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FAQ’s

Is timing important when loading plates?

Timing is also important when loading plates. The reagents should be loaded in the same order and with the same timing as previous reagents. This is especially true of the substrate and stop solutions. This ensures that each well is exposed to substrate for the same amount of time before the reaction is stopped.

For what classes of antibodies are ANCA clinically significant?

Clinically significant ANCA are predominantly of IgG class. There are reports of IgA and IgM class antibodies, but these instances are very rare.

Can I mix kit reagents from different lot numbers?

It is important NOT to mix or substitute reagents from different lot numbers. This is not recommended as each assay kit contains reagents or components that have specific lot numbers. This ensures that all the components are performing optimally, as QC testing is performed on each specific lot. If the components are interchanged with other kits or vendors, it can result in a suboptimal performance.

What conditions can affect the reagents stability?

Reagent stability can be affected by a number of conditions. This can be minimized by adhering to storage conditions as indicated in the product insert. Inspect reagents for signs of instability or contamination. The presence of a precipitate or discoloration in the solution is an indicator of contamination. Also avoid frothing of reagents. Do not pour reagents back into the original bottle after use; only pour off what is needed for the test. Check the expiration date to make sure it has not lapsed.

When is it recommended to not use the substrate?

It is recommended to not use the substrate if it turns yellow as this interferes with the reaction and may result in a high background. A slight yellow color can be observed with alkaline phosphatase substrates. This slight hue is not a concern. You should only be concerned if the OD of the substrate alone goes above 0.3 OD at 405nm. Also, check the expiration date to make sure it has not lapsed as this may affect the stability of the substrate.

What is the best method to seal and store plates?

It is important to ensure that plate pouches are sealed correctly and undamaged. Always return any unused portion of plate as quickly as possible, as for most plates prolonged exposure to outside air can cause the solid phase to decay much more rapidly. When resealing the plate, make sure that the desiccant packet is replaced in the pouch, sealed tightly and returned to storage at 4 degrees centigrade. An unsealed pouch reduces shelf life quickly.

How can I optimize washing of plates?

Washing can be optimized by using distilled or deionized water when diluting wash buffer powdered concentrate. Also check the expiration date to make sure it has not lapsed. When using an automated plate washer or manifold dispenser, be sure all pins on the wash head are clear.Why is it important to adhere to timing during the test?It is important to adhere to the timing during the test as variations may cause results to vary greatly. Check the product insert for timing and follow it closely as incorrect incubation time may result in an invalid assay.

How does variation in incubation time affect the results of an assay?

Variations in incubation time may cause results to vary greatly. This is especially true of short incubation times. Binding will continue if the incubation time is allowed to lapse, yielding high results while shorter incubation times may cause low results. It is important to have all reagents ready so that plates can be loaded as quickly as possible, not to exceed 5 minutes. These assays were developed according to the incubation times listed in the product insert and variation will affect results negatively.

How can I remedy timing problems?

To remedy timing problems, always be ready with required equipment (pipettes, tips, test tubes, reservoirs, etc.). Also warm all reagents to room temperature prior to starting the assay so they can be used immediately (except when special conditions indicated in the product insert demand cold reagents are used at the start of 4 degree centigrade incubation period). If multiple assays are run, time each plate individually so incubation times do not lapse. Allow for washing and pipetting time by spacing assays 2-5 minutes apart.

Why is it important to adhere to temperature while performing the test?

It is important to adhere to temperature as stated in the product insert while performing the test as it is essential for accurate, reproducible results. Allow time for reagents to equilibrate to room temperature (except when special conditions indicated in the product insert demand cold reagents are used at the start of 4 degree centigrade incubation period). Cold reagents will cause lower results and uneven warming of the wells may yield an “edge effect.” If this is the case, samples located in wells toward the edge of the plate will warm faster and therefore falsely yield higher readings than those located in the center.

How does room incubator temperature affect the outcome of a test?

It is important to make certain that the laboratory and work surfaces are within the temperature range recommended in the product insert. Ensure that the room and/or incubator temperature are within the required temperature range, preferably in the middle of the range. If the laboratory is especially warm, it is recommended that a controlled room temperature incubator be used for room temperature assays. These assays have been developed based upon the given temperature range and results differ when conditions vary.

How does the bench top temperature affect the assay?

A frequently overlooked factor to consider is the temperature of the bench top on which the assay is being performed. For example, if the laboratory has heat turned down or off overnight, the temperature of the bench top will remain lower than the air temperature for some time after the heat is turned on. A cold bench top will cause the signal to be much lower and may contribute to the “edge effect.” Also locations near heat sources, in direct sunlight or close to warm equipment will cause the bench top to warm and may affect results.

What is “edge effect”?

“Edge effect” occurs when the reagents or ambient temperatures are colder than that specified in the product insert. This causes uneven warming of the wells and hence, the samples located in wells toward the edge of the plate will warm faster and therefore falsely yield higher readings than those located in the center.

What guidelines can I follow to pipette accurately?

Important during the processes of loading plates with calibrators, samples and reagents and making sample dilutions. Some of the processes that can increase the accuracy and precision of an ELISA assay are –

– Do not use pipettes below or above the recommended volume range of the manufacturer as they are not designed to be accurate outside of the range.
-Ensure that any pipettes being used are calibrated properly.
-Read the product insert carefully.

What is the best method for dispensing a reagent or serum out of a pipette tip?

When dispensing a reagent or serum out of the pipette tip, some liquid adheres to the inside of the tip and the full volume is not dispensed, especially with viscous solutions. To avoid this, draw the reagent or serum up into the pipette tip and, keeping the tip immersed, successively eject to the first stop of the pipette, and then proceed to draw the reagent back up. This effectively rinses the pipette tip. The liquid can then be dispensed accurately by ejecting only to the first stop of the pipette. Successive uses of this pipette tip(s) for the same reagent or sample do not require additional rinsing. It is important not to scratch the bottom of wells when dispensing reagents onto the micro titer plate. Place the pipette tip on the side of the well to prevent droplets from remaining on the end of the tip, while avoiding contact with the bottom of the well. Take care to press pipette tips firmly onto the pipette. Check tips visually to be sure that liquids are being dispensed and aspirated completely.

How can I avoid cross-contamination?

To avoid cross-contamination, do not drag pipette tips over into other wells. Always use separate reservoirs and change pipette tips with each new reagent. Always use clean tips. Avoid mixing reagents.

What is the best way for loading the micro titer plate?

When pipetting and loading the micro titer plates, it is important not to scratch the bottom of the well with the pipette tips. Avoid cross-contamination and pipette slowly to prevent bubbles in the pipette tip due to turbulence. These bubbles will prevent liquid from being dispensed completely when the pipette is ejected to its first stop. After loading is complete, gently agitate plates briefly to be certain that all of the liquid in each well is at the bottom. An air pocket may form at the bottom of a well due to the surface tension of the liquid. Brief agitation will eliminate this.

Why is location of plates important during the assay?

It is important to choose the location of your plate carefully. Do not move or agitate plates during incubation. Also avoid areas of high intensity and stay away from air currents or machines that radiate heat.

What is an ideal location for ELISA plates?

An incubator set to the middle of the given temperature range is an ideal location for ELISA plates. Read the product insert for incubation temperature for the assay.

Why is it important not to move the plates during incubation?

It is important to avoid moving or agitating the plates during incubation as this may cause the reagents to spill out of the wells and may increase development rates. Be sure to avoid areas where other workers will bump or move plates.

Why is it important to avoid areas of high intensity or sunlight?

Do not incubate plates in areas of high intensity. Avoid placing plates near air vents or other objects such as refrigeration units that will emit warm or cool air. Direct exposure to sunlight will increase the temperature of the plate or the reagent. Enzyme substrate is also light sensitive and backgrounds will increase if the plate is exposed to light during the substrate incubation period.

Why is it recommended to use an automated plate washer?

It is recommended to use an automated plate washer for the wash steps to ensure that complete and even amounts of wash buffer are administered. Check for clogged pins in the wash head, rinse with deionized water if necessary. Also, decontaminate the washer weekly or monthly. After washing, invert plate and tap on an absorbent paper towel. Tapping technique is a critical step in the washing process. Firmly tap plates before adding new reagents. The number of repetitions necessary depends on how firmly or gently the plates are tapped. For example, more repetitions are required when gentle tapping is done. Check the product insert for information about which method is appropriate for your particular kit. Always visually check the plate for remaining liquid. More tapping may be necessary if liquid is remaining in the wells. After washing, proceed to the next step immediately to avoid drying out the plate. If an automated plate washer is not available, manual washing may be done.

What are the recommendations for manual washing?

During manual washing, it is recommended to make sure that all wells are filled completely. It is important though, to avoid creating bubbles which limit contact of wash buffer with the well surface. Always fill each well completely with wash buffer. After filling wells, decant plate by inverting and “flicking” contents into a sink. Repeat this process 4 times. After washing, invert plate and tap on an absorbent paper towel. Tapping technique is a critical step in the washing process. Firmly tap plates before adding new reagents. The number of repetitions necessary depends on how firmly or gently the plates are tapped. For example, more repetitions are required when gentle tapping is done. Check the product insert for information about which method is appropriate for your particular kit. Always visually check the plate for remaining liquid. More tapping may be necessary if liquid is remaining in the wells.

Why is it important to adhere to timing during the test?

It is important to adhere to the timing during the test as variations may cause results to vary greatly. Check the product insert for timing and follow it closely as incorrect incubation time may result in an invalid assay.

What are the recommendations for manual washing?

During manual washing, it is recommended to make sure that all wells are filled completely. It is important though, to avoid creating bubbles which limit contact of wash buffer with the well surface. Always fill each well completely with wash buffer. After filling wells, decant plate by inverting and “flicking” contents into a sink. Repeat this process 4 times. After washing, invert plate and tap on an absorbent paper towel. Tapping technique is a critical step in the washing process. Firmly tap plates before adding new reagents. The number of repetitions necessary depends on how firmly or gently the plates are tapped. For example, more repetitions are required when gentle tapping is done. Check the product insert for information about which method is appropriate for your particular kit. Always visually check the plate for remaining liquid. More tapping may be necessary if liquid is remaining in the wells.

Why do I have poor precision in my results?

Many things can cause a lack of precision, including: -Wavelength correction was not used. Read plates at 405nm for assays using AlkPhos conjugate, and some assays may be read at 450nm, with a correction wavelength of 630nm. -Too much time taken while dispensing samples, calibrators and reagents. -Perform loading procedures as quickly as possible, preferably in less than 5 minutes. -Incomplete filling of the wells during washing. -Extra wash buffer remaining in wells. Be sure to remove all liquid from the wells by tapping plates after each set of washes. Check wells for any remaining liquid. -Evaporation of liquid from wells during incubation. If long incubation times (>1 hour) or circulating air in the incubation area are encountered, a plate sealer should be used (do not use during enzyme substrate incubation). -Problems with pipetting volume. Ensure that correct quantities are being dispensed while making dilutions and loading wells. Check pipette volumes often to ensure correct reading on the dial. Also, check that pipette tips are firmly pressed onto fittings. -Samples may have been diluted in serum diluents used for another kit.

Why do I get high backgrounds?

Here are a few possibilities: -Temperature is above the recommended range. In warm laboratories, it is highly recommended that controlled room temperature incubators be used. -Incubation is extended beyond the recommended time. -Substrate has been contaminated, stored improperly or exposed to light prior to use. This will be indicated by a strong yellow color in the substrate when removed from the bottle. -Exposure of plate to light while color is developing. Enzyme substrate is light sensitive. -Liquid from adjacent wells has been transferred. Take care to prevent mixing of solutions in adjacent wells while pipetting and manipulating plates.

What should be done in the event that wavelength correction is not available?

Read the plate at 405 nm. The dual wavelength is used to subtract out any interference from a soiled micro titer plate (ie. oil from hands on bottom of a plate). The effect is not typically significant, so plates can be read at one wavelength if a dual reader is not available.

Can I substitute the primary wavelength filter for another?

No. The recommended primary reading wavelength is based upon the peak absorbance of the substrate’s development color. The signal will vary significantly if another wavelength is used.

There is low or no signal after performing the assay. Why would this happen?

Here are some possible explanations: -On the plate reader, a 450nm or other primary wavelength filter was used instead of the 405nm filter. -Reagents added in a different order than the protocol calls for. Check the product insert carefully for the order of steps to be performed. -Sample dilutions may not have been mixed. Thorough mixing of sample is necessary before loading the plate. -Plate was washed after addition of substrate. Stop solution should be added directly to the substrate without washing the plate first. -Assay was performed below the recommended temperature range and/or the reagents and samples were not brought up to temperature before starting the assay (for room temperature assays). -Standards were not added or standards from another kit were added for the assay being performed. -Storage conditions were less than optimal and one or more of the components of the kit were damaged or decayed.

What is the recommended method of storage for samples that cannot be tested at the time of collection?

It is recommended to store samples at –20° C or less. -Avoid freeze-thaw cycles that may be damaging to samples. Use a fixed temperature freezer. Frost-free freezers warm up periodically and may degrade samples.

Are plate sealers necessary?

Plate sealers will be beneficial for incubation times of more than 1 hour. They may also be used to prevent evaporation when plates are exposed to circulating air.

Can a plate sealer be reused?

No. Over each well, the plate sealer may contain residue from the previous step. Contact with this residue will cause contamination and will reduce the level of precision in the assay.

Why am I getting false positive results?

The following are possible explanations: -Standard curve was calculated using calibrator values from another kit, or calibrators from another kit have been run. . A different curve fit method may be required. If the bottom calibrator lies above the curve the patient samples can be calculated too high because of the Calibrator D being above the curve. Use a different curve fitting program like a 2, 3, 4 parameter curve or a cubic spline curve. Alternatively, calibrator D can be used as a cutoff calibrator in instances were the Calibrator D is above the curve for a lin – lin curve fit. -Incubation times or temperatures were not followed as per product insert protocol. • Screen assays and single point determination assays sometimes give false positive results. Run the assay using an entire standard curve to obtain more accurate results. -Values assigned to calibrators may not be appropriate for the representative sample population being tested. It may be necessary, and it is highly recommended, to use normal human sera from the sample population at a Mean + 3 Standard Deviations cutoff to reassign values to calibrators.

Is it necessary to use a multichannel pipette?

In general, yes. It is recommended to use a multichannel pipette whenever possible to reduce the time needed to load plates. Precision will increase by reducing the loading time.

May the wash buffers from other kits be used for this one?

Not all of the wash buffers included in the kits are the same. Check the product Code Number located on the label to ensure that the two wash buffers are of the same type.

Can I change the method of washing, for example, using less wash buffer or cutting down on the number of washes?

No. We recommend that the number of washes and the volume of wash buffer indicated in the product insert be strictly adhered to. buffer indicated in the product insert be strictly adhered to. Lowering the volume of wash buffer or number of washes will affect the accuracy and precision of the assay.

My standard curve does not follow a linear pattern or all of the calibrators have nearly the same optical density. Why would this occur?

Here are some possible explanations:

  • Wavelength used for reading the plate was not at the peak absorbance of the development color. For example, the commonly used 405nm and 450nm wavelength filters are often confused with one another.
  • Washing was not complete. This may arise from clogged tips in the washer or incorrect washing technique. If two reagents from successive steps become mixed in the wells due to incomplete washing, unspecific binding will occur and the plate will have a uniformly high optical density.
  • Conjugate or enzyme substrate is not performing properly.
  • Pipette tips that were used for one reagent were used to load another reagent or an unclean reservoir was used to pipette from. This may cause unspecific binding, and give an artificially strong positive signal.
  • Calibrators from another kit were used.
  • Plate was loaded too slowly. Do not exceed 5 minutes during this step, since the incubation times must be adhered to within a +/- 5-minute range of the suggested incubation time.
  • Plate was not read within the acceptable time limit (see product insert). Incubation times and/or temperatures deviate outside the range recommended in the product insert.
My standard curve does not seem to produce the correct results. How can I best calculate results?

If the positive and negative controls are in range, and Calibrator A is above 1.0 the assay meets the basic acceptance criteria. However, assay conditions may have produced a standard curve that is acceptable but not ideal. When the bottom point of the calibration curve is significantly above or below the curve IMMCO recommends use of a best fits curve to calculate EU/ml values.

A 2 parameter curve, cubic spline or other non-linear curve fit will yield more appropriate results. Linear curves that are not aligned closely with the bottom point are prone to overestimate or underestimate patient values. When a best fits curve is used the patient values are more accurately predicted by the equation of the line.

Results like those described above may stem from a variety of issues, including a calibration of pipettes, bubbles dispensed into wells, incomplete washing/aspiration, incubation times and temperature of incubation. Each of these elements should be considered when troubleshooting similar problematic assay results.

I am seeing depressed OD or EU/ml values. What could be the cause?

Possible causes include environmental factors, procedural factors, and contamination, exposure and stability issues. Not allowing reagents to reach room temperature before performing assay Shorter than indicated incubation times Incorrect wavelength of filter (correct filter: 405nm for all Alk.Phos. conjugates and 450 nm for HRP conjugate) Pouring conjugate or substrate into another vial or reservoir and then pouring back into original vial for later use Failure to re-sealing the micro plate pouch correctly Contamination of reagents.

What is the difference between a standard curve and a single cutoff calibrator? Is one better?

A standard curve is better at predicting the value of a patient sample because it is more accurate to calculate value from a 4 point curve than from a single cutoff calibrator. Cutoff calibrators are beneficial in screen assays where you are only interested in determining whether a patient is positive or negative. Fewer wells are used for a cutoff calibrator and more a screen positive sample one would use the appropriate antibody specific kits that have 4 point standard curves. With the exception of screen assays all IMMCO kits are supplied with a standard curve, and the calibrator D can be used separately as a cutoff calibrator. No. It is necessary to run a set of calibrators and obtain a standard curve for each plate. Variations in assay conditions for each plate prevent the use of one standard curve for several plates.

Can plasma samples rather than serum be used for detecting antibodies?

Antibodies to specific antigens are present both in serum and in plasma, so the assay can be used for either sample. However, the sample dilution has to be optimized for serum. Optimal dilution for plasma has not been tested. Qualitative results are likely to be accurate. We cannot indicate how well quantitative results will correlate. Standardization of this method would need to be performed by the client lab.

Can IMMCO test serum from animals for specific antigens by ELISA?

Even though the ELISA test performed on animal serum is similar to that for humans, we at IMMCO have not studied and validated this method. IMMCO has only validated the system for human serum. Samples from other species have not been tested.

Is the IMMCO ELISA kit compatible with my automated ELISA instrument?

In general, IMMCO Elisa’s are easily run on automated systems. However, before running an IMMCO assay on an instrument for the first time, consider the following:

  • Do the requirements for sample/reagent volumes exceed the volumes provided in the IMMCO kit?
  • Is it difficult to perform accurate 30 minute incubations when dispensing reagents to the micro plate?
  • Is the reader capable of reading at 405nm?
  • If the answer to these questions is “no” the laboratory can confidently initiate any validation / testing required.

If the answer to any of these questions is “yes” contact IMMCO technical support to discuss proper implementation on the system in question. The following exceptions should be noted:

  • Product code 1175, IRA 96 RT, and CCP 8001 are read at 450nm because they utilize an HRP conjugate system.
  • Product codes 1158, 1180G, 1180M, 1183G, 1183M, 1184G, 1184M, 1185G, 1185M, 1186G, and 1186M have cold temperature incubation steps and are difficult to automate on standard ELISA analyzers.
Can a standard curve from one plate or from the product insert be used to calculate the values of samples on multiple plates?

No. It is necessary to run a set of calibrators and obtain a standard curve for each plate. Variations in assay conditions for each plate prevent the use of one standard curve for several plates.

Does IMMCO have external QC for Anti Parietal Cell for ELISA kit?
QC used should be the standards in the insert and the positive control range.
Individual run OD may be impacted by environmental factors such as temperature, but should yield similar EU/ml.
Internal QC acceptance standards include r-squared values > 0.95.
What are the kinds of samples that can be used for this assay?

Freshly drawn and properly refrigerated serum, and in some cases, plasma (read product insert) can be used for this assay. Do not use icteric, hemolyzed, grossly lipemic, or specimens with obvious microbial contamination. These may cause false reactions and strips are unreadable. Plasma may be used in the ANA Western Blot Immunoassay, but serum is preferred as the risk of cross contamination and false positives are high.

What is the best method for storing samples?

Samples can be stored at 2-8 degrees centigrade for 3-5 days or at -20 degrees centigrade or lower for longer periods. Avoid repeated freezing and thawing of samples. Optimum conditions for specimen handling are provided in the product insert.

What storage conditions are needed for the assay kit or product?

Each assay kit and its components have recommended storage temperatures that are stated on the label and product insert. Read the label/product insert for the recommended storage condition. Most of the reagents can be stored refrigerated at 2-8 degrees centigrade. Do not freeze. All reagents must be brought to room temperature (22-30 degrees centigrade) prior to use.

Can I use components from different kits?

This is not recommended as each assay kit contains reagents and components that have specific lot numbers. This ensures that all the components are performing optimally as QC testing is performed on each specific lot. If the components are interchanged with other kits or from other vendors, it may result in a suboptimal performance.

Do assay kits have an expiration date?

All kits have an expiration date and are clearly marked on the kit box label and individual components. In order for the kit to perform optimally, it should not be used past the expiration date.

Can the kit expire even if the expiration dates on individual component labels have not?

The kit expiration date may not be the same as the expiration dates on the individual reagents. The reagents may have an expiration date that may exceed the kit expiration date and vice versa. This is because when the components are manufactured, they are stored in-house under different conditions, and hence the individual reagent expiration dates reflect this alternate storage. The kit expiration date is assigned for optimal performance of all reagents when stored at the temperature recommended on the labels.

Why does the Negative Control show a positive band?

Strong bands on a Negative Control strip may be due to contaminated negative control or due to cross contamination from a well containing a positive control or serum.

Why does the positive control appear like a negative control?

A positive control can appear as a negative control or with no color if the control was not added (omitted) or if the positive control vial was confused with the negative control. This may also result if the control is pipeted incorrectly (see product insert).

Why do the strips appear completely blank at the end of the test?

The strips may appear completely blank if the conjugate or substrate was omitted. No color will develop if the proper volume of reagents were not added, incubation time and temperature was not observed. This may also occur if the strip was not adequately shaken or the reagents were not brought to room temperature prior to use. Also, please refer to the product insert for tips on good technique to avoid common mistakes, or contact IMMCO technical service department for assistance.

What is the cause of weak color development on the str?

Weak color development may be due to a number of reasons for example, if substrate, antibody and conjugate were not added at the correct point in the assay. Also it is important to use all the components that belong to the specific kit used. Interchanging components with different kits can result in false test results. Incubation times also play a role. Bands may be lower in intensity and color, than expected if the optimum temperature is not used, for example, if the strip was left to incubate on a cold bench or drafty area during ambient incubation. It is also important that all reagents are brought to room temperature prior to use (see product insert). Usually, leaving the kit out on the bench for about half an hour before setting up the assay is sufficient when the reagents have been stored at 4 degrees centigrade. DO NOT freeze reagents.

What is the cause for a high background and poor contrast between bands and background?

The most likely cause for high background and poor contrast is improper washing. Complete washing of the strips between incubations is crucial to obtain valid results. (see product insert). Also adding too little Wash Buffer can result in high background, while adding too much is not a problem. High background could also result if the strip was incubated for too long or at a higher than recommended temperature. The incubation time in the substrate can have an effect, i.e. longer substrate incubations can result in a higher than normal background. This should be monitored closely and the reaction stopped when the controls have reacted to a useful degree (see Product Insert). Also, proteins present in the patients serum may interfere and cause a high background. We recommend to ultra centrifuge the serum and retest. Please contact IMMCO technical service department for any assistance.

Can the volume of sample used in the assay be altered?

It is recommended that the sample volumes not be altered (see product insert). Assay kits are designed for optimal performance at the recommended volumes. In case a smaller volume of the sample is used, then the substrate and conjugate should be reduced by the same factor. The use of non-recommended volumes can alter the test results.

Can the assay protocol be modified?
The assay protocol should not be modified as the Assay Design kits have been optimized to give the best results possible. Modifying the format will give false results (see product insert).
What are the sources of background noise in fluorescence microscopy?

Fluorescence microscopy contrast can be affected by signal to noise ratio. Some of the sources that can increase noise and weaken the signal are: -Unwanted light source. This is the amount of light illuminating a sample that passes through the slide and cover slip or room light. -Vibrations that may arise due to other equipment or air ducts. -Cleanliness of the microscope. -Using an objective that is not specifically designed for high performance. -Using a port that does not give the optimal transmission for green. -Using a filter that does not maximize the number of sample photons collected. – If the illumination time is not minimized.

How can we reduce background noise?

Background noise can be reduced by minimizing the sources that cause it. This can be achieved by the following:

  • Minimize the amount of unwanted light for example, room light. Fluorescence microscopy is generally performed in a darkened room. This can be done by reflecting the light by a white light above the stage in an inverted scope back to the sample.
  • Unwanted room light can be reduced by closing the ocular slider. This can be done by placing a 35mm. plastic film canister upside down over the microscope slide. A hole is placed in the bottom of the film container to allow light to pass through. Also isolate the imaging portion of the microscope from the remainder of the laboratory.
  • Computer monitors also contribute to room light. Other sources like light leaks can be reduced by using black felt at these sites.
What is photo bleaching?

Photo bleaching is the chemical destruction of fluorescent molecules as a result of excitation, fluorescence, microscopy, photo bleaching, and testing”

What are the means of controlling photo bleaching?

Photo bleaching can be controlled by several means:

Fluorescence, Microscopy, Photo Bleaching & Testing”

  • Reducing the illumination level by inserting neutral density filters into the light path or by reducing the lamp output.
  • Using a less sensitive flurophore, for example, polyfluorinated dyes and Alexa
  • Dyes are more stable than conventional dyes.
  • Inhibiting photochemical decomposition may also reduce photo bleaching. Antiphotobleaching compounds such as n-propylgallate, ascorbic acid, and dithionite act as electron donors for triplet fluorophores and thereby lower the concentrations of the reactive triplet during photoexcitation
What is prozone phenomenon?

Prozone phenomenon usually occurs in specimens with high titer antibodies. In immunofluorescence (IFA) testing, when an undiluted or relatively low dilution sample with high levels of specific antibodies produces a negative or weak reaction it is known as prozone phenomenon. Unless the laboratory utilizes an appropriate procedure to screen for prozone, many of these cases would be reported as false negative. Laboratories may guard against prozone by screening specimens at two dilutions, one at the typical screening dilution and one at a two-fold to four-fold dilution greater. Prozone occurs in specimens with high titer antibodies. If prozone is present, the higher screening dilution produces greater (positive) specific fluorescence than the lower screening dilution, which may appear weak, difficult to read or negative.” “fluorescence, microscopy, prozone, testing”

Can I add Evan’s Blue counterstain directly to the conjugate?

IMMCO supplies conjugates in two formats: one with no counterstain and one containing an optimized formulation of Evan’s Blue counterstain. IMMCO does not recommend adding counterstain to the vialed conjugate contained in the kits because this approach is very imprecise with the low volumes involved. The approximate volume of counterstain that would need to be added to the conjugate is approximately 0.02 ml. IMMCO instead recommends purchase of conjugate with counterstain (either separately or with the kit) or addition of counterstain to the reconstituted wash solution as indicated in the instructions for use. “IFA, counterstain, conjugate, testing”

What impact does a variety of conditions have on IFA?

Some of the conditions that might impact IFA testing are :

  • Cross-reactivity in general has limited or minimal impact on the test, however any significant cross reactivity will be identified in the individual product insert.
  • Interference may occur if the serum specimen is either contaminated, hemolytic or icteric, refer to individual product insert.
  • Time of submission, in general antibodies in serum are durable. IMMCO requests that samples be shipped overnight.

However, stability studies have shown autoantibodies to be stable at room temperature for one month or more. Requests for other types of specimens are indicated in the special test request form and product insert.

What are acceptable/unacceptable specimens?

Refer to the individual Test Request forms. A specimen is unacceptable if the vacutainer tube/vial used for collection of serum/biopsy is inappropriate, proper biopsy site for various tests and conditions are not met with. If the blood is hemolytic / icteric it is unacceptable as it interferes with the fluorescence.

 

What causes delay in specimen processing?

Specimen processing can be delayed if all applicable areas are not filled out on the requisition form. This includes :

patient information, insurance/billing information, specimen collected incorrectly (refer to test request form), physician information, test code, clinical information if applicable. IMMCO Diagnostics is committed in providing quality services and will make all efforts to try and resolve matters efficiently and as quickly as possible.

Why is my test showing all negative results?

There are a number of possible reasons, for example, the protocol was not followed, reagents were not added in order, all reagents were not added, for example, if the conjugate is missed there will be no fluorescent reaction, incubation times are not followed. Also, make sure that the microscope is calibrated properly; the technician may be new to reading IFA. These problems are generally addressed by following good laboratory practices such as regular microscope calibration and internal standardization of new assays with known positive and negative controls.

Why is my test showing all positive results?

There are a number of possible reasons, for instance, the microscope may not be calibrated properly, the technician may be new to reading IFA slides, a non-specific fluorescence may be confused with specific fluorescence, there may be infiltration of a positive control or specimen into the well. These problems are generally addressed by good laboratory practices, such as calibration of the microscope, use of correct IM filters, standardization of new assays with known positive and negative controls, “”IFA, results, testing”

Why do expected negative results look positive?

In such a case, check to make sure that the microscope is properly calibrated and the filter is adequate. If the fluorescent light is too strong it may enhance the intensity. Generally this can be resolved by checking the microscope for proper calibration, filters and light intensity. Also, check the reagents supplied in the IMMCO Diagnostics Kits. Technicians may review the product inserts on tips on good technique to avoid common mistakes””IFA, results, testing”

How do I calibrate my IF microscope?

Calibration of microscopy systems may be facilitated by use of Optical Standard slide .IMMCO Diagnostics supplies calibrators such as the IMMCO Diagnostics Optical Standard slide. “calibrate, microscope, IFA, testing”

What are some common problems that customers experience?

Some of the common problems that can occur are : “problems, IFA, testing” ” Reagents running off of wells,” “If this occurs use blotters to prevent runoff, dry the slide before reaction occurs, and while washing shake off the excess water. If using automatic washers make sure it is dispensing properly otherwise the well is not going to get a nice bead on the slide. ” Reaction looks different in different sections of the well. “This may be due to the conjugate or other components running off the well or the dispensing is not appropriate, which effects the beads in the wells .”

I do not understand the proper appearance of a positive reaction?

Refer to IMMCO Atlas at www.IMMCO Diagnostics .com for examples of positive reactions.”atlas, reaction, testing, IFA”

How do I tell specific from nonspecific reactions?

Refer to Interpretation Of Results in the Products section on the website www.IMMCO Diagnostics.com

Can plasma samples rather than serum be used for detecting antibodies?

Antibodies to specific antigens are present both in serum and in plasma, so the assay can be used for either sample. However, the sample dilution has to be optimized for serum.

Where are the proximal tubules of kidney located in the substrate of the LKM, COMVI-VII slide?
Please refer to Interpretation of Results “interpretation, IFA, COMVI, LKM” “file Names ; 2152_3well.jpg,2194_well.jpg, 577px-histology-kidney.jpg “
What is a fluorochrome?

“Flurochromes are highly resonant molecules with fluorescent properties. Upon illumination with a suitable wavelength of excitation the fluorescent molecules absorb energy to an excited state. On relaxation of the excited state, fluorescence is produced by radiative transition to the resting or the ground state. As the fluorochrome has the property of emitting fluorescence on excitation, it can be directly examined under the fluorescent microscope. “

What is photobleaching?

Photobleaching is one of the characteristics and limitations of fluorescence microscopy as the fluorescent preparations may fade on irradiation. The mechanism of photobleaching is not entirely known. Some of the factors may include: -photochemical decomposition involving oxidative and non-oxidative mechanisms. -“the rate at which photobleaching occurs may be dependent on the characteristics of the fluorochrome. Some undergo rapid photobleaching, whereas others are relatively stable.”

How can photobleaching be diminished?

IMMCO mounting medium has been optimized to minimize photobleaching. It is formulated with antifading agents to best preserve fluorescence.

What are the types of fluorescence that can be observed on examination under the fluorescent microscope?

The fluorescence observed on examination under the fluorescence microscope are as follows: -Desired or Specific staining: This is true staining as a result of antigen/antibody reaction. The intensity of the reaction depends upon the antigen/antibody concentration and the degree of the antibody to the flurochrome (fluorescein/protein ratio). -Undesired but Specific staining: This type of reaction may be due to the presence of cross-reactive antibodies in the conjugate to the tissue substrate other than to the target antigen. This can be minimized either by using affinity purified conjugates to the target antigen or by absorption of conjugate with cross-reactive antigen. -“Autofluorescence: this is due to the presence of natural substances in the specimen that fluoresce on excitation with UV light. Sometimes this may conceal or be confused with specific fluorescence. In tissue sections, collagen and elastic fibers quite often exhibit this phenomenon. Examination of sections unstained may help to distinguish autofluorescence from other types of fluorescence.” -Induced fluorescence: This refers to the conversion of a nonfluorescent substance in the tissue into a fluorescent compound by treatment with a nonfluorescent reagent. Formaldehyde-induced fluorescence is the most common example of this phenomenon. -Non-specific staining: This is attributed to the hydrophobic and ionic interactions of highly fluorescinated conjugate with the antigenic substrate.

What is counterstain?

“Counterstain is a reagent used to minimize non-specific fluorescence. It may be present in the conjugate or as a separate component. Unless otherwise requested, IMMCO provides conjugate with Evan’s Blue counterstain added to the formulation. If desired, IMMCO can provide conjugate and counterstain as separate components. When used as a separate component, the technician adds 2-3 drops of Evan’s Blue counterstain in the final wash buffer.”

Can I add Evan’s Blue counterstain directly to the conjugate?

IMMCO supplies conjugate IMMCO provides conjugates in two formats: one with no counterstain and one containing the counterstain volumes involved. The approximate volume of counterstain that would need to be added to the conjugate is approximately 0.02 ml. IMMCO recommends the use of conjugate with counterstain

Can the condition of the specimen impact IF test results?

“It is possible that certain conditions may have an impact on IF test results. Such conditions include specimen storage, condition of the specimen (hemolytic, icteric, contaminated sera). In general, auto antibodies are durable and serum samples will provide reliable results if stored under proper conditions (limited exposure to temperature extremes, long term storage at 2-8ºC or frozen, avoidance of multiple freeze/thaws). Improper storage will lead to degradation of the specimen over time. Cross-reactivity in the assay has been tested using disease control specimens and normal human sera. Any known cross-reactivity has been listed in the product insert. It is not recommended that compromised sera (e.g., grossly hemolyzed, icteric, microbially contaminated) be used in the immunoassay. “

What is prozone phenomenon?

“In immunofluorescence (IF) testing, when an undiluted or relatively low dilution sample with high levels of specific antibodies produces a negative or weak reaction it is known as prozone phenomenon. Unless the laboratory utilizes an appropriate procedure to screen for prozone, many of these cases would be reported as false negative. Laboratories may guard against prozone by screening specimens at two dilutions, one at the typical screening dilution and one at a two-fold to four-fold dilution greater. Prozone occurs in specimens with high titer antibodies. If prozone is present, the higher screening dilution produces greater (positive) specific fluorescence than the lower screening dilution, which may appear weak, difficult to read or negative.”

Why is my test producing all negative results?

-Test protocol not followed (e.g. reagents not added in order or all fail to add one of the reagents). If the conjugate was omitted, for example, there will be no fluorescent reaction.” -Incubation time guidelines were not followed. Certain tests require longer incubation timed (e.g. overnight 18-24 hour incubation). -The fluorescence microscope is not adjusted or calibrated properly. Make sure the proper light source and filters are being used. Such problems are generally addressed by following good laboratory practices such as development of training and maintenance procedures including regular microscope calibration and internal standardization of new assays with known positive and negative controls. IMMCO offers optical standard (OS) slides to facilitate calibration of fluorescence microscopes.

Why is my test producing all positive results?

There are a number of possible reasons, including: -Technician training. Technicians inexperienced in the reading of IFA slides, non-specific fluorescence may be confused with specific fluorescence. The technician must be looking for fluorescence related to a specific reaction pattern rather than general, visible fluorescence.” -Infiltration of a positive control or positive specimen into the wells of other specimens. Generally, specimens and controls form a bead on the slide surface and remain within the well. Technicians should be alert to the fact when samples flow beyond the boundaries of their well and into others it may cause a positive result through cross-contamination. -Poor washing / cross contamination. IMMCO recommends a rinse in a fresh beaker of wash buffer followed by a soak in a coplin jar for 10 minutes. These wash buffers should be replaced between assay runs to eliminate potential for cross-contamination.

What are some common problems experienced by customers performing immunofluorescence assays?

Some of the common problems that can occur are: -Reagents run of from wells of the slide. Make sure the reagents are confined to the wells at each step and there is no run off. To ensure this IMMCO recommends wiping around the wells after each wash with a kim wipe or the use of a blotter. These blotters could be obtained from IMMCO. If using instrument for dispensing make sure it is dispensing properly within the well with complete coverage of the area of the substrate. -Reactions are variable from well to well. This may be caused by reagents running off the well or not being distributed throughout the well. It may also be caused by cross-contamination of specimens and/or controls from one well to another well on the slide. -Background high. This may be either associated with the serum. Some lipemic or incompletely defibrinated serum samples result in high backgrounds. Other reason could be due to incomplete washing of the slides.

What is the desired or expected positive immunofluorescence staining?

Descriptions of reaction patterns are indicated in the product insert and positive controls are provided with kits to present an appropriate reaction pattern, however, the controls provided in the kit may not represent all the reactions that could be observed on a particular substrate. To help with identification of specific fluorescence reaction patterns on a given substrate you may contact our technical services department that may be able to provide images of other reactions. In addition you may visit IMMCO web site for help.

Can plasma samples rather than serum be used for detecting antibodies?
Antibodies to specific antigens are present both in serum and in plasma, so the assay can be used for either sample. However, the sample dilution has to be optimized for serum. Optimal dilution for plasma has not been tested. Qualitative results are likely to be accurate. We cannot indicate how well quantitative results will correlate. Standardization of this method would need to be performed by the client lab.
What is fluorescence microscopy?

The two different methods employed in immunofluorescence are: direct immunofluorescence (IF) and indirect IF. “Direct IF is generally used for immnunopathological examination of skin, kidney or other tissues for the detection of in vivo bound immune deposits.” Indirect IF is used routinely for detection of circulating antibodies in the serum of patients with various autoimmune disorders.

What are the advantages of fluorescence microscopy?

A fluorescence microscope is basically a conventional light microscope with added features and components. It uses higher intensity light to illuminate the sample. “fluorescence, microscopy, IF, testing” “This light excites fluorescence molecules in the sample, which then emit light of a longer wavelength. The light emanating from the fluorescent molecule” is green for FITC. “Fluorescence microscopy is used routinely for studying the distribution of substances present in small amounts in the tissues, serum and other body fluids. Immunofluorescence microscopy is simple and, in the clinical laboratory, it is the mainstay for detecting small amounts of auto antibodies. In fluorescence microscopy the specimen to which a fluorochrome is bound is illuminated with light of a short wavelength, part of this light is absorbed by the specimen and remitted by fluorescence at a wavelength that is longer than that of the incident light. In order to visualize the fluorescence under the microscope the incident light is filtered out by a secondary set of filters, known as barrier filters, which are placed between the specimen and the eye. The resulting fluorescence is green if the flurochrome used is FITC.”

What are the disadvantages of fluorescence microscopy?

“The disadvantages of fluorescence microscopy are that it requires a fluorescence microscope, requires expertise in reading the slides and the fluorescence fades upon illumination (photobleaching).”

What fluorochromes are commonly used in fluorescence microscopy?
“The most common fluorochrome used is fluorescein isothiocyanate (FITC). It produces a green fluorescence when excited with ultra violet light, producing an excitation at 495nm. and emission at 525nm. FITC is used in IMMCO Diagnostics immunofluorescence assays (IFA).” Other fluorochromes that can used are : Rhodamine which produces a red fluorescence with excitation at 552nm. and emission at 570nm. Texas red which produces a red fluorescence at 596nm. and emission at 620nm. Phycoerythrin which produces orange/red fluorescence at different excitations.
What is ANCA?

ANCA stands for ant-neutrophil cytoplasmic antibodies. These antibodies can be visualized using immunofluorescence techniques to react human sera on human polymorphonuclear neutrophils (PMN) as a substrate. Refer to the ANCA product insert for the proper procedure. These antibodies are associated with certain autoimmune vasculitic and gastrointestinal disorders. These antibodies target a number of antigens and produce reaction patterns based on the specificity of the reaction. The types of immunofluorescence reactions that may be visualized on human polymorphonuclear neutrophils (hPMN) cells include cytoplasmic (cANCA), perinuclear (pANCA), atypical pANCA, ANA, and negative reactions.

Why should a patient be tested for the presence of ANCA?

ANCA are markers for various vasculitic disorders including: -Wegeners granulomatosis -Microscopic polyangitis -Churg Strauss syndrome ANCA are markers for inflammatory bowel disease (IBD) including: -Crohn’s Disease -Ulcerative Colitis ANCA are present in hepatobilliary disorders such as: -Primary sclerosing cholangitis -Autoimmune hepatitis “ANCA help to differentiate various subtypes of vasculitis. cANCA is found preferentially in Wegener’s granulomatosis (small blood vessel vasculitis with respiratory tract involvement), while pANCA is found in Microscopic polyangitis and Churg-Strauss syndrome. ” “The levels (titers) of ANCA in patient serum are indicators of disease activity. They are therefore useful in predicting the disease progression and severity, and in monitoring the efficacy of treatment. Early detection of ANCA has therapeutic significance as it may lead to effective early treatment.”

What are neutrophils?

Neutrophils a type of white blood cell that constitute 60-75% of the total white blood cell population. The other major white blood cell is the lymphocyte which constitutes 20-45%. The remaining white blood cells (basophils, monocytes and eosinophils) together constitute less than 10%.

What are the organelles against which ANCA react?

ANCA react to various antigens of the neutrophils and are associated with vasculitis, inflammatory bowel disorders, autoimmune hepatitis, and primary sclerosing cholanagitis. The antigens are primarily associated with alpha granules in the cytoplasm of the neutrophils and monocytes.

What is the disease association of ANCA?

cANCA and pANCA are associated with vasculitis. Atypical pANCA is associated with IBD, primarily with ulcerative colitis.

What is vasculitis?

Vasculitis is inflammation of blood vessels affecting small, medium and large blood vessels. The commonly affected area of the body is skin (cutaneous vasculitis), however, it may also affect other organs such as kidney, lungs, etc. (systemic vasculitis).

What are the symptoms of vasculitis?

Cutaneous vasculitis causes a skin rash, papules and pain. Systemic vasculitis may cause general symptoms such as not feeling well, weight loss, and joint pain.” -Any organ may be affected by vasculitis. When kidney is involved, blood and protein may appear in the urine. -Weakness and numbness of the hands and feet may be caused by inflammation of blood vessels. -Abdominal pain and bleeding. -Vasculitis affecting the myocardium may result in symptoms similar to a heart attack. In the brain, vasculitis may cause a stroke.

How is vasculitis diagnosed?

Initial screening of suspected vasculitis patients is performed by laboratory testing for ANCA by IFA in patient sera. In addition, the following may be observed: -Erythrocyte sedimentation rate (ESR) is generally elevated. -Complete Blood Count will often show anemia, elevated white cell and platelet counts. -Urinalysis to screen for kidney involvement. Kidney biopsies are helpful if there are indications of kidney involvement. -Angiograms can help diagnose vasculitis affecting large blood vessels.

What is the pathophysiological significance of ANCA testing?

The close association between ANCA levels in serum and disease activity suggests a patho-physiological role for ANCA.

What is the clinical significance of testing for the presence of both cANCA and pANCA?

cANCA are predominantly present in patients with Wegener’s granulomatosis, whereas pANCA are predominantly present in patients with Microscopic polyangitis and Churg-Strauss syndrome.

Is it necessary to always test patient sera on both the formalin and ethanol fixed neutrophils?

No. If the patient is suspected of vasculitis, the patient sera can be screened by testing on ethanol fixed neutrophils for the presence of cANCA or pANCA. If pANCA are observed they can be easily confirmed by testing the sera on formalin fixed neutrophils.

What kinds of ANCA reaction patterns are observed?

Three types of ANCA reactions are observed on the neutrophils. These include cytoplasmic (cANCA); perinuclear (pANCA) and atypical pANCA. In addition antinuclear antibodies (ANA) also provide nuclear reactions on the neutrophils.

What is cANCA?

Cytoplasmic reactions on ethanol fixed slides indicate the presence of cANCA antibodies. A sample positive control reaction appears at right. Note that the hPMN cells produce a fluorescent reaction of the cytoplasm of the cell while the lobes of the nucleus are dark (A). Lymphocytes are negative (B). In this image, the lymphocyte has been enhanced to make it more noticeable. ” cANCA antibodies reacted on formalin fixed slides also produce a cytoplasmic pattern. “The primary antigen targeted by cANCA antibodies is proteinase 3 (PR3). PR3 antibodies are associated with vasculitic disorders such as Wegener’s granulomatosis, microscopic polyarteritis and Churg-Strauss syndrome. Other cANCA specificities include bactericidal permealizing increasing factor (BPI) (5%). While the disease association of PR3 and BPI have been established, certain specificities of cANCA may not have clinical relevance.”

What is pANCA?

Perinuclear reactions on ethanol fixed slides may indicate the presence of pANCA antibodies or anti-nuclear antibodies (ANA). A sample positive control reaction appears at right. Note that the hPMN cells produce a fluorescent reaction around the multi-lobed nucleus (A). Lymphocytes are negative (B). In this image, the lymphocyte has been enhanced to make it more noticeable.” “pANCA, perinuclear, testing” “It may be difficult to discern the reaction pattern of pANCA from ANA. A model ANA reaction on ethanol fixed hPMNs appears at right (C). The reaction is similar to the pANCA pattern and may be more intense depending on the titer of the ANA. Note the lymphocytes are positive (D). While the reaction may differ somewhat from pANCA and one can see a difference in the reaction of lymphocytes present, we do not recommend differentiating pANCA from ANA using the ethanol fixed hPMN substrate alone. Specimens that produce a perinuclear reaction on ethanol slides should be tested and compared with their reactions on a formalin fixed hPMN substrate.” “A reference reaction for pANCA on formalin has been provided at right. On a formalin fixed substrate, pANCA reactions become cytoplasmic (E) while ANA reactions become weak or negative. Note the lymphocytes remain negative (F). Comparison of reactions on ethanol and formalin fixed substrates is the best method for discriminating between pANCA and ANA. One may also consider reacting ANA positive reactions on HEp-2 cells to determine the titer and specificity of ANA. ” “The primary antigen targeted by pANCA antibodies is myeloperoxidase (MPO). MPO antibodies are associated with a variety of immune mediated diseases, including microscopic polyarteritis, ” “necrotizing and crescentic glomerulonephritis, Churg-Strauss syndrome, polyarteritis nodosa,” “Wegener’s granulomatosis and chronic inflammatory bowel diseases. Other cANCA specificities include elastase, cathepsin G, and lactoferrin.

Why do the antibody reactions directed against cytoplasmic granules appear perinuclear on ANCA IFA?

The pANCA reactions are considered artifacts of ethanol fixation of neutrophils. Upon ethanol fixation the positively charged pANCA antigen migrate and associate with the negatively charged nucleus. This results in a perinuclear reaction.

How can the pANCA reaction pattern on IFA be distinguished from other anti-nuclear antibody (ANA) reactions?

The pANCA reactions can be distinguished from ANA reactions on formalin fixed neutrophils. On formalin fixed neutrophils the pANCA reaction will convert to a cytoplasmic reaction, whereas ANA reactions will stay nuclear and are either diminished or disappear. Use of formalin (a crosslinking fixative), prevents the migration of the cytoplasmic granules towards the nucleus and thus, pANCA reactions appear cytoplasmic. ” “pANCA, reaction, patter, testing” Samples with pANCA reaction patterns may also be tested on HEp-2 cells to identify specific ANA reactions.

What is Atypical ANCA?

Atypical pANCA, sometimes referred to as “xANCA,” is a distinct perinuclear ANCA pattern characterized by an uneven perinuclear or “snowdrift” pattern and negative by MPO ELISA. Atypical pANCA is present in cases of inflammatory bowel disease. Approximately 80% of ulcerative colitis (UC) patients and 25% of Crohn’s disease (CD) patients are positive for atypical pANCA. In conjunction with testing for anti-saccharomyces cerevisiae antibodies (ASCA), atypical pANCA IFA is a powerful tool for discriminating cases of UC from CD.

What is xANCA?

xANCA is another name for atypical pANCA.

Why are ANCA slides available using different fixatives?

The two primary techniques used for fixation

Why are lymphocytes on the slides? What do they do?

Lymphocytes are present to help a technician distinguish between ANCA and ANA reactions. Patient sera that are positive for cANCA or pANCA but negative for ANA will not produce a fluorescence reaction on the lymphocytes while ANA positive sera will produce a fluorescence reaction on the lymphocytes.

What reactions can I see with ethanol fixed slides?

One can see cANCA, pANCA, atypical pANCA, ANA and negative reactions on ethanol fixed hPMNs. Multiple specificities of ANA may be distinguished, however, this substrate is not recommended for detection of ANA, quantitation of ANA and identification of ANA specificities.

What reactions can I see with formalin fixed slides?

The primary reaction visualized on formalin fixed slides is a cytoplasmic pattern. Both cANCA and pANCA produce cytoplasmic patterns on formalin fixed hPMNs. On this substrate the ANA reaction patterns become weak or negative.

Can ANA and ANCA be present together in the same specimen?

It is very unusual but possible for ANA and ANCA to occur together. This can be identified on formalin fixed hPMN slides, on which pANCA reactions become cytoplasmic, and HEp-2 substrate slides or particular ANA specificities.

With the availability of antigen specific immunoassays such as MPO antibody and PR3 antibody ELISAs, is there a need for immunofluorescence methods of detecting ANCA?

Yes. The recommendation of the ANCA standardization committee is that the initial screening for ANCA should be performed by immunofluorescence as 5-10% ANCA positive samples in patients with vasculitis are only positive by immunofluorescence and negative on the antigen specific immunoassays.

Why is my specimen cANCA positive by IFA and PR3 negative by ELISA?

There are two possible reasons for this: either a negative specimen was misread as cANCA positive or the specimen is positive for cANCA with specificity other than PR3. “IFA, PR3, ANCA, testing” “There are a number of possible reasons for the first instance, for example, the microscope may not be calibrated properly, the technician may be new to reading ANCA or unfamiliar with IFA, there may have been infiltration into the well from another positive specimen or the positive control, etc. These problems are generally addressed by following good laboratory practices, such as regular microscope calibration training and internal standardization of new assays with known positive and negative samples. Such efforts may be facilitated with dilution of an end-point control. Such a control can be produced by diluting out the positive control supplied with the kit to the titer indicated on the certificate of analysis. Calibration of microscopy systems may be facilitated by the use of third party calibrators such as the IMMCO Diagnostics Optical Standard slide.” If the positive cANCA result was read correctly it is likely that the specificity of the cANCA reaction is not PR3. Studies have found commercially available PR3 assays to be 80-90% sensitive for cANCA positive specimens. This means that 10-20% of cANCA positives will be negative by PR3 ELISA. Current literature has not shown clinical relevance for specificities of cANCA other than PR3 and BPI.

Why is my specimen pANCA positive by IFA and MPO negative by ELISA?

There are two possible reasons for this: either a negative specimen was misread as pANCA positive or the specimen is positive for pANCA with specificity other than MPO. “There are a number of possible reasons for the first instance, for example, the microscope may not be calibrated properly, the technician may be new to reading ANCA or unfamiliar with IFA, an ANA reaction may have been confused with pANCA, there may have been infiltration into the well from another positive specimen or the positive control, etc. These problems are generally addressed by following good laboratory practices, such as regular microscope calibration training and internal standardization of new assays with known positive and negative samples. Such efforts may be facilitated with dilution of an end-point control. Such a control can be produced by diluting out the positive control supplied with the kit to the titer indicated on the certificate of analysis. Calibration of microscopy systems may be facilitated by the use of third party calibrators such as the IMMCO Diagnostics Optical Standard slide.” If the positive pANCA result was read correctly it is likely that the specificity of the pANCA reaction is not MPO. Studies have found commercially available MPO assays to be approximately 90% sensitive for pANCA positive specimens. This means that 10% of pANCA positives will be negative by MPO ELISA such as elastase or lactoferrin. The clinical relevance of pANCA specificities other than MPO and atypical pANCA is not well established.

Why is my specimen cANCA negative by IFA and PR3 positive by ELISA?

In such a case, the likelihood is an error in performing the immunofluorescence technique. There are several possibilities: one step of the assay may have been missed, there may have been an air bubble on the well during delivery of the specimen or reagents or the cells on the slide may have been damaged. An experienced technician will quickly identify if the third case is true as the hPMN cells can generally be discerned even on a negative patient specimen and broken cells will generally leave observable debris in the well. Technicians may wish to review the tips on good technique in the document to avoid common pitfalls.

How do I tell the difference between a weak positive and a negative reaction?

We recommend testing such specimens on HEp-2 IFA substrate. ANA negative specimens may be atypical pANCA or BPI positive. Additional ELISA assays to detect other specificities of ANCA may be performed (e.g. BPI), however, many of these assays are available for research use only and the clinical significance of these specificities may not be established.

Do I need to test a specimen with a positive pANCA reaction on a formalin fixed slide?

It is recommended that laboratories use best practices including internal standardization of the assay using known positives and negative, train the individuals that will be reacting and reading slides and perform regular maintenance and calibration of their microscopy systems. Experienced immunofluorescence technicians will be well prepared to properly read slides and distinguish between specific and non-specific fluorescence. Standardization may be facilitated with dilution of an end-point control. Such a control can be produced by diluting out the positive control supplied with the kit to the titer indicated on the certificate of analysis. Calibration of microscopy systems may be facilitated by the use of third party calibrators such as the IMMCO Diagnostics Optical Standard slide.

Do I need to test a specimen with a positive pANCA reaction on a formalin fixed slide?

While it is possible to distinguish between pANCA and ANA reactions on ethanol fixed slides alone, such reading is fraught with the possibility for error. For this reason we strongly recommend reacting specimens producing perinuclear reactions on both ethanol fixed and formalin fixed substrates. To facilitate this, we offer a number of options for your laboratory, including COMVI slides which allow patient specimens to be reacted on both substrates on a single slide.

Why doesn’t the reaction I am seeing look like the sample cANCA and pANCA reactions?

The ANCA reactions produced using the controls provided with the kit and found in this document are ideal reactions. While it is not unusual for reactions of patient specimens to closely resemble these images, specificity, antibody titer, masking reactions and background may complicate reading. The following problems stemming from environmental conditions and performance of the assay may also affect your results: -Weak reactivity “Possible causes: slides and reagents not allowed to equilibrate to room temperature, insufficient incubation time” cANCA and pANCA reactions in the same well “Possible causes: specimens allowed to bleed from well to well, washing technique used caused cross reaction from well to well” Positive and negative reactions in the same well “Possible causes: specimens allowed to bleed from well to well, washing technique used caused cross reaction from well to well, bubble prevented delivery of reagents or specimen to the entire well” Difficulty reading slide due to bubbles or blurriness Possible cause: air trapped between coverslip and slide when mounting the coverslip Broken cells “Possible causes: washing of slide performed with direct, forceful stream of wash buffer, coverslip applied forcefully, physical contact with well, slide not read within 48 hours, prolonged exposure to environmental conditions outside of recommended storage conditions (2°-8°C).” Diffuse pANCA reaction “Possible causes: slides not read within 48 hours of reaction, prolonged exposure to environmental conditions outside of recommended storage conditions (2°-8°C)” High background “Possible causes: poor washing technique, infiltration of other specimens into well, extended incubation times, slides not read within 48 hours of reaction, prolonged exposure to environmental conditions outside of recommended storage conditions (2°-8°C)” No cells in the well “Possible causes

It looks like the cells on the slide are damaged or disintegrating. Why is this happening?

There may be several reasons for this happening. This can occur as the result of improper technique or environmental effects. “cells, slides, ANCA, damaged, testing” “Most commonly this results from pressure applied to delicate cells. Cells can be damaged by washing forcefully with a stream of wash buffer from a squeeze bottle applied directly to a well, by forceful application of a coverslip or even through brushing the well with a fingernail.” “It is also common to see this if slides have not been read within 48 hours of reaction. The cells will begin to lose integrity after reaction. Unless the slides are specially treated, reactions will diffuse within a period of days. Similar effects may be seen if the slides have suffered prolonged exposure to environmental conditions outside of recommended storage conditions (2°-8°C).”

Why is the negative control well giving a positive reaction?

Generally, this results from cross reaction or carryover from well to well. This will occur when specimens are allowed to bleed into the negative control well during reaction or washing or when washed-off specimens become concentrated in a Coplin jar. To prevent this, we recommend use of two Coplin jars in washing IFA slides: one to quickly rinse controls and reagents off the slide.

Why is the slide blurry?

This may occur as a result of coverslipping technique. Sufficient mounting medium should be applied to cover the wells before the coverslip is applied. One should take care to avoid or gently squeeze out air bubbles. If this occurs, the coverslip can be removed and remounted, however, care must be taken to prevent damage to the cells.

Why am I seeing both cANCA and pANCA reactions in a well?

Generally, this results from cross reaction or carryover from well to well. This will occur when specimens are allowed to bleed from well to well during reaction or washing or when washed-off specimens become concentrated in a Coplin jar. To prevent this, we recommend use of two Coplin jars in washing IFA slides: one to quickly rinse controls and reagents off the slide and a second to soak the slide for 10 minutes.

Can ANCA of varying specificities occur in the same specimen?

The majority of ANCA reactions are mono-specific. However, very rarely, ANCA of more than one specificity may be present in the same specimen.

Under the microscope the field looks reddish brown. Why?

This results from prolonged exposure to environmental conditions outside of recommended storage conditions (2°-8°C). If the well is turning red/brown, the slide has likely been exposed to temperatures higher than room temperature.

Under the microscope some sections of the well seem to be positive and some seem to be negative. Why?

The most common reason for such a reaction is that reagents or specimens have not been delivered to the well properly during processing. This can result from pipetting technique, especially if air bubbles are delivered or allowed to form on the well.

There seems to be a lot of debris. Why?

This often occurs if the hPMN cells have been damaged or if reactions have been allowed to diffuse. To prevent this we recommend that the slides be washed gently in Coplin jars, that the coverslip be applied gently and that slides be read within 48 hours of being reacted.

Why is the positive control reaction so weak?

The most likely cause is that the slides or reagents have not been allowed to equilibrate to room temperature.

Why doesn’t the reaction look as strong as when I first looked at it?

Reactions will fade over time, especially if slides are left exposed to light.

Could you provide some tips on good technique for running this test?
Guidelines for running the ANCA assay appear below: “tips, guidelines, technique, ANCA, testing” Precautions 1 “Exercise Universal Precautions when performing this assay. Although potentially infectious materials in this kit have been tested for HBsAg, HCV, HIV-1 and 2 and HTLV-I and found negative, these reagents and patient specimens must be handled as though they carry infectious biological agents. Lab coats and gloves must be worn while performing this procedure.” 2 “Reagents included with this kit contain sodium azide (NaN3) which may react with lead and copper plumbing to form highly explosive metal azides. Upon disposal of liquids, flush with large volumes of water to prevent azide buildup. Sodium azide may be toxic if ingested. If ingested, report incident immediately to laboratory director or poison control center.” 3 Instructions should be followed exactly as they appear in the product insert to ensure valid results. Do not interchange kit components with those from sources other than the same catalog number. 4 Do not use this kit beyond the expiration date. 5 “Only serum specimens should be used for this procedure. Grossly hemolyzed, lipemic or microbially contaminated specimens may interfere with the performance of this test and should not be used. ” Storage 1 “Store specimens at 2°‑8°C for no longer than one week. For longer storage, serum should be frozen at -20°C.” 2 Avoid repeated freezing and thawing of samples. 3 “After slides are reacted, if slides are not to be read immediately, they should be stored at 2°‑8°C in a dark place until read.” 4 Slides should be read with in 48 hours of reaction. Preparation 1 Allow reagents and patient specimens to equilibrate to room temperature for 10-15 minutes. 2 Allow pouches containing substrate slides to equilibrate to room temp