Both Positive and Negative Controls not visible through the fluorescent microscope.

Fluorescence microscope may not be functioning properly.

Check microscope with previously stained slide. If there is still nothing visible, check filters for correct wavelength and/or replace or realign the bulb.

Antigen wiped off during removal from pouch or during testing.

Overly vigorous use of PBS squeeze wash bottle during rinses.

Microbial contamination of serum specimen will digest off the substrate.

PBS pH too acid or too alkaline may cause loss of antigen adhesion to slide.

Glassware dirty or not thoroughly rinsed free of detergents.

Do not touch surface with hands or pipettes at any time.

Follow rinse directions carefully. Do not focus PBS stream directly onto the antigen well.

Take care to avoid contamination during specimen collection, seperation and handling.

When making PBS always mix well and leave overnight to disolve. Check pH before use. Adjust pH if necessary.

Clean and sterilize glassware properly or use sterile, disposable containers.

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

Reagents used in wrong sequence or one or more reagents not added.

Review procedure and repeat test following staining steps precisely. Prepare and use abbreviated checklist of steps.

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

Use of wrong control or reagents.

Check reagent labels and repeat test.

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

Cross-contamination of Controls and/or specimens.

Running of controls and/or specimens between wells.

Splashing of excess control and/or specimens from well to well due to over vigorous rinsing with wash bottle.

Always change pipettes or pipettor tips between use of controls and specimens. Always return proper caps to reagent vials and specimen containers.

Use smaller drops to stay within well perimeters.

Follow rinse directions carefully. Alternative rinse method is to shake off excess reagent and gently agitate slide in jar of fresh PBS.

Variation of endpoint titer of known positive control either weaker or stronger than expected and/or specimen results inconsistent.

Inconsistency in timing intervals and incubation temperatures.

Serum dilutions made improperly. Serum added at top of test tube and not mixed well into PBS. Serum amount not correct for dilution.

Pipettor delivery not accurate.

Conjugate dilution made improperly. (When not using a kit with the conjugate included.)

Reagents outdated or mixed between kits.

Use consistent, uniform technique run to run and person to person. Recheck times and temperatures on your abbreviated checklist.

Recheck serum dilution scheme. Prepare fresh dilutions with proper technique (mixing serum and PBS well at each step). Repeat test.

Pipettor calibration must be periodically checked.

Recheck dilution calculation. Prepare fresh solution and repeat.

Always check expiration dates of reagents and never mix reagent from kit to kit or substitute from another manufacturer.

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

Check fluorescence microscope. Most common error is improperly aligned bulb.

Deterioration of all reagents particularly the antigen substrate due to improper storage such as excessively hot room temperature.

Deterioration of serum specimen due to improper storage (excessive heat or multiple freeze/thaw cycles).

Conjugate dilution made too weak (when not using a kit with conjugate included).

Conjugate activity lost due to excessive exposure to strong light and/or heat during storage or use in test.

Antigen deterioration from punctured pouch or slides removed from pouch too long before testing.

Settling of antigen substrate in center of well causes compression of cells which results in quenching.

pH of any reagent (conjugate, PBS mounting medium) too acid (below pH 7.0).

PBS not prepared correctly.

Evans Blue Counterstain solution is too strong blanching out or quenching specific positive fluorescence.

Reagents not brought to room temperature prior to use in test.

Substrate not incubated long enough with specimen or conjugate.

Excessive wash times may leach out antigenic material.

Mounting medium dried out between slide and coverslip.

Excess mounting medium doesn't allow tight contact between antigen substrate and coverslip for best light pathway.

Slides left overnight before reading may have deterioration in fluorescent intensity, especially if exposed to excessive light and/or heat.

Recheck filter wavelengths. Replace or realign bulb.

Store kit with all components as instructed in refrigerator.

Handle serum specimens sterilely and refrigerate until tested. For long term storage aliquot and freeze at -20° C, or lower.

Recheck dilution calculations. Prepare fresh solution and repeat test.

Obtain fresh conjugate. Always store away from direct light. Cover moist chamber with paper towel to block light during restain.

Check pouch for punctures. Puffiness indicates integrity not compromised. Remove slides from sealed pouch 5 to 30 minutes before use in test. Restain.

Check antigen under light microscope. Replace substrate.

Use components furnished. Do not substitute reagents.

Follow directions for PBS preparation carefully. Always mix well to dissolve and pH before using. Distilled water gives more consistent results that deionized water.

When not using a kit with counterstain included, establish directions for its use as part of the last buffer rinse or as part of the conjugate diluent.

Allow time for reagents to equilibrate to room temperature. Cold reagents take longer to react.

Recheck correct time for all steps. Do not shorten incubation periods.

Recheck wash times. Do not leave substrate in buffer wash longer than instructed.

Add additional mounting medium and/or recoverslip.

Drain excess mounting medium by holding edge of slide against absorbent paper. Do not push down on coverslip.

Store slides in refrigerator away from direct light and read as soon as possible.

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

Presence of Rheumatoid Factor.

Pretreatment of serum, using ion exchange chromatography or IgG immunoprecipitation, to remove IgG interference.

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

Competition from IgG specific antibodies.

Pretreatment of serum, using ion exchange chromatography or IgG immunoprecipitation, to remove IgG interference.

Weaker results than expected with IgM testing.

Incubation time too short.

Inadequate incubation temperature.

Separation system inadequate (especially with extremely high IgG antibody titers).

Serum should be incubated 60-90 minutes and conjugate incubated 30-60 minutes.

Incubate at 37° C

Check separation system procedure from manufacturer.

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

Over-reading -- some specimens may fluoresce more than the negative control, but less than 1+, or nonspecific fluorescence misinterpreted as true staining.

Experienced personnel must be available to read and interpret results of fluorescent tests.

Nonspecific staining with all cells apple green.

Some sera may have heterophile or autoantibodies against nuclear or cytoplasmic cellular components that may add to or obscure the specific staining pattern (i.e. ANA, AMA, ASMA).

Try to titer out beyond the nonspecific staining interference. May have to report results as 'Unable to interpret due to the presence of interfering antibodies or nonspecific fluorescence".

Nonspecific staining with entire antigen substrate appearing green.

FITC conjugate at too strong a dilution or conjugate may have free fluoescein present.

Mounting medium may autofluoresce.

Check microscope filter for wavelength band.

If not using a kit with conjugate included, establish QC procedure to evaluate conjugate properly.

If not using a kit with mounting medium included, purchase from a reliable manufacturer or establish QC procedures to properly evaluate mounting medium.

Replace broad band filter with a narrower band more suitable to FITC conjugates.

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

Antigen substrate inadequately rinsed and washed between incubation steps.

Drying of antigen substrate after rinse and wash steps.

Check fluorescence microscope. Optical pathway may be dirty.

Check if coverslip is too thick or if two coverslips are stuck together.

Frequent changes of fresh PBS for the rinse and wash steps are necessary.

Do not allow antigen to dry at any time during or between staining steps. Handle only one or two slides at a time from wash step before applying conjugate and returning to moist chamber.

Clean objectives and eyepiece lens.

Use #1 coverslip. Carefully remove double coverslip and remount with single coverslip.

Nonspecific haze or film with only certain specimens.

Lipemic or chylous serum may bind nonspecifically to antigen substrate masking specific antigen/antibody reaction.

Many other proteins and antibodies are present in serum and may react.

Hemolyzed serums may have nonspecfific fluorescing porphyrins.

Obtain fasting specimen to reduce lipids.

Obtain a fresh fasting specimen.

Obtain fresh non-hemolyzed serum specimen.

Nonspecific dense staining particularly around perimeter of well.

Drying out of specimen or conjugate on antigen substrate during incubation steps before excess is removed.

Conjugate may run off slide edge due to capillary action.

Increase size of reagent drop to well area and/or increase amount of water in moist chamber.

Shake off excess PBS and carefully wipe around edges of slide before adding conjugate.

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

Pushing down with excessive pressure of coverslip on substrate to get rid of bubbles or excess mounting medium.

Microbial contamination of serum specimen.

PBS not prepared correctly.

Use of water in place of PBS.

Drain off excess mounting medium by holding edge of slide against absorbent paper. If bubbles are excessive, remove coverslip and remount.

Take care to avoid contamination during specimen collection, separation and handling.

Follow directions for PBS preparation carefully. Always mix well to dissolve and PH before using.

Use properly prepared PBS.

Uneven staining within well.

Fluorescent staining in bubbles of air in mounting medium between substrate and coverslip is dimmer.

Don't agitate mounting medium bottle causing bubbles. Follow coverslipping procedure carefully.

Scrapes or tears in antigen surface.

Excessive movement of coverslip or scraping with pipet tips.

Handle coverslips gently. Do not touch antigen surface.

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

Foreign matter or bacterial/fungal contamination of buffer and reagents may obscure or distort antigen substrate.

Check PBS (particularly the squeeze wash bottle) and other reagents for precipitate or turbidity. Change PBS rinse/wash solutions frequently.

Results differ from those reported by another laboratory.

Microscope optics, filters, light sources, reagents and personnel may vary enough to result in differences of twofold or more.

Each laboratory must establish their own normal ranges. Results between laboratories should not be compared.