Anti-Nuclear Antibody Screening Using HEp-2 Cells

The overall goal of the following experiment is to use an indirect immunofluorescent assay to screen for anti-nuclear antibodies. This is achieved by incubating patient serum with hep two substrate slides. The unbound patient serum is washed off the bound auto antibodies are then incubated with specific fluorescein labeled conjugate, and the unbound reagent is washed off.

When viewed through a fluorescence microscope, auto antibody positive samples will exhibit an apple green fluorescence corresponding to areas of the cell or nuclei where auto antibody has bound in a pattern of fluorescence indicative of the antigen being present. Ultimately, the presence of arna can be used to aid in the diagnosis of connective tissue diseases. The main advantage of the indirect immunofluorescent method over other methods like solid phase Eliza, is that the Hep two cell substrate contains over 100 antigens expressed in their native configuration.

This allows the identification of almost all clinically relevant O antibodies, which is not possible with solid face techniques as the indirect immunofluorescent method is a very specialized technique. Individuals new to this method need time and practice to become proficient in the procedure of slide processing. Interpreting the microscope images and learning to identify relevant patterns is also a skill that takes time to master.

With the right reagents and tools, the results are more consistent and easier to interpret. Demonstrating the procedure will be Cassandra Bryant, a technologist from the Immunofluorescent Asay Development Laboratory. Remove reagents from packaging and allow each item to come to room temperature, prepare reagents and dilute patient serum according to the direction insert slides are barcoded and can be easily integrated into automated systems.

This procedure illustrates manual slide processing. High throughput laboratories, however, may opt for automated slide processing equipment with barcode scanning capabilities. Inova instruments are connected through a centralized intelligent network that provides control over workflow results and reporting.

For IFA. This results in positive patient identification from processing and elimination of transcription and related errors. Dispense one drop of positive control and one drop of negative control onto the appropriate slide.

Wells pipette 20 to 25 microliters of diluted patient serum to the remaining wells. Process one slide at a time. Place the slide in a staining container with a damp paper towel on the bottom, cover the container and incubate the slide for 30 minutes.

The humid conditions will prevent the substrate from drying out, which could result in artifactual staining. During this incubation period, the anti-nuclear antibodies in the patient's serum will bind to antigens of the cells that are fixed onto each well. After the incubation period, rinse off the serum using a gentle stream of wash buffer in order to avoid damaging the substrate.

Angle the slide slightly so that the stream is not directly pointed on the substrate. This orientation of the slides will also help to prevent crossover of samples between wells. Tap off excess wash buffer and place the slide into a Copeland jar containing wash buffer.

The incubation time for the wash step should be approximately five minutes. Remove the slides from the wash buffer and gently tap. To remove the excess wash buffer, apply one drop of fluorescent conjugate onto each well.

For a NA testing, the use of an IgG FC specific conjugate is recommended. Incubate the slides for 30 minutes in the humidified container and be sure to replace the staining cover. The conjugate is light sensitive and the cover will protect the slides from light exposure.

During this incubation period, the conjugate will bind to the patient's anti-nuclear antibodies that have bound to the cell antigens. This conjugate binding results in the presence of fluorescence in the wells following incubation. Wash the slide with wash buffer.

As before, place a cover slip on a paper towel and apply mounting medium in a continuous line to the bottom edge of the cover slip. Remove each slide from the wash buffer and tap the slide gently. To remove the excess wash buffer, touch the lower edge of the slide to the edge of the cover slip.

Gently lower the slide onto the cover slip in such a way that the mounting medium on the cover slip flows to the top edge of the slide without air bubble cover slipping, including the optimal amount of mounting medium is a technique that takes practice to perfect view. Slides with a fluorescent microscope located in a dark room, scanning of the entire well should be performed with a 20 x or 25 x objective. To assess cell distribution and uniformity of fluorescence switch to a 40 x objective.

To make the final interpretation regarding positivity and pattern, look at the positive and negative controls. The negative control may not appear completely dark, but will often display low level non-specific fluorescence. The positive control will display bright apple green fluorescence in the nucleus.

Positivity can be graded by using a reactivity grading scale from one plus to four plus. In addition to manual interpretation, slides can be loaded and scanned by the automated fluorescence microscope. No dark room is necessary.

After creating a project by selecting the appropriate slide type, the instrument acquires and stores high resolution digital images of cells in each well. Additionally, Nova view measures fluorescent light intensity and categorizes results as positive or negative and provides pattern recognition for positive samples. Images are viewed by the operator on a high resolution computer monitor, allowing for final interpretation, revision, and confirmation of Nova View.

Result reports can be generated on confirmed results. Auto-antibody binding on the constituent protein structures within the nucleus results in five major nuclear patterns, including homogeneous, speckled centromere, nucleolar, and nuclear dot. To identify a homogeneous pattern as shown here, identify mitotic or dividing cells.

Mitotic cells show solid uniform fluorescence, which is often more pronounced than in resting cells. The resting cell nuclei should be uniform with diffuse staining. This characteristic pattern is most likely the result of autoantibodies to double stranded DNA.

A key characteristic of the speckled pattern is the coin slot appearance of the metaphase mitotic cells. The chromosomal region of these cells is negative. The resting cells display a speckling pattern throughout the nuclei.

The speckling can be defined as coarse or fine. Course speckling is the result of auto antibodies to SM and RNP. Fine speckling can be due to auto antibodies to S-S-A-S-S-B as well as RNA polymerase.

The DFS pattern is referred to as dense, fine speckled, and is indicative of auto antibodies to DFS 70. Mitotic cells show speckled staining while resting cells display uniformly distributed fine speckles throughout the nucleus. In these cases, confirmatory tests should be performed as DFS 70 autoantibodies are prevalent in healthy individuals versus patients with connective tissue disease.

To identify the centromere pattern, scan the wells and identify mitotic or dividing cells. The dividing cells have numerous discreet speckles in close association with one another, often called the metaphase bar. The resting cells show approximately 40 to 60 discrete speckles distributed throughout the nucleus.

The centromere pattern is associated with autoantibodies to centromere proteins with the nucleolar pattern. Staining of the chromosomal region in mitotic cells is variable. The nucleolar pattern is associated with homogeneous or speckled staining of the nuclei, along with weak, speckled, or homogeneous staining of the nucleoplasm of the resting cell nuclei.

This pattern is associated with autoantibodies to RNA polymerase three fibrillin and PM SCL antibodies. The nuclear dot pattern is associated with negative metaphase, mitotic cells, and few discrete dots in the resting cell nuclei. This characteristic pattern is often the result of autoantibodies to SP 100 PML or P 80 colan.

These antibodies are associated with primary biliary cirrhosis and autoimmune hepatitis. In the image shown, the nuclear dot pattern displays cytoplasmic staining due to auto antibodies to mitochondrial antigens. Anti-nuclear antibody detection and pattern recognition serves as an important tool to aid in patient diagnosis.

Understanding the significance of various patterns enables clinicians and laboratory personal to perform appropriate follow-up testing. The most important factors to correctly identify anti-nuclear antibody patterns are selecting a high quality cell substrate and processing the slides with consistent good technique. Slide reading is traditionally performed by fluorescent microscopy in dark rooms and is done by trained technologists who are familiar with the various patterns in the context of cell cycle and cell morphology.

In the last few years, digital imaging systems have been developed for the automated reading of the slides, such instruments automated workflow and increase the consistency of reading and interpretation. Moreover, with the use of barcoded slides, sample traceability throughout the process eliminates potential transcription errors and increases data integrity and patient safety. After watching this video, you should have a good understanding on how to perform each step of the indirect immunofluorescent procedure and how to identify clinically relevant antinuclear antibody patterns.