Further Information - Leica BIOSYSTEMS BOND Mode D'emploi

• Consult Federal, State or local regulations for disposal of any potentially toxic components.
• 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
PD-1 (CAL20) primary antibody was developed for use on the automated BOND system (includes Leica BOND-MAX system and Leica
BOND-III system) in combination with BOND Polymer Refine Detection. The recommended staining protocol for PD-1 (CAL20) primary
antibody is IHC Protocol F. Heat induced epitope retrieval is recommended using BOND Epitope Retrieval Solution 2 (AR9640) for 20
minutes.
Results Expected
Normal Tissues
Clone CAL20 detects the PD-1 protein in the cytoplasm/membrane of a proportion of immune cells. Staining was observed within
immune cells in most tissue types, and particularly in lymph node, tonsil, spleen and thymus gland. Staining was also observed in
alveolar macrophages. (Total number of normal cases evaluated = 135)
Tumor Tissues
Clone CAL20 stained 3/101 lymphomas (including 2/6 angioimmunoblastic T-cell lymphomas, 1/55 diffuse large B-cell lymphomas, 1/6
anaplastic large cell lymphomas, 0/16 T-cell lymphomas, 0/7 Hodgkin lymphomas, 0/5 extra nodal marginal zone B cell lymphomas,
0/3 follicular lymphomas, 0/2 Burkitt lymphomas and 0/1 lymphoma NOS) and 1/5 thyroid tumors (including 1/1 follicular carcinoma,
0/3 follicular adenomas and 0/1 papillary thyroid carcinoma). No staining (other than infiltrating immune cells) was detected in bowel
tumors (0/8) (including 0/6 adenocarcinomas and 0/2 adenomas), metastatic tumors (0/5), breast tumors (0/5) (including 0/3 invasive
ductal carcinoma and 0/2 fibroadenomas), hepatocellular carcinomas (0/4), brain tumors (0/4) (including 0/3 meningiomas and 0/1
astrocytoma), lung tumors (0/4) (including 0/2 squamous cell carcinomas, 0/1 adenocarcinoma and 0/1 small cell carcinoma), ovarian
tumors (0/3) (including 0/1 granulosa cell tumor, 0/1 adenocarcinoma and 0/1 endometrioid adenocarcinoma), squamous cell carcinomas
of the esophagus (0/3), adenocarcinomas of the stomach (0/3), adrenal gland tumors (0/2) (including 0/1 cortical adenoma and 0/1
adrenocortical carcinoma), transitional cell carcinomas of the bladder (0/2), bone tumors (0/2) (including 0/1 osteosarcoma and 0/1
chondrosarcoma), squamous cell carcinomas of the cervix (0/2), endometrial adenocarcinomas (0/2), clear cell carcinomas of the
kidney (0/2), adenocarcinomas of the prostate (0/2), salivary gland tumors (0/2) (including 0/1 pleomorphic adenoma, 0/1 adenoid cystic
carcinoma), skin tumors (0/2) (including 0/1 squamous cell carcinoma, 0/1 melanoma), seminomas (0/2), an oral adenocarcinoma
(0/1), a pancreatic adenocarcinoma (0/1), a squamous cell carcinoma of the tongue, and a prostate hyperplasia (Total number of cases
evaluated = 169)
PD-1 (CAL20) is recommended for the detection of PD-1 protein in normal and neoplastic tissues, as an adjunct to
conventional histopathology using non-immunologic histochemical stains.
Product Specific Limitations
PD-1 (CAL20) has been optimized at Leica Biosystems for use with 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 antigen enhancement, and must
be determined empirically. Negative reagent controls should be used when optimizing retrieval conditions and protocol times.
Troubleshooting
Refer to reference 3 for remedial action.
Contact your local distributor or the regional office of Leica Biosystems to report unusual staining.

Further Information

Further information on immunostaining 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.
Bibliography
1. Clinical Laboratory Improvement Amendments of 1988, Final Rule 57 FR 7163 February 28, 1992.
2. Villanova PA. National Committee for Clinical Laboratory Standards (NCCLS). Protection of laboratory workers from infectious
diseases transmitted by blood and tissue; proposed guideline. 1991; 7(9). Order code M29-P.
3. Bancroft JD and Stevens A. Theory and Practice of Histological Techniques. 4th Edition. Churchill Livingstone, New York. 1996.
4. Wang C, Hillsamer P and Kim CH. Phenotype, effector function, and tissue localization of PD-1-expressing human follicular helper T
cell subsets. BMC Immunology. 2011; 12:53.
5. Roncador G, Garcia Verdes-Montenegro J, Tedoldi S et al. Expression of two markers of germinal center T cells (SAP and PD-1) in
angioimmunoblastic T-cell lymphoma. Haematologica 2007; 92:1059-1066.
6. Dorfman DM, Brown JA, Shahsafaie MS et al. Programmed Death-1 (PD-1) is a marker of germinal center-associated T cells and
angioimmunoblastic T-cell lymphoma. American Journal of Surgical Pathology 2006; 30:802-810.
Date of Issue
03 April 2020
PA0216
Page 3