Immunohistochem test2
Cause : Primary reagents
Ab was or any other reagent was defective . Solution : Change your reagents or order new antibody . Cause Primary
Antigen
Antibody A Antigenic determinants on antigen Antigens Binding sites of cell components wall Antibody
IgM Antibodies
Antigen binding sites J chain virtualmedicalcentre.com
Antigens and Epitopes
Antigens are almost always proteins . Sometimes epitopes are referred to as antigenic determinants
Antigens and Epitopes
Antigens are almost always proteins . Sometimes epitopes are referred to as antigenic determinants .
Principle of Fluorescence Example Fluoroscein will fluoresce when
Example Fluoroscein a fluorescent dye ) will fluoresce when hit by light with any wavelength between 450 nm and 520 nm . However , the closer the wavelength is to 495 nm the more fluorescence will be produced . The optimal wavelength is called the excitation peak .
Combined HIER & EIER
The use of enzyme digestion alone for epitope retrieval reduce the quality of tissue morphology because of a ling exposure time . Therefore it may be useful to combine bod methods of epitope retrieval to minimize the extent of ene pretreatment required . In general , HIER is performed firs followed by rinsing , and then enzyme digestion for a short from 1-5 minutes ) .
Two types of IF
Primary Antibody Direct Immunofluorescence Fluorochrome Indirect Immunofluorescence Secondary Antibody
Cause the stain
Primary Antibody or any other reagent was omitted . Solution : Repeat the stain using the manufacture's staining system . Do the stain again but use the reagent you omitted
Fixatives for paraffin processed tissue
Proper tissue handling , beginning with fixation and continuing through processing and microtomy , is pivotal to producing consistent results when performing immunohisto chemical staining on paraffin sections . It is important for the immunohistochemist to understand the basics of how fixation , processing , temperature , and pH can affect the quality of a stain . Preanalytical factors strongly influence immunohistochemical results and consistency .
Ideal Fluorochrome Properties
Properties An absorption peak at an excitation wavelength available on the fluorescence detection instrument ( fluorescent microscope ) . • Bright fluorescence • A narrow emission spectrum • Good photostability Fluorescent properties that aren't significantly altered by conjugation to an antibody or environment of the sample .
Advantages of Direct IF
Shorter staining times . You only need to apply one antibody . Simpler protocols
Principle of Fluorescence The excited state of the electron doesn't last Generally
long . Generally , less than 10 seconds . • The electron loses energy and falls back to its original energy level . The energy given off during that process is in the form of a photon .
There are 2 approaches using no
new antibody development , the first using no retrieval , multiple retrieval solutions , and multiple enzyme pretreatments ; this approach may be necessary when validating a " homegrown " antibody or an antibody that has no previously documented use in the clinical laboratory . The second approach is researching the antibody and planning a development protocol using the wealth of information available Resources for a starting point are the antibody or detection vendor , antibody specification , journal articles , and text books [ Dabbs 2006 , Shi 2000 ] . These resources provide basic methods 262
Antibody
of the antigen is known as an epitope , and is the site at which the antibody attaches to tissue . An antibody may target more than 1 antigen , but it is specific for only 1 epitope . Proteins very good antigens because of their large size . The fact that anti are bodies ( eg , immunoglobulins ) are proteins makes them very potent antigens .
Stokes shift Determines
what color light is given off by the fluorochrome . A fluorochrome with a small Stokes shift which is excited by green light may fluoresce green , however a fluorochrome with a larger Stokes shift that is excited by green light may fluoresce a yellow or orange light .
Principle of Fluorescence
. The emitted fluorescence has a lower energy than the absorbed light , so the wavelength of the emitted light is longer than that of the excitation light . A range of wavelengths can excite the electrons of a fluorochrome ( fluorescent dye ) .
solution Molar Solutions ( M )
74.55 74.55 * CI = weight Example atoms the weight water ( = A 1 1M Mole of g Chloride Potassium is in sum ( formula of give ) solution to KCL 74.55 : the of Potassium the a a Molar is molecule final substance contains dissolved 35.45 is 39.10 combined Weight ) volume 1 chloride of expressed a 1 L ( in atomic of dissolved gram of KCI substance gram in distilled the grams ) in weights L 1 molecular is molecular all . of molecular weight ) enough water .
General immunology
( antibody ) and cellular components . The overview of the The immune system exerts its control through humoral or antibody system and how it relates to the discussion of immu immune system presented here will emphasize only the humoral nohistochemical techniques .
Dilutions for the Clinical Laboratory ( cont'd )
. Another example : One ( 1 ) part of concentrated acid is diluted with 99 parts of water . The total solution volume is 100 parts ( 1 part acid + 99 parts water ) . The dilution is written as 1/100 or said " one in one hundred " .
IHC stain Troubleshooting your
5. 2. Inadequate After you Cause : for sheet Cause : : Cause Higher your incubation Cause the the Cause stain reagents the : reagent stain : . counterstain interpretation the Staining your the of Counterstain . slide use and Ab was was lower of order Ab , temp Excessive Ab Primary Primary or concentration and Excessively you time using Primary ( slides the little omitted Antibody no to and the counterstaining the diluted new or the temps diluted manufacture's . there for Solution . : Too correct with other and Remember along excessively staining other any time reagents . go can or any or staining a is correct Use reagent vice little or problem . amount a much of different . buffer the wrong versa . time was that with reagent system . was background defective and too . concentrated buffer . temperature counterstain . time counterstain . that Solution the Do omitted . Solution Solution . . reagents stain : : Dilute Check : again Solution your compromises the Change . Determine Inappropriate relationship use but : the data
Direct method
A labeled antibody of known specificity is used to identify antigens in the patient's tissue . An example is the detection of immune complex deposition in renal biopsy specimens using direct immunohistochemical techniques . In this method , the antibody may be labeled with FITC or other fluorescent dyes for fluorescence microscopy , or it may be labeled with an enzyme such as HRP , AP , or glucose oxidase for subsequent reaction with a chromogen and visualization with the light microscope f12.1 .
Solutions
A solution is a homogenous mixture of two or more substances . Can be in any form of matter : solid , liquid or gas Solutions are essential in most laboratory - based biomedical research Examples : buffers , reaction mixtures , cell culture media , cell lysates , etc.
compound . An HRP system in a clinical laboratory because it is routinely serves as a " work horse consistent when producing large quantities of slides . AEC is soluble in organic solvents and requires an aqueous mounting slides may be mounted with synthetic resins . medium , whereas DAB stained
AP is demonstrated by the e use use of of naphthol - AS - phosphate ( substrate ) and a chromogen such as fast red - violet LB , fast - red TR , or fast - blue BBN . AP is sensitive can lead to staining inconsistency . Sen Semipermanent to heat and light , which chromogens compatible with AP allow the use of a permanent mounting medium but require either no dehydration and air drying before coverslipping or a quick dehydration to xylene . If not properly dehydrated , the AP chromogen can break down and precipitate across the tissue i12.8 . A red chromogen signal can assist in and identifying antigen sites in those tissues containing melanin to igment which may obscure a brown signal i12.9 . If hematoxylin is used as a counterstain , a formula such as with Mayer hematoxylin is recommended because it does not contain , alcohol . If Harris or another hematoxylin solution containing alcohol is used with AEC or the AP chromogens , the reaction product will be dissolved and thus removed from section . A false - negative result will occur . Immunohistochemical
9. Cause : reusing the same
AR buffer . Solution : make new AR buffer or get new buffer . 10. Cause : Failure to achieve a high enough temperature for AR . Solution : Readjust temperature or use a closed heating system like a pressure cooker , or autoclave . General Background ( too much background staining ) 1. Cause : Excessive incubation time with chromagen . Solution : reduce incubation time * 2. Cause : Chromogen was prepared incorrectly . It may be too concentrated . Solution : Next time use the chromogen at its proper dilution . 3. Cause : Secondary or link Ab cross - reacts with antigens from the tissue ( endogenous ) . Solution Might need to use a different secondary . Check species of secondary in relationship to the primary .
Ideal Fluorochrome Properties
An absorption peak at an excitation wavelength available on the fluorescence detection instrument ( fluorescent microscope ) . • Bright fluorescence • A narrow emission spectrum Good photostability Fluorescent properties that aren't significantly altered by conjugation to an antibody or environment of the sample .
Antigen
An antigen is a molecule hydrates , or other polymers , and is made up of proteins capable of producing , carbo immune response in animals or cell cultures for the an bind antibodies at different production of antibodies . Antigens in tissues sites : the membrane cytoplasm cell nation of sites . Many compounds are antigenic large size , proteins are ideal for immunization nucleus , organelles , or a combi . Because of the and for binding , polymers also can antibody but polysaccharides , nucleic acids , and other be antigenic . The most common antigens that induce production by the body are bacteria and viruses .
Antigens and Epitopes
An antigen is a molecule that is capable of binding to an antibody . An epitope is the structural part of an antigen that reacts with an antibody .
Polyclonal Antibodies
Are a heterogenous mixture of antibodies directed against various epitopes of the same antigen . • The antibodies are generated by different B - cell ( white blood cells ) clones of an animal and consequently are dissimilar . The antibodies in polyclonal mixtures have different specificities and affinities .
Avidin - biotin methods
Avidin has a very high affinity for the vitamin biotin ( > 1 million times greater than the affinity of antibody for most antigens ) and the binding is essentially irreversible . Avidin isolated from egg white was used first in the ABC methods , but streptavidin , isolated from the microorganism Streptomyces avidinii , is more commonly used today . In the ABC method , the primary antibody is followed by a biotinylated secondary antibody ( linking antibody ) . The third step in the ABC method is the application of a preformed avidin - biotin enzyme complex . This method has low back ground economy , and sensitivity reported to be up to 40x that of other immunoperoxidase methods [ Hsu 1981 ] . The antibodies may be used at higher dilutions than in other techniques f12.3 .
Units of Metric System
Base Units Distance = meter ( m ) Mass or Weight = gram ( 9 ) Volume = liter ( L ) Prefixes are used to indicate larger or smaller quantities of the base units above
Storage of antibodies
Both the storage container and the temperature at which the antibody is stored are very important . Polypropylene , polycarbonate , or borosilicate glass is recommended for storage because these materials have low protein absorptive properties . If the antibody solution contains very low concentrations of protein , 0.1 % -1.0 % bovine serum albumin may be added to reduce loss through polymerization and absorption on the container [ Boenisch 2001 ] . The manufacturer's recommendations for the storage temperature should be carefully followed . Most prediluted antibodies and kits are stored at 4 ° C - 8 ° C to avoid repeated freezing and thawing . Once lyophilized antibodies have been diluted , they are usually divided into aliquots and stored at -70 ° C .
Direct
Used less frequently The primary antibody being used is conjugated to a fluorescent dye .
Processing
Complete fixation before processing is achieve optimal results for stains performed necessary to necessary for consistent staining in the tissue infiltration or molecular level . Thorough dehydration and at either a protein are section False negative or variable to proper staining at the staining in the middle of a section in edges is a common artifact produced fixed and the center is alcohol contra when the outer edge is formalin fixed , the result of underfixation before processing i12.3 The paraffin that is used in both the processor and embedding center should have a low melting point . Paraffin containing dimethyl sulfoxide may require extended deparaffinization times to ensure complete removal . Incomplete removal of paraffin may obstruct antibody - binding sites , producing variation of staining intensity Antigens are primarily proteins that can be altered by high temperatures . Tissue exposed to high temperatures during processing can accelerate the damage . Reprocessing tissue may alter the epitope sites , causing irreversible damage and leading to false - negative results
Disadvantages of Indirect IF
Cross - reactivity potential You have to find a primary and secondary that were raised in different species . Great chance of getting background staining
Dilutions for the Clinical Laboratory
Dilution = making weaker solutions from Laboratory stronger ones frozen frozen orange juice with concentrate . You Example : Making orange juice from three ( 3 ) cans mix one can of of water .
Dilutions for the Clinical Laboratory ( cont'd ) Dilutions original
Dilutions are always part of original substance diluted as one ( 1 ) . If expressed with the original substance more than one initially used , it is necessary to substance part to convert the original dilution is expressed . one ( 1 ) when the Example : 2 parts Ab and 8 parts diluent for a total of 10 parts . Expressed as 2/10 ( reduce your fraction ) = 1/5
Metric System English system of measurement is not enough for most scientific
English system of measurement is not accurate enough for most scientific measurements Because metric system is a decimal system , it can be used for very small quantities with accuracy International System of Units ( SI ) is a form of the metric system adopted for use by the worldwide scientific community .
Percent Solutions Calculating Percent Solutions Use the following formula
Example # 11 Prepare 100 mL of a 0.9 % solution of Sodium Chloride ( Solute ) and dissolved in distilled water ( solvent ) . % desired 0.9g / mL 100 X 100ml 0.9 g / ml 100ml 100 100X 0.9g ( 100 ) = 90g 100 X = 0.9g of Sodium Chloride and 100ml of dichillad wstar
Principle of Fluorescence Example Fluoroscein fluoresce
Example Fluoroscein ( a fluorescent dye will fluoresce when hit by light with any wavelength between 450 nm and 520 nm . However , the closer the wavelength is to 495 nm the more fluorescence will be produced . The optimal wavelength is called the excitation peak .
Cause incubation
Excessively diluted or excessively concentrated reagents . Inappropriate incubation time and temps can go along with that too . Solution : Determine the right concentration of reagents . Remember time and temperature relationship . Higher the temp lower the time and vice versa .
IF
Fluorescence occurs when molecules light and emit light of a different with luminescent properties absorb wavelength . after the absorption of light . • The emission of light occurs rapidly
Principle of Fluorescence
Fluorescent materials give off light because of their atomic structure . Electrons around an atom are arranged in different energy levels ● Each energy level as a predetermined amount energy . . When an electron absorbs the energy from light , it becomes " excited " and jumps to a higher , less stable energy level .
Buffer solutions
For reproducible results , the pH of the buffer used during staining must be maintained and checked often . For each new of commercially prepared buffer , or each time a buffer is prepared , the immunohistochemist should check and document its pH before use . Typically , 2 buffers are used during the staining process : PBS and trisbase solution ( TBS ) . Both can be used with an HRP detection system , but only TBS should be used with an AP detection system . The addition of a wetting agent will assist with even distribution of the reagents during staining and improve rinsing steps ; at the same time , it will slow down bacterial growth . Common wetting agents used are Tween - 20 , Triton - X , and brij - 35 . The buffer should be used at room temperature during staining .
Tissue handling
Frozen tissue fixation & processing Before performing any immunologic procedure , the stability of the antigen must be aqueous solutions , the antigens must be considered . Because most antigens are soluble fixed in situ when the technique permits CASCP 2020 ISBN 978-089189-6760
Advantages of Indirect IF
Greater sensitivity Great amplification because more than one secondary antibody can attach to the primary antibody . Secondary antibodies are relatively cheaper and available in a wider array of colors .
Summary
IF is the visualization of antigens within cells using antibodies as fluorescent probes .
Polyclonal antisera blood
If blood was drawn from your arm and IgM antibody was isolated from it and injected into a rabbit , your IgM antibody would act as an antigen and stimulate the rabbit to make antihuman IgM antibody . Serum from the immunized rabbit could then be used as a polyclonal antihuman IgM antibody reagent . When lymphocytes are exposed to antigen , some of the lymphocytes proliferate , and each lymphocyte forms a cell line . All cells in a single clone , or cell line , produce an identical antibody , but various clones produce antibodies of different classes with specificities to different molecular sites ( epitopes ) on the antigen . The result of antigenic stimulation is the production of a mixture of antibodies from many clones of lymphocytes . This pool of antibodies is known as polyclonal antiserum . Polyclonal antiserum is highly sensitive it binds to
IgM Antibodies
IgM is a pentamer , which means that it contains 5 subunits . • The pentamers are connected by the J chain . • When a host is first injected with an immunization , the IgM antibodies are the first to respond . . Within a couple weeks , the IgG antibodies become more predominate .
IgM Antibodies
IgM is a pentamer , which means that it contains 5 subunits . • The pentamers are connected by the J chain . • When a host is first injected with an immunization , the IgM antibodies are the first to respond . Within a couple weeks , the IgG antibodies become more predominate .
Methods of visualization Immunofluorescence Immunofluorescence the oldest of
Immunofluorescence tochemical techniques , is still widely Immunofluorescence , the oldest of pathology's immunohis used . This technique makes antigens in tissue sections or in live cell it possible to visualize suspensions . A fluorochrome is a dye that absorbs light and then emits TR its own light at a longer wavelength . This phenomenon of can absorption and emission of light is called fluorescence . When the fluorochrome is attached or conjugated to an antibody , the sites of reaction between antigen and the labeled antibody can be visualized easily . The most commonly used fluorochromes in immunofluo rescence techniques are fluorescein isothiocyanate ( FITC ) and rhodamine . Both of these dyes absorb light that is not visible to the human eye ( UV ) and emit light that is visible using a fluo rescence microscope . Virtually any antigen can be detected with immunofluorescence . The combination of sensitivity , specificity and simplicity makes the method very useful . In most routine histopathology laboratories , frozen kidney and skin biopsy spec imens are examined with immunofluorescence techniques , but tumor differentiation is performed using enzyme immunohisto chemical techniques suitable for light microscopy . immunohistochemistry
Immunoglobulins
Immunoglobulins Blood is collected and the removal of certain cells and fibrins takes place . • The remaining useful substances antibodies , is the antiserum or plasma . Basically , you want to get down to the immune response cells within the blood . Centrifuge
Negative controls
In most laboratories negative controls are run by substi tuting for the primary antibody either nonimmune serum from the same species as the primary antibody or the diluent used for the primary antibody . If diluent buffer is used , the negative control will not detect nonspecific binding of animal components to the tissue , and any staining observed will be the serum peroxidase activity or binding of result of either endogenous other antibody reagents to the specimen [ Bourne 1989 ) .
Indirect method
In the indirect method , the patient's serum is added to tissue presence of antibodies to those sections containing known antigens to test the patient for the antigens An example is the test for the presence of antinuclear antibodies . The patient's serum may also be added to a known bacterium to detect the presence of bacterial antibodies in the patient . A labeled antibody ( anti human Ig ) must be used to detect the bound antibodies from the patient's serum . These indirect methods most frequently involve immunofluorescence microscopy .
Disadvantages of Direct IF
Lower signal ( weaker staining ) Higher cost Less flexibility
IHC
It is a histological technique for identifying cells and cellular components ( antigens ) by means of antigen - antibody interaction at the site of the binding
Common Metric Prefixes
Kilo ( k ) = 1000 x base unit Centi ( c ) = .01 x base unit Milli ( m ) = .001 x base unit Micro ( u ) = .000001 x base unit Nano ( n ) = 10-9 x base unit Pico ( p ) = 10-12 x base unit
Dilutions for the Clinical Laboratory ( cont'd )
Laboratory ( cont'd ) Dilutions are expressed as the volume of the solution being diluted per the total final volume of the dilution . In the orange juice example on the previous slide , the dilution would be expressed as 1/4 , for one can of O.J. to a TOTAL of four cans of diluted O.J. When saying the dilution , you would say , in the O.J. example : " one in four " .
Quality control
Measures of QC and thorough documentation are fundamental in a diagnostic laboratory . QC measures serve many roles in the laboratory and are especially useful for troubleshooting . Antibody and control tissue results must be documented and maintained . Each laboratory should define a quality system that enables the immunohistochemistry technician to have easy access to previous results and final protocols , especially in a laboratory where manual staining is performed . Tracking of lot numbers , expiration dates , dilutions protocols , pH monitoring , and verification of control results are basic QC measures taken in the immunohistochemistry laboratory . Laboratory accreditation agencies also have Both paper and specific QC and documentation electronic documentation are useful , but if a final protocol can requirements . 978-089189-6760
Converting between English and Metric Systems
Mile x 1.6 kilometers Pound x 0.454 = kilograms Quart x 0.95 = liters Kilometer x 0.6 = miles Meter x 3.3 = feet Meter x 39.37 = inches Centimeter x 0.4 = inches Gram x .0022 = pounds Liter x 1.06 = quarts
Monoclonal Antibodies
Monoclonal antibodies are a homogeneous population of immunoglobulin directed against a single epitope . • The antibodies are generated by a single B - cell clone from one animal . ● They are commonly produced in mice and rabbits .
Metric System
Most countries use the metric system for measurement Examples : Gasoline by liter Body weight in kilograms Distance in meters or kilometers U.S. uses English system of measurement in everyday life Examples : Gasoline in gallons Weight in pounds Distance in miles
Blocking reactions
Most immunoperoxidase methods include 2 blocking reactions . The first is the use of 3 % hydrogen peroxide , usually prepared in absolute methanol , to block tissue endogenous peroxidase activity . If the tissue contains many red blood cells , this blocking step is essential i12.13 . The second block is for nonspecific background staining that may occur as a result of antibody ( protein ) attachment to highly charged collagen and connective tissue elements
Converting within Metric System
Move decimal to left Move decimal to right King Henry Died BY Drinking Chocolate Milk ** Micro Д Д Д ( kilo ) ( hecto ) ( deca ) BASE ( deci ) ( centi ) ( milli ) ** ( micro ) Example : Convert Kilograms to Grams : Move decimal 3 places to right Example : Convert Centimeters to Meters : Move decimal 2 places to left
Percent Solutions Solutions
Multiply % desired : 100 = g or mL needed Volume desired Multiply
Limitations of IF
Photobleaching - destruction of a fluorophore due to reactive oxygen molecules in the air or tissue . Can be prevented by using mounting media such as Antifade .
Principle of Fluorescence
Principle It is important to know the excitation and emission properties of fluorochromes when selecting which ones to use together .
Rabbit monoclonal antibodies
Rabbit monoclonal antibody production was first reported by Spieker - Polet in 1995. Since that initial report , the production of rabbit monoclonal antibodies is making its way into the clinical laboratory . This technology follows the same principle of monoclonal hybridoma production , but uses rabbit fusion partner cells , " which fuse with rabbit B cells . These antibodies are proven to provide both sensitivity of a rabbit antibody and specificity of targeting a single epitope i12.1 , 12.2 .
Specificity
Refers to how often the antibody identifies the correct epitope and only that epitope .
Multilink biotinylated secondary antisera
Secondary antibodies that are typically called " multlink " or " universal " refer to cocktails of antibodies raised in differe species . These secondary antibody mixtures are commonly biotinylated antimouse IgG and IgM and antirabbit IgG . Other species such as goat can also be added . The laboratory technician should know the species in the secondary antibody Buffe mixture to ensure that the targeted antibody is compatible . One advantage of this type of secondary antibody is that it avoids the problem of having to stock various specific link antibodies ( biotinylated goat antirabbit or goat antimouse IgG and IgM ) .
DAB reaction product intensification
Several methods have been used to intensify the DAB Heavy metals can be used for reaction product intensification the incubation medium with reagents the DAB reaction product by supplementing , or f following such as 1 % cobalt chloride . However , there is a risk of increasing background staining with heavy metal intensification , and some of the m should not be used with the immunohistochemical detection nuclear antigens Elias 1990 ] , especially if weak hematoxylin co terstaining is used . Imidazole is an effective intensification reagent . Elias Qual 1990 ] states that the addition of 0.01M imidazole to the DAB incubation medium at pH 7.6 is superior to standard DAB an DAB - cobalt chloride - nickel sulfate or DAB - cobalt chloride procedures , both in sensitivity and detection efficiency Imidazole also significantly inhibits the pseudoperoxidase activity of hemoglobin . Osmium tetroxide may also be used to intensify the DAB mentioned t reaction product , but unlike the other reagents prod reaction is osmiophilic and will be darkened by mild always used after the DAB reaction The DAB poststaining unles storage ; prevent fading of the final however darkening , osmification . This also helps reaction product during long -term possibility of the re carefully done this background staining present as well as reagent has the intensifying product 978-089189-6760
Stokes shift
Stokes ' shift Emission Absorption 45 500 550 600 650 700 Wavelength ( nm )
Saturated Solution
The SATURATED SOLUTION is a solution solute dissolved in the solvent . Any additional containing the MAXIMUM concentration of a solute WILL NOT dissolve .
Solvent
The SOLVENT is able to dissolve other substances .
Monoclonal Antibodies
The animal is injected with an antigen and blood is periodically drawn to ensure an ideal antiserum is obtained . . When the ideal antiserum is obtained , the animal is sacrificed and an immortalized cell line is made from it using B - cells fused with myeloma cells . • The cell line is called a hybridoma and it is used in cell cultures to create more B - cells that produce specific antibodies . • Monoclonal antibodies have a high specificity .
Antibody specification sheet provided
The antibody specification commercial antibody contains valuable sheet provided with a information regarding nician should be familiar the antibody or antiserum . The immunohistochemistry tech with the specification sheet for many reasons . The following are typically included , but are not always available : a monoclonal antibody listing the clone and isotope ( IgG or IgM ) to ensure that the secondary antibody will bind to the primary antibody the expiration date under recommended storage conditions long - term or short - term storage temperatures ; some antibodies require long - term storage at -20 ° C ; measures to ensure that the antibody storage condition is optimal will assist with antibody stability and maximum reactivity for the life of the antibody the protein concentration and suggested working dilution ranges pretreatment ( antigen retrieval ) solution , pH , and / or method , if recommended expected positive and negative tissue types , including normal and tumor reference to publications and applications for the antibody ; these are useful if additional staining methods are needed antibody type in vitro , research use only , or analyte specific reagent ( ASR )
Concentration
The concentration of a solution is how much of the solute is present per unit of volume It can be recorded and reported in multiple ways depending on the solution and the scientist Molarity - Moles / Liter ; mols / L ( M ) Percent ( Mass / volume ) - grams / L ; g / L or mg / ml Normality - moles of active ions / L ( N )
Principle of Fluorescence
The emitted fluorescence has a lower energy than the absorbed light , so the wavelength of the emitted light is longer than that of the excitation light . • A range of wavelengths can excite the electrons of a fluorochrome ( fluorescent dye ) .
The fundamental Enzyme immunohistochemistry
The fundamental Enzyme immunohistochemistry similar to fluorescein - labeled principle of enzyme - labeled antibodies antibodies . The enzyme , in the ( may be the same or presence of a substrate and a chromogen separate reagents ) , provides the indicator system to visualize the location of the antibody . Various enzymes such as alkaline phos phatase ( AP ) , B - galactosidase glucose oxidase , and horseradish peroxidase ( HRP ) are used as markers . HRP and AP are the enzymes most commonly chosen for antibody visualization . HRP , in the presence of hydrogen peroxide ( substrate ) and a chromogen such as 3,3 ' - diami nobenzidine ( DAB ) or 3 - amino 9 - ethylcarbazole ( AEC ) will identify the sites of antibody binding by forming a colored 2020 978-089189-6760
Immunoglobulins
The heavy chains differ in antigenic and structural properties , which helps to determine what class they are . ( ex . IgG vs. IgM ) The two light chains are either type kappa or lambda . Disulfide bonds bind the heavy and light chains .
IHC
The integral reagent common to all immunohistochemical techniques is the antibody . Antibodies belong to a group of proteins called immunoglobulins ( Ig ) .
Polyclonal Antibodies
The most frequent animal species used for polyclonal antibody production is the rabbit . They're easy to maintain and human antibodies appear more rare to rabbit proteins than most animal species . The New Zealand White rabbit is the most frequently used species .
Indirect IF
The primary antibody is unlabeled A secondary antibody which is directed towards the constant portion of the primary antibody is conjugated with a fluorescent dye .
Polyclonal Antibodies
The rabbit ( host animal ) is injected with an antigen . Usually via the use of a needle . • An immunization period of between 3 to 8 months takes place . . At the end of the immunization process blood is collected from the host animal and the antiserum is obtained via the use of a centrifuge . • The antiserum then goes through a purification process .
Immunoglobulins
The two most common types of antibodies in immunohistochemistry are IgG and IgM .
Antibody Regions
The two regions are the variable and constant . The variable region determines the binding site for the antibody and allows it to bind .
Two types of IF
The two types of IF are - Direct immunofluorescence Indirect immunofluorescence
Microtomy
The water bath should contain clean slide preparation deionized immunohistochemical water for water bath adhesives should be avoided . Albumin and other slide and because of potential nonspecific staining over the worn to avoid squamous cell contamination tissue . Gloves should be . Positively charged during the slides are recommended to ensure tissue adhesion epitope retrieval and rinsing steps . Staining quality is strongly affected by the quality of the tissue section . Sections should be free of wrinkles and folds . Good technique will decrease produced by reagents incomplete rinsing during Section thickness routinely should be picked up in thickness ; 3- to 4 um the patient tissue and control tissue staining i12.4 , being trapped potential staining artifacts underneath the tissue or 112.5 , i12.6 can affect staining intensity , therefore should be cut at the same sections are recommended Tissue sections the same area of the slide , typi cally the center to ensure proper coverage of reagents . Avoid on the edges of the slide because this area is placing the tissue commonly missed by reagents or it dries out . Areas of incom plete coverage or drying may display false - negative , or most likely nonspecific , staining . To preserve precious tissue when the block is returned to the chuck for additional sections , the technician should avoid further trimming by carefully realigning the previously sectioned block with the blade . Air drying overnight in front of a cool fan is optimal but is not realistic for a diagnostic laboratory . Once prepared , the tissue slide can be placed into a 55 ° C oven for 30 minutes . A convection oven that circulates the air can increase water evapo ration and reduce drying time . Avoid drying in high temper ature ovens > 60 ° C ; many nuclear and cell surface markers are sensitive to dry heat in combination with high temperature , or weak staining . Oven dry time should leading to false - negative be controlled and exact to reduce the risk of variable staining outcomes .
Epitope enhancement or retrieval
There are 2 common categories of retrieval methods : the retrieval ( HIER ) and the second is enzyme - induced epitope first is heat - induced epitope retrieval ( EIER ) . These methods are used when it is necessary to break down the hydrogen bonds that are formed during fixation because immunoreac aldehydes Overfixation tivity can be compromised by some fixatives , especially the of tissue by formaldehyde can result in an antibody not having access to its epitope , thereby leading to a false - negative result . The detectability of many antigens in formalin fixed tissue is greatly improved by epitope retrieval methods . Some of the advantages of epitope retrieval are Lear 1995 ] : ability to further dilute antibodies exposure of epitope sites not previously detectable more intense reactions with decreased incubation times more uniform staining decreased background staining day - to - day consistency of stains improved standardization
Unlabeled or soluble enzyme immune complex method
This is a 3 step method using primary antibody , linking or enzyme - antienzyme complex . complexes must be secondary antibody , and soluble The primary and enzyme - antienzyme made in the same animal species for the secondary antibody to techniques are based on the original link them together . These peroxidase - antiperoxidase ( PAP ) technique of Sternberger 1979 ] . The most common techniques in this category involve the use of either a PAP or AP - anti - AP immune complex f12.2 .
Temperature Conversion
To convert from Fahrenheit to Centigrade : C⁰ = 5/9 ( F⁰ - 32 ) To convert from Centigrade to Fahrenheit : F ° = 9/5 ( Cº ) + 32
IF
Used widely in research and clinical diagnostics Can be used on fresh or fixed specimens Antibodies are chemically conjugated to fluorescent dyes Two common fluorescent dyes are fluorescein isothiocyanate ( FITC ) and tetramethyl rhodamine isothiacyanate ( TRITC ) .
Immunohistochemical staining methods Various
Various techniques are used to detect the presence of antigen in the patient's tissue or the presence of antibody in the patient's serum . References to " direct " and " indirect " methods are typically used in conjunction with immunofluorescence tech niques whereas the " unlabeled " or " soluble enzyme complex " and the " avidin - biotin - complex " ( ABC ) methods typically refer to visible light microscopic methods . The methods that will be described are : direct indirect unlabeled or soluble enzyme immune complex avidin - biotin polymeric and monomer
Methods of HIER
agent , so a number of enzymes have been methods . As Elias [ 1990 ] stated , there is no universal r used . Some of proteolyse 0.1 % pronase in 0.5M the ficin ; 0.1 % protease in PBS hydrochloric acid . tris solutions that have been used are buffer , pH 7.5 ; 0.6 % 0.4 % pepsin in 0.01N enzymes may be needed for epitope enhancement of proteolytic Different , pH 7 A , and antigens . Digestion times for these proteolytic enzymes various from 1-60 minutes or more . It is important to know the vary tration of the enzyme solution being used . concen Although proteolytic enzyme digestion usually reduces increase nonspecific staining if weaken specific staining and create fragmentation or loss of nonspecific staining , it may not used carefully . It may false - negative results , and also cause tissue sections The term " overdigestion " of the tissue is used to results describe distorted tissue morphology and poor staining This can be caused when the incubation time is extended or concentration of the enzyme is high . Commonly the result is loss of cellular detail , in combination with nonspecific staining or no staining . Excessive digestion can promote loss of tissue from the slide during the staining process . Measures should be taken to ensure that when EIER is performed manually , the digestion time is controlled for staining consistency . Temperature can influence the rate at which the digestion is achieved ; the temperature should be the same each time an enzyme is used . A temperature of 37 ° C is commonly used for enzyme incubation . This step should be performed in humidity chamber to avoid drying of the tissue
Dehydration , or drying categories of
and fixation by chemical reagents are the 2 major categories of fixation . Dehydration unfolds and changes the solubility of the protein . Chemical fixation denatures and stabilizes proteins by coagulation , by forming additive compounds , or by a combination of the 2 actions . Acetone and alcohol are coagulating , nonadditive reagents ; formaldehyde is a noncoagulating , additive fixative that crosslinks proteins ; and mercuric chloride is both a coagulating and additive fixative . The coagulant fixatives that do not form additive compounds permit good penetration of the antibody and do not block immunoreactive determinants . However , the treatment of cryostat sections with acetone does not achieve complete fixation . Extended immunohistochemical procedures may produce deleterious morphologic changes , including chromatolysis and loss of membranes [ Farmilo 1989 ) Frozen sections can be cut at 4-5 µm , placed in cold 10 % neutral - buffered formalin for 5 minutes , then transferred to phosphate buffered saline ( PBS ; pH 7.4 ) and held for routine immunohistochemical staining . Muscle sections should be cut , air dried for 1 hour , and immediately stained with antibodies targeted for dystrophin or other muscular dystrophy related proteins . Elias [ 1985 ] states that there appears to be an inverse relationship with regard to preservation of antigenicity and irreversibly blocks tissue antigenic determinants , therefore it is morphology . Glutaraldehyde preserves morphology best , but not ideal for immunohistochemical applications .
HIER The
antibody specification This method advocated this revolutionized immunohistochemistry paraffin embedded tissue The first method for antigen not known exactly how and time and temperature . " Although tiveness of retrieval include the pH , Bancroft & Gamble ( 20081 , " major the recommended The commercial method for antigen sections was introduced by Shi [ 1991 ] retrieval in formaldehyde why the heating method works there are many theories , it is volume of fluid , heating factors that govern the effec recovery . According to sheet typically states fixed , in the early 1990s . in a metallic tallic salt solution solutions , pH , length of retrieval solution is also very HIER , many studies have shown that the Although high heat is the most important heating , and methods of heating . which address various antigen retrieval 100 ° C . Many articles have sales appeared in the literature since then , and heating in a microwave oven to immersing formalin fixed tissue sections component of composition of the solutions proposed by Shi 1 % zinc sulfate ) have the disadvantage of potential toxicity , so 1991 ) ( saturated lead thiocyanate and important . The original retrieval numerous other less toxic solutions have been proposed . Two of glycine HCl ( 0.05M , pH 3.5 ) , and 3M urea solution . Shi Other retrieval solutions that have been used are distilled water , and ethylenediaminetetraacetic acid ( EDTA ; 1mM , pH 8.0 ) . the most widely used are sodium citrate buffer ( 0.01M , pH 6.0 ) important than the composition , particularly for nuclear and ( 1995 ) found that the pH value of the retrieval solution was more cell surface antigens ; they suggested that the critical influence of pH in antigen retrieval may provide an explanation of reported inconsistencies in HIER immunohistochemistry . They recom mended that retrieval solutions of high pH be used for most of the antibodies in surgical pathology , and stated that tris - hydro chloric acid or sodium acetate buffer solutions at pH 8-9 may be suitable for most antigens ; however , certain nuclear antigens estrogen receptor [ ER ] , retinoblastoma protein [ Rb , and MIB - 1 , monoclonal Ki - 67 ) show optimal staining at low pH . The disadvantage of a high pH is the extent of damage to the tissue sections after heating in a strongly alkaline solution . High pH can promote tissue detachment , resulting in holes in the tissue , or in extreme cases , complete removal from the slide i12.7 . The observations of Shi ( 1995 ) further support the concept that the antigen retrieval methods loosen or break the cross linkages caused by formalin fixation . Many other methods of heating are also available , including modified pressure cooker laboratory microwave oven , vegetable steamer , or circulating water bath . A standard method should be established in each laboratory , but at least 2 methods should be available . A higher - volume laboratory will most likely require multiple retrieval solutions and methods , because the increased variety of antibodies will require different pH solutions and retrieval techniques . Technology has allowed for automated immunohisto chemical stainers to perform the steps of heating , deparaf finization , and antigen retrieval , reducing human error and ensuring that the temperature and exposure time are consistent each time a stain is performed , thus improving reproducibility
IgG Antibodies
antige variable ..... antigen binding region binding site ...... site ****** light constant region heavy chain
Polyclonal
antiserum is highly sensitive because it binds to multiple epitopes , but this also means that it is not as selective , resulting in some nonspecific staining ( background ) . The used to stain tissue must be carefully selected to ensure no false titers positive staining . Examples polyclonal antiserum include human . Because of the variability of the immune only in a limited number of immuno are difficult to stan response from rabbit , goat , horse , sheep , and of hosts that are used to produce 1 animal to another , polyclonal antibodies same dardize and can be used immunized animals " assays . Pooled antibodies made from many of the same species are less likely to than pools made from only a few animals exhibit major batch - to batch variations
Polymeric detection
become a standard for clinical The dextran polymer and monomer technology has quickly immunohistochemical staining . such as HRP or AP have been The polymer enzyme molecules fused with the secondary antibody . Monomer technology uses a single strand , allowing for greater sensitivity and penetration at the antibody - binding site . Turnaround times have been improved with the elimination of serum and avidin - biotin blocking steps necessary for the ABC method . In addition , this chemistry provides sensitivity without the risk of nonspecific staining when using the ABC method . Staining steps would be antibody , polymer , and chromogen .
Polyclonal
by tissue culture or by quantities mice . Although other animal toneal antibodies for the transplantation into the peri species can be histopathology laboratory quantities . The can be characterized , standardized are produced used , monoclonal cavities of most commonly in mice . high homogeneity the absence of or lot - to - lot of the undesirable characteristics affinity and selectivity ) of clonal antibodies display the most antibodies no batch - to - batch are purer than advantages of monoclonal , and produced Monoclonal antibodies in unlimited antibodies include nonspecific antibodies , and variability . Mouse monoclonal polyclonal antibodies , and mono desirable attributes ( high polyclonals without displaying most ( lack of chemical homoge neity and lack of continuous supply ) . Rabbit monoclonal
Prediluted & concentrated antibodies Prediluted antibody refers
commercially available ready - to - use solution . The vendor has already taken a concentrated antibody and diluted it down to an " optimal " dilution . Because it has been diluted , the shelf - life is routinely shorter and it cannot be frozen to extend it ; thus prediluted antibodies are less economical if not used before the date of expiration . Prediluted antibodies must be validated for reactivity before use on patient tissue . The laboratory cannot assume that the antibody is ready to use with that laboratory's method and serial dilutions ( 1 : 2 , 1 : 4 , 1 : 8 , and 1:16 ) to validate must be performed . The exception is a Food and Drug Administration ( FDA ) -approved antibody test , which mandates the antibody concentration in addition to times , temperature , and detection reagents . This is usually manufactured and distributed as an FDA - approved kit , or the manufacturer provides specific protocols to follow A concentrated antibody is one that is supplied in an " undi luted " form Dilutions must be performed to find the optimal dilution for antigen detection , and the user has more control of fine tuning the dilution to be compatible with the laboratory's fixation and processing protocols . These undiluted antibodies are more economical because they allow for the modification of titers used in staining . In addition , they routinely have a longer shelf - life when stored properly ; most concentrated antibodies
basic formula to use to make 1 mL ( 1,000 µL ) of a
dilution
A Positive positive control controls must performed
each time it is A Positive positive control must be run slides should es automated can be used to check reliability of the performed . This applies to both commercially methods . Although with each antibody stain manual and prepared control reagents , these not be used as s r routine patient controls prepared in the laboratory under tissue , including ty type fixation varies purchased The optimum control is one conditions as the diagnostic fixation and processing . Because the reactions so dramatically , determine if a commercially slide is a true - negative overfixation . The method described negative reaction reaction or if is the by Battifora ( 1986 ) controls is excellent , though somewhat of preparing sausage result of on the diagnostic control slides and affects and timing of the exact same are not ideal to prepared the laboratory can make time consuming and difficult to the most efficient execute . With careful planning , use of available tissue to fulfill requirements of using controls containing both performed using an instrument positive and negative tissue types . Tissue microarray technique is a more effective means for constructing multitissue control blocks ( both positive and negative tissues than manual methods Technology can reduce the distortion of the tissue block and extend the use of a control tissue due to smaller precise core sizes [ Bancroft 2008 ) . Best practice is to use a multitissue control that contains both positive and negative tissue for an antibody . This control specimen should be placed on the same slide as the patient's specimen to ensure that all of the steps , reagents and temper ature are exactly the same for the patient and control specimens during the staining process . Control specimens can be precut before cutting the patient specimen . When cutting controls , the end of the slide that is for the patient should not be placed into the water bath , because this can reduce the charge of the slide .
result with a given antibody . Not all antibodies are affected in same way . Dr. Timo Drevyanko [ 1983 ] wrote that delayed concerns on the staining results when diag replaced
fixation may have an effect on the staining results when diag nosing colorectal carcinoma with microsatellite testing . Specific concerns may be related to false - negative colon microsatellite marker results when negative staining is interpreted as a positive result . It was suggested that there is a correlation between extended cold ischemic time and loss of antigen stability . Engel & Moore [ 2011 ] outlined preanalytical variables that can affect immunohistochemistry results in the categories of fixation and processing and are antigen dependent . Key factors that influence staining quality include < 12 hour fixation delay , fixative prop erties of concentration , pH and buffered solution , time in fixative , and storage of mounted slides . The immunohistochemist should know which fixative was used before staining with an antibody . If the laboratory uses multiple fixatives , each antibody protocol should be validated with each type of fixative before staining patient tissue . If this is the case , it is desirable to have a multiple tissue control block containing tissue fixed with the different fixatives to ensure adequate results . It is also likely that different antibody dilutions and staining times may be required based on the tissue fixative .
Techniques such as immunofluorescence available
immunofluorescence and enzyme immunoassays have been available as diagnostic tools for over 50 years , but the development of immunodiagnostic methods including immunohistochemistry , was much slower because of the unpredictable quality of polyclonal antibodies . Since the introduction of immunohistochemistry techniques in the 1980s , a number of dramatic advances have been made in immunology and immunodiagnostic methods ; these advances have had a substantial impact on all phases of laboratory medicine . In most laboratories , the use of immunohistochemical stains has become as routine as the use of any other special stain and in many instances , these stains have completely replaced the older histochemical or empirical methods . The development of immunohistochemical staining standards and guidelines set by the College of American Pathologists ( CAP ) is related to predictive markers that are used for therapeutic patient care , and which qualify the patient for specific drug treatment . The laboratory's role is pivotal ensuring that all specimens are processed and treated according to the recommended guidelines .
Antibody An antibody
immunoglobulins ( Igs ) , are proteins that are produced by B or other agents in the body . Antibodies , commonly known as in response to the presence of foreign molecules , organisms , antibody is a host protein ( immunoglobulin ) produced lymphocytes in response to antigenic stimulation . An immu noglobulin is a " Y " -shaped protein molecule that is composed of both heavy and light chains . Classes of antibodies differ in structure and function , with each immunoglobulin antigenically distinct . An immunoglobulin is composed of 2 identical heavy chains ( y , a , mu , 6 , or e ) that determine the immunoglobulin ( Ig ) subclass ( IgG , IgA , IgE , IgD , and IgM ) . The light chain is either K or λ . These proteins are expressed on the cell surface or membrane and secreted into blood and other fluids by plasma cells . The upper arms of the Y are the regions of the antibody that bind to the short arms of their specific antigen . This region
Principle of Fluorescence It is
important to know the excitation and emission properties of fluorochromes when selecting which ones to use together .
can be " snap " frozen using liquid nitrogen and stored at -70 ° indefinitely . If undiluted
in the immunohistochemistry laboratory . Microliter ( μL Pipettes capable of measuring in microliters ( µL ) are a must a total of 20 parts , or 1 part antibody plus 19 parts of diluent . 1:20 dilution . This is equivalent to 1 part antibody solution in dilutions are needed . Dilutions are expressed as ratios , as in a diagnostic slides or the smallest amount feasible when very high to dilute only the amount of antibody needed for the current If undiluted antibodies come in solution form , it is best and lambda ( A ) are used interchangeably , with both equal to 0.001 mL . Pipettes that will cover a range , such as 0.5-10 μL , 10-100 μL , and 100-1,000 µL , are the most useful . For higher dilutions , it is best to prepare a more concentrated stock dilution , and then prepare further dilutions from this stock dilution .
Monoclonal Antibodies
injected into antigen antibody fused together produces T mouse plasma cell tumor hybridoma endless supply of monoclonal antibodies
Immunofluorescence method
insensitive method , and to the maximal extent by today's standards , is a relatively antigenic reactivity must be preserved possible . Frozen sections of unfixed tissue constitute the classic preparation of tissue for immunofluores cence , because antigenic reactivity is minimally impaired and fluorescent antibody staining is strongest ; however , in such sections , soluble antigens are lost ( Sternberger 1979 ) . Formalin fixed paraffin sections are rarely used in immunofluorescence because of inconsistent results ; it is suggested that fixation and processing impair antigenic reactivity beyond the sensitivity of immuno fluorescence
interpretation of counterstain . Counterstain
interpretation of the slide . Solution : Use a different counterstain . Dilute your counterstain . Counterstain for the correct amount of time . Cause : Antigen levels in the tissue are low and the staining is minimal . Solution : Longer incubation time or use a more sensitive staining system . May need validate staining times and titers . Use different AR . 7. Cause : Used an inappropriate fixative that damaged the antigen Check data sheet and use the proper fixative . Cause : Immunoreactivity was diminished or destroyed during dewaxing or high temps . Solution : Don't dewax at over 60 degrees . Cause : reusing the same AR buffer . Solution : make new AR buffer or get new buffer . Cause : Failure to achieve a high enough temperature for AR . Solution : Readjust temperature or use a closed heating system like a pressure cooker , or autoclave . General Background ( too much background staining ) 1. Cause : Excessive incubation time with chromagen . Solution time 2. Cause : Chromogen was prepared incorrectly . It may be too concentrated . Solution : Next time use the chromogen at its proper dilution . Cause : Secondary or link Ab cross reacts with antigens from the tissue ( endogenous ) . Solution : Might need to use a different secondary . Check species of secondary in relationship to the primary .
EIER In
methods . As Elias 11990 ] stated , there is no have been universal agent , so a number of enzymes solutions that have been used are 0.1 % used . Some p buffer , pH 7.5 ; 0.6 % ficin ; 0.1 % pronase of in 0.5M 0.4 % pepsin in 0.01N hydrochloric enzymes may be antigens . Digestion times for these needed for epitope protease in PBS , pH acid . enhancement Different 7 . pro proteolytic of va from 1-60 minutes or more . It is tration of the enzyme solution being important used . to enzymes vi know the c Although proteolytic enzyme digestion nonspecific estaining , it may increase nonspecific usually reduce staining not used carefully . It may weaken specific false - negative resul results , and also cause tissue sections . The term " overdigestion " of fragmentation staining and c or lo describe distorted tissue morphology This can be caused when the concentration of the enzyme i is high . incubation and poor the e tiss tissue is wel stainin re time is extended Commonly the re with nonspecific staining loss of tissue brun of cellular detail , a combination staining . Excessive digestion can promote slide during the staining process . Measures should be taken to performed manually , the consistency Temperature can influence ensure that when ETER digestion time is controlled for the rate at which de should be the same d of 37 ° C is commo should be performed a digestion is achieved ; the temperature time an enzyme is used A temperature used for enzyme incubation . This step humidity chamber to avoid drying of the tissue . In the EIER method , a proteolytic enzyme is used to expose epitope sites . Proteolytic enzyme digestion is the older of the 2 epitope enhancement methods , but is used much less frequently since the advent of the heat - induced epitope enhancement Histotechnology Se
Antibody protocol development
most formalin fixed tissue samples . This Routinely a standard staining protocol will be used for will also assist with laboratory reproducibility requiring unique detection , special pretreatments , and incu necessary to have unique protocols , the number of antibodies and consistency . Although it will be bation times should be kept to a minimum . An example of a standard laboratory protocol is as follows 1. retrieval EDTA , pH 8.0 2. heating method : modified pressure cooker ( 115 ° C for 25 minutes or 124 ° C for 40 minutes ) ; combination of time and temperature is based on the stringency of retrieval needed 3. peroxidase quenching : 3 % hydrogen peroxide ( H₂O₂ ) , 10 minutes 4. detection system : universal polymer , HRP DAB staining times : antibody 30 minutes , polymer 10 minutes , and chromogen 5 minutes 6. counterstain : Mayer hematoxylin , 3 minutes
before staining , thus knowledge is gained of the
recommended retrieval solution , pH and method , type detection used , and types . Knowing expected staining positive and negative tissue patterns and location of expression , such as nuclear or cyto plasmic , can assist when reviewing staining results . Upon receiving the new undiluted antibody , the labo ratory will typically begin with a series of testing variables First perform serial dilutions ( 1 : 100 , 1 : 200 , 1 : 400 , and 1 : 800 ) of the antibody using a known positive and negative tissue control . Usually the manufacturer will indicate an approximate dilution : however , several different dilutions should be used with the standard laboratory protocol to determine the correct dilution for your laboratory . It is important to use a diluent that contains carrier proteins and the proper pH for antibody stability . Review the results for an optimal dilution looking for the best signal - to - noise ratio . If the 1 : 100 targeted cells are staining faintly , with no nonspecific staining , then stain further with more concentrated dilutions of 1:25 , 1:50 , and 1 : 100 . To ensure no errors in dilution preparation and reproducibility , always repeat in the new series the previous 1 : 800 : 1,600 overstained with high dilution that is closest to optimal signal and high background then start with . If the 1 : 800 appears and 1 : 3,200 to further negative or suboptimal with changing only 1 variable at a time . solution is first ; if unsuccessful , the Routinely changing results , then the laboratory should begin validate . If the initial staining yields the pH of the retrieval enzyme will but also omitting the retrieval require not solution is introduction of an enzyme step recommended . The introduction of an concentration only a redetermination of the antibody an evaluation of the time in the enzyme solution . It is recom 20 , and mended that when an should , 5- , be do not 30 minute periods in the to ensure that the require any type of staining i12.10 , protocol and least 10 ensure the targeted positive and 10 dilution are 112.11 , 112.12 . retrieval , and using retrieval may retrieval is adequate . Some enzyme solution enzyme is introduced 10- , evaluated antibodies introduce false - positive deter negative Once the appropriate mined for a given antibody , at cells are CASC neoplasms should be tested to ISBN 978-089189-6760
Regulations responsibility
regarding predictive marker staining of tissue places responsibility on the clinical laboratory for documenting the time in fixative . For example , it is recommended that breast tissue from all invasive carcinomas be formalin fixed at room temperature for a minimum of 6 hours and a maximum of 72 hours [ Wolff 2014 ) before HER2 testing . Breast tissue should be grossly examined and sectioned promptly to begin formalin fixation , and the time of fixation must be documented . A fixative can positively or negatively influence the staining
concerns about mercury as an environmental hazard replaced with substitutes like
replaced with substitutes like acetic zinc formalin is found to yield results comparable hazard , it is formalin . Acetic to B - 5 in zine being histochemical staining [ Bonds 2005 ] . imme positive Bouin fixative composed of glacial picric acid is not ideal for staining Crisp staining detail is lost complete removal of picric acid is difficult yet immunohistochemical acetic acid , formalin , and staining . The vital for adequa with this fixative . markedly from laboratory to labo Because fixation varies ratory vimentin provides an excellent way of determining when tissue has been overfixed . Vimentin staining is usually excellent in paraffin sections of tissue that has been optimally fixed in formalin but is progressively lost as the length of time in the fixative increases . When the vimentin stain completely negative , the tissue is most likely overfixed and all antibody reactions should be interpreted with caution ; if preservation of vimentin is uneven then immunostains on parallel sections should be read in the area of most intense vimentin staining [ Battifora 1991
4. Cause : Slides were inadequately rinsed . Solution : increase rinsing times additional rinses .
rinsed . Solution : increase rinsing times or add additional rinses 5. Cause : Insufficient saline or detergent ( surfactants ) concentration in wash buffers . Solution : change concentration of buffers . Ex . Make a 3 % dilution a dilution . 6. Cause : You used the wrong blocking agent . Solution : Use the correct blocker . Ex . Block endogenous peroxidase when using PAP . Block endogenous biotin when using ABC Other consideration in Troubleshooting IHC Instrumentation -Is your automated system working correctly ? -Do you have error messages ? -Make sure your machine has proper updates Are there any strange noises ? -Is the machine clean and up to date with routine maintenance ? -Are bulk reagents full and detection kit reagents ?
Block reaction
secondary antibody will bind to this is the primary antibody , nonspecific If the first protein solution applied to the tissue binding can occur . The nonspecifically bound primary antibody , and react with the substrate - chromogen way to resulting in nonspecific positive staining . The best prevent this type of nonspecific staining is to add an innocuous protein solution to the tissue before the primary antibody applied . The protein will bind to the charged sites and then nonspecific binding of the primary will not occur . Nonimmune serum from the same animal species in which the secondary antibody is produced is the most common source of the blocking reagent . The nonimmune serum is applied just before the primary antibody . After 10-20 minutes of incubation , the excess is tapped off ( do not rinse off ) and the primary antibody is applied . Casein and nondry fat milk have also been used blocking agent .
Herman demonstrated remarkably
that zinc formalin preserved immunoreactivity remarkably well and that it can be used successfully on automated tissue processors . This report , and the search for better antigen retention and preservation , has led many laboratories to switch from 10 % neutral - buffered formalin to some formulation of a zinc formalin solution for routine processing . Zinc formalin is discussed in Chapter 4 , " Fixation Mercuric chloride and Bouin are rarely used . B - 5 is noted for producing excellent nuclear morphologic detail in hemato poietic tissues such as bone marrow or lymph nodes . Because of
heavily used technique for assisting the pathologist in making In the clinical laboratory , immunohistochemistry is
the pathologist in making the bleshooting for variables introduced by fixation , processing and patterns . The major role an immunohistochemist plays is trou results are accurate and consistent with expected staining care . Rigorous quality control ( QC ) is vital to ensure that methods used , to achieve reproducible , high - quality patient to understand the basics of immunology and the reagents and origin of a tumor , prognosis , and treatment . It is important a diagnosis . Immunohistochemistry is used to determine the than tissue types . The immunohistochemist should be familiar with both the controls used and the staining patterns of an antibody to troubleshoot unexpected results . Techniques such as immunofluorescence
Solubility
to be dissolved in other substances The SOLUBILITY is the ability of a solute ( especially water ) .
Applications of IF in Pathology
• Analysis of antigens in fresh , frozen or fixed tissues . . Observation of bacterial or parasitic specimens . • Detection and localization of the presence or absence of specific DNA sequences .
Stokes shift
• Determines what color light is given off by the fluorochrome . A fluorochrome with a small Stokes shift which is excited by green light may . fluoresce green , however a fluorochrome with a larger Stokes shift that is excited by green light may fluoresce a yellow or orange light .
IgM Antibodies
• IgM is a pentamer , which means that it contains 5 subunits . The pentamers are connected by the J chain . When a host is first injected with an immunization , the IgM antibodies are the first to respond . • Within a couple weeks , the IgG antibodies become more predominate .
Immunoglobulins
• Immunoglobulins are present in the blood of immunized animals . • Think about immunizations or Igs that are received from your mother . IgG can cross the placenta and there are Igs in breast milk
Immunoglobulins
• The five major classes of immunoglobulins are immunoglobulin G ( IgG ) , IgA , IgM , IgD , and IgE . • Each immunoglobulin is composed of two identical heavy chains and light chains IgGx IgG ) IgAx IgA2 IgMk IgM )
Dilutions for the Clinical Laboratory ( cont'd )
◆ Notice that dilutions do NOT have units ( ml , etc ) but are expressed as one number to another number Example : 1/10 or " one in ten "
