The slide to be stained for Melan A (MLA) comprised:
1. Angiomyolipoma, 2. Malignant melanoma, 3, Granulosa cell tumour, 4, Adrenal gland, 5. Kidney.
All tissues were fixed in 10% neutral buffered formalin.

Criteria for assessing a MLA staining as optimal included:

• A strong, distinct granular cytoplasmic staining in virtually all the adrenal cortical cells (clone A103).
• A strong, distinct cytoplasmic staining of the majority of the neoplastic cells of the malignant melanoma.
• A moderate to strong, distinct cytoplasmic staining of the neoplastic cells of the angiomyolipoma and the luteinized cells in the granulosa cell tumour.
• No or only minimal staining of the kidney.

115 laboratories submitted stains. At the assessment 24 achieved optimal marks (21%), 34 good (29%), 38 borderline (33 %) and 19 poor marks (17 %).

The following Abs were used:
mAb clone A103 (Dako, n=78; Ventana, n=11; Novocastra/Leica, n=9; NeoMarkers/Thermo, n=5; Monosan, n=1)
mAb clones A103 + M2-7C10 + M2-9E3 (Zymed, n=2)
mAb clones M2-7C10 + M2-9E3 (NeoMarkers/Thermo, n=2; BioCare, n=1)
mAb clones M2-7C10 + M2-9E3 + T311 (BioCare, n=1)
mAb clones HMB45 + M2-7C10 + M2-9E3 + T311 (BioCare, n=3; Zytomed, n=1)
pAb RB-9054 (Neomarkers/Thermo, n=1)

Optimal staining for MLA in this assessment was only obtained with the mAb A103 (24 out of 104).

All optimal protocols were based on heat induced epitope retrieval (HIER) using Tris-EDTA/EGTA pH 9.0 (17/42)*, Target Retrieval Buffer pH 9, (Dako) (4/16), Bond Epitope Retrieval Solution 2 (Bond, Leica) (2/3) or Citrate pH 6.0 (1/12) as HIER buffer. The mAb was diluted in the range of 1:10 – 1:100 depending on the total sensitivity of the protocol employed or as a Ready-To-Use Ab (Dako IR633).
Using these protocol settings 44 out of 58 (76 %) laboratories produced a sufficient staining (optimal or good).
* (number of optimal results/number of laboratories using this buffer)

The most frequent causes of insufficient staining were:
- Too low concentration of the primary antibody
- Insufficient HIER (too short HIER time and/or a non-alkaline buffer)
- Less successful ready-to-use (RTU) mAb clone A103
- False negative staining combined with a false positive staining of endogenous biotin

In this assessment the prevalent feature of an insufficient staining was a false negative reaction of the granulosa cell tumour and the angiomyolipoma. In general, almost all laboratories were able to detect MLA in the malignant melanoma. This observation was in concordance with previous assessment of MLA and stresses that a highly sensitive IHC system is mandatory to demonstrate MLA in tumours with low MLA expression as granulosa cell tumours, desmoplastic melanoma etc.
An applicable critical staining quality indicator using the clone A103 was the ability to demonstrate a strong granular cytoplasmic reaction in virtually all the epithelial cells throughout the adrenal cortex. However this reaction pattern can only be applied when a non-biotin based detection system is used, as the adrenal cortical cells are rich on endogenous biotin and a false positive reaction will thus mimic the specific reaction and eliminate the potential as a reliable control.

In the last 3 assessments it has been shown that the most successful clone for MLA is A103 and the protocol settings and Ab. format are important parameters to obtain a sufficient result. From the 3 assessments virtually only an alkaline buffer as Tris-EDTA/EGTA pH 9 or similar and a relative high conc. of the primary Ab. ranging from 1:10 – 100 could be used to obtain a sufficient result, while protocols based on other HIER buffers and a reduced concentration of the Ab. typically gave an insufficient result. Also the RTU formats of the clone A103 have shown a variable performance, which seems to be related to the sensitivity of the total RTU system and the calibration of this.

Table 1 shows the cumulated data of the performance and protocol settings of the clone A103 in the latest three MLA assessments.

Table 1. Cumulated data for clone A103 in three runs

*HIER in an alkaline buffer such as Tris-EDTA/EGTA pH 9 or equivalent and an appropriate dilution of the antibody listed in the three assessments.

This was the 4th assessment of MLA and as shown in table 2 the pass rate and proportion of sufficient stainings has been slightly improved in the last 3 runs, but is still on a low level.

Table 2. Pass rate in four runs

In the previous assessment of MLA (run 20), 90 laboratories participated out of which 47 (52%) obtained an insufficient mark. Each was given a specific recommendation to improve their protocol. 40 of them submitted a new MLA stain in run 24. 18 followed the recommendation, of which 10 improved to good or optimal (56 %). 17 laboratories did not follow the recommendation, and 1 of these (6 %) obtained a sufficient staining in run 24. 5 laboratories changed their entire IHC system and 3 of these obtained a sufficient staining.

In total 112 laboratories obtaining an insufficient result in run 7, 16 and run 20 have been given specific recommendations how to improve their protocol. 89 laboratories of them submitted a new MLA stain in the subsequent run. 44 followed the recommendation, of which 28 improved to good or optimal (64 %). 40 laboratories did not follow the recommendation, and 3 of these (9 %) obtained a sufficient staining in the subsequent run.

Conclusion
The mAb clone A103 seems be to the most sensitive and robust marker for MLA. HIER in an alkaline buffer such as Tris-EDTA/EGTA pH 9 is highly recommended for optimal performance. Normal adrenal gland is an appropriate control: Virtually all the cortical epithelial cells must show a strong distinct granular staining. Biotin based detection systems can not be recommended for MLA due to the risk of false positive reaction due to endogenous biotin.