| Home |
| About EUVAS |
| Active Trials |
| Completed Trials |
| Meetings |
| Guidelines |
| Publications |
| Nomenclature |
| ANCA testing |
| Disease scoring |
| Histopathology |
| Links |
| Contact details |
This assay description closely follows the methodology agreed upon as the standard method at the 1.st. International Workshop on ANCA in 1988 (5), later described in great detail to avoid problems with the leucocyte substrate due to cell activation (6).
10 ml of blood is drawn into a syringe and the content is immediately transferred to a conical flask containing 10 glass beads for defibrinating the blood by circular move-ments. The defibrinated blood is transferred into a 10 ml polypropylene tube and mixed with 250 IU of heparin. 2 ml aliquots of the stabilized blood are layered onto 5 ml dex-tran-metrizoate gradients (24.7 ml dist. H2O, 89.4 ml 6% dextran T-500 and 20 ml 75% sodium metrizoate) in 10 ml polypropylene tubes, left for 45 min. at room temperature to sediment red cells. The leucocyte-rich plasma fractions are combined for washing in a 10 ml tube, centrifuged at 200 x g for 10 min. at room temperature, the pellet resuspended in phosphate buffered saline (PBS) pH 7.4 with 1% human serum albumin, centrifuged as before and the supernatant discarded. The cells are finally resuspended in PBS/albumin, transferred to a conical tube and centrifuged as above. The cell pellet in the bottom is re-suspended in 150-200 ml of the PBS/albumin by tapping the tube, and the concentrated buffy coat suspension is used for making cell smears. A fine-tipped Pasteur pipette drawn out over a gas flame to produce a capillary pipette is now used to deposit 1 mm diameter droplets on carefully defatted objective slides, immediately followed by smearing using a ground-edged slide. Alternatively the leucocytes can be deposited by cytocentrifugation, aiming at getting leucocytes with a suitable density for reading the results. After drying by air at room temperature slides are fixed for 5 min. in 40 °C cold 99% ethanol. These slides can be used directly or can be stored dry at -200 for use within a week. A negative control serum, positive C-ANCA and P-ANCA control sera and patient sera are added to the slides at a dilution of 1:20 in PBS, left for incubation at ambient temperature for 30 min. in a humid chamber. Washing of the slides in PBS is done twice and finally FITC-labelled anti-human IgG conjugate is added at appropriate dilution, the slides incubated for 30 min. in the humid chamber and the two washing steps repeated. After removal of surrounding buffer a 2:1 mixture of PBS pH 8.0 and glycerol is added as a drop, and the cells covered by a cover glass to avoid evaporation. Slides are then read in an epi-illumination fluorescence microscope at 400 x magnification using an objective with a high numerical aperture.
The single most important factor is to avoid activation/degranulation of the neutrophils in the buffy coat during cell isolation and washing. This is best accomplished by following the diligent handling procedure described above, and by running each step just after the other until the leucocyte smears have been fixed in ethanol. Extra hands are mandatory when the concentrated leucocyte droplets have to be smeared. FITC-conjugates with a low fluorescein/protein ratio (< 2.5) are necessary to avoid non-specific staining of neu-trophils and eosinophils. The smears should contain enough lymphocytes to allow control for presence of non-neutrophil specific antibodies.