The overall goal of this procedure is to demonstrate an improved, indirect immunofluorescent assay that uses a Novel HEp-2, dense fine speckled 70 knockout substrate to screen antinuclear autoantibodies and simultaneously confirm monospecific and mixed dense fine speckled 70 patterns. The Novel HEp-2 substrate uses a standard, indirect immunofluorescence procedure and interpretation criteria for screening of clinically relevant antinuclear antibodies, which are also known as ANAs. The main advantage is the ability to distinguish the dense fine speckled 70 pattern from disease-associated, homogeneous speckled, or challenging mixed patterns at the screening step.
Demonstrating the procedure will be Sofia Badanin, a technician from our quality control laboratory. For substrate slides, use glass slides with 12 wells on each slide, containing either the conventional HEp-2 cells, or a mixture of conventional and dense fine speckled 70 knockout HEp-2 cells. Allow the pouches containing the substrate slides to equilibrate to room temperature for optimal performance and to prevent condensation of moisture on the slide surface prior to sample addition.
This should take between 10 and 15 minutes. Once equilibrated to room temperature, carefully remove the substrate slides using one's fingers, avoiding contact between the slides and the sides of the pouch. Label the slides and place them in an incubation chamber that his been lined with moistened paper towels to prevent drying of the slides during sample and conjugate incubation.
Dispense approximately 50 microliters of the negative and positive controls, as well as the diluted patient sera onto individual slide wells before placing the slides into the incubation chamber. The ANA test is the first step in screening for an autoimmune disease. It is critical that cross-contamination of patient samples on the slide is avoided to minimize false positive or false negative results.
To prevent well cross-contamination, avoid overfilling the wells. Place the lid on the incubation chamber and incubate the slides for 30 minutes at room temperature. If there is any ANA present in the serum of the patient, it will bind to the cells present in each will during this time.
Once the incubation period is over, remove the slide from the incubation chamber. Hold the slide at the tab end and gently rinse for 10 to 15 seconds with approximately 10 milliliters of the reconstituted phosphate buffered saline wash buffer. Transfer the slide immediately into a Coplin jar with PBS wash buffer and soak for 10 minutes.
Repeat the process with all remaining slides. Remove only one slide at a time from the Coplin jar. To remove the excess PBS wash buffer from the slide, gently tap or blot the slide on a paper towel.
Onto each well, gently apply one drop of fluorescein isothiocyanate, labeled anti-human IgG fluorescent conjugate. Then, incubate the slides in the closed staining incubation chamber for 30 minutes. Once the incubation period is over, remove the slide from the incubation chamber.
Hold the slide at the tab end and gently rinse as before, with approximately 10 milliliters of PBS wash buffer. Transfer the slide immediately into a Coplin jar with PBS wash buffer and soak for 10 minutes. Place the cover slips that are provided in the kit on a dry paper towel.
To the bottom edge of the cover slip, apply between 3 and 4 drops of the mounting media in a line. Next, take out one slide at time from the Coplin jar containing the wash buffer. To remove the excess wash buffer, gently tap or blot the slide on the paper towel.
Avoid leaving an excess amount of wash buffer on the slide, applying too much pressure on the cover slip or introducing air bubbles. All of these can impact the cell morphology, the resulting fluorescent patterns and the intensity of the reactions. Lower the slide gently onto the cover slip, by placing the lower edge of the slide to the edge of the cover slip.
This allows the mounting media present on the cover slip to flow to the top edge of the slide and minimizes the formation of air bubbles, which may interfere with reading each well. View the slides with a fluorescent microscope, using a fluorescein isothiocyanate filter set located in a darkroom. Examine the sample for specific bright green fluorescence under the microscope at a magnification of 200-fold or greater to make the final interpretation regarding positivity and pattern.
Observe the positive and negative controls. The positive control will display bright green fluorescence in the nucleus. The negative control will display no specific green fluorescence under a fluorescence microscope.
Record the positive or negative result for each well and include the ANA pattern for positive cases. HEp-2 dense fine speckled 70 knockout substrate preserves all major nuclear and cytoplasmic patterns. Dense fine speckled pattern is characterized by the presence of uniformly distributed speckled staining throughout the interphase nucleus and chromatin in the mitotic phase.
Presence of both conventional HEp-2 cells that express dense fine speckled 70 antigen and engineered HEp-2 cells devoid of the antigen facilitates accurate distinction of the dense fine speckled 70 pattern, while retaining all advantages of conventional HEp-2 substrates. Sera positive for major disease-associated ANA patterns were mixed with a dense fine speckled 70 pattern positive serum in equal proportions and tested on both conventional HEp-2 and the HEp-2 dense fine speckled 70 knockout substrates. Here, red arrows indicate cells in the mitotic phase.
Yellow arrows indicate the conventional HEp-2 nuclei and blue arrows indicate the engineered HEp-2 nuclei. For all the depicted combinations of reactions, the dense fine speckled 70 knockout substrate shows a clear intensity difference between the unmodified cells and engineered cells on the same field of view. This aids in distinguishing mono-specific and mixed dense fine speckled 70 reactions.
Autoantibodies to nuclear and cytoplasmic antigens are highly prevalent in systemic autoimmune diseases. Dense fine speckled pattern resulting from auto antibodies towards DFS70, also known as PSIP1 protein, have been reported to be highly prevalent in healthy populations. Moreover, these DFS70 autoantibodies occur both in isolation as well as with other disease-associated ANAs.
This makes it critical for clinical labs to accurately distinguish this pattern from other disease-associated patterns. For example, in mixed homogeneous speckled pattern, which is associated with Lupus, could be misinterpreted as DFS70 pattern necessitating multiple confirmatory assays. Indirect immunofluorescence method using HEp-2 substrates is considered the gold standard screening method for ANAs.
However, accuracy is highly dependent on the skill of the technician, careful execution of the individual steps and accurate interpretation of resulting patterns. Conversely, the comparative evaluation of conventional HEp-2 and DFS70 knockout substrates demonstrates the utility of this new substrate in distinguishing the DFS70 pattern with high confidence, while screening for ANAs. Further interpretation of both monopositive and mixed DFS70 patterns was relatively simplified using this improved HEp-2 substrate.
The new substrate uses a standard, indirect immunofluorescence protocol and established interpretation criteria for all clinically relevant ANA patterns.
