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Guide for Troubleshooting Immunofluorescence

● Both Positive and Negative Controls not visible through the fluorescent ● Increased incidence of weak positive specimens. (All specimens show microscope. increased fluorescent intensity.)

● Nothing seen on slide. ● Nonspecific staining with all cells apple green. ● Nonspecific staining with entire antigen substrate appearing green. ● Variation of endpoint titer of known Positive Control either weaker or stronger than expected and/or specimen results inconsistent. ● Nonspecific haze or film over entire antigen substrate in all controls and specimens. ● Positive Control stains at a weaker reaction than expected. (All specimens show decreased fluorescent intensity.) ● Nonspecific haze or film with only certain specimens. ● Negative Control stains positive and/or Positive Control stains negative. ● Nonspecific dense staining particularly around perimeter of well. ● Negative Control stains positive and/or repeated specimens not giving ● Antigen substrate cells distorted or nucleus of cells with punched out reproducible results. appearance.

● Cells visible but negative staining with all tests including Positive Control. ● Uneven staining within well. Scrapes or tears in antigen surface. ● More positive results than expected (false positives) with IgM testing. ● ● Irregular fluorescent staining pattern not closely resembling that seen ● More negative results than expected (false negatives) with IgM testing. with the Positive Control. ● Weaker results than expected with IgM testing. ● Results differ from those reported by another laboratory.

Welcome to MBL Bion’s Troubleshooting in Immunofluorescence Guide. Our team of immunofluorescence specialists have worked together to identify common problems, possible causes, and possible solutions for the most commonly occurring issues in the staining and interpretation of IFA slides.

455 State Street, Suite 100 Des Plaines, IL 60016-2204 • T: 847.544.5044 F: 847.544.5051 • mblintl.com Problem Both Positive and Negative Controls not visible through the fluorescent microscope.

Possible Causes Possible Solutions Check microscope with previously stained slide. If there is still nothing visible, Fluorescence microscope may not be functioning properly check filters for correct wavelength and/or replace or realign the bulb.

Problem Nothing seen on slide.

Possible Causes Possible Solutions

• Do not touch antigen surface with hands or pipettes at any time. • Antigen wiped off during removal from pouch or during testing. • Follow rinse directions carefully. Do not focus PBS stream directly onto the • Overly vigorous use of PBS squeeze wash bottle during rinses. antigen well. • Microbial contamination of serum specimen will digest off the antigen • Take care to avoid contamination during specimen collection, separation and substrate. handling. • PBS pH too acid or too alkaline may cause loss of antigen adhesion • When making PBS, always mix well and leave overnight to dissolve. Check to slide. pH before use. Adjust pH if necessary. • Glassware dirty or not thoroughly rinsed free of detergents. • Clean glassware properly.

Problem Variation of endpoint titer of known Positive Control either weaker or stronger than expected and/or specimen results inconsistent.

Possible Causes Possible Solutions

• Inconsistency in timing intervals and incubation temperatures. • Use consistent, uniform technique run to run and person to person. • Serum dilutions made improperly. Serum added at top of test tube and • Recheck serum dilution scheme. Prepare fresh dilutions with proper technique not mixed well into PBS. Serum amount not correct for dilution. (mixing serum and PBS, well at each step). Repeat test. • Pipettor delivery not accurate. • Pipettor calibration must be periodically checked. • Conjugate dilution made improperly. (When not using a Kit with the • Recheck dilution calculation. Prepare fresh solution and repeat. conjugate included.) • Always check expiration dates of reagents and never mix reagents from kit to • Reagents outdated or mixed between kits. kit or substitute from another manufacturer.

Problem Positive Control stains at a weaker reaction than expected. (All specimens show decreased fluorescent intensity.)

Possible Causes Possible Solutions

• Check fluorescence microscope. Most common error is improperly • Recheck filter wavelengths. Replace or realign bulb. aligned bulb. • Store kit with all components as instructed in refrigerator. • Deterioration of all reagents particularly the antigen substrate due to Handle serum specimens sterilely and refrigerate until tested. For long term improper storage such as excessivel heat. • storage aliquot and freeze at -20 ˚C, or lower. • Deterioration of serum specimen due to improper storage (excessive heat or multiple freeze/thaw cycles). • Recheck dilution calculations. Prepare fresh solution and repeat test. • Conjugate dilution made too weak (when not using a kit with conjugate • Obtain fresh conjugate. Always store away from direct light. Cover moist included). chamber with paper towel to block light during restain. • Conjugate activity lost due to excessive exposure to strong light and/or • Check pouch for punctures. Puffiness indicates integrity not compromised. heat during storage or use in test. Remove slides from sealed pouch 5 to 30 minutes before use in test. • Antigen deterioration from punctured pouch or slides removed from • Check antigen under light microscope. Replace substrate. pouch too long before testing. • Settling of antigen substrate in center of well causes compression of • Use components furnished. Do not substitute reagents. cells which results in quenching. • Follow directions for PBS preparation carefully. Always mix well to dissolve • pH of any reagent (conjugate, PBS, mounting medium) too acid (below and pH before using. Distilled water gives more consistent results than pH 7.0). deionized water. • PBS not prepared correctly. • When not using a kit with counterstain included, establish directions for its • Evans Blue Counterstain solution is too strong quenching specific use as part of the last buffer rinse or as part of the conjugate diluent. positive fluorescence. • Allow time for reagents to equilibrate to room temperature. Cold reagents • Reagents not brought to room temperature prior to use in test. take longer to react. • Substrate not incubated long enough with specimen or conjugate. • Recheck correct time for all steps. Do not shorten incubation periods. • Excessive wash times may leach out antigenic material. • Recheck wash times. Do not leave substrate in buffer wash longer than instructed. • Mounting medium dried out between slide and coverslip. • Add additional mounting medium and/or recoverslip. • Excess mounting medium doesn’t allow tight contact between antigen substrate and coverslip for best light pathway. • Drain excess mounting medium by holding edge of slide against absorbent paper. Do not push down on coverslip. • Slides left overnight before reading may have deterioration in fluorescent intensity, especially if exposed to excessive light and/or heat. • Store slides in refrigerator away from direct light and read as soon as possible.

Problem Negative Control stains positive and/or Positive Control stains negative.

Possible Causes Possible Solutions Use of wrong control or reagents. Check reagent labels and repeat test.

455 State Street, Suite 100 Des Plaines, IL 60016-2204 • T: 847.544.5044 F: 847.544.5051 • mblintl.com

2 Problem Negative Control stains positive and/or repeated specimens not giving reproducible results.

Possible Causes Possible Solutions

• Always change pipettes or pipettor tips between use of controls and specimens. • Cross-contamination of Controls and/or specimens. Always return proper caps to reagent vials and specimen containers. • Running of controls and/or specimens between wells. • Use smaller drops to stay within well perimeters. • Splashing of excess control and/or specimens from well to well due to Follow rinse directions carefully. Alternative rinse method is to shake off excess over vigorous rinsing with wash bottle. • reagent and gently agitate slide in jar of fresh PBS.

Problem Cells visible but negative staining with all tests including Positive Control

Possible Causes Possible Solutions

Reagents used in wrong sequence or one or more reagents not added. Review procedure and repeat test following staining steps precisely.

Problem More positive results than expected (false positives) with IgM testing.

Possible Causes Possible Solutions Pretreatment of serum, using ion exchange chromatography or IgG Presence of Rheumatoid Factor. immunoprecipitation, to remove IgG interference.

Problem More negative results than expected (false negatives) with IgM testing.

Possible Causes Possible Solutions Pretreatment of serum, using ion exchange chromatography or IgG Competition from IgG specific antibodies. immunoprecipitation, to remove IgG interference.

Problem Weaker results than expected with IgM testing.

Possible Causes Possible Solutions • Incubation time too short. • Serum should be incubated 90 minutes and conjugate incubated 60 minutes. • Inadequate incubation temperature. • Incubate at 37°C • Separation system inadequate (especially with extremely high IgG antibody • Check separation system procedure from manufacturer. titers).

Problem Increased incidence of weak positive specimens. (All specimens show increased fluorescent intensity.)

Possible Causes Possible Solutions Over-reading — some specimens may fluoresce more than the negative Experienced personnel must be available to read and interpret results of control, but less than 1+, or nonspecifc fluorescence misinterpreted as fluorescent tests. true staining.

Problem Nonspecific staining with all cells apple green.

Possible Causes Possible Solutions Some sera may have heterophile or autoantibodies against nuclear or Try to titer out beyond the nonspecific staining interference. May have to cytoplasmic cellular components that may add to or obscure the specific report results as “Unable to interpret due to the presence of interfering staining pattern (i.e. ANA, AMA, ASMA). antibodies or nonspecific fluorescence”.

Problem Nonspecific staining with entire antigen substrate appearing green.

Possible Causes Possible Solutions

• If not using a kit with conjugate included, establish QC procedure to evaluate • FITC conjugate at too strong a dilution or conjugate may have free conjugate properly. fluorescein present. • If not using a kit with mounting medium included, purchase from a reliable • Mounting medium may autofluoresce. manufacturer or establish QC procedures to properly evaluate mounting medium. • Incorrect microscope filter for wavelength band. • Check that microscope filter is apprepriate for FITC

455 State Street, Suite 100 Des Plaines, IL 60016-2204 • T: 847.544.5044 F: 847.544.5051 • mblintl.com

3 Problem Nonspecific haze or film over entire antigen substrate in all controls and specimens.

Possible Causes Possible Solutions • Fresh PBS for the rinse and wash steps is necessary. • Antigen substrate inadequately rinsed and washed between incubation • Do not allow antigen to dry at any time during or between staining steps. steps. Handle only one or two slides at a time from wash step before applying • Drying of antigen substrate after rinse and wash steps. conjugate and returning to moist chamber. • Check fluorescence microscope. Optical pathway may be dirty. • Clean objectives and eyepiece lens. • Check if coverslip is too thick or if two coverslips are stuck together. • Use #1 coverslip. Carefully remove double coverslip and remount with single coverslip.

Problem Nonspecific haze or film with only certain specimens.

Possible Causes Possible Solutions

• Lipemic or chylous serum may bind nonspecifically to antigen substrate • Obtain fasting specimen to reduce lipids. masking specific antigen/antibody reaction. • Obtain a fresh fasting specimen. • Many other proteins and antibodies are present in serum and may react. • Obtain fresh non-hemolyzed serum specimen. • Hemolyzed serums may have nonspecific fluorescing porphyrins.

Problem Nonspecific dense staining particularly around perimeter of well.

Possible Causes Possible Solutions

• Increase size of reagent drop to well area and/or increase amount of water • Drying out of serum specimen or conjugate on antigen substrate during in moist chamber. incubation steps before excess is removed. • Shake off excess PBS and carefully wipe around edges of slide before adding • Conjugate may run off slide edge due to capillary action. conjugate.

Problem Antigen substrate cells distorted or nucleus of cells with punched out appearance.

Possible Causes Possible Solutions • Drain off excess mounting medium by holding edge of slide agains absorbent paper. • Pushing down with excessive pressure of coverslip on substrate to get If bubbles are excessive, remove coverslip, remount, and gently apply covership. rid of bubbles or excess mounting medium. • Take care to avoid contamination during specimen collection, separation and • Microbial contamination of serum specimen. handling. • PBS not prepared correctly. • Follow directions for PBS preparation carefully. Always mix well to dissolve and pH before using. • Use of water in place of PBS. • Use properly prepared PBS.

Problem Uneven staining within well.

Possible Causes Possible Solutions Fluorescent staining in bubbles of air in mounting medium between Don’t agitate mounting medium bottle causing bubbles. Follow coverslipping substrate and coverslip is dimmer. procedure carefully.

Problem Scrapes or tears in antigen surface.

Possible Causes Possible Solutions Excessive movement of coverslip or scraping with pipet tips. Handle coverslips gently. Do not touch antigen surface.

Problem Irregular fluorescent staining pattern not closely resembling that seen with the Positive Control.

Possible Causes Possible Solutions

Foreign matter or bacterial/fungal contamination of buffer and reagents Check PBS (particularly the squeeze wash bottle) and other reagents for may obscure or distort antigen substrate. precipitate or turbidity. Change PBS rinse/wash solutions frequently.

Problem Results differ from those reported by another laboratory.

Possible Causes Possible Solutions Microscope optics, filters, light sources, reagents and personnel may Each laboratory must establish their own normal ranges. Results between vary enough to result in differences of twofold or more. laboratories should not be compared.

455 State Street, Suite 100 Des Plaines, IL 60016-2204 • T: 847.544.5044 F: 847.544.5051 • mblintl.com

4 MBL Bion Form 1.11, 2.2 Rev. 10/14 MC-IVD-003