The DAT is an accepted method of detection of anti-erythrocyte antibodies and an essential part of the diagnosis of IMHA [2
]. The first aim of this study was to compare the results obtained in the DAT versus the gel test. The high number of samples, 247 canine and 74 feline samples, creates a relatively powerful study, as for such comparative investigations 40 samples has been described as sufficient [8
]. Analysis of the correlation between the log titres of the DAT results and the ordinal gel test results revealed that the latter can be interpreted as binary test results. Therefore kappa statistics were chosen as the appropriate method to compare the two tests in the absence of a "gold standard" for the diagnosis of IMHA [8
]. The kappa values for the comparison of the canine BCIDL (0.86) as well as the kappa of 0.94 for the BCIDL canine samples with Ht below 0.35% can be interpreted as "almost perfect agreement" and the kappa for the feline BCIDL samples (0.58) and kappa for the UVLD canine samples (0.48) as "moderate agreement" using the classification of Landis and Koch [9
The use of an extended DAT protocol such as performed by both laboratories in this study in their diagnostic support for a university referral hospital has been questioned for samples derived from first opinion clinical practice [10
]. A still unresolved issue is the use of multiple dilutions of antisera to prevent false negative DAT outcomes due to the prozone effect [5
]. Five of 17 UVDL samples had prozones with a titre around the cut-off value defined for the DAT and would have been missed if higher dilutions of antiserum had not been used.
A second issue concerns the inclusion of multiple monovalent reagents to increase the sensitivity of the DAT [5
]. Indeed in the BCIDL canine samples, six positive DATs would have been missed if the test had relied on polyvalent antisera only. This finding corroborates a previous study by this laboratory in which 11 of 77 dogs with IMHA would have had a negative DAT had the extended test protocol not been used [5
The third issue related to the DAT is the clinical relevance of performing an extended DAT with full determination of the pattern of antibody reactivity instead of use of a polyvalent reagent only, such as in the gel test. In human IMHA the antibody reactivity pattern is of diagnostic relevance [14
]. In dogs, however, the clinical relevance remains debated. C3b and IgM reactivity have been reported with higher frequency in secondary IMHA [5
] and IgM reactivity at 4°C was also more often seen in the dogs with secondary IMHA in the present study (Table ). The clinical significance of cold agglutinating antibodies is also widely debated. There is a belief that they are less significant as they are rarely active at body temperature [2
], but few studies have investigated the complete temperature gradient of the reactivity of these immunoglobulins.
Despite the fact that the DAT is an accepted test for the diagnosis of IMHA [2
], it is by no means internationally standardized. To realise such standardisation between laboratories will be very difficult, if not impossible. A commercially produced test, such as the gel test, might be better suited to this task. Therefore, in addition to the comparative study of the two diagnostic procedures, the second goal of the present study was to investigate the value of the gel test as a diagnostic tool for IMHA. In the absence of a gold standard we defined clinical categories of idiopathic IMHA, secondary IMHA and no IMHA on the basis of a combination of diagnostic criteria present at clinical presentation and as used by others [2
]. Additionally, these case definitions included follow-up and pathology data in order to prevent misclassification. It can be argued that this clinical classification is based on subjective criteria; however, until better methods have been found, it is consistent with current clinical practice.
The gel test identified fewer positive samples than the DAT despite the fact that in essence it is also a test reliant on the use of a polyvalent antiserum (Figure ). The positive DAT samples that were missed by the gel test (n = 27) were mainly from dogs with secondary IMHA (n = 20). Only three cases of idiopathic IMHA were missed. Since the gel test is faster and more easily performed than the conventional DAT it might be used as a screening test for IMHA, but if negative, IMHA cannot be excluded and an extended DAT should be performed. This observation contrasts, however, with the findings in human samples where the micro-column gel test is found to be more sensitive than the DAT [21