Leica BIOSYSTEMS Novocastra Mode D'emploi page 4

Quality Control
Differences in tissue processing and technical procedures in the user's laboratory may produce significant variability in results,
necessitating regular performance of in-house controls in addition to the following procedures.
Controls should be fresh autopsy/biopsy/surgical specimens, formalin-fixed, processed and paraffin wax-embedded as soon as possible
in the same manner as the patient sample(s).
Positive Tissue Control
Used to indicate correctly prepared tissues and proper staining techniques.
One positive tissue control should be included for each set of test conditions in each staining run.
A tissue with weak positive staining is more suitable than a tissue with strong positive staining for optimal quality control and to detect
minor levels of reagent degradation.
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Recommended positive control tissue is tonsil
If the positive tissue control fails to demonstrate positive staining, results with the test specimens should be considered invalid.
Negative Tissue Control
Should be examined after the positive tissue control to verify the specificity of the labeling of the target antigen by the primary antibody.
Recommended negative control tissue is cerebellum.
Alternatively, the variety of different cell types present in most tissue sections frequently offers negative control sites, but this should be
verified by the user.
Non-specific staining, if present, usually has a diffuse appearance. Sporadic staining of connective tissue may also be observed in
sections from excessively formalin-fixed tissues. Use intact cells for interpretation of staining results. Necrotic or degenerated cells often
stain non-specifically. False-positive results may be seen due to non-immunological binding of proteins or substrate reaction products.
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They may also be caused by endogenous enzymes such as pseudoperoxidase (erythrocytes), endogenous peroxidase
(cytochrome C), or endogenous biotin (eg. liver, breast, brain, kidney) depending on the type of immunostain used. To differentiate
endogenous enzyme activity or non-specific binding of enzymes from specific immunoreactivity, additional patient tissues may be stained
exclusively with substrate chromogen or enzyme complexes (avidin-biotin, streptavidin, labeled polymer) and substrate-chromogen,
respectively. If specific staining occurs in the negative tissue control, results with the patient specimens should be considered invalid.
Negative Reagent Control
Use a non-specific negative reagent control in place of the primary antibody with a section of each patient specimen to evaluate
non-specific staining and allow better interpretation of specific staining at the antigen site.
Patient Tissue
Examine patient specimens stained with NCL-L-bcl-2 last. Positive staining intensity should be assessed within the context of any
non-specific background staining of the negative reagent control. As with any immunohistochemical test, a negative result means that
the antigen was not detected, not that the antigen was absent in the cells/tissue assayed. If necessary, use a panel of antibodies to
identify false-negative reactions.
Results Expected
Normal Tissues
Clone bcl-2/100/D5 stained the cytoplasm and membrane of lymphocytes in mantle zones and T cell areas of the spleen, lymph node,
tonsil and thymus. Staining was also observed in renal tubules, basal keratinocytes, basal epithelial cells of the cervix and fallopian tube,
stromal fibroblasts of breast and ovary, smooth muscle cells of the myometrium and syncytiotrophoblasts in placenta. (Total number of
normal cases evaluated = 55).
Abnormal Tissues
Clone bcl-2/100/D5 stained 88/113 diffuse large B-cell lymphomas, 8/26 Hodgkin's disease, 13/15 follicular lymphomas, 11/12
chronic lymphocytic lymphomas, 7/7 mantle cell lymphomas, 3/6 T-cell anaplastic large cell lymphomas, 4/4 angioimmunoblastic
T-cell lymphomas, 3/3 T-cell lymphomas, 3/3 T/NK lymphomas, 1/1 B-cell acute lymphoblastic lymphoma, 1/1 primitive B/T cell acute
lymphoblastic lymphoma, 1/1 peripheral T-cell lymphoma, and 1/1 marginal zone lymphoma, 2/4 lung tumors (including 1/1 non-small
cell carcinoma, 1/1 adenocarcinoma, 0/1 squamous cell carcinoma and 0/1 large cell carcinoma), 2/4 ovarian tumors (including 1/1
serous cystadenocarcinoma, 1/1 clear cell carcinoma, 0/1 malignant germ cell tumor and 0/1 mucinous cystadenocarcinoma), 1/4
liver tumors (including 1/1 metastatic carcinoma, 0/2 hepatocellular carcinomas and 0/1 cholangiocarcinoma), 3/3 thyroid papillary
carcinomas, 2/2 breast infiltrating ductal carcinomas, 2/2 metastatic tumors of unknown origin, 1/2 kidney renal cell carcinomas, 1/2
cervical squamous cell carcinomas and 1/1 thymus atypical carcinoid. No staining was observed in brain tumors (0/2), esophageal
tumors (0/2), stomach tumors (0/2), tumors of the tongue (0/2), testicular tumors (0/2), tumors of the colon (0/2), rectal tumors (0/2), skin
tumors (0/2), soft tissue tumors (0/2) and a tumor of the larynx (0/1). (Total number if tumor cases evaluated = 236).
NCL-L-bcl-2 is recommended for the assessment of bcl-2 oncoprotein expression in normal and neoplastic tissues.
General Limitations
Immunohistochemistry is a multistep diagnostic process that consists of specialized training in the selection of the appropriate reagents;
tissue selection, fixation, and processing; preparation of the IHC slide; and interpretation of the staining results.
Tissue staining is dependent on the handling and processing of the tissue prior to staining. Improper fixation, freezing, thawing, washing,
drying, heating, sectioning or contamination with other tissues or fluids may produce artifacts, antibody trapping, or false negative
results. Inconsistent results may be due to variations in fixation and embedding methods, or to inherent irregularities within the tissue.
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Excessive or incomplete counterstaining may compromise proper interpretation of results.
The clinical interpretation of any staining or its absence should be complemented by morphological studies using proper controls and
should be evaluated within the context of the patient's clinical history and other diagnostic tests by a qualified pathologist.
Antibodies from Leica Biosystems Newcastle Ltd are for use, as indicated, on either frozen or paraffin-embedded sections with specific
fixation requirements. Unexpected antigen expression may occur, especially in neoplasms. The clinical interpretation of any stained
tissue section must include morphological analysis and the evaluation of appropriate controls.
BCL-2-L-CE
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