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United States Government Work, uncopyrighted, public-domain,
DRAFT COPY ONLY. This document does not necessarily represent the views
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This document is provided "as is", without warranty of any kind,
express or implied, including but not limited to the warranties
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or in connection with the document or the use or other dealings
made with the document.
See also: Tissue Stains: 49,
51, 52,
55, 69,
72, 81.
PRINCIPLE OF THE TEST.
The first peroxidase anti-peroxidase kits were developed in 1980.
The kit systems utilize the concept of Sternberger's Technique, which
is a modification of the basic immunoperoxidase technology. Commercially
constructed immunoenzymatic staining kits are utilized in our laboratory
for PSA/PSAP, S-100, UCHL1/L26, CEA, Kappa/Lamba, Cytokeratin,
High-Molecular-Weight Cytokeratin, HMB-45, and Chromogranin.
SPECIMEN REQUIRED.
All human tissue excised at surgery, outpatient clinics, and postmortems,
fresh or in fixative, along with a filled-out Tissue Examination Form
(U. S. Standard Form 515, USSF515).
REAGENTS, INSTRUMENTATION.
Dako Rabbit anti-human chromogranin A, prediluted.
Dako Mouse Anti-human melanoma (HMB45) prediluted.
Dako PAP Kit System 40.
Dako PAP Kit System 20.
Dako AP-AAP-Kt-t System.
Dako Monoclonal and Polyclonal Kits.
Xylene substitute.
Absolute ethanol.
95% ethanol.
Methanol.
Hydrogen peroxide.
Tris buffer, phosphate buffered saline (PBS).
Mayer's hematoxylin.
Ammonium hydroxide.
Glycerol gelatin.
DAB chromogen substrate (see specific procedure).
STEP-BY-STEP DESCRIPTION.
1. The first peroxidase anti-peroxidase kits were developed in 1980.
The kit systems utilize the concept of Sternberger's Technique, which
is a modification of the basic immunoperoxidase technology. Commercially
constructed immunoenzymatic staining kits are utilized in our laboratory
for PSA/PSAP, S-100, UCHL1/L26, CEA, Kappa/Lamba and Cytokeratin.
HMB-45 and Chromogranin. If immunohistochemical studies involving reagents
labelled by the manufacturer as "analytic-specific reagents" comprise part
of the specimen evaluation, then the federally-required clarifying statement
is part of the report.
2. Immunoenzymatic staining kits are used for staining paraffin embedded
tissue, frozen tissue, bone marrow and bone smears, cytospin slides,
imprints and positive and negative controls.
3. Kits:
Dako Rabbit anti-human chromogranin A, prediluted.
Dako Mouse Anti-human melanoma (HMB45) prediluted.
4. Reagents:
Dako PAP Kit System 40.
Dako PAP Kit System 20.
Dako AP-AAP-Kt-t System.
Dako Monoclonal and Polyclonal Kits.
5. The above kits contain (depending on specific kit) normal goat serum,
anti-serum, non-immune rabbit and mouse serums, goat anti-rabbit IgG
dilution, rabbit and mouse PAP and control tissue depending on the
specific kit.
6. Other reagents needed that are not supplied by the kit6 are:
6.1. Xylene substitute.
6.2. Absolute ethanol.
6.3. 95% ethanol.
6.4. Methanol.
6.5. Hydrogen peroxide.
6.6. Tris buffer, phosphate buffered saline (PBS).
6.7. Mayer's hematoxylin.
6.8. Ammonium hydroxide.
6.9. Glycerol gelatin.
6.10. DAB chromogen substrate (see specific procedure).
CONTROLS
7.
When new antibodies are introduced into the laboratory,
various mixtures of the reagents are prepared and tested.
Then the optimal preparation is selected for use,
after review by two staff pathologists.
The positive controls (use of primary antibody) will ascertain if specimens
are correctly fixed, embedded, deparaffinized, rehydrated and stained.
Negative controls help to evaluate non-specific staining. Some of
the kits provide control slides that are intended only for the initial
use of the kit. However, laboratory generated control slides are also
available.
Records of reactivity of control tissue blocks are kept.
8.
When Dako AP M P Kit System is utilized, the immune complex of (APAAP)
alkaline phosphatase-anti-alkaline phosphatase is used instead of PAP
reagent. Mouse serves as the source for the primary antibody as well
as for the AP M P complex. As in the PAP techniques, tissue or cell
preparations are incubated with the primary antibody, link antibody
and AP M P immune complex. The rinses and washes are interposed and
alkaline phosphatase substrate applied and results in a bright red
color to the antigen.
9.
If immunohistochemical studies involving reagents
labelled by the manufacturer as "analytic-specific reagents"
comprise part of the specimen evaluation, then
the federally-required clarifying statement is part of the report.
NOTES
11. Exposure to drafts from heating and air conditioning systems may
necessitate covering the slide and placing in a humid environment.
Otherwise, the slides can remain uncovered.
12. All incubations are carried out at room temperature. More intense
staining can be achieved by incubating with antisera and/or substrate
for either longer times or at 37oC.
13. Excessive fixation, especially with formalin, can mask the antigen.
To unmask, post-fix in B-5 for two hours or Bouin's fluid overnight.
14. If the procedure must be interrupted, it is best to do so while
the slides are in any of the buffer washes in which they may be left
for up to 48 hours if necessary. The staining process can then be
resumed.
15. Treatment of tissue sections with hydrogen peroxide may be omitted
if the sections are known not to contain endogenous peroxidase activity.
16. Quenching with normal serum may be omitted if no excessive non-specific
background staining is likely to occur, such as when using monoclonal
antibodies.
17. Filter column should be replaced after two to three uses with fresh
serum separator filter or Whatman #l filter paper and funnel.
18. Do not use kits and primary antibodies that state "Quick Staining"
on the label.
Procedure.
19.
Remove the following items from the refrigerator,
and let set half hour at room temperature:
19.1. pertinent Dako kit
19.2. prediluted primary solution
19.3. PBS solutions pH 7.4 and pH 7.6
20.
Obtain control slides (all control slides are paraffin sections, that are
kept in a dedicated file area room temperature. This storage does
not result in detectable loss of antigenic activity.
21.
Incubations are conducted at room temperature in a moisture chamber
(clear plastic box lined with paper towels soaked with water). Tissue
sections are cut at five microns and dried in oven at 50ø to 60ø for
one hour before staining procedure.
22.
Begin the procedure:
1. Slide preparation
Prepare for each immuno-peroxidase stain requested two patient-specimen-
slides designated with patient I.D. and test type and two positive
control slides.
Labeling slide example: (Keratin WSS)
Patient Specimen Positive Controls
John Doe WSS Keratin + Control - Control
Negative John Doe WSS Keratin
2. Paraffin is removed from the tissue sections by treatment
with three changes of clearing solvent (five minutes for each change).
3. Immerse in two changes of absolute ethanol for one minute
4. Immerse in one change of 95% ethanol for one minute.
5. Immerse in one change of 80% ethanol for one minute.
6. Rinse in top water for one minute.
7. Drop 3% hydrogen peroxide on the slides,
and let stand for five minutes.
8. Wash in running water for five minutes.
9. Treat for 20 minutes with undiluted blocking serum.
10. Shake off excess blocking serum and treat
with the primary antiserum for 20 minutes.
11. Wash for five minutes in two changes of PBS buffer at pH 7.6.
12. Wipe off excess buffer; then add goat anti-rabbit IgG or anti-mouse
for 20 minutes.
13. Wash in two changes of PBS buffer at pH 7.6 for five minutes
each.
14. Add rabbit or mouse PAP for 20 minutes.
15. Wash in two changes of PBS buffer at pH 7.6 for five minutes
each.
16. Antibody localization in the tissue sections is detected by
placing them in a filtered and freshly prepared solution of DAB (one
DAB tablet in 10 ml of PBS at pH 7.4 with eight drops of 3% hydrogen peroxide).
17. Wash in distilled water.
18. Stain with Gill or Mayer hematoxylin for 30 seconds to one
minute.
19. Wash in tap water for one minute.
20. Blue in bluing agent.
21. Wash in tap water.
22. Pass through a series of increasing percent ethanol solutions
to the clearing solvent.
23. Cover-slip.
Positive areas will be stained brown, and nuclei are stained blue.
RESULTS.
When Dako PAP Kit System 40 and 20 and Dako PAP Kit System Monoclonal
and Polyoclonal are utilized, tissue or cell preparations are first
treated with hydrogen peroxide or absolute methanol and hydrogen peroxide
mixture to suppress endogenous peroxidase activity. Following this,
incubation with normal serum is imposed, which results in quenching
of non-specific protein binding to certain tissue elements. Antibody
to the target antigen (primary antibody), antibody to the primary antibody
(link antibody) and PAP reagent are then applied sequentially with
interposed washing steps. Because the antibody of the PAP reagent
is from the same species of animal as the primary antibody, they will
be unbound by the link antibody at the site of the tissue antigen.
Oxidation of amino-ethylcarbozol results with the formation of a reddish
water insoluble product with the antigen.
REFERENCES.
1. Miller KD.
Immunohistochemical Techniques.
Chapter 21. pp. 421-464.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;:421-464.
ISBN 0-443-06435-0, 796 pages.
Table 20.1. Table of Clusters of Differentiation. pp.442-444.
bcl2 ..... Follicular lymphoma.
CD2 ..... T cells.
CD3 ..... T cells.
CD4 ..... T-helper cells.
CD5 ..... T cells.
CD8 ..... T-suppressor cells.
CD15 ..... Hodgkin cells.
CD20 ..... B cells
CD21 ..... Follicular dendritic cells
CD23 ..... Lymphocytic lymphoma.
CD30 ..... Hodgkin cells.
CD56 ..... Small cell carcinoma.
CD68 ..... Monocytes, macrophages.
CD79a ..... B cells.
CD117 ..... Gastric stromal tumor.
Chromogranin ..... Carcinoid tumor.
CK 5/6 ..... Epithelioid mesothelioma.
CK 20 ..... Transitional cell carcinoma.
Cytokeratin ..... Adenocarcinoma.
Cytokeratin LP34 ..... Prostatic basal cells.
Desmin ..... muscle.
Epithelial membrane Antigen ..... Anaplastic large cell lymphoma.
GFAP ..... Astrocytoma.
IgA ..... B cells.
IgD ..... B cells.
IgG ..... B cells.
IgM ..... B cells.
S100 ..... Malignant melanoma.
Vimentin ..... Mesenchymal cells.
2. Krenacs L, Bagdi E, Krenacs T.
Immunocytopathology of Lymphomas.
In: loc. cit.
3. Barr N, Wu N, Taylor CR.
Immunocytopathology of Routine Histopathology.
Chapter 24. pp. 537-552.
In: loc. cit.
4. Sternberger LA, et al.
The Unlabeled Antibody-Enzyme of Immunohistochemistry.
Preparation and properties of soluble antigen-antibody complex
(horseradish peroxidase-anti-peroxidase) and its use in identification
of spirochetes.
J Histochem Cytochem. 18:315, 1970.
5. Dako Corporation.
Immunoenzymatic Staining, General Instructions.
Dako Corporation.
6. Prophet EB, Mills B, Arrington JB, Sobin LH, eds.
Laboratory Methods in Histotechnology.
Washington, DC: Armed Forces Institute of Pathology. 1992;:.
ISBN 1-881041-00-X, 278 pages.
8. Mikel UV, ed.
Advanced Laboratory Methods in Histology and Pathology.
Washington, DC: Armed Forces Institute of Pathology. 1994;:.
ISBN 1-881041-13-1, 254 pages.