slide to be stained for
Haemangiosarcoma, 3. Small intestinal
lymphangioma, 4. Liver.
Criteria for assessing a
staining as optimal included: A strong and distinct predominantly
membranous but also cytoplasmic staining of normal vascular
endothelial cells (including hepatic sinusoidal endothelial cells
and lymphatic vessels), the haemangiosarcoma and the small
intestinal lymphangioma, and a weaker staining of activated B- and
59 laboratories submitted stainings. At the
assessment 22 achieved optimal staining (37 %), 17 good (29 %), 12
borderline (20 %) and 8 (14%) poor staining.
The following Abs were used:
mAb clone JC70A (DakoCytomation, n=56; Cell Margue, n=1)
mAb clone 1A10 (Novocastra, n=1; Ventana, n=1)
In this assessment optimal stainings could
only be obtained with the mAb clone JC70A.
In the optimal protocols all used HIER, the majority with Tris-EDTA/EGTA
pH 9 as the heating buffer (16 out of 27 were optimal), the
remainder Citrate pH 6 (1 out of 9 was optimal) or Target Retrieval
Solution (DakoCytomation; 3 out of 5 were optimal). None of
the 13 laboratories using proteolytic pretreatment obtained an
optimal staining, and only one was assessed as good.
mAb JC70A was used in the range of 1:20 – 150 depending on the total
sensitivity of the used protocol.
The majority of laboratories with
insufficient staining were able to
detect CD31 in the endothelial cells of the large vessels in all
materials of the multi tissue-block while CD31 in
the hemangiosarcoma and the lymphangioma could not be
revealed or were only focally demonstrated. In all
optimal stainings the hepatic sinusoidal endothelial cells and the activated
lymphocytes were demonstrated, while these cells were very weakly
stained or negative in the insufficient stainings.
The most frequent causes of insufficient
stainings were (often in combination):
- Inappropriate choice of epitope retrieval: Proteolytic
- Insufficient epitope retrieval (typically citrate pH 6 as the
heating buffer combined with a low sensitivity of the
- Inappropriate choice of primary Ab
- Too low concentration of the primary Ab