P030 : THE EFFECTS OF HYPOTONIC DIALYSIS AND SPIN COLUMNS ON SERUM ANTI-HLA IgG LEVELS

Abstract

Aim Our laboratory recently noticed an influx in positive allo- and auto-crossmatches presumably due to non-HLA interfering factors. We investigated the use of hypotonic dialysis (HD) and size exclusion spin columns (SC) to remove these factors while mitigating the effects on IgG antibodies. Methods 50 kDa dialysis membranes and 300 kDa size exclusion spin columns were used to remove IgM and other interfering factors from serum. Sera from six patients with moderate-titer antibody were treated three ways: DTT (5 mM @ 37 °C for 30 min), HD using Spectra/Por® Float-a-lyzer G2 according to the product insert, and NanoSep 300 SC (Pall Life Sciences) according to product insert. The IgG fractions of these separation methods were tested for IgG levels using OneLambda LabScreen single antigen (SA) bead kits. Class I (CI) and Class II (CII) antibody levels were analyzed separately using a two-tailed t -test, due to the inherent variability seen in the CI SA assay. Only allele specificities above our laboratory’s positive cutoff (MFI > 1000) were investigated. Results Luminex SA CI results showed variability between patients tested. No significant change in MFI was seen for CI DTT vs dialyzed samples ( p = 0.128) or for DTT vs SC-treated samples ( p = 0.209). CII samples showed a significant change for both methods: DTT vs dialyzed ( p = 0.016) and DTT vs SC ( p = 1.38x10-6). Spin columns decreased the MFI for all 181 CII specificities investigated for the six patients tested. Fig. 1 illustrates how the MFI for one patient’s CII sample was affected by all studied methods. Conclusions We have determined that SC treatment alters patient serum too significantly for our laboratory to use. HD appears to be potentially useful, though it does often raise the MFI of some existing specificities and may increase the amount of unacceptable antigens detected. More testing is required to determine the clinical relevance of MFI increases. Flow XMs will also need to be performed with dialyzed serum to determine if sera treated in such a way may be used in crossmatches.