slide to be stained for
1. Lymph node with Hodgkin’s lymphoma, mixed cellularity (MC), 2. Lymph node with Hodgkin’s lymphoma, nodular
sclerosing (NS), 3. Spleen with acute myeloid leukaemia (AML), 4.
Lung adenocarcinoma, 6. Tonsil.
Criteria for assessing a
CD15 staining as optimal included:
A strong and distinct
predominantly membranous staining and reaction of the Golgi
apparatus of the Reed-Sternberg and Hodgkin’s cells in both cases of
Hodgkin’s lymphoma, a membranous and cytoplasmic staining of the
neoplastic cells of the AML, and a focal cytoplasmic reaction of the
neoplastic cells of the lung carcinoma, whereas the malignant mesothelioma should be
dot-like nuclear staining in neutrophils and the adenocarcinoma was accepted
(see Fig. 3).
71 laboratories submitted
stainings. At the assessment 23 achieved optimal staining (32 %), 13
good (18 %), 11 borderline (16 %) and 24 poor staining (34%).
The following mAbs were
clone C3D-1 (DakoCytomation,
clone MMA (Becton
Dickinson, n=16; Cell Margue, n=1; NeoMarkers, n=1)
clone BY87 (Ventana, n=3;
clone H198 (Becton
clone Tu9 (Quartett
In this assessment optimal
stainings could be obtained with the mAbs MMA (15/18), C3D-1 (7/45)
and Tu9 (1/1). In the optimal protocols all used HIER, predominantly
(22/23) with Tris-EDTA/EGTA pH 9 as the heating buffer. MMA was used
in the range of 1:10 – 1:40 and C3D-1 in 1:10 – 1:25, depending on
the total sensitivity of the used protocol. Tu9 was used in 1:3.
The majority of
laboratories were able to detect CD15 in the neoplastic cells of the
AML, whereas the demonstration of CD15 in the two Hodgkin’s diseases
only was achieved with an optimal protocol.
The most frequent
causes of insufficient stainings (often in combination) were:
- Inappropriate choice of primary Ab
epitope retrieval or proteolytic pretreatment
HIER: Too short heating time often in combination with citrate pH 6
as the heating buffer
- Too low
concentration of the primary antibody.