The Emergence of Rapid Counter Immunostaining in the Controlled Narrow Excision of Malignant Melanoma—How We Do It

doi:10.4236/jct.2013.46123

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Journal of Cancer Therapy, 2013, 4, 1082-1084

http://dx.doi.org/10.4236/jct.2013.46123 Published Online August 2013 (http://www.scirp.org/journal/jct)

The Emergence of Rapid Counter Immunostaining in the

Controlled Narrow Excision of Malignant

Melanoma—How We Do It

Jordan Troxel, Grace Brummer, Kristin Cox, S. Ray Peterson

Department of Dermatology, Central Utah Clinic, Provo, USA.

Email: [email protected]

Received May 22nd, 2013; revised June 25th, 2013; accepted July 3rd, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Mohs Micrographic Surgery (MMS) is widely employed in the treatment of non-melanoma skin cancer and is a pre-

ferred treatment for many cutaneous malignancies, particularly in high risk locations and tumors [1,2]. It has also been

used in the narrow excision of malignant melanoma with local control rates equivalent to standard margins [3]. It has

gained acceptance in the treatment of noninvasive melanoma where standard 0.5 cm margins may be inadequate for

local control [4]. The frozen section processing used in MMS has been assumed by some to be inadequate in assessing

melanocyte populations or residual melanoma within excision margins. This difficulty has likely led to a majority of

surgeons with fellowship training to process margins with slow, permanent hematoxylin and eosin sections (“slow-

mohs”) or to simply resort to standard 0.5, 1.0, or 2.0 cm margins with traditional excision and outside pathology con-

firmation of clear margins. A recent survey of practicing fellowship-trained Mohs surgeons revealed roughly one-third

(35.9%) of Mohs surgeons felt comfortable interpreting MART-1 immunostains, and far fewer were actually perform-

ing immunostains in their labs [5]. Some Mohs surgeons currently refer melanoma to a colleague experienced in proc-

essing and reading melanoma with available rapid immunostaining. The development of rapid immunohistochemistry,

which can be implemented into a traditional frozen section laboratory, has greatly improved the ease of interpreting

margins in the excision of melanoma. Although the process is considerably more complicated than staining with H&E

or Toluidine Blue (T-Blue), it easily falls within the skill-set and equipment of most busy frozen section laboratories.

The additional cost of biologic reagents may be fully recovered by proper billing of immunohistochemical laboratory

work and interpretation of slides.

Keywords: Moh’s Surgery; Malignant Melanoma; Rapid MART-1

1. Introduction

Multiple immunohistochemical staining protocols for the

melanoma antigen recognized by T-cells (MART-1) us-

ing frozen sections have been developed and presented

over the last decade. Historically, melanoma was proc-

essed with en-face frozen H&E staining, or with perma-

nent paraffin sections requiring overnight processing and

multiple days between each Mohs layer. In 2004, Bricca

[6] and associates presented a reproducible 1-hour MART-

1 protocol for melanoma frozen sections. Later, Asadi [7]

and colleagues reported a modified 20-minute protocol.

Currently, the American College of Mohs Surgery has a

suggested modified protocol available to its members

online [8]. The third protocol resembles a combination of

the Bricca and Asadi protocols.

While all three methods produce reliable staining, each

has its respective strengths and weaknesses. The 1-hour

protocol gives consistent dependable results, however the

lab processing time is dramatically longer than that of a

routine section stained with H&E. The 20-minute rapid

protocol requires considerably less time, but demands the

lab technician’s full attention, excluding other casework.

The clarity of the slides produced using this rapid proto-

col also may contain excessive chromogen “chatter” where

much of the melanocytic detail may be lost. The weak-

ness inherent in the Mohs College protocol is that it is

only available to its members, and remains unpublished

in the public domain.

We present an experimental 35-minute MART-1

staining protocol for melanoma frozen sections that

The Emergence of Rapid Counter Immunostaining in the Controlled Narrow

Excision of Malignant Melanoma—How We Do It

1083

combines the best of the previously presented protocols,

while eliminating many of the aforementioned obstacles

(Tables 1 and 2). In addition, we propose the use of

Table 1. Happy medium protocol.

1. Cut thin (2 - 4 µm) sections-minimum two

copies of each piece

2. Mount on a positively charged slide

facilitates better adhesion

3. Air dry-2 minutes—room temperature

4. Heat on a 60˚C hot plate (or equivalent)—3

minutes

5. Fix in Acetone(in a Coplin jar) —2 minutes

6. Air dry-2 minutes—room temperature

Mounting and

Preparation

7. Rehydrate in Tris-Buffered Saline (TBS)

(in a Coplin Jar)—2.5 minutes

1. Add protein blocking agent—3 minutes

2. Shake off, do not rinse

3. Apply “Ready to Use” MART-1 antibody

—

6 minutes

4. Rinse in Tris Buffered Saline (TBS)—2

minutes

5. Apply Polymer HRP (horse-radish

peroxidase)—6 minutes

6. Rinse in TBS–2 minutes

(during this processing-mix up DAB

Chromogen for step 7)

7. Apply pre-mixed

(1 drop substrate/1 drop solution/1 ml distilled

water) DAB Chromogen—2 minutes

Staining*

8. Rinse in distilled water for 1.5 - 2 minutes

(shorter time = darker chromogen)

1. Dip in T-Blue—10 seconds

2. Rinse with running water-30 - 45 seconds

3. Dip in 95% reagent alcohol-15 seconds

4. Dip in 3 changes of 100% reagent

alcohol—15 seconds each

Counterstain

(with Toulidine Blue)+

5. Dip in 3 changes containing a clearing

agent—15 seconds each

1. Dip in Hematoxylin—2 seconds

2. Rinse with running water-30 - 45 secondsǂ

3. Dip in 95% reagent alcohol—20 seconds

4. Dip in 3 changes of 100% reagent

alcohol—20 seconds each

Counterstain

(with Hematoxylin)

5. Dip in 2 - 3 changes of a clearing agent—20

seconds each

*Steps in this section take place in a humidity chamber-to achieve humidifi-

cation by pouring 90˚C - 100˚C (boiling) water in the chamber beneath the

slides and shutting the lid. +We perform the counterstain in a linear auto-

mated stainer—can be hand dipped using the same time increments. ǂIf you

require bluing in the H & E protocol, it will need to be added here.

T-Blue as a reasonable alternative to the standard use of

Hematoxylin, as a counterstain, although Hematoxylin

may certainly be used. We have also substituted T-Blue

in all of the published protocols with reproducible stain-

ing compatible with the suggested reagents. Lastly, we

suggest the implementation of an automated stainer dur-

ing the counterstaining process for consistent, reproduci-

ble, easily readable slides. As an added benefit, the use of

the automated stainer unburdens the histo-technician

freeing him/her for additional lab work.

We have also found inexpensive ways to implement

this protocol. In addition to readily available supplies, we

were able to utilize commonly found items in lieu of

more costly lab equipment. Our 60˚C hot plate is a toast-

er oven in combination with a certified thermometer. A

small percolator of boiling water replaced the need for an

additional 100˚C hot plate that is used in the humidifica-

tion process. Likewise, we eliminated the problematic

issue of obtaining negative control tissue, that can be

costly and difficult to store, by utilizing non-melanoma

tissue from cases being treated at the same time as the

melanoma(s). Positive/negative controls are subjected to

the same protocol as the melanoma tissue, replacing the

MART-1 antibody with distilled water for the negative

control. By setting this as our standard operating proce-

dure, we were able to satisfy CLIA requirements for pos-

itive/negative controls.

Lastly, we introduced a linear automated stainer to

perform the counterstain. With our stainer set with T-

Blue, it delivers a pleasing alternative to the traditional

Hematoxylin counterstain. This simple step provides

more opportunity for the histotechnician to continue pro-

cessing other tissue while not sacrificing reproducible

slide quality.

2. Conclusion

Most practices currently performing standard Mohs

processing are well equipped to add rapid MART-1 im-

munostaining regardless of training or experience. Addi-

tional publications have elucidated simple interpretation

of normal melanocytes, atypical melanocytic prolifera-

tions, or malignant melanoma and other cells which will

stain positive with MART-1 [9,10]. Processing mela-

noma with MART-1 staining, due to the complexity and

intensive hands-on nature of the staining process, can

initially be a rate-limiting event, but may quickly become

routine, reproducible, and valuable in the treatment of

melanoma. As most practices have a single histotech,

multi-tasking is essential to the efficiency and flow of the

lab. Finding efficient, reproducible practices are central

to the continued adeptness of the Mohs laboratory. Our

experience with this protocol has produced well-defined,

consistent readable slides in which the melanocytic detail

is markedly distinct (Figure 1). Counterstaining with

The Emergence of Rapid Counter Immunostaining in the Controlled Narrow

Excision of Malignant Melanoma—How We Do It

1084

[2] Ad Hoc Task Force, et al., “AAD/ACMS/ASDSA/ASMS

2012 Appropriate Use Criteria for Mohs Micrographic

Surgery: A Report of the American Academy of Derma-

tology, American College of Mohs Surgery, American

Society for Dermatologic Surgery Association, and the

American Society for Mohs Surgery,” Dermatologic Sur-

gery, Vol. 38, No. 10, 2012, pp. 1582-1603.

doi:10.1111/j.1524-4725.2012.02574.x

[3] J. A. Zitelli, C. Brown and B. H. Hanusa, “Mohs Micro-

graphic Surgery for the Treatment of Primary Cutaneous

Melanoma,” Journal of the American Academy of Der-

matology, Vol. 37, No. 2, 1997, pp. 236-245.

doi:10.1016/S0190-9622(97)80131-4

Figure 1. Example of frozen sections stained using our

Happy Medium Protoc ol with a T-Blue counterstain. [4] J. H. Kunishige, D. B. Brodland and J. A. Zitelli, “Surgi-

cal Margins for Malanoma in Situ,” Journal of the Amer i-

can Academy of Dermatology, Vol. 66, No. 3, 2012, pp.

438-444. doi:10.1016/j.jaad.2011.06.019

Table 2. Product resource list**.

1. Humidity trays/staining chamber—Evergreen Scientific

www.evergreensci.com

2. Tris Buffered Saline-Dako—www.dakocytomation.com

3. Polymer based detection system—Leica Microsystems fax:

847-236-3009

4. MART-1 primary antibody, Coplin staining

jars—ThermoFisher—800-828-1628

5. Positively charged slides, TBS mounting media, Toulidine Blue,

Histoclear Clearing Agent, Reagent grade

alcohol—Avantik—www.avantik-us.com

[5] J. S. Trimble and B. S. Cherpelis, “Rapid Immunostaining

in Mohs: Current Applications and Attitudes,” Derma-

tologic Surgery, Vol. 39, No. 1, 2013, pp. 56-63.

doi:10.1111/dsu.12015

[6] G. M. Bricca, D. G. Brodland and J. A. Zitelli, “Immu-

nostaining Melanoma for Frozen Sections: The 1-Hour

Protocol,” Dermatologic Surgery, Vol. 30, No. 3, 2004,

pp. 403-408. doi:10.1111/j.1524-4725.2004.30110.x

[7] A. K. Asadi, G. B. Ayala, L. H. Goldberg, J. Vujevich

and M. H. Jih, “The 20-Minute Rapid MART-1 Immu-

nostain for Malignant Melanoma Frozen Sections,” Der-

matologic Surgery, Vol. 34, No. 4, 2008, pp. 498-500.

doi:10.1111/j.1524-4725.2007.34095.x

**We receive no commercial support from the above suppliers.

MART-1 has become a valuable tool in the controlled

excision of malignant melanoma. [8] “MART 1 Staining Protocol for Melanoma,” 2013.

http://www.mohscollege.org/members/resources.php

[9] A. Hendi, D. G. Brodland and J. A. Zitelli, “Melanocytes

in Long-Standing Sun-Exposed Skin,” JAMA Dermatol-

ogy, Vol. 142, 2006, pp. 871-876.

doi:10.1001/archderm.142.7.871

REFERENCES

[1] Ad Hoc Task Force, et al., “AAD/ACMS/ASDSA/ASMS

2012 Appropriate Use Criteria for Mohs Micrographic

Surgery: A Report of the American Academy of Derma-

tology, American College of Mohs Surgery, American

Society for Dermatologic Surgery Association, and the

American Society for Mohs Surgery,” Journal of the Ame-

rican Academy of Dermatology, Vol. 67, No. 4, 2012, pp.

531-550. doi:10.1016/j.jaad.2012.06.009

[10] A. Hendi, D. A. Wada, M. A. Jacobs, J. E. Crook, K. R.

Kortuem, B. R. Weed, C. C. Otley and L. E. Gibson, “Me-

lanocytes in Nonlesional Sun-Exposed Skin: A Multicen-

ter Comparative Study,” Journal of the American Acad-

emy of Dermatology, Vol. 65, 2011, pp. 1186-1193.

doi:10.1016/j.jaad.2010.10.039