Leica BIOSYSTEMS BOND Ready-to-Use ISH Lambda Probe PB0669 Mode D'emploi page 4

• Minimize microbial contamination of reagents or an increase in non-specific staining may occur.
• Retrieval, incubation times or temperatures other than those specified may give erroneous results. Any such change must be
validated by the user.
Instructions for Use
Lambda Probe was developed for use on the automated BOND system (includes Leica BOND-MAX system and Leica BOND-III system)
in combination with Anti-Fluorescein Antibody and BOND Polymer Refine Detection. The recommended staining protocol for Lambda
Probe is ISH Protocol A. Enzyme retrieval is recommended using the BOND Enzyme Pretreatment Kit, Enzyme 1, for 15 minutes.
Appropriate tissue and reagent controls should always be used. The protocol for the tissue and reagent controls should correspond to
that of the Lambda Probe.
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.
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.
Negative Tissue Control
Should be examined after the positive tissue control to verify the specificity of the labeling of probe to the target.
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.
Negative Reagent Control
Use RNA Negative Control Probe PB0809 in place of the Lambda Probe with a section of each patient specimen to evaluate non-specific
staining and allow better interpretation of specific staining at the target.
Positive Reagent Control
Use RNA Positive Control Probe PB0785 in place of the Lambda Probe with a section of each patient specimen to provide information
on the preservation of nucleic acids in the tissue as well as accessibility of nucleic acids to the probe. If the RNA Positive Control Probe
fails to demonstrate positive staining, results with the test specimens should be considered invalid.
Patient Tissue
Examine patient specimens stained with Lambda Probe last. Positive staining intensity should be assessed within the context of any
non-specific background staining of the RNA Negative Control Probe PB0809.
Results Expected
Normal Tissues
PB0669 detected Lambda light chain mRNA in B-cells in spleen and tonsil as well as the occasional B-cell in a wide range of tissues
including breast, cervix, liver, salivary gland, parotid gland, adrenal gland, larynx, small intestine and large intestine. Precursor B-cells
were also positive for lambda mRNA in bone marrow. Additional staining was also seen in epithelial cells of the thyroid, prostate, larynx,
and esophagus, tubules of the kidney, secretory cells in the adrenal glands and Leydig cells. (Total number of normal cases evaluated =
109).
Abnormal Tissues
PB0669 stained 4/6 plasma cell myelomas. No staining was seen in T-cell lymphomas (0/3), diffuse large B-cell lymphomas (0/2),
Hodgkin's lymphomas (0/2), a Burkitt's lymphoma (0/1), lung tumors (0/3), ovarian tumors (0/3), liver tumors (0/2), thyroid tumors (0/2),
esophageal tumors (0/2), breast tumors (0/2), metastatic tumors of unknown origin (0/2), brain tumors (0/2), testicular tumors (0/2), skin
tumors (0/2), a colon tumor (0/1), a stomach tumor (0/1), a rectal tumor (0/1), a tumor of the larynx (0/1) and a tumor of the thymus (0/1).
(Total number of abnormal cases evaluated = 41).
PB0669 is recommended for the detection of Lambda light chain mRNA.
Product Specific Limitations
Lambda Probe has been optimized at Leica Biosystems for use with Anti-Fluorescein Antibody, BOND Polymer Refine Detection and
BOND ancillary reagents. Users who deviate from recommended test procedures must accept responsibility for interpretation of patient
results under these circumstances. The protocol times may vary, due to variation in tissue fixation and the effectiveness of enzymatic
digestion, and must be determined empirically. RNA Negative Control Probe should be used when optimizing retrieval conditions and
protocol times.
Troubleshooting
Reference 6 may aid in remedial action.
Test samples should be complemented by the appropriate tissue and reagent controls.
Contact your local distributor or the regional office of Leica Biosystems to report unusual staining.
Further Information
Further information on in situ hybridization with BOND reagents, under the headings Principle of the Procedure, Materials Required,
Specimen Preparation, Quality Control, Assay Verification, Interpretation of Staining, Key to Symbols on Labels, and General Limitations
can be found in "Using BOND Reagents" in your BOND user documentation.
PB0669
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