slide to be stained for
Liver, 3. Juvenile xanthogranuloma, 4.
Myeloid leukaemia FAB M4,
Criteria for assessing a
staining as optimal included: A strong and distinct cytoplasmic granular staining of macrophages
in the appendiceal germinal centres, hepatic Kupffer cells and
macrophages in, e.g., the meningioma, the juvenile xanthogranuloma
and the myeloid leukaemia. The neoplastic cells of the menigioma
should be negative. A weak reactivity in epithelial cells was
accepted with the clone KP1.
64 laboratories submitted stainings. At the
assessment 25 achieved optimal staining (39 %), 22 good (34 %), 7
borderline (11 %) and 10 (16 %) poor staining.
The following Abs were used:
mAb clone KP1 (DakoCytomation, n=36; NeoMarkers, n=1; Ventana, n=1;
mAb clone PG-M1 (DakoCytomation, n=23)
mAb clone 54H12 (Novocastra, n=1)
In this assessment optimal stainings could be
obtained with the mAb clones KP1 and PG-M1.
11 out of 39 laboratories (28 %) using the clone KP1 and 14 out of
23 (61 %) using the clone PG-M1 achieved an optimal staining.
In the optimal protocols all used HIER, the majority with Tris-EDTA/EGTA
pH 9 as the heating buffer (22 out of 46 were optimal), but also Citrate
pH 6 (2 out of 9 were optimal) and Target Retrieval Solution (1
out of 3) (DakoCytomation)
could be used.
None of the 6 laboratories using proteolytic pretreatment (KP1: n=4;
PG-M1: n=2) obtained an optimal staining.
KP1 was used in the range of 1:2.000 – 15.000 using Tris-EDTA/EGTA
pH 9 (depending on the total sensitivity of the protocols
1:200 using Citrate pH 6 as the heating buffer.
PG-M1 was used in the range of 1:100-1:500 with both heating
The majority of laboratories were able to
detect CD68 in the myeloid leukaemia, whereas CD68 in the juvenile xanthogranuloma were only demonstrated in sensitive protocols. The
staining patterns for KP1 and PG-M1 were almost identical. However,
PG-M1 typically provided a more distinct staining reaction and a
better signal-to-noise ratio. The multi-tissue block only comprised
a myeloid leukaemia M4. Thus, any difference as regards the
reactivity with KP1 and PG-M1 in other subtypes of myeloid
leukaemias could not be evaluated.
Using KP1 the appendiceal enterocytes are faintly stained. However,
in many insufficient protocols a too high concentration of KP1 was
used giving a strong unspecific background reaction in enterocytes
as well as various other cell types.
The most frequent causes of insufficient
- Inappropriate choice of epitope retrieval (i.e., proteolytic
- Too low concentration of the primary antibody
- Too high concentration of the primary antibody (KP1).
Controls: Liver and appendix are
appropriate controls. In the liver the hepatic Kupffer cells must
show an as strong as possible positive staining while the liver
cells must be negative. In the appendix the germinal centre
histiocytes must show an as strong as possible positive staining
while the enterocytes must be negative or show not more than a weak