Troubleshooting Flashcards
Problem: Both specimen and positive control are unstained.
- Primary Ab was not applied to tissue.
- Reagents were applied in the wrong order or the incorrect counterstain was used with AEC
- Wrong secondary Ab was used.
- Tissue sections were allowed to dry during staining.
Problem: specimen unstained, positive control stained.
- Substrate- chromogen was improperly prepared.
- Primary Ab too dilute or not properly prepared.
- Insufficient incubation time.
- To much buffer was left on the slides during staining.
- The Epitope enhancement was incorrectly done.
Problem: Specimen has weak staining but the positive control stained.
- The paraffin was incompletely removed.
- Too many cells contain endogenous peroxidase and haven’t been adequately “quenched”.
- Excessive adhesive was used.
- The slides were not washed well with reaction buffer.
- Incubation time of either the primary or secondary Ab was too long or the labeled reagent was too high.
Problem: Excessive background staining in the patient tissue but none in the control.
Free Ag is present in tissue due to: necrosis, autolysis or degeneration.
Problem: One area within specimen(s) show(s)
appropriate ‘reactivity’, while other areas do
not, may appear as a “tie dyed” effect.
Incomplete deparaffinization, slides were not placed onto the machine level or there was an inconsistent reagent volume during processing.
A section labeled with an antibody has no background but the antigen of interest stains very weakly. This is an example of:
high specificity, low sensitivity.
Define sensitivity.
Sensitivity refers to how well an antisera can demonstrate all of the antigen of interest present in a specimen
Define specificity.
Specificity refers to how well the antisera demonstrates the antigen of interest versus any non-specific staining.
A brain biopsy reveals a poorly differentiated malignant tumor. The differential diagnosis is primary versus metastatic brain cancer. The most useful antibody panel to resolve this differential is:
glial fibrillary acid protein, cytokeratin AE1/3, synaptophysin
Archival unstained sections are stained for ER and PR because the original slides were lost; the newly stained slides were negative for both ER and PR, despite having been reported previously as positive. What is the likely cause of the discrepancy?
The sections are too old
A section of intestine is labeled with an anti-keratin antibody and visualized with alkaline phosphatase. The section shows strong staining along the entire brush border of the intestinal epithelium. What artifact must you be aware of while interpreting this stain?
The intestinal brush border cells contain endogenous alkaline phosphatase
An undifferentiated malignant neoplasm stains positive for leukocyte common antigen (LCA) and negative for carcinoembryonic antigen (CEA) and cytokeratin (CK). The origin of these malignant cells is:
Lymphoreticular.
What does LCA test for?
Antibodies to leukocyte common antigen are used to distinguish malignant tumors of lymphoreticular origin from tumors of anaplastic, non-lymphoid origin.
B-lymphocytes can produce specific antibodies without T-lymphocyte help only if the antigen is:
A. processed by macrophages
B. a cluster designation
C. greater than 10,000 kd
D. T-independent
T-independent. B-cells can produce antibody without the help of T-lymphocytes following stimulation by T-cell-independent antigens, which consist primarily of non-protein antigens such as polysaccharides and lipids.
The group of components present in serum that is responsible for cytolytic destruction of cells bound with specific antibodies is called:
Complement
