slide to be stained for
alpha-smooth muscle actin
1. Uterine leiomyosarcoma, 2.
Breast fibrocystic disease, 3. Gastric gastro
intestinal stromal tumour (GIST), 4. Small intestinal GIST, 5.
Criteria for assessing an ASMA
staining as optimal included:
A strong and distinct cytoplasmic reaction of
the appendiceal smooth muscle cells (vessels and muscular layers)
and myofibroblasts, the myoepithelial cells of the glands and ducts of the breast fibrocytic disease and the uterine
leiomyosarcoma, and a focal cytoplasmic reaction of the two GISTs.
71 laboratories submitted stainings. At the
assessment 30 achieved optimal staining (42 %), 14 good (20 %), 19
borderline (27 %) and 8 poor staining (11 %).
The following appropriate mAbs were used:
clone 1A4 (DakoCytomation, n=55; Sigma, n=4; BioGenex, n=1; BioMakor,
n=1; NeoMarkers, n=1)
clone asm1 (Novocastra, n=1; Ventana, n=1)
Seven laboratories used an inappropriate mAb, clone HHF35
(Enzo, n=5; DakoCytomation, n=2), which is a pan-actin Ab.
In this assessment optimal stainings could
only be obtained with the mAb clone 1A4. In the optimal protocols
all used HIER, 29 with Tris-EDTA/EGTA pH 9 as the heating buffer and
one with Target Retrieval Solution pH 6. 1A4 was typically used in a
dilution of 1:100–600 (depending on the total sensitivity of the
used protocol). Twelve laboratories used the mAb clone 1A4
without pre-treatment and two used proteolytic pre-treatment. None
of these protocols resulted in optimal stainings, twelve
were assessed as insufficient.
Besides being less specific for ASMA, clone HHF35 revealed a
rather poor sensitivity for this actin (no staining was optimal, one
was good, the others insufficient).
The majority of laboratories were able to
detect ASMA in the appendiceal muscular layers and breast
myoepithelial cells. In the insufficient stainings (primarily
performed without HIER) the two GIST’s were typically negative and
the leiomyosarcoma only weakly labelled.
In the periphery of normal germinal centres and
lining the surface
epithelial cells of the appendix tiny
myofibroblasts were identified in the
optimal stainings only, indicating that these cells might serve as
an appropriate positive control for detection of ASMA.
The most frequent causes of insufficient
- Inappropriate choice of primary Ab
- Inappropriate epitope retrieval
- Insufficient HIER, i.e. too short heating time
- Too low concentration of the primary antibody.