Table 1.
Effect of removal of sialic acid residues and/or removal of polylactosamines on ANCA staining pattern
| Patienta | Sex | Age (yr) | IIF | MPO/PR3 (U/ml) | hLAMP-2 | ANCA | |||
|---|---|---|---|---|---|---|---|---|---|
| ANCA | GEnC | Neuraminidase | PNGaseF | Endo-β-Galactosidase | |||||
| ANCA-negative/hLAMP-2 autoantibody positive | |||||||||
| A | F | 60 | − | + | − | + | Membrane ++ | No difference | C+ |
| B | F | 65 | − | NA | − | + | Atypical C/ membrane ++ | No difference | C+ |
| C | F | 33 | − | + | − | + | Atypical C+ | No difference | C++ |
| D (39) | M | 33 | − | + | − | + | Atypical C+ | No difference | Atypical C++ |
| E | F | 22 | − | + | − | + | Atypical C++ | C++ | Negative |
| F | F | 85 | − | + | − | + | C | No difference | C± |
| G | F | 70 | − | NA | − | + | C | No difference | C |
| H (70) | F | 17 | − | + | − | + | C++ | No difference | C++ |
| ANCA-negative/hLAMP-2 autoantibody negative | |||||||||
| I | M | 29 | − | NA | − | − | No difference | NA | NA |
| J | F | 73 | − | + | − | − | No difference | NA | NA |
| K | M | 59 | − | + | − | − | No difference | No difference | No difference |
| ANCA-positive/hLAMP-2 autoantibody negative controls | |||||||||
| L | M | 78 | P | NA | MPO 22 | − | No difference | NA | NA |
| M (57) | F | 57 | C | NA | PR3 20 | − | No difference | No difference | No difference |
| N (51) | M | 58 | C | − | PR3 50.55 | − | No difference | NA | NA |
| O (9) | F | 76 | C | − | PR3 360 | − | No difference | No difference | No difference |
| P | M | 45 | C | − | PR3 39.8 | − | No difference | NA | NA |
All 11 ANCA-negative patients (patients A–K) had negative results in standard, commercially available indirect immunofluorescence assays; however, patient sera with antibodies to hLAMP-2 exhibited binding to GEnC by IIF using either frozen sections of human kidney or immortalized glomerular endothelial cells (n=8, A–H). Using sera from those eight patients for IIF, removal of either N-glycans (n=1, E) or both, removal of sialic acid and polylactosamines (n=7, A–D, F–H) from normal human granulocytes, enhanced fluorescence intensity and revealed either a membrane bound staining (n=2) and/or an (atypical) cANCA pattern (n=7). The remaining three patients did not exhibit hLAMP-2 antibodies by ELISA and treatment of granulocytes did not alter their ANCA negativity by IIF assays (patients I–K). The ANCA staining pattern of five patients with either cANCA/anti-PR3 antibodies (n=4) or pANCA/anti-MPO antibodies (n=1) was not altered by carbohydrate removal (patients L–P), nor did treatment with the enzymes change negative results from sera of healthy controls (n=3). IIF, indirect immunofluorescence; F, female; −, negative; +, positive; membrane ++, membrane accentuated staining; C, cytoplasmic ANCA; NA, not available; M, male; atypical, atypical staining pattern; no difference, no difference in staining pattern to untreated cells; P, perinuclear ANCA.
Numbers in parentheses next to the patients’ assignation (A–P) refer to patients in Supplemental Table 3 published previously by Kain et al. (18).