An Investigation of the Ability of the Glutaraldehyde Test to Distinguish between Acute and Chronic Inflammatory Disease in Horses

The Glutaraldehyde test (GT), a rapid and inexpensive test, has been utilized empirically for many years in bovine practice for diagnosing inflammatory diseases. GT is used primarily to demonstrate increased serum concentrations of fibrinogen and globulin. Glutaraldehyde binds with free amino groups in fibrinogen and immunoglobulin to create a clot in a first degree chemical reaction. The clotting time of the GT estimates the content of proteins produced in response to inflammation. The applicability of GT for diagnosing inflammation in the horse has never been investigated. The objective of this study was to determine the ability of GT to distinguish between acute and chronic inflammatory disease in horses. Thirty-seven horses with suspected inflammatory diseases were evaluated using the GT, history, complete clinical examination and routine blood analysis. GT-times, laboratory results and clinical outcome were compared statistically. Horses that were determined to be acutely affected (based on history, clinical examination and routine blood analysis) tended to have a negative GT (75%). Results of the GT did not correlate with blood fibrinogen concentration. Positive GT also predicted a fatal outcome in 69% of the clinical cases. The results of this trial indicate that GT can be a useful screening test to distinguish between acute and chronic inflammatory disease in horses.


Materials and methods
Thirty seven horses admitted for investigation of suspected inflammatory disease were evaluated using the GT (Glutarvac a ), a complete clinical examination, CBC and routine serum biochemistries that included total protein, albumin, globulin and fibrinogen. Blood for the GT and laboratory analysis was collected at the same time either upon arrival at the hospital or the following day. Horses having a history of clinical signs of inflammatory disease of total duration six days or less were arbitrarily classified as acutely inflamed. Horses with a history of clinical signs greater than six days were arbitrarily classified as chronically inflamed. The clinical examination leading to the diagnosis and etiology was also used to reinforce the distinction between acute and chronic disease ( Table 1). The GT was performed by adding equal amounts of fresh blood and glutaraldehyde in a test tube, mixing by slowly turning the test tube and visually observing and noting the time re-  Group # GT-times Empiric categorization 1 0 < GT-time < 3 min. High increase in concentration of fibrinogen and/or immunoglobulin 2 3 < GT-time < 6 min.
Moderate increase in concentration of fibrinogen and/or immunoglobulin 3 6 < GT-time < 15 min. Low increase in concentration of fibrinogen and/or immunoglobulin 4 GT >15 min.
No increase in concentration of fibrinogen and/or immunoglobulin  Table 2). The results of the GT and fibrinogen, globulin and albumin/globulin ratio were compared using regression and correlation. The association of the GT results with fatality was analyzed using chi-square. All data from the blood analysis were also tested for correlation with GT using principal component analysis.

Results
In Table 1, diagnoses, estimated duration of the diseases and outcome of the clinical cases are summarized.

Discussion
The results of this study indicate that the GT can be used to quickly differentiate chronic from acute inflammatory disease in horses. The high proportion of test negatives of horses having acute inflammation indicates that horses with inflammatory disease and negative GT are likely to be acutely, rather than chronically, inflamed. Among GT positive horses, 77% were chronically inflamed as shown in Table 5. The GT was not reliable in predicting the blood concentration of fibrinogen in acute or chronic inflammatory diseases.
Useful clinical information could be obtained by dividing GT-times into categories (groups) as listed in Table 2 (Liberg et al. 1975a, Liberg et al. 1975b). Comparison of category and respectively globulin concentration and albumin/globulin ratio within a category seemed to correlate, although this tendency was not statistically significant. This could be due to the small number of data points. A larger number of horses included in a future study like ours would probably eliminate this statistical uncer- tainty. The correlation above has been observed in cattle (Sandholm 1974a, Liberg et al. 1975a, Liberg et al. 1975b, Nielsen 1975, Eriksen 1984. The difference between other studies of other species and this study was that horses in Group 1 had only moderately increased globulin concentration and moderately decreased albumin/globulin ratio, Group 2 horses had a mildly increased globulin concentration and mildly decreased albumin/globulin ratio, and horses in Group 3 had a globulin concentration and albumin/globulin ratio within normal range. If the clinical examination indicates systemic infection (eg. increased rectal temperature) and the GT is positive, the probability is high (77% likelihood) for chronic inflammatory disease. A positive GT acts then as an indicator for further laboratory analysis of blood to determine chronicity and etiology of the disease. If the test is negative, the disease is most likely acute or the systemic inflammatory response is either insignificant or absent. The GT can also be used as an additional diagnostic test to indicate prognosis because a positive test predicted fatal outcome in 69% of the clinical cases we studied. The test performance regarding the predictability of a fatal outcome might increase if only severe inflammatory diseases are included as compared to a study also including mild cases (selection bias). Also, the lack of controls will add bias to the percentages and will eliminate false positives. Because the study did not include a group of controls and a group of horses suffering from non-inflammatory diseases, the data presented can only be considered valid for horses with inflammatory disease. For this reason, the conclusions are not valid for the entire population of horses. The selection of horses among patients submitted to a large referral hospital also might introduce spectrum bias as the hospitalized horses are more likely to be severely affected than horses treated in practice. A positive GT in horses indicated the probability of increased serum concentration of globulin and a decreased albumin/globulin ratio, but the GT was not correlated with the blood concentration of fibrinogen. Taking into consideration the low cost and rapid application of the GT and correlation of a positive test with increased concentration of globulin, the GT is a useful screening test for horses suspected to suffer from inflammatory disease. a) Glutarvac Test tube; Jorgen Kruuse A/S, Marslev, Denmark.