At present histomorphologic examination with immunohistochemistry remains the most reliable and practical means of detecting melanoma micrometastases in sentinel lymph nodes [6, 21]. MART-1 and Melan-A are two antibodies to different epitopes of the same antigen and are complementary to each other. As previously reported, MART-1 and Melan-A demonstrated predominantly cytoplasmic staining of melanoma cells . The monoclonal antibody (T311) to tyrosinase, has been reported to demonstrate high sensitivity and specificity for melanoma . It has also demonstrated an immunostaining pattern in sentinel lymph nodes identical to MART-1 and Melan-A. We combined these three antibodies as 'MCW melanoma cocktail' (Table 1).
In contrast to S-100, these immunomarkers did not obscure nuclear details. In addition, they did not stain the dendritic cells which are generally prominent in the sentinel lymph nodes after the diagnostic biopsy of a primary lesion. This facilitated unequivocal interpretation of singly scattered melanoma cells which were otherwise impossible to identify in sections immunostained for S-100 due to high noise to signal ratio resulting from numerous S-100 immunoreactive dendritic cells in the background. Thus, a highly sensitive immunomarker such as S-100 is less effective for interpretation of sentinel lymph nodes for melanoma micrometastases (especially when present as single cells) than MART-1 and Melan-A . Certain morphological features help to differentiate melanoma cells and other non-melanoma cells such as S100 immunoreactive dendritic cells in sentinel lymph nodes. However, as demonstrated in our previous study, it is not uncommon to face an equivocal situation when S-100 protein is used as an immunomarker for the interpretation of single melanoma micrometastases in sentinel lymph nodes . Based on our previous study with HMB45  and an initial study with micropthalmia transcription factor , these immunomarkers were not considered suitable for inclusion in the cocktail due to the immunoreactivity of non-melanoma cells in the sentinel lymph nodes.
During further evaluation, we confirmed that the single cells immunostained with HMB45 were mast cells and not melanophages. Although not reported in the literature, mast cells in mastocytosis and reactive mast cells in desmoid tumors also showed immunoreactivity for HMB45 with granular cytoplasmic immunostaining pattern. The same cells in the immediately adjacent section were confirmed as mast cell by CD117 (personal experience). Lack of similar staining in the negative control sections excludes their non-specific nature. Mast cells are relatively frequent in sentinel lymph nodes, likely secondary to the previous biopsy in the draining field (personal experience). Occasionaly these cells may present an interpretation challenge, but they can usually be interpreted as benign . To avoid this interference by HMB45 and its lower sensitivity in general, HMB45 was not incorporated in the cocktail.
Out of 188 sections immunostained with the cocktail and interpreted by all five pathologists collectively, 41 were positive for melanoma micrometastases (Figure 2A and 2B). Az values amongst the five pathologists interpreting these sections independently did not show statistically significant differences, demonstrating excellent interobserver agreement with the cocktail (Table 2). None of the 81 sections of mammary carcinoma sentinel lymph nodes showed immunoreactivity for the cocktail in any of the non-melanoma cells (Figure 2C) and were interpreted as negative for melanoma micrometastases by all five observers.
The cocktail showed favorable detection rate for melanoma micrometastases as compared to MART-1 and Melan-A alone. Of 78 sections, 25 sections were positive for melanoma micrometastases with the cocktail. However, only 21 were positive with MART-1 and Melan-A individually. Thus, MART-1 and Melan-A could not individually detect melanoma micrometastases in 16% (4/25) of slices that were positive for melanoma micrometastases with the cocktail. As expected, combining a monoclonal antibody to tyrosinase with MART-1 and Melan-A in the cocktail increased the spectrum to encompass three epitopes of two melanoma antigens (MART-1/Melan-A and tyrosinase). If indicated in the future, additional monoclonal antibodies showing immunostaining patterns in sentinel lymph nodes that are similar to these three antibodies which also posess high specificity for melanoma may be added to this cocktail, by widening the spectrum for many melanoma specific antigen epitopes. However, care should be taken to not include any antibody that may immunostain other non-melanoma cells in the sentinel lymph nodes. Otherwise, such antibodies may contribute to the deterioration in the specificity of the cocktail compromising its application for evaluation of the sentinel lymph nodes of melanoma.
During the standardization, all 35 melanomas (including one spindle cell melanoma and one clear cell melanoma) were immunoreactive with 'the cocktail'. Non-immunoreactivity of special variants of melanoma to various melanoma immunomarkers is a challenge. During interpretation of sentinel lymph nodes, the diagnosis of melanoma is known and the aim is to detect the melanoma micrometastases of the known primary. If the sentinel lymph nodes of special variants of melanoma are negative by 'the cocktail', it would be prudent to confirm the immunoreactivity of the primary lesion for 'the cocktail'. If the primary melanoma is non-immunoreactive for 'the cocktail', the negative interpretation of the sentinel lymph nodes with cocktail in this situation may be confirmed with other suitable immunomarker to which the primary melanoma is immunoreactive. The cocktail was not compared with S-100 in this study, as its role in evaluation of sentinel lymph nodes of melanoma was already demonstrated to be unreliable even in comparison to MART-1 and Melan-A individually in the previous study . We did not compare tyrosinase alone with the cocktail because the questionable contribution of such a study would not be offset by the additional cost involved. Published studies do not report 100% immunostaining with anti-tyrosinase antibody (T311). Tyrosinase alone or any other immunomarker used individually, would be less sensitive than a cocktail. To evaluate the exact impact of the cocktail in comparison to all of its component immunomarkers individually for evaluation of sentinel lymph nodes, additional studies dedicated to comparison of individual component immunomarkers with the cocktail may be called for.
Capsular nevi in the form of a few cohesive cocktail immunoreactive spindle cells with bland elongated nuclei (Figure 3) were observed in 5% (9/188) of sections (10 % (9/88) of sentinel lymph nodes and 21% (5/24) of cases. As compared to this, none of the 81 slices of sentinel lymph nodes from 30 cases of mammary carcinoma showed capsular melanocytic nevi. The sentinel lymph nodes of melanocytic nevi were from different sites and none were from the axillary region. The exact explanation for this difference in the prevalence of capsular nevi could not be deciphered and further evaluation is in process. The association of higher frequency of capsular melanocytic nevi with congenital nevi  is one possibility. In our study, except for the difference in their regional variation, another possible explanation is the cytokine effect of the melanoma draining to the sentinel lymph node. Hypothetically, an association between the higher prevalence of melanocytic capsular nevi and the predisposition for melanoma may be likely. Although, these melanocytic capsular nevi appear benign morphologically, a low probability that they may represent subtle metastases was also considered. Further evaluation of these and other possibilities in larger series is recommended.
Using immunohistochemistry, Yu et al  reported capsular benign melanocytic nevi in 4% (9/235) of sentinel lymph nodes of melanoma from 9% (8/94) cases (some cases had more than one sentinel lymph node). Capsular melanocytic nevi appear to be the most significant benign lesion that could pose a diagnostic challenge in the interpretation of melanoma micrometastases in sentinel lymph nodes. For similar reasons, non-in-situ molecular techniques, such as RT-PCR [6, 8, 24–29] would have higher tendencies for false positive results. Morphology appears to be the single most important tool facilitating their correct interpretation . Other approaches, including the observation that capsular nevi are non-immunoreactive or weakly immunoreactive for HMB45, have been suggested for differentiating capsular nevi from melanoma micrometastases . However, it is important to note that a significant number of melanomas are also non-immunoreactive for HMB45. Thus, strong immunoreactivity for HMB45 rules out capsular nevus, but non-immunoreactivity may not be contributory.
Rather than immunostaining three adjacent sections with individual antibodies in the cocktail, only one slide is required which leads to cost savings. Although, the cost of the three antibodies used in the cocktail has to be incurred, the material and labor cost savings for one instead of three slides is obvious. For proper sampling, some studies recommend the evaluation of three levels at 200 μ intervals . In that case, if each of the three antibodies in the cocktail had to be used individually, it would require the immunostaining, interpreting, and correlating of nine sections. In contrast to this, the cocktail requires only three sections, each at intervals of 200 μ. Thus, use of the cocktail in concert with any protocol with reference to number of sections to be evaluated and proportion of sentinel lymph node to be sampled [30, 31] would achieve obvious cost saving.
The most ideal positive control would be the sausage of three controls immunoreactive individually for one of the antibodies but non-immunoreactive for other two respective antibodies in the cocktail. However, this is a difficult positive control to get. The positive control used by us showed subtle differences when immunostained with each individual antibody in the cocktail with some areas slightly darker than others. However, when the same control was immunostained with the cocktail, the entire control showed relatively homogeneous immunostaining. The change in this immunostaining pattern would suggest the deterioration of one of the component which can be correlated with the same control immunostained with the individual antibodies. In addition, another precaution taken by us was that the cocktail was prepared after periodically evaluating individual antibodies with the positive control. This later approach is the best alternative.