Fluorescent antinuclear antibody test (FANA) : Principle, Procedure & Test results

Fluorescent antinuclear antibody test (FANA) is an indirect immunofluorescence staining technique used for the detection of antinuclear antibodies present in human serum (FANA).

Fluorescent antinuclear antibody test (FANA) : Principle, Procedure & Test results
Fluorescent antinuclear antibody test (FANA) : Principle, Procedure & Test results
Fluorescent antinuclear antibody test (FANA) : Principle, Procedure & Test results

Principle of Fluorescent antinuclear antibody test

The antibodies in the patient’s serum combines with the tissue antigen to form a complex. This complex then binds with the fluorescent anti-human globulin conjugate and thus fluoresce in ultra-violet illumination.

Fluorescent antinuclear antibody test Performance specifications

  • Then test should be perform with freshly coated slides.
  • The slides should be thoroughly washed after each step to get rid of non-specific fluorescence which might interfere with reading of the result.
  • Reading of the result should be done by experienced personnel

Sample Collection

  • Use only serum for this test
  • Collect 2 mL of venous blood in a plain red topped vacutainer tube
  • Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10
  • Take note not to use lysed or grossly lipemic or contaminated serum.
  • It is advisable to process the specimen on the same day. If analysis is not done on the same day of collection, separate the serum and store it at 2–8 °C for up to 5 days or at –20°C for longer periods.

Required Reagents/consumables      

  • Phosphate buffered saline at pH 7.2
  • Fluorescein conjugated anti-human immunoglobulin (polyvalent)
  • Acetone
  • Mounting medium - 50% glycerol in 0.05 M sodium barbitate pH 8.6

Required Equipment for TPHA Test

  • Micro pipettes/cell droppers
  • Micro titer plates are required

Preparation of Glass Slides

On the glass slides five circles are marked using diamond marker and the slides are dropped in distilled water containing 0.025% sodium meta silicate and it is kept in room temperature for half an hour. Then the slides are cleared well with tissue paper, then with methanol dipped tissue paper and again with clean tissue paper. Finally slides are wrapped with aluminum foil and it is kept in hot air oven for sterilization at 160°C for 1 hour.

Coating of Slides

  • Inoculate into the wells of the specially made slides with HEp - 2 cells on the previous day which is incubated in 10% Carbon dioxide atmosphere overnight.
  • Dilute the serum in saline from 1 in 4 up to 1 in 32 (four-fold dilution).
  • Include positive control
  • Include negative sera control at the same dilution as test. Mark the slide with diamond marker.
  • Then add 10 μL of the 1 in 8, 1 in 16 and 1 in 32 diluted samples to three
    wells of the slide coated with cell lines.
  • Keep in a moist chamber for 30 minutes. Wash thrice with PBS in slide chamber.
  • Then add 5 μL of 1 in 20 diluted (diluted in PBS pH –7.2) rabbit antihuman Fluorescent Isothiocyanate (FITC) conjugate polyvalent serum to each of the wells.
  • Keep for 30 minutes in moist chamber. Wash thrice in PBS and mount with glycerol mounting fluid and observe under 20X of fluorescent microscope with blue filter.

Fluorescent antinuclear antibody test results

Staining character

Staining character

Antibodies to

Rim and homogeneous

 

dS DNA

DNA - histone complex

Classes of histones

dS DNA

DN protein

LE - Cell ab

Variable large speckles

 

H3

 

Histone

 

Speckled        

Small nucleolar RNP

RNA protein

Complex protein

Smith Ag

Nuclear RNP

NSp I, NSpII

Nucleolar & Cytoplasm

Ribosomal fraction

Cytoplasmic RNP

Variable speckled in some cells

33 KD a protein

PCNA

Descrete speckled

Protein

Centromere

 

Precautions          

  • Handle all samples as potentially infectious.
  • Handle all reagents with care and avoid contact with eye, mouth and skin
  • Do not perform mouth pipetting.
  • Discard used reagents and sample as per disposal procedure

References

Sankara Nethralaya’s, Fluorescent antinuclear antibody test (FANA)  test, Manual of Medical Laboratory Techniques page 377

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