Specimen Collection and Preparation for Analysis
For tissue specimens, routinely processed, formalin fixed, paraffin embedded tissues are
suitable for use with this reagent when used with Ventana detection kits and automated
Cell Conditioning Solution (CC1) slide stainer (see Materials and Reagents Needed, But Not Provided section). The
recommended tissue fixative is 10% neutral buffered formalin. Variable results may occur
Catalog number 950-124 as a result of prolonged fixation or special processes such as decalcification of bone
marrow preparations.
Each section should be cut the appropriate thickness and placed on a positively charged
INDICATIONS AND USE
glass slide. Slides containing the tissue section may be baked for at least 2 hours (but not
Intended Use
longer than 24 hours) in a 70掳 C 卤 5掳 C oven.
This reagent is intended for in vitro diagnostic use.
Ventana庐 Medical Systems' (Ventana) Cell Conditioning Solution (CC1) is a pre diluted
WARNINGS AND PRECAUTIONS
solution used as a pretreatment step in the processing of tissue samples for
1. Take reasonable precautions when handling reagents. Use disposable gloves when
immunohistochemistry (IHC) reactions carried out on Ventana BenchMark庐 and
handling suspected carcinogens or toxic materials.
BenchMark XT automated slide stainers.
2. Avoid contact of reagents with eyes and mucous membranes. If reagents come in
contact with sensitive areas, wash with copious amounts of water.
Summary and Explanation
3. Do not smoke, eat or drink in areas where specimens or reagents are being handled.
CC1 is a tris based buffer which must not be diluted. CC1 is poured into the appropriate
4. Patient specimens and all materials contacting them should be handled as
position (CC1 bottle) of the automated fluidics module on the automated slide stainer. The
biohazardous materials and disposed of with proper precautions. Never pipette by
instrument applies CC1 automatically as required by the procedure being run on the
mouth.
automated slide staining system.
5. Avoid microbial contamination of reagents, as this could produce incorrect results.
6. This reagent may cause skin and eye irritation. It may also be irritating to mucous
Principles and Procedures
membranes and upper respiratory tract. An allergic respiratory or skin reaction may
Fixation of tissue by formalin results in the formation of covalent bonds between the
be possible in sensitized individuals.
aldehyde and amino groups present in the tissue. The formation of these bonds denatures
7. CC1 is not flammable.
protein and can result in the loss of antigenicity. In addition, the formaldehyde can form
8. When used according to instructions, this product is not classified as a hazardous
methylene bridges cross linking tissue proteins thus reducing the penetration of the tissue
substance. The preservative in the reagent is ProClin 5000, containing the active
to large molecules such as antibodies.
ingredients 5-chloro-2-methyl-4-isothiazine-3-one and 2-methyl-4-isothiazolin-3-one.
CC1 is a tris based buffer with a slightly basic pH, which, at elevated temperatures is
Symptoms of overexposure to ProClin 5000 include skin and eye irritation, and
capable of hydrolyzing the covalent bonds formed by formalin in tissue. Removing these
irritation of mucous membranes and upper respiratory tract. The concentration of
bonds allows renaturation of protein molecules and increases antibody accessibility. Often
ProClin 5000 in this product is 0.05% and does not meet the OSHA criteria for a
these changes result in significant gains in antibody binding and improved signal to noise
hazardous substance. Systemic allergic reactions are possible in sensitive
ratios. The automated slide stainer automatically heats the slide to the appropriate
individuals.
temperature and time as selected by the user.
INSTRUCTIONS FOR USE
MATERIALS AND METHODS
CC1 is poured into the appropriate position (CC1 bottle) of the automated fluidics module
Reagents Provided
on the Ventana automated slide stainer. CC1 is applied to tissue specimens following the
1 - 2 L bottle of CC1; contains a tris based buffer and a preservative.
removal of paraffin and prior to the application of other reagents used in the detection of the
target analyte as required by the protocol being run. The instrument applies CC1
Reconstitution, Mixing, Dilution, Titration
automatically. The user is given the option of selecting mild (30 minutes), standard (60
This reagent is ready to use directly from the bottle and must not be diluted.
minutes), or extended (90 minutes) cell conditioning.
Prior to initial use of the CC1 in the user鈥檚 laboratory, appropriate staining should be verified
Materials and Reagents Needed But Not Provided
by staining a number of positive and negative tissues with known performance
The following reagents and materials may be required but are not provided with this kit:
characteristics for antibodies which require antigen enhancement. Assay verification on a
1. Ventana Negative Control Reagent or Rabbit Negative Control
daily basis may be accomplished through the proper use of positive and negative controls.
2. Microscope slides, positively charged
Ventana recommends positive controls be placed on the same slide as the patient tissue
3. Positive and negative tissue controls
sample. Users can adjust assay parameters to optimize staining results with the mild,
4. Drying oven capable of maintaining a temperature of 70掳C 卤 5掳C
standard, and extended options as well as adjusting the incubation time of the selected
5. Bar code labels (appropriate protocol)
primary antibody by adjusting the digestion conditions.
6. 10% neutral buffered formalin
7. Staining jars or baths
Step by Step Procedure
8. Timer
Ventana reagents have been developed for use on a Ventana automated slide stainers in
9. Xylene
combination with Ventana detection kits and accessories. Recommended staining protocols
10. Ethanol or reagent alcohol
for the automated slide stainers are described in the package insert of the primary antibody
11. Deionized or distilled water
of interest. The parameters for the automated procedures can be displayed, printed and
12. BenchMark or BenchMark XT automated slide stainers
edited according to the procedure in the Operator鈥檚 Manual. Other operating parameters for
iVIEWTM DAB, AEC, V Red (ALK PHOS) or Enhanced V Red detection kits*
13. the automated slide stainers have been preset at the factory. For more detailed instructions
14. Ventana Endogenous Biotin Blocking Kit* and additional protocol options refer to your Operator鈥檚 Manual.
15. Primary Antibody
Ventana EZ PrepTM solution*
16. BenchMark or BenchMark XT Automated Slide Stainers
17. Ventana LCS, Ventana鈥檚 coverslip solution 1. Apply slide bar code label, which corresponds to the protocol to be performed.
18. Ventana Protease I, II or III* 2. Load the primary antibody, appropriate detection kit and required accessory reagents
19. Ventana Hematoxylin Counterstain* onto the reagent tray and place them on the automated slide stainer. Check bulk
20. Ventana Bluing Reagent* fluids and waste.
21. Ventana Reaction Buffer 3. Load the slides onto the automated slide stainer.
22. Mounting medium 4. Start the staining run.
23. Cover glass 5. At the completion of the run, remove the slides from the automated slide stainer.
24. Light microscope (20-80X) 6. For iVIEW DAB and Ventana Red kit, wash in a mild dishwashing detergent or
* As needed for specific applications. alcohol to remove the coverslip solution; dehydrate, clear, and coverslip with
permanent mounting media in the usual manner.
Storage and Handling 7. For AEC chromogen, do not dehydrate and clear. Mount AEC with aqueous mounting
Store CC1 at room temperature (20 to 28掳 C). Keep out of direct sunlight. Do not freeze. medium. The stained slides should be read within two to three days of staining, and
The user must validate any storage conditions other than those specified in the package are stable for at least two years if properly stored at room temperature (20 to 28掳 C).
insert.
This reagent is expiration dated. When properly stored, the reagent is stable to the date Quality Control Procedures
indicated on the label. Do not use reagent beyond the expiration date for the prescribed Positive Tissue Control
storage method. A positive tissue control must be run with every staining procedure performed.
2003-11-30 Page 1 of 3 16307EN Rev A
The positive staining tissue components are used to confirm that the reagents were applied
and the instrument functioned properly. This tissue may contain both positive and negative LIMITATIONS
staining cells or tissue components and serve as both the positive and negative control General Limitations
tissue. Control tissues should be fresh autopsy, biopsy or surgical specimens prepared or 1. IHC testing is a multiple step diagnostic process that requires specialized training in
fixed as soon as possible in a manner identical to the test sections. Such tissues may the selection of the appropriate reagents, samples, fixation, processing, preparation
monitor all steps of the procedure, from tissue preparation through staining. Use of a tissue of the slide, and interpretation of the staining results.
section fixed or processed differently from the test specimen will provide control for all 2. For IHC testing, false positive results may be seen because of nonimmunological
reagents and method steps except fixation and tissue processing. binding of proteins or substrate reaction products. They may also be caused by
A tissue with weak positive staining should be used for optimal quality control and for pseudoperoxidase activity (erythrocytes), endogenous peroxidase activity
detecting minor levels of reagent degradation. (cytochrome C), endogenous alkaline phosphatase activity, or endogenous biotin
Known positive tissue controls should be utilized only for monitoring the correct (example: liver, brain, breast, kidney) depending on the type of immunostain used.
performance of processed tissues and test reagents, not as an aid in determining a specific 3. Tissue staining is dependent on the handling and processing of the tissue prior to
diagnosis of patient samples. If the positive tissue controls fail to demonstrate positive staining. Improper fixation, freezing, thawing, washing, drying, heating, sectioning, or
staining, results with the test specimens should be considered invalid. contamination with other tissues or fluids may produce artifacts or false negative
results. Inconsistent results may result from variations in fixation and embedding
Negative Tissue Control methods, or from inherent irregularities within the tissue.
The same tissue used for the positive tissue control may be used as the negative tissue 4. Excessive or incomplete counterstaining may compromise proper interpretation of
control. The variety of cell types present in most tissue sections offers internal negative results.
control sites, but this should be verified by the user. The components that do not stain 5. The clinical interpretation of any positive staining, or its absence, must be evaluated
should demonstrate the absence of specific staining, and provide an indication of non within the context of clinical history, morphology and other histopathological criteria.
specific background staining. If specific staining occurs in the negative tissue control sites, The clinical interpretation of any staining, or its absence, must be complemented by
results with the patient specimens should be considered invalid. morphological studies and proper controls as well as other diagnostic tests. It is the
responsibility of a qualified pathologist to be familiar with the reagents and methods
Unexplained Discrepancies used to produce the stained preparation. Staining must be performed in a certified
Unexplained discrepancies in controls should be referred to your local Ventana office licensed laboratory under the supervision of a pathologist who is responsible for
immediately. If quality control results do not meet specifications, patient results are invalid. reviewing the stained slides and assuring the adequacy of positive and negative
See the Troubleshooting section of this insert. Identify and correct the problem, then repeat controls.
the patient samples. 6. Ventana provides reagents at optimal dilution for use when the provided instructions
are followed. Any deviation from recommended test procedures may invalidate
Negative Reagent Control expected results. Appropriate controls must be employed and documented. Users
For IHC, a negative reagent control must be run for every specimen to aid in the who deviate from recommended test procedures must accept responsibility for
interpretation of results. A negative reagent control is used in place of the primary antibody interpretation of patient results.
to evaluate nonspecific staining. The slide should be stained with Negative Control Reagent 7. Reagents may demonstrate unexpected reactions in previously untested tissues. The
(mouse) or Rabbit Negative Control, as appropriate. If an alternative negative reagent possibility of unexpected reactions even in tested tissue groups cannot be completely
control is used, dilute to the same concentration as the primary antibody antiserum with eliminated because of biological variability of antigen expression in neoplasms, or
Ventana Antibody Diluent. The diluent alone may be used as an alternative to the other pathological tissues. Contact your local Ventana office with documented
previously described negative reagent controls. The incubation period for the negative unexpected reactions.
reagent control should equal the primary antibody incubation period.
When panels of several antibodies are used on serial sections, a negative reagent control Specific Limitations
on one slide may serve as a negative or nonspecific binding background control for other 1. CC1 must be examined for microbial contamination prior to use. The signs
antibodies. indicating contamination or instability of this product are: turbidity of the solution,
odor development or precipitation. At the first sign of possible reagent contamination
Assay Verification or instability, call your local Ventana Office.
Prior to initial use of a staining system in a diagnostic procedure, the specificity of the 2. CC1 has been optimally formulated for use on Ventana automated slide stainers.
system should be verified by testing it on a series of samples with known staining Dilution of this product will result in poor instrument performance and possible loss
performance characteristics representing known positive and negative samples (refer to the of staining.
Quality Control Procedures previously outlined in this section of the product insert, to the
Quality Control recommendations of the College of American Pathologists Laboratory SUMMARY OF EXPECTED RESULTS
Accreditation Program, Anatomic Pathology Checklist, and the NCCLS Approved Refer to the appropriate Ventana primary antibody package insert for expected patient
Guideline). These quality control procedures should be repeated for each new primary sample results. Appropriate sample control results verify the reagents and system are
antibody lot, or whenever there is a change in assay parameters. working properly.
1. Cell conditioning solution CD1 is a tris based buffer used a s a pretreatment step in
the processing of tissue samples for immunohistochemistry or in situ hybridization
Interpretation of Results
The Ventana automated staining procedures cause colored reaction products. Refer to the reactions. This reagent is a stand alone reagent that can not be tested for sensitivity
appropriate detection kit package insert for expected color reactions. A qualified pathologist or specificity; it is a qualitative chemical enhancement to the target antigen and may
must evaluate positive and negative controls before interpreting results. be used as a tool to enhance staining. CC1 was tested with a variety of antibodies
representing Breast, Lymphoma, and Leukemia panels at various incubation times
Positive Tissue Control with specific tissue types. The antibodies include PR, ER, ki67, p532 CD5,CD3,CD,
The stained positive tissue control should be examined first to ascertain that all reagents CD10, CD79a, Bcl-2, CD15, CD30, Cyclin D1, Synaptophysin, Keratin, CD8, CD4
are functioning properly. The presence of an appropriately colored reaction product within and Vimentin. The tissue types include Breast Carcinoma, Tonsil, Mantle Cell
the target cells is indicative of positive reactivity. Refer to the package insert of the Lymphoma, Spleen, Adrenal Tumor, Schwancma, Nerve Sheath Tumor, Colon
detection kit used for expected color reactions. Intensity of the counterstain will be Carcinoma, Thyroid Carcinoma, Thymus, Lymphoma, and Hodgkins Lymphoma.
depended on the incubation time selected. 2. Inter and intra run reproducibility of staining was determined by taking the difference
in staining intensity from each of three slides per case between three runs. The
For IHC counterstaining, the incubation length and potency of the hematoxylin used will
intensity of staining from slide to slide within and between runs when read by three
range from a pale to dark blue coloration of cell nuclei.
qualified pathologists did not vary by more than one reading grade for all cases.
Excessive or incomplete counterstaining may compromise proper interpretation of results.
If the positive tissue control fails to demonstrate positive staining, any results with the test
specimens should be considered invalid. TROUBLESHOOTING
1. If the positive control exhibits weaker staining than expected, other positive controls
run during the same instrument run should be checked to determine if it is because
Patient Tissue
of the primary antibody or one of the common secondary reagents.
Patient specimens should be examined last. Positive staining intensity should be assessed
2. If the positive control is negative, it should be checked to ensure that the slide has the
within the context of any background staining of the negative reagent control.
proper bar code label. If the slide is labeled properly, other positive controls run on
For IHC tests, a negative result means that the antigen in question was not detected, not
the same instrument run should be checked to determine if it is because of the
that the antigen is absent in the cells or tissue assayed. If necessary, use a panel of
primary antibody or one of the common secondary reagents. Samples may have
antibodies to aid in the identification of false negative reactions.
been improperly collected, fixed or deparaffinized.
The morphology of each sample should also be examined utilizing a hematoxylin and eosin
3. If excessive background staining occurs, high levels of endogenous biotin may be
stained section when interpreting any results. The patient's morphologic findings and
present. A biotin blocking step should be included.
pertinent clinical data must be interpreted by a qualified pathologist.
2003-11-30 Page 2 of 3 16307EN Rev A
4. If all of the paraffin has not been removed, the deparaffinization procedure should be
repeated.
5. If specific antibody staining is too intense, the run should be repeated with incubation
time shortened by intervals of 4 minutes until the desired stain intensity is achieved.
6. If tissue sections wash off the slide, slides should be checked to ensure that they are
positively charged.
7. For corrective action, refer to the Step By Step Procedure section, the automated
slide stainer Operator鈥檚 Manual or contact your local Ventana office
REFERENCES
Beesely JE. Immunocytochemistry and in situ Hybridization in the Biomedical Sciences.
Birkh盲user.2001.
College of American Pathologists Laboratory Accreditation Program, Anatomic Pathology
Checklist, 2001.
NCCLS. Quality Assurance for Immunocytochemistry: Approved Guideline. NCCLS
document MM4-A- (ISBN 1-56238-396-5). NCCLS, 940 West Valley Road, Suite 1400,
Wayne, PA 19087-1898 USA, 1999.
Herman GE, Elfont EA. The taming of immunohistochemistry: the new era of quality control.
Biotech Histochem 66(4): 194-199, 1991.
Roche PC, Hsi ED. Immunohistochemistry-Principles and Advances. Manual of Clinical
Laboratory Immunology, 6th edition. (NR Rose Ed.) ASM Press, 2002.
Nadji M, Morales AR. Immunoperoxidase: part 1. The technique and its pitfalls. Lab Med
14: 767, 1983.
INTELLECTUAL PROPERTY
iVIEWTMand EZ PrepTM and are trademarks of Ventana Medical Systems, Inc.;
BenchMark庐 and Ventana庐 are registered trademarks of Ventana Medical Systems, Inc.
Covered by the following patents: U.S. Pat. Nos. 6045 759, 6192 945 B1, 6416 713 B1 and
foreign counterparts.
CONTACT INFORMATION
North America
Ventana Medical Systems, Inc.
1910 Innovation Park Drive
Tucson, Arizona 85737
U.S.A.
800 227 2155 (U.S. only)
520 229 1910
Europe
Ventana Medical Systems, S.A.
Parc d鈥橧nnovation 鈥? BP 30 144
Rue G. de Kaysersberg
F - 67404 Illkirch Cedex
France
33 (0) 3 90 40 52 00
EU Representative
MDCI Ltd.
Arundel House
1 Liverpool Gardens
Worthing
West Sussex BN11 1SL
UK
Japan
Ventana Japan K.K.
Landmark Tower, 35F
2-2-1, Minatomirai, Nishi-ku
Yokohama, 220-8135
Japan
81 (0) 45-228-5071
Australia, New Zealand
Ventana Medical Systems, Pty Ltd
5/39 Grand Boulevard
Montmorency VIC 3094
Australia
61 (0) 3 9431 6064
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