IHC Guidebook - Troubleshooting - Chapter 16
Contents
1. Different fixatives may affect standardization of cells Immunoreactivity diminished or destroyed during embedding process Immunoreactivity diminished or destroyed during dewaxing at high oven temperature Immunoreactivity diminished or destroyed on pre cut tissue sections Immunoreactivity diminished or destroyed by the enzyme blocking reagent altering a specific epitope Solution Replace defective or expired antibody repeat staining protocol replacing one reagent at a time with fresh in date reagents Store products according to each product specification sheet or package insert f using a neat or concentrated antibody and directed by the manufacturer to store frozen the reagent may be aliquoted to avoid repeated freezing and thawing Do not freeze ready to use RTU or customer diluted products Follow manufacturer recommendations on product specification sheets package inserts and reagent labels Particularly a feature of low affinity antibodies Polyclonal primary antiserum Attempt staining at lower dilutions higher concentrations Monoclonal primary antibody Replace with higher affinity antibody of identical specificity Re optimize incubation times for washing buffer and link antibody Repeat staining using water based counterstain and mounting media m Use a permanent chromogen such as DAB DAB that is not affected by organic solvents Use a counterstain that m Will not excessively stain tissue2. Lesser amounts are found in the Gl tract lung spleen pancreas brain mammary gland adipose tissue lymphoid tissue and cells grown in culture media containing biotin as a nutrient Cause is not understood It is possibly due to antibodies to muscle antigens in primary and negative reagent control serum Binding of the Fc portion of Ig by Fc receptors on the cell membrane of macrophages monocytes granulocytes and some lymphocytes Phagocytosis of antigens may render phagocytes positive for the same Tissue from persons infected with Hepatitis B virus and expressing Hepatitis B surface antigen may exhibit undesired staining Miscellaneous Some manufacturers produce antibodies and reagents for in vitro use only These products may contain preservatives usually sodium azide which is a known poison Solution This observation is rare and should not interfere with interpretation of specific staining Use alternate or prolonged peroxidase blocks or use another enzyme label such as alkaline phosphatase Eosinophils and mast cells are particularly resistant to peroxidase quenching Use a peroxidase blocker m Use special stains eosin will stain eosinophils a bright red orange Add levamisole to the alkaline phosphatase chromogen reagent or use another enzyme label such as horseradish peroxidase Intestinal alkaline phosphatase is not quenched by the addition of levamisole Pretreat the tissue with 0 03 N HCI Use a bi
3. Ensure automated stainer is programmed correctly and is running to manufacturer s specification Non specific staining of fatty tissue rarely interferes with interpretation of specific staining and can usually be disregarded Reoptimize the dilution of the primary antibody and negative control serum m Use a higher dilution of the primary antibody and negative control serum m Increase the incubation time m Replace the antibody Proteolytic digestion or antigen retrieval will break down cross linking and render some tissue antigens reactive Refer to the primary antibody and or the negative reagent control specification sheet for appropriate pre treatment Chapter 16 Troubleshooting Possible cause of poor staining Focal cytoplasmic staining is seen particularly in intermediate and superficial layers of the epidermis May be caused by passive absorption of plasma proteins into degenerating epidermal cells Unquenched endogenous peroxidase activity may be seen in all hemoprotein containing specimens including hemoglobin in erythrocytes myoglobin in muscle cells cytochrome in granulocytes and monocytes and catalases in liver and kidney Unquenched endogenous alkaline phosphatase activity may be seen in leucocytes kidney liver bone ovary bladder salivary glands placenta and gastro intestinal tissue Endogenous protein bound biotin water soluble B vitamin High amounts of biotin are found in adrenal liver and kidney
4. antibody specification sheet will list tissue that will exhibit positive and negative staining patterns in the Performance Characteristics section NOTE abnormal tissue will not necessarily be labeled Both negative and positive tissue controls should be processed using the same fixation embedding mounting drying epitope retrieval and immunostaining protocols as the patient tissue Comments The information can be located in different places based on your automated solution If the location is not known then contact your automated platform supplier Chapter 16 Troubleshooting Information You Need to Know Target retrieval has been performed under the right conditions LIS Protocols align with workstation test Staining process has been performed under the right conditions Information Located at the Instrument Target retrieval Target retrieval solution Find the location of the specific data for the target retrieval procedure this information can be a part of the slides log file under completed slides Check that the right target retrieval solution has been used Sub optimal results can be seen if a high pH target retrieval solution is used for an antibody that according to the specification sheet requires a low pH target retrieval solution Check that the target retrieval solution has been within the expiration limits when used for the slide Target retrieval temperature Check that the temperatur
5. be requested from the supplier Check that the wash buffer used is the buffer specified for the assay or has been validated for the assay by the laboratory Check that the wash buffer used for the slide has been within the expiry limits when used for the slide in question General If a positive control has been run for the assay on the slide in question an evaluation of the effect of any deviations can be made based on the positive control Based on this evaluation it can be determined if the slide can be used even though not processed inside the allowed limits Special care shall be taken if the time has been reduced because the level of positivity in the samples is not known and can differ from the positive control and thereby potentially result in a false negative result Check of leveling of the slide during the staining process How this is done will vary based on the automated platform used The information can be obtained either from the user guides for the automated platform or requested from the the supplier It is important to check the level of Troubleshooting Chapter 16 Comments If problems with the automated platform is indentified it is recommended to check other samples processed in the same period on the same platform or platforms in order to identify potential relations To confirm or discharge the automated platform as the reason to problems with the staining quality it is a good idea to search for related sample
6. not be identified by the negative control as it will be present both on the positive control and the negative control In order to identify this contamination it is necessary to make an investigational test where the first dispense of substrate chromogen can be removed and examined by mass spectrometry or ELISA for small quantities of visualization component Contamination with bacterial and or fungal growth can be seen in automated IHC platforms when the recommended maintenance schedule is not followed and or some parts has been defective which increase the risk of bacterial and or fungal growth Inspection of the visual parts of the instrument as well as keeping the maintenance schedule can prevent the contamination from happening If growth is expected normal microbiological methods can be used to determine the presence of both bacteria and fungus After identification the automated platform has to be cleaned according to specifications listed in the user guides or the supplier can be contacted for advice on how to clean Contamination caused by inadequate wash of the automated platforms probe After the probe aspirates reagent it has to be washed before transferred to another reagent bottle If this wash is not adequate then the second bottle can be contaminated with reagent from the previous bottle This will be identified as an unspecific reaction for a given marker e g CD20 staining in the nuclei if the first aspiration wash from Ki67 This contam
7. sections Can be diluted so as not to obliterate the specific signal m Reduce incubation time of the counterstain Repeat substrate chromogen treatment with correctly prepared reagent Staining intensity may be decreased when excess DAB DAB is present in the working reagent Check compatibility of buffer ingredients with enzyme and substrate chromogen reagents Repeat staining Commercial phosphate buffers may contain additives that will inhibit alkaline phosphates activity Avoid sodium azide in diluents and buffers A concentration of 15 mM L sodium azide which is routinely added to IHC reagents to inhibit bacterial growth will not impair HRP conjugated labels Utilize a higher sensitivity staining system Prolong incubation time of primary antibody Re optimize incubation times and concentrations of ancillary reagents Perform antigen retrieval if applicable using a range of pH buffers see Chapter 3 Re optimize concentration of the primary antibody and ancillary reagents Antibody concentration of the primary antibody may be too high Check manufacturer s specifications regarding recommended fixatives known to be effective with antibody and protocol in use Use a paraffin wax with a melting temperature 55 58 C Wax used for embedding should not exceed 60 C Oven temperature not to exceed 60 C NOTE The intensity of immunostaining may be diminished when tissue is exposed to prolonged heat at this stage in the p
8. Part Il The Potentials and Pitfalls Ypa ko A Better Path Chapter 16 roublesnooting Helle Grann Wendelboe MSc Anette Lykke MLS Dip Gale Pace MI ASCP BSc l p 4 DE a STA he MT nee tale i lt Mia _ a i i h i a a 3 P3 x ata iP By f s TE l k i j p Meee cme RE O r i M N F Aa y i f E li Fa P l h lt pe Fs h a k T i E s b he x n E L i y r ei ia E k m ea 2 i is it a i j i a j t 2 Cai ja L Trouebleeshooteing n Discovering why something does not work effectively and making suggestions about how to improve it Cambridge Advanced Learner s Dictionary Click here for all chapters Eee Agilent Technologies Troubleshooting Chapter 16 Chapter 16 1 Introduction Immunohistochemistry IHC is a multi step process that requires specialized training in the processing of tissue the selection of appropriate reagents and interpretation of the stained tissue sec tions In general IHC staining techniques allow for the visualiza tion of antigens by sequential application of a specific antibody to the antigen a secondary antibody to the primary antibody an enzyme complex and a chromogenic substrate The enzymatic activation of the chromogen results in a visible reaction product at the antigen site Because of its highly complex nature the causes of unexpected negative reactions undesired specific staining or undesired back
9. as to procedure at each step in the Total Test Species important to note in research studies Organ tissue source Collection Surgical specimen biopsy Post mortem specimen Fine needle aspirate Peripheral blood include anti coagulant Brushing Biologic fluid Cell culture Other Tissue preparation Paraffin embedded Plastic embedded Cryostat section Oo OF OF 0 0 8 O Cytospin Cell smear Mono layer cultured cells Other Tissue fixation Oo O OF OF 0 O0 O O Type of fixative O Total length of time in fixative including during transport grossing and on the tissue processor O Size of specimen size of block wheterh additional blocks are available if needed Tissue mounting slide mount Tissue thickness Gelatin glue commercial adhesive or starch in the water bath Other Oo OF QO o0 Blocking of endogenous components that may produce spu rious staining Background staining is defined as unexpected or undesirable staining seen on the test or control tissue which does not repre sent the target antigen Frequent causes of background staining are endogenous enzyme activity and endogenous biotin Peroxidase is an enzyme of the oxido reductase class that re acts with a substrate containing hydrogen peroxide as the elec tron acceptor To block this activity a variety of hydrogen per oxide reagents can be applied to cells producing this enzyme Alkaline phosphatase is an enzy
10. ation of the validated procedure is not affected Re optimize concentration of counterstain and incubation time Ensure automated stainer is programmed correctly and is running to manufacturer s specifications Standardize routine fixation matching test specimens to control tissues Proteolytic digestion or antigen retrieval will break down cross linking and render some tissue antigens reactive Chapter 3 Refer to the primary antibody specification sheet for additional information Serum proteins diffuse through tissue and are fixed in place Cut new tissue block if available using sharp blade Ignore physically damaged portions of stained tissue sections Fix tissue biopsy for longer period of time or fix smaller pieces to ensure complete penetration Unfixed tissue tends to bind all reagents non specifically Reduce incubation time Repeat incubation with correctly prepared chromogen reagent Absorb link antibody with tissue protein extract or species specific normal serum from tissue donor Repeat staining Determine correct concentration for each reagent Incubation temperature and incubation time will affect results To determine optimal incubation protocol vary both the time and temperature for each reagent in the IHC staining protocol Repeat incubation with correctly prepared chromogen reagent Chapter 16 Troubleshooting Possible cause of poor staining Slides inadequately rinsed Insufficient saline or de
11. ck with a biotin block or switch to a staining system that Is not dependent on the streptavidin biotin reaction Red blue color observed m Indicates non specific or undesired binding of the secondary antibody to the tissue sections This primarily occurs when the secondary antiserum has not been prepared for use on a specific species tissue m To determine if this is the problem absorb out non specific proteins by adding 2 5 or 10 uL of normal serum from the species of tissue to be stained per 100 uL of the secondary antibody Red blue color observed m May indicate non specific binding of the primary antibody carrier protein Perform a protein block with normal serum from the host of the link antibody or a protein block add 0 05 0 1 TWEEN 20 to wash buffer to decrease protein attachment m Antigen retrieval lipofusion artifact may appear as granule staining in liver and cardiac tissue or as specific staining in pancreatic sections POCO E OOOOH OT EEEE TEE EEO ESOHHTEEEO EEO OHOETEEOHEESEHE TESTO TS OSSOE ETOH ES OSEHEESESSOESEOT ESOT OT ETOH TECESESTESESESSESEEEESEOEE Red blue color observed on Negative Control Tissue m Monoclonal antibody Possible contamination m Polyclonal antibody Possible contamination or undesired antibody in the host lg fraction m Antigen retrieval lipofusion artifact may appear as granule Staining in liver and cardiac tissue or as specific staining in pancreatic sections Chapter 16 Troubleshoo
12. d staining NOTE If your state regulatory agency requires written documentation that a reagent can be used for automated staining and this indication is not listed on the specification sheet you may wish to contact the manufacturer s technical support group for further information Includes a suggested dilution for the antibody and the recommended diluent The dilution is a suggested starting point but may require further optimization depending on specimen preparation method temperature of the laboratory or automated instrumentation Information You Need to Know Controls Positive Control Tissue Information Located on the Specification Sheet Package Insert Staining procedure Controls Positive and negative control tissues should be run simultaneously using the same protocol as the patient specimens The positive control tissue should include prostate and the cells structures should display reaction patterns as described for this tissue in the Performance characteristics section Negative control The recommended negative control reagent is Dako Negative Control Mouse IgG1 Code X0931 diluted to the same Ig concentration as the primary antibody Unless the stability of the diluted antibody and negative control has been established in the actual staining procedure dilute these reagents immediately prior to use Positive and negative controls should be run simultaneously with patient specimens Performance cha
13. e user guides or can be provided by the supplier Check that the incubation times the slide actually received are within the allowed limits for the assay performed Variation in incubation time can result in variation in staining results Too long incubation time can potentially lead to increased level of background staining which can influence the interpretation of the slide Too short incubation times can lead to false negative results Wash Find the location of the executed wash incubation times for the slide in question This location can vary based on which automated platform has been used The location of this information is accessible either from the user guides or can be provided by the supplier Check that the incubation time the slide actually received in the wash steps are within the allowed limits for the assay performed Wide variation in wash times can be allowed for most assays However too short wash time potentially can result in unwanted specific as well as non specific background due to inadequate wash Extensive wash times can for special assays lead to reduced intensity Check that the wash buffer volume has been within the acceptable limits How this is done can be different based on the automated platform used Most platforms have estimated buffer consumption per run which can be checked against the actual usages Furthermore description of how to check the wash volume per slide may be provided in the user guides or can
14. e Specification Sheet Package Insert Intended use For in vitro diagnostic use Specimen preparation Paraffin sections The antibody can be used for labeling formalin fixed paraffin embedded tissue sections Tissue specimens should be cut into sections of approximately 4 um Pre treatment Pre treatment of formalin fixed paraffin embedded tissue sections with heat induced epitope retrieval HIER is required Optimal results are obtained by pretreating tissues with HIER using diluted EnVision FLEX Target Retrieval Solution High pH 50x Codes K8000 K8004 Deparaffinization rehydration and epitope retrieval can be performed in Dako PT Link Code PT100 PT101 For details please refer to PT Link User Guide The tissue sections should not dry out during the treatment or during the following immunohistochemical staining procedure For greater adherence of tissue sections to glass slides the use of FLEX IHC Microscope Slides Code K8020 is recommended After staining the sections must be dehydrated cleared and mounted using permanent mounting medium Staining procedure Visualization The recommended visualization system is EnVision FLEX High pH Code K8000 K8010 using a 20 minute incubation at room temperature Follow the procedure enclosed with the selected visualization system s Automation The antibody is well suited for immunohistochemical staining using automated platforms such as Dako Autostainer Autostainer Pl
15. e has been held within the limits of the target retrieval equipment throughout the course of the target retrieval process A too high temperature can lead to impaired morphology and over retrieval of the antigen epitopes Low temperature can lead to inadequate retrieval of the epitopes and thereby reduced staining intensity or lack of stained epitopes The temperature data for the slide can be located at different places in the instrument software based on which automated solution is used It is recommended to consult the user guides for the automated platform or to contact the supplier High altitude installations need to provide information in the datalog that appropriate temperature was achieved Target retrieval time Find the location of the specific data for the target retrieval time The target retrieval time for the slide can be located at different places in the automated platforms software based on which automated solution is used It is recommended to consult the user guides for the automated platform to find the location or to contact the supplier Check that the time the slide actually received target retrieval is within the allowed limit for the assay performed If positive control has been run for the assay on the slide in question an evaluation of the effect of any deviations can be made based on the positive control Based on this evaluation it can be determined if the slide can be used even though not processed inside the all
16. e sections should be approximately 4 6 um cryostat section 4 6 um or less Seen in frozen sections cell smears and non paraffin embedded tissue incomplete permeabilization of cells allows unattached reagents to become trapped within the cells and resistant to removal by wash buffer Possible cause of poor staining Negative control serum insufficiently diluted Contaminating antibodies in the negative control serum are cross reacting with proteins from the specimen tissue Negative reagent control serum contaminated with bacterial or fungal growth Limited Background Protein trapped beneath the tissue during the mounting process will allow partial lifting of the section Pooling of IHC reagents beneath the section or partial detachment of the tissue from the slide may occur Undissolved granules of chromogen Incomplete dezenkerization of tissue fixed with B5 or mercury containing reagents Incomplete dezenkerization of tissue fixed with B5 or mercury containing reagents Bacterial or yeast contamination from mounting waterbath Partial drying of tissue prior to fixation Unaffected areas show normal staining Instrument malfunction Hydrophobic and ionic interactions between immunoglobulins and lipoid substances in fatty tissue Primary antibody and negative reagent control serum are insufficiently diluted Both the primary antibody and negative control serum contain contaminating antibodies to epithelial e
17. ecific or undesired binding of the secondary antibody to the tissue sections This primarily occurs when the secondary antiserum has not been prepared for use on a specific species tissue m To determine if this is the problem absorb out non specific proteins by adding 2 5 or 10 uL of normal serum from the species of tissue to be stained per 100 uL of the secondary antibody POCO OTOH EO HE HSE HOES E ETOH SE SEOEO EEO HOEEOOTHESEHOEEE OTOH SE HOOT SEOH OEE OOTOEEEHOES EOE HSETOTTEHEHOEEHEOTE TEESE EEEHSOLESEEED Brown red color observed m May indicate non specific binding of the primary antibody carrier protein Perform a protein block with normal serum from the host of the link antibody Add 0 05 0 1 Tween 20 to wash buffer to decrease protein attachment m Antigen retrieval lipofusion artifact may appear as granule staining in liver and cardiac tissue or as specific staining in pancreatic sections POCO EEE OO HSE EHE TELE EE SEE HEO EE SEET OT EESO ETO HHE TEE OEESCHTEHEESEOT EE EOSEE SETS TETHOET EERE TESOEET EHS ETEESEHEEEEESEESOED EEE EEES Brown red color observed on Negative Control Tissue m Monoclonal antibody Possible contamination m Polyclonal antibody Possible contamination or undesired antibody in the host lg fraction m Antigen retrieval lipofusion artifact may appear as granule staining in liver and cardiac tissue or as specific staining in pancreatic sections Red blue color observed m Indicates endogenous alka
18. es like different automated platform or other reagents in the process It is recommended that controls are applied on every slide to ensure that it has received the exact same treatment as the sample being evaluated which supports the trouble shooting for the samples to a higher degree than a separate run or daily control will do Information You Need to Know Information Located at the Instrument Check whether the reagent has been stored according to recommendation from the supplier Check that the reagent volume usages have been adequate compared to the calculated use This can be done by checking the bottle history together with the weight of an empty container and compare this to the container used making it possible to estimate the consumption Staining temperature If temperature control is used during the staining process then check that the temperature has been held within the limits of the staining process The location of the information can be found in the user guides for the automated platform or provided by the supplier Check that the operating conditions for the automated platform are within specifications The specified operating conditions for automated platforms are listed in the user guides Incubation time Find the location of the executed incubation times for the slide in question This location can vary based on which automated platform is used The location of this information is accessible either from th
19. f the antibody diluent can cause a diminution in the sensitivity of the antibody Addition of NaN should be avoided This problem is primarily seen with monoclonal antibodies Chapter 16 Troubleshooting Possible cause of poor staining Primary antibody defective one or several secondary or ancillary reagents defective Do NOT use product after expiration date stamped on vial Dissociation of primary antibody during washing or incubation with link antibodies Use of alcohol based counterstain and or alcohol based mounting media will remove aqueous based chromogens Excessive counterstaining may compromise proper interpretation of results Incorrect preparation of substrate chromogen mixture Incompatible buffer used for preparation of enzyme and substrate chromogen reagents Use of PBS wash buffer with an alkaline phosphatase staining system Sodium azide in reagent diluent or buffer baths for immunoperoxidase methodologies Antigen levels are too low for detection by the employed visualization system May be due to loss of antigenic differentiation in some tumors or loss of antigenicity due to sub optimal tissue fixation Steric hindrance due to high antigen level and possible prozone effect Use of inappropriate fixative Use of certain fixatives may damage or destroy antigens or epitopes in the tissue specimen Use of non cross linking fixatives may allow the elution of antigens soluble in IHC reagents
20. ground may be difficult to isolate The information contained in this chapter should enable the user rapidly to pinpoint and resolve problems encountered during the staining procedure section 1 is a compilation of common problems encountered when using IHC staining reagents including the underlying causes of staining failure and the recommended corrective actions The chart is divided into sections describing inade quate staining general background staining and limited back ground staining Section 1 Common Problems Inadequate Staining little or no specific staining Possible cause of poor staining Solution section 2 presents a method of systematically adding one IHC reagent at a time to determine at which stage in a staining protocol non specific or undesired staining may be occurring section 3 is a simple chart used to define the type of tissue specimen the IHC reagents and the staining protocol already in use by the laboratory personnel The user is encouraged to copy this chart and use it to help troubleshoot any problems that may be encountered in the IHC staining process section 4 is a guide to reading a manufacturers specification sheet for IVD in vitro diagnostic antibodies The guide includes general information for use in immunchistochemistry including fixation rec ommended visualization systems recommended titer and diluent pre treatment methods and selection of required controls section 5 is a guide t
21. has been used for in the bottle history can be located different places dependent of automated platform used If the bottle history shows successful use of the reagent prior to this then the right reagent is in the bottle Comments If any of the checks performed for the target retrieval step show irregularities it is recommended to re run the sample and or to get the automated equipment serviced by the manufacturer If a failure of either use of the target retrieval reagents or the target retrieval platform has been identified remember to search for other slides which potentially have been submitted to the same failure and perform a quality check of these related slides Check of the instrument overall performance is not based on a single slide However if a given automated platform is the main denominator between failing slides then it should be considered to make a check of the automated platform and potentially get it serviced by the supplier Important to check this whenever new protocols or tests have been added to the server of the system LIS may not automatically update and map new test protocols from the server If you use reagents that you dilute from concentrate then it is important to check that the dilution has been done correctly This can be done by validating the new dilution against a previous dilution still within the expiry limits It is recommended that these checks are done to eliminate other variabl
22. he surrounding conditions for optimal staining Improper closing can result in inability to start or create staining conditions which are sub optimal do to the air getting into the instrument through the open cabinet Instrumentation should be installed away from direct sunlight Sunlight can affect the staining conditions and viability of reagents Acknowledgements References Sections in whole or parts thereof from the previous editions 1 Wood G et al Suppression of endogenous avidin binding activity in tissues and its relevance to biotin avidin detection systems J of this Guidebook are used in the 6th edition We sincerely Histochem Cytochem 1981 29 1196 204 thank and acknowledge the contribution of the authors Spe 2 Sayaki H et al Azure B as a counterstain in the immunohistological l evaluation of heavily pigmented nevomelanocytic lesions Appl nical Support Group Immunohistochem 1995 3 268 71 cial acknowledgements to Karen N Atwood and Dako Tech 3 Federal Register January 24 2003 68 12 42CFR Part 493 4 College of American Pathology Anatomic Pathology Checklist October 2005 5 College of American Pathology Anatomic Pathology Checklist July 2013 p 34 38 Click here for all chapters
23. hematoxylin If Result Action does not match the observed staining Go to next step v Positive Control Tissue DAB AEC chromogen and counterstain If Result Action does not match the observed staining Go to next step v Positive Control Tissue Peroxidase Block Secondary Antibody Streptavidin HRP DAB AEC Counterstain If Result Action does not match the observed staining Go to next step v Result Action Brown endogenous pigment such as melanin observed m To distinguish melanin pigment from DAB chromogen Azure B can be used as a counterstain The melanin stains blue green while the DAB remains brown m An alternate method is to use AEC as the chromogen However if high levels of pigment exist in the tissue the red chromogen may be partially obscured Since bleaching protocols to remove melanin may compromise tissue antigenicity it should be avoided if at all possible secococoooocococoocococoocococococcoocococcocooococococococcococosoocococoococococoococococcococoooococooocoocococoooococoooooo Brown red color observed m Indicates endogenous peroxidase activity in the tissue sections It is present in all hemoprotein containing tissue including erythrocytes muscle liver kidney granulocytes and monocytes m Block with 3 hydrogen peroxide or other peroxidase blocking reagent Using a new bottle of hydrogen peroxide perform a 3 H 0 peroxidase block followed by DAB a
24. ination can be confirmed by having the probe go into a bottle of wash buffer or other neutral fluids after the first aspiration and then measure the content of the previous reagent by either mass spectrometry or ELISA Contamination of the tissue by the probe dropping a drop of unrelated reagent onto the slide This will be recognized as a false positive reaction either being in the wrong structure or wrong location Use of positive tissue controls including multiple organ types on each slide will make it possible to identify contamination of this origin Waste separation Automated platform separates hazardous from non hazardous waste Failure of this separation does not impact the staining process and thereby should not influence the staining result Bulk fluid supply The automated platforms normally have a function which makes it possible to check whether the supply of bulk fluids is working adequately This check can be a prime of the bulk fluid trough the system securing that there are no leakages or clots preventing the fluid from flowing Check of the bulk fluid supply is described in the user guides or can be requested from the supplier General If in doubt about the performance of the instrument thorough observation of the automated platform during the staining process can give an indication of whether the individual steps is being executed as expected Check that the instrument is closed correctly as the performance depends on t
25. ive Loss of antigenicity in unfixed tissue General Background Excessive incubation with substrate chromogen reagent Substrate chromogen reagent prepared incorrectly Secondary or link antibody cross reacts with antigens from tissue specimen Secondary or link antibody and or tertiary reagents too concentrated Substrate chromogen reagent prepared incorrectly Troubleshooting Chapter 16 Solution Excess residual reagent will dilute the next consecutive reagent Repeat staining making sure to wipe away excess washing buffer and blocking serum Many tissue antigens require proteolytic enzyme digestion or heat induced antigen retrieval performed prior to staining Chapter 3 The need for pre treatment depends on the type and extent of fixation specific characteristics of the antigen and the type of antibody used Use the pretreatment method recommended by the manufacturer No single pre treatment is suitable for all applications Do not reuse buffer Prepare new sections and deparaffinize according to standard laboratory protocol using fresh xylene or xylene substitute m When using a waterbath or steamer allow sufficient time for the retrieval buffer to equilibrate to a temperature range of 95 99 C At high altitude greater than 4 500 feet the buffer will boil at less than 95 C Utilize a closed heating system such as a pressure cooker autoclave or Pascal or utilize a low temperature protocol if standardiz
26. lements possibly cytokeratins Excessive formalin fixation of tissues may increase protein cross linking resulting in tissue hydrophobicity Troubleshooting Chapter 16 Solution Use properly diluted negative reagent control serum For polyclonal antibodies dilute the negative reagent control serum until the protein concentration is equal to that of the primary antibody For monoclonal antibodies dilute the negative reagent control serum until the Ig concentration is equal to that of the primary antibody Replace the negative reagent control serum repeat staining protocol Replace product with non contaminated serum Avoid the use of commercial adhesives glue starch or gelatin in water baths when mounting tissue sections Avoid allowing water from an initial section mounting to flow over an area where additional sections will be mounted This is particularly important when using charged or silanized slides Ensure that chromogen in tablet or powder form is completely dissolved or switch to a liquid chromogen Remove embedding medium thoroughly using fresh reagents Perform dezenkerization with fresh reagents Clean and refill waterbath Immerse tissue promptly in fixative or holding reagent Keep moist during the entire staining process Use a humidity or moist chamber during incubation steps m When using an automated staining instrument addition of wet towels to the sink may prevent drying of slides
27. line phosphatase activity in the tissue sections It is present in liver kidney GI tract bone bladder ovary salivary gland placenta leukemic necrotic or degenerated cells m Block with levamisole Intestinal alkaline phosphatase may be quenched by the addition of 0 03 N HCI prior to the addition of the alkaline phosphatase Troubleshooting Chapter 16 Reagents Positive Control Tissue streptavidin AP Fast Red Fuchsin or BCIP NBT Counterstain If Result Action does not match the observed staining Go to next step v Positive Control Tissue Biotin Block if required Secondary Antibody Streptavidin AP Fast Red Fuchsin or BCIP NBT Counterstain If Result Action does not match the observed staining Go to next step VW Positive Control Tissue Biotin Block if required Negative Re agent Control Secondary Antibody Streptavidin AP Fast Red Fuchsin or BCIP NBT Counterstain If Result Action does not match the observed staining Go to next step v Negative Control Tissue Perform complete staining protocol Result Action Red Blue color observed m Indicates endogenous biotin activity in the tissue sections Protein bound biotin may be found in adrenal liver kidney Gl tract lung spleen brain mammary gland adipose tissue lymphoid tissue and cells grown in culture media containing biotin RPMI NCTC MEME m Blo
28. me having various isoforms which are produced in the leukocytes liver bone intestine placenta and Regan carcinoma Addition of levamisole to the chromogen substrate will inhibit endogenous alkaline phos phatase activity with the exception of the intestinal isoform If necessary this can be blocked with a weak acid wash such as 0 03 0 5 N HCI Biotin a B vitamin may be protein bound to tissue and can interfere with proper interpretation of staining patterns when using a streptavidin or avidin reagent To block this binding a biotin avidin block Peroxidase block 3 H O Methanol H O Sodium azide Peroxidase Block e g Dako Code S2003 Other Alkaline phosphatase block 0 O 0 Q O0 o Levamisole o 0 03 N HCI not for use on cryostat tissue O Other Biotin block O Biotin Block e g Dako Code X0590 o Other e g skimmed milk Protein block o Protein Block e g Dako Code X0909 o Normal sera or lg from host species of the secondary antibody o Other Chapter 16 Troubleshooting Section 4 Specification Sheets Below is an example of the information supplied in a typical Dako package insert for an IVD in vitro diagnostic concentrated antibody The information and placement in the package insert will vary Information You Need to Know Regulatory Status of the Primary Antibody Tissue Preparation Choosing the Visualization System Diluting the Primary Antibody Information Located on th
29. nd an appropriate counterstain COCO EE HEHE SESE SETH HHEESEEEES OSHS OEE H EE OESOE ESTOS ESSE ESOS H ESHEETS OEE EOESHEE OES EESHEEEESEOTESOEHEETEOEESOESEEEEEOES Brown red color observed m Indicates endogenous biotin activity in the tissue sections Protein bound biotin may be found in adrenal liver kidney GI tract lung spleen brain mammary gland adipose tissue lymphoid tissue and cell grown in culture media containing biotin RPMI NCTC MEME m Block with a biotin block or switch to a staining system that is not dependent on the streptavidin biotin reaction Chapter 16 Troubleshooting Reagents Positive Control Tissue Peroxidase Block Biotin Block if required Secondary Antibody Streptavidin HRP DAB AEC Counterstain If Result Action does not match the observed staining Go to next step v Positive Control Tissue Peroxidase Block Biotin Block if required Negative Reagent Control Secondary Antibody Streptavidin HRP DAB AEC If Result Action does not match the observed staining Go to next step v Negative Control Tissue Perform complete staining protocol Background Staining Encountered with Alkaline Phosphatase Positive Control Tissue Fast Red Fuchsin or BCIP NBT Counterstain If Result Action does not match the observed staining Go to next step v Result Action Brown red color observed m Indicates non sp
30. o check that the automated platform used to perform the staining has operated correctly during the stain ing process A Both the positive controls and the specimen tissue show little or no specific staining except for counterstain The tissue section may show little or no background staining Primary antibody or labeled reagent omitted Reagents used in wrong order Repeat the procedure using the manufacturer s staining system specification sheet or the standard operating procedure reagent checklist as established by the individual laboratory Excessively diluted or excessively concentrated reagents inappropriate incubation time and or temperature Determine correct concentration for each reagent see Chapter 4 and Chapter 5 Depending on the degree of staining obtained if any a 2 to 5 fold increase in concentration may be needed Incubation temperature and incubation time are inversely proportional and will affect results To determine optimal incubation protocol vary either the time or temperature for each reagent in the IHC staining system Generally incubation times can be extended if little or no background is detected Overnight incubation at higher dilution may also be effective Primary antibody diluted with inappropriate buffer Use of PBS or TBS as an antibody diluent Lack of stabilizing or carrier protein Detergent in diluent Check formula and compatibility of antibody diluent A change of ion content and or pH o
31. ormulation Block with serum from the host of the secondary or link antibody Avoid serum that contains auto immune immunoglobulins Alternatively a serum free protein block lacking immunoglobulins may be substituted for the serum block Prepare new sections and deparaffinize according to standard laboratory protocol using fresh xylene or xylene substitute Use a secondary antibody that has been absorbed against a species specimen or use a secondary antibody produced in a host that exhibits little or no cross reactivity with the tissue source Ensure automated stainer is programmed correctly and is running to manufacturer s specification Standardize routine fixation Proteolytic digestion or antigen retrieval will break down cross linking and render some tissue antigens reactive Refer to the primary antibody specification sheet for additional information Fix tissue biopsy for longer period of time or fix smaller pieces to ensure complete penetration Serum proteins diffuse through tissue and are fixed in place Re cut tissue using sharp blade Ignore physically damaged portions of stained tissue sections Some IHC reagents may bind to these products resulting in a light stain over the entire slide surface Some slides may be unevenly coated and will exhibit the above problems on only a portion of the tissue or glass Avoid delays in fixation of the tissue Cut tissue sections thinner Formalin fixed paraffin embedded tissu
32. otin block or chose another non biotin based staining system Should not interfere with interpretation of specific staining m Use F ab or F ab fragments for the primary and secondary antibodies rather than intact antibodies Add detergent to the wash buffer Rare Should not interfere with interpretation of specific staining Utilize a non peroxidase staining system Utilize an in vivo product for application on viable cells For use on cell cultures only sodium azide may be dialyzed out of some reagents Contact Dako Technical Support for additional information Troubleshooting Chapter 16 Section 2 Systematical Approach Using a Troubleshooting Flow Chart This flow chart can be used to determine source s of non specif ic staining that has been encountered when using an IHC staining protocol Each step Slide box to the left is a suggestion for rea gents to be tested on the indicated tissue type with known posi tive or negative expression pattern In the first step Slide 1 the tissue is only counterstained with hematoxylin If the described result to the right does not match the observed staining pattern when using the suggested setup proceed to the next step in the flow chart In the next step Slide 2 a chromogen is added to the staining protocol before counterstaining and so forth Background Staining Encountered with Peroxidase Reagents Reagents Positive Control Tissue Counterstain with
33. owed limits Special care shall be taken if the time has been reduced because the level of positivity in the samples is not known and can differ from the positive control and thereby potentially result in a false negative result Protocols from the server Verify that correct protocols are received from the server Find the appropriate test name for the protocol Verify that this name is mapped in the list of IHC or ISH test protocols Contact the vendor technical support or bioinformatics department for assistance Staining process Reagents Find the location of the specific data for the executed protocol this can be separate or a part of the slides log file under completed slides The location of the information can be looked up in the user guides for the automated platform or provided by the supplier Check that the right protocol including the right reagents have been used to stain the slide in question Sub optimal results may be seen if another reagent than the validated for the assay either by the laboratory itself or the supplier and the laboratory in combination is used Check that the right reagent volume has been applied according to the protocol Check that all the reagents used have been within the expiration limits when used for the slide Check that the label of the reagents used is actually in agreement with liquid in the bottles used on the automated platform This can be done by looking at what the bottle previously
34. racteristics Normal tissues In prostate glandular epithelial cells show a moderate to strong cytoplasmic and or membranous staining reaction Abnormal tissues In 92 102 prostate adenocarcinoma glandular epithelial cells showed a moderate to strong cytoplasmic and or membranous staining reaction section 5 Automated Platform Performance Checks Information You Need to Know The right protocol has been applied to the slide Information Located at the Instrument Right protocol Find the location for the completed slides in the automation software and look up the slide ID of the slide in question and check that the applied protocol is the correct one Troubleshooting Chapter 16 Comments Use of a negative reagent control ANP 22570 is no longer required by the College of American Pathologists CAP based on Clinical Laboratory Improvement Amendments revised July 31 CLIA 2012 for each patient or patient block in a staining run when using polymer detection systems The latest guidelines from CAP 5 leave it up to the individual laboratory to evaluate whether the selected detection method poses a negligible risk for non specific staining that the negative reagent control for the polymer based detection methods can be eliminated CLIA 2003 Sec 493 1273 3 Mandates that fluorescent and immunohistochemical stains must be checked for appropriate positive and negative reactivity each time they are used Most IVD
35. rotocol Paradoxically heating of the section in aqueous solution is used to recover antigenicity in the AR process See Chapter 3 Refer to the primary antibody specification sheet for additional information The intensity of immunostaining may be diminished when pre cut tissue sections are exposed to air Use freshly cut sections and re seal paraffin embedded blocks More common on frozen sections apply the primary antibody prior to the enzymatic block to ensure its reaction In such cases the blocking reagent can be applied at any point after the primary and before the enzyme labeled components Possible cause of poor staining Excessive wash buffer or blocking serum remaining on tissue section prior to application of IHC reagents Antigen retrieval protocol is inappropriate or has been omitted Repeated reuse of antigen retrieval buffer Sections incorrectly dewaxed Failure to achieve the optimal temperature required for heat induced antigen retrieval Excessive or incomplete counterstaining Instrument malfunction Specimen held for too long in a cross linking fixative usually in formalin causing masking of antigenic determinants Control appropriately fixed Sectioned portion contains crush artifact caused by grossing tissue with dull scalpel or razor Sectioned portion of specimen contains necrotic or otherwise damaged elements Uneven fixation of section portion of specimen not penetrated by fixat
36. s e g other samples treated on the same automated platform run or in the same slide position this can help lead in the right direction when doing the troubleshooting If problems with the automated platform is identified a service of the instrument is recommended the slide as a slide which is not in level during staining can lead to the area of interest not receiving the necessary reagents This will result in inconsistent staining Chapter 16 Troubleshooting Information You Need to Know Information Located at the Instrument Automated IHC staining platform Automated IHC staining platform has been performing as expected Reagent volume m Make sure that the volume your automated platform is set to use is adequate to cover the entire area of your sample over the total duration of the incubation time It is important to take into account the potential evaporation of the reagent over time Drying out of the tissue during the staining process can result in staining effects including no staining inconsistent staining extensive background and other staining artifacts Contamination Contamination of the probe on the automated platform can lead to mixing of reagents in the probe and or on the slide This can lead to false positive staining or background on the slide The inclusion of negative tissue controls can help identify if a contamination has taken place A contamination of the visualization system in the substrate chromogen will
37. tergent in wash buffer Blocking serum or wrong blocking serum used Sections incorrectly dewaxed Non specific binding of the secondary antibody with an animal tissue specimen Instrument malfunction Specimen held for too long in a cross linking fixative usually in formalin causing masking of antigenic determinants due to aldehydes cross linking and increased hydrophobicity of tissue Sectioned portion of specimen not penetrated by fixative Loss of antigenicity in unfixed tissue Unfixed tissue tends to bind all reagents nonspecifically Sectioned portion contains crush artifact caused by grossing tissue with dull scalpel or razor Serum proteins diffuse through tissue and are fixed in place Sectioned portion of specimen contains necrotic or otherwise damaged elements Excessive or unevenly applied subbing agent on poly L lysine charged or silanized slides Antigen diffusion prior to fixation causing specific background outside the expected antigen site Tissue sections too thick Incomplete permeabilization of tissue sections Solution Gently rinse slide with wash buffer bottle and place in wash bath for 5 minutes Gentle agitation of the wash bath may increase effectiveness when used with cytoplasmic or nuclear staining protocols High sensitivity staining systems may require higher concentrations of saline or detergent in the wash buffer Refer to the staining system specification sheet for optimal f
38. ting Negative Reagent Control Reagents Result Action m Human tissue Perform the peroxidase blocking protocol Negative Control Reagent from Slide 2 under Background Staining Encountered with Perform complete staining protocol Peroxidase Reagents m Perform a biotin block if required protein block if required apply the appropriate negative reagent control see below apply biotinylated secondary antibody apply streptavidin HRP reagent and DAB m Prepare a negative reagent control Polyclonal non immunized sera from the same species diluted to the same protein concentration as the primary antibody Monoclonal negative reagent control that matches the isotype as the primary antibody Additionally the diluent used to manufacture a monoclonal primary antibody and isotypic negative control should contain the same ions Diluents containing sodium or phosphate ions may change the sensitivity of some monoclonal antibodies Calculation lg or total protein concentration of primary antibody divided by dilution factor of primary antibody x lg or total protein concentration of negative reagent control divided by x dilution factor of negative rea gent control Troubleshooting Chapter 16 Section 3 Troubleshooting Chart Tissue Specimen Successful staining of tissue with an IHC marker is dependent on the type and preparation of the spec imen The chart below provides a convenient check list
39. us and Autostainer Link as well as PT Link for pre treatment Staining procedure Dilution The recommended dilution of Monoclonal Mouse Anti Human PSMA Clone 3E6 Code M3620 is 1 50 Dilute the antibody in Dako Antibody Diluent Code S0809 Incubate pretreated tissue sections for 20 minutes at room temperature These are guidelines only Optimal conditions may vary depending on specimen and preparation method and should be validated individually by each laboratory Comments Indicates that a product meets the FDA requirements as a clinical diagnostic product Likewise a CE icon indicates the reagent meets European Union requirements Indicates the type of specimen that was used during validation studies In many cases this would include formalin fixed tissue and frozen sections Use of other fixatives requires validation by each individual laboratory This section also indicates the optimal epitope retrieval procedure and warns against procedures that may destroy the epitope Specimen preparation and staining procedure sections can and will change periodically to reflect changes in technology So remember to retain copies of each version of the reagent specification sheet Version numbers are usually found on each page Indicates the recommended visualization system to be used with the antibody Conditions will differ if other detection systems are used It also indicates that the antibody can be used for automate
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