The
slide to be stained for CD23 comprised: 1: tonsil, 2: marginal zone
lymphoma, 3: B-CLL, 4: follicular cell lymphoma, 5: mantle cell
lymphoma.
Criteria for assessing a CD23 staining as optimal included: A
moderate to strong, distinct membranous staining of normal,
activated
B-cells in the mantle zone of the tonsil, a strong staining of the
follicular dendritic reticulum cells in the germinal centres and a
distinct staining of the neoplastic cells of the
B-CLL. The
neoplastic cells
of
the mantle cell lymphoma should be negative.
59
laboratories submitted stainings. At the assessment 35 achieved
optimal staining (59 %), 10 good (17 %), 10 borderline (17 %) and 4
poor staining (7%).
46
laboratories used mAb 1B12
(Novocastra,
n=39, Ventana,
n=3, NeoMarkers,
n=3,
or
Maixin Bio,
n=1).
8 used mAb MHM6
(DakoCytomation), 5 used mAb
BU38
(Binding Site,
n=4 or
Ancell,
n=1).
In
this assessment optimal staining could be obtained
only
with
mAb
1B12 (33/42) and MHM6 (2/8).
Almost all laboratories were able to detect CD23 in the follicular
dendritic reticulum cells, but the
staining
of CD23 in normal
mantle zone
B-cells and the neoplastic cells of the
B-CLL could only be obtained
in optimized
protocols.
All
laboratories achieving an optimal staining used HIER, most
frequently (31/34) with Tris-EDTA/EGTA pH 9 as the heating buffer.
MWO, pressure cooker and water
bath could be used as the heating
device.
The
optimal dilution of mAb 1B12 was in the range of 1:20 – 200,
that of
mAb MHM6 was 1:50.
The
most frequent causes of insufficient stainings were:
-
Insufficient HIER: too short efficient heating time (MWO< 15 min.,
water bath <40 min.)
-
Usage of proteolytic pre-treatment
- Too
dilute concentration of the primary antibody
-
Inappropriate choice of mAb.
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