Hemolysis may cause false negative results and underdiagnosis of celiac disease when measuring anti-tissue transglutaminase antibodies in serum by immunoassays

According to the Phadia 250 user manual [1], hemolysis and lipemia can potentially cause incorrect results. However, the manufacturer, Thermo Fisher Scientific (TMO), states that the interfering sample deviates less than ±20% in concentration when compared to the reference sample [2]. In order to measure the actual deviation in our test procedure, we investigated the influence of hemolysis and lipemia on the results of cyclic citrullinated peptide antibody, immunoglobulin (Ig)G (anti-CCP), tissue transglutaminase antibodies, IgA (anti-tTG) and deamidated gliadin peptide-antibodies, IgG (Anti-DGP). In clinical use, the three analyses are negative when the result is <7 kU/L, in the grey zone from 7–10 kU/L and positive when the result is >10 kU/L. In total, 20 serum samples were collected to measure anti-tTG and anti-DGP and 14 serum samples were collected to measure anti-CCP. We selected samples without measurable hemolysis or lipemia and with values 5 kU/L, since interference in these samples could potentially change the clinical interpretation. We then used these samples to prepare two degrees of hemolysis with a hemoglobin concentration of 3.5–5.5 g/L (H1) and 9–1.1 g/L (H2) and two degrees of lipemia with a lipid concentration of 8.5–10 g/L (L1) and 18.5–21 g/L (L2). The low degree of hemolysis (H1) was selected because TMO states that there is no interference with a hemolysis degree of 4.53 g/L on anti-CCP and anti-tTG and no interference with a hemolysis degree of 4.94 g/L on anti-DGP. TMO has not investigated lipid interference on anti-CCP and anti-tTG, but has done so on anti-DGP, which showed no interference with a lipemia degree of 10 g/L (L1) [3]. The high degrees, H2 and L2, were selected based on the serum index in the user’s guide for Cobas 8000 [4] where the maximal degree of hemolysis and lipemia for IgA and IgG analyses is 10 and 20 g/L, respectively. If there is no interference at the maximal degrees, it can be assumed that hemolysis and lipemia do not interfere with the analyses. Heparinized whole blood mixed from three anonymous samples was used to prepare the hemolysate by using the method described by Snyder et al. [5]. However, Snyder et al. used distilled water to dilute the washed erythrocytes. We used 0.9% NaCl 1:5, as it is isotonic with serum and thus will correspond to the conditions under which hemolysis occur in vitro. The hemolysate was then added to each sample in appropriate amounts to obtain the two degrees of hemolysis. We used a lipid solution by the brand IntralipidVR for preparing the tests with lipemia, since this comes close to the natural lipids in vivo and has been used in several studies [5–9]. After preparing the hemolytic and lipemic samples, their actual hemolysis and lipemic degrees were analyzed at Cobas 6000, to make sure the degrees were in the right intervals. We then analyzed the samples at Phadia 250. Lipemia (L2) did not have any substantial influence on levels 25 kU/L, with fluctuations between an increase of 14% and a decrease of 12% for all three analytes. The anti-CCP and anti-DGP values of the samples with hemolysis (H2) fluctuated between an increase of 11% and a decrease of 4% in levels 25 kU/L. However, as shown in Figure 1, anti-tTG was substantially affected by even the low degree of hemolysis. The interference by the highest degree of hemolysis (H2) was substantial and the values decreased up to 80% in the levels 25kU/L. In total, 12 out of 20 samples showed a decrease of more than 20% from the reference sample, after adjusting for the dilution from the hemolysate. These deviations can cause false negative anti-tTG results and potentially an underdiagnosis of celiac disease. This finding is consistent with previous studies [10,11]. There is one good explanation for the influence of hemolysis on anti tTG values: endogenous tTG released from red cells may immunoprecipitate with anti-tTG antibodies and interfere with their detection [8,9]. We therefore recommend that anti-tTG results between 1 and 7 kU/L are tested for hemolysis and if hemolysis is detected, the result should not be released. Finally, we recommend analyzing an alternative analyte such as anti-DGP as well as anti-tTG. Thus, if the anti-tTG result is affected by hemolysis or the patient is IgA deficient, the clinician still receives the anti-DGP result. In the latest guideline from The European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN), anti-endomysial antibodies (EMA) IgA are recommend for confirming the diagnosis, especially in pediatric patients [12]. We prefer using anti-DGP IgG as it also gives a valid result in IgA deficient patients.