. Oncol Rep 16:1143–1147

Morales AR, Nassiri M, Kanhoush R, Vincek V, Nadji M (2004)Experience with an automated microwave-assisted rapid tissue


TheJourna

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Histoch

emistry&

Cytoc

hemistry

processing method: validation of histologic quality and impact onthe timeliness of diagnostic surgical pathology. Am J Clin Pathol121:528–536

Nadji M, Nassiri M, Vincek V, Kanhoush R, Morales AR (2005)Immunohistochemistry of tissue prepared by a molecular-friendlyfixation and processing system. Appl Immunohistochem MolMorphol 13:277–282

Reineke T, Jenni B, Abdou MT, Frigerio S, Zubler P, Moch H,Tinguely M (2006) Ultrasonic decalcification offers new perspec-tives for rapid FISH, DNA, and RT-PCR analysis in bone marrowtrephines. Am J Surg Pathol 30:892–896

Salem AA, Douglas-Jones AG, Sweetland HM, Mansel RE (2003)Intraoperative evaluation of axillary sentinel lymph nodes using

touch imprint cytology and immunohistochemistry: I. Protocolof rapid immunostaining of touch imprints. Eur J Surg Oncol29:25–28

Tsutsumi Y, Serizawa A, Kawai K (1995) Enhanced polymer one-stepstaining (EPOS) for proliferating cell nuclear antigen (PCNA) andKi-67 antigen: application to intra-operative frozen diagnosis.Pathol Int 45:108–115

Viberti L, Croce S, Pecchioni C, Bongiovanni M, Sapino A (2001)[Rapid immunohistochemistry applied to intraoperative diagno-sis: a critical analysis]. Pathologica 93:544–548

Vincek V, Nassiri M, Nadji M, Morales AR (2003) A tissue fixativethat protects macromolecules (DNA, RNA, and protein) andhistomorphology in clinical samples. Lab Invest 83:1427–1435


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Development of an Ultrasound-emitting Device for