Aust. N.Z.J. Obstet. Gynaec. (1979) 19: 23
Measurement of Diamine Oxidase Activity in Vaginal Fluid - An Aid to Diagnosis of Ruptured Fetal Membranes M. M. Wishartl", D. T. Jenkins2, and M. L. Knott3 Department of Pathology, King Edward Memorial Hospital for Women, Perth, Western Australia
Summary: Measurement of diamine oxidase (DAO) activity in vaginal fluid as an aid to diagnosis of premature rupture of the fetal membranes was studied. In 76 patients following artificial rupture of the membranes and in 70 patients with known intact membranes, the accuracy was 100%. One hundred and fortynine patients were admitted to hospital with suspected ruptured membranes; using the criteria outlined in this paper, 51 had ruptured membranes and in 98 the membranes were intact. The overall accuracy was 96%.
Management of premature rupture of the fetal membranes is a controversial issue and the clinical diagnosis is often difficult. Many procedures have been described as diagnostic aids for this condition and these were reviewed by Friedman and McElin (1969). None of these procedures proved entirely satisfactory on its own, but a combination of any 3 of the following (careful history, the nitrazine pH test, amniotic fluid crystallization test and/or the nile blue stain) produced a diagnostic accuracy of approximately 93%. Elmfors et al. (1974) described the measurement of diamine oxidase (DAO) activity in vaginal fluid to diagnose ruptured membranes and implied 100% accuracy with this method. In the present study, we have investigated the reliability of this assay. 1. Clinical Pathologist. 2. Registrar. 3. Medical Technologist. *Present Address: Perth Pathology Service, 7 Burgay Court, Osborne Park, Western Australia.
PATIENTS AND METHODS
The specimen was collected by placing a strip of Whatman's no. 1 filter paper measuring 8.0 X 1.0 cm in the vagina for approximately 30 seconds. A speculum was not required for this procedure, but it was important that the whole strip was moistened. The strip was eluted in 6 ml of 1/15M phosphate buffer, pH 7.4 and DAO activity in vaginal fluid was measured using C14 putrescine as the substrate according to the method of Tufvesson and Tryding (1969). Permablend 5 g/1 toluene was the schtillant used and radioactivity was measured using a Packard Tri-Carb Liquid Scintillation Spectrometer 2450. Activity was calculated according to the definition that one unit of enzyme is that amount of diamine oxidase that catalyses the degradation of a pmole of substrate per minute under defined conditions. It was not possible to express activity per ml of secretion as the amount of fluid absorbed by the filter paper strip was unknown.
24
AUST. AND
N.Z.
JOURNAL OF OBSTETRICS AND
The enzyme is not present in normal vaginal secretions or urine. The specificity of the assay is high and in the absence of contamination with blood, the presence of any DAO activity in vaginal secretions is an indication of ruptured membranes. The sensitivity of the test is also high, measuring down to 1 p per 100 p l of amniotic fluid. Initially 146 patients were studied to assess accuracy of the method; in 76, artificial rupture of the membranes had been performed (DAO activity was measured within 2 hours of this procedure), and in the remaining 70 the membranes were known to be intact. Following this the assay was performed on 149 patients delivered at this Hospital with suspected premature rupture of the membranes. All patients were assessed clinically by one of us (M.M.W. or D.T.J.) and the case histories reviewed for any subsequent clinical evidence of ruptured membranes. If delivery did not occur within 72 hours and there was no further suggestion of drainage of liquor, the membranes were considered to be intact. A result was regarded as a false negative when the assay indicated intact membranes but clinical assessment strongly suggested that the membranes were ruptured and a repeat assay confirmed that clinical suspicion. RESULTS
Diamine oxidase activity was present in all 76 patients whose membranes had been ruptured artificially (range 1-780 p units). No activity was detected in any of the 70 patients with known intact membranes. These results give an accuracy rate of 100%. There was positive DAO activity in the vaginal fluid (range 1-513 p units) in 48 of the 149 patients admitted with suspected premature rupture of the membranes: 3'7 of these delivered within 72 hours of admission, 1 I delivered more than 72 hours after admission and in 8 of these 11 labour was pharmacologically inhibited but clinical evidence of ruptured membranes persisted, giving a presumed accuracy of 94%. Three patients initially gave false negative results; a positive result was obtained in 2 on single repeat assay and the remaining patient after 3 further assays. There was no DAO activity in the vaginal fluid in the remaining 101 patients in the group with suspected membrane rupture; 90 of these delivered more than 72 hours after the assay was performed and 11 delivered within 72 hours ( 8 following artificial rupture of the membranes for complications of pregnancy and 3 after the spontaneous onset of labour). The presumed accuracy in this group was 97%. DISCUSSION
Diamine oxidase is an enzyme present in the plasma of normal adults and increases during preg-
GYNAECOLOGY
nancy. It is also present in amniotic fluid, although its activity is variable and levels are lower than in maternal plasma (Tornqvist et al., 1971). I t is produced, at least partly, by decidual cells (Swanberg, 1950). Correlation of clinical outcome with assay results can be difficult. It has been shown that 95% of pregnancies at or near term will deliver within 72 hours of rupture of the membranes and that although the latent period between rupture of the membranes and delivery increases with increasing prematurity, 81% of premature infants will still deliver within 72 hours (Eastman and Hillman, 1961). The 100% accuracy claimed by Elmfors et al. (1974) for this method was not reproduced in the 149 patients in the present study where the overall accuracy obtained was 96%. The 3 patients with initial false negative results all drained liquor intermittently and all the specimens were collected at an interval greater than 2 hours after liquor was seen clinically. It became obvious that DAO activity in vaginal fluid was present for not more than 2 hours after liquor was discharged into the vagina. This limits the usefulness of the method. Since many patients drain liquor intermittently and as the assay takes 4-5 hours to complete, the specimen, which has to be processed immediately, must be collected at a time suitable to the laboratory in order for the assay to be completed within a normal working day. As sophisticated equipment is required, this test cannot be performed by many small laboratories. Although not included in the present study, there were 2 patients in whom fetal death had occurred and in whom there was no DAO activity in the presence of obvious ruptured membranes. It seems, therefore, that a functioning fetal-placental unit is required if DAO activity is to be measured. We consider that measurement of DAO activity in vaginal fluid is a useful diagnostic aid (with an overall accuracy of 96%) in cases of suspected premature rupture of the membranes and a positive result is an absolute indication that the membranes are ruptured. References Eastman, N. J., and Hellman, L. M. (1961), Williams Obstetrics, 12 Edn., Appleton-Century-Crofts, New York, p. 432. Elmfors, B., Tryding, N., and Tufvesson, G. (19741, J . Obstet. Gynaec. Brit. Cwlth, 81: 301. Friedman, M. L., and McElin, T. W. (1969), Amer. J. Obstet. Gynec., 104: 544. Swanberg, H. (1950), A d a Physiol. scand., 23: Supp. 19. Tornqvist, A., Jonassen, F., Johnson, P., Fredholm, A. M. (1971), Acta obstet. gynec. scand., 50: 79. Tufvesson, G., and Tryding, N. (1969), Scund. 1. din. Lab. Invest., 24: 163.
Measurement of Diamine Oxidase Activity in Vaginal Fluid - An Aid to Diagnosis of Ruptured Fetal Membranes M. M. Wishartl", D. T. Jenkins2, and M. L. Knott3 Department of Pathology, King Edward Memorial Hospital for Women, Perth, Western Australia
Summary: Measurement of diamine oxidase (DAO) activity in vaginal fluid as an aid to diagnosis of premature rupture of the fetal membranes was studied. In 76 patients following artificial rupture of the membranes and in 70 patients with known intact membranes, the accuracy was 100%. One hundred and fortynine patients were admitted to hospital with suspected ruptured membranes; using the criteria outlined in this paper, 51 had ruptured membranes and in 98 the membranes were intact. The overall accuracy was 96%.
Management of premature rupture of the fetal membranes is a controversial issue and the clinical diagnosis is often difficult. Many procedures have been described as diagnostic aids for this condition and these were reviewed by Friedman and McElin (1969). None of these procedures proved entirely satisfactory on its own, but a combination of any 3 of the following (careful history, the nitrazine pH test, amniotic fluid crystallization test and/or the nile blue stain) produced a diagnostic accuracy of approximately 93%. Elmfors et al. (1974) described the measurement of diamine oxidase (DAO) activity in vaginal fluid to diagnose ruptured membranes and implied 100% accuracy with this method. In the present study, we have investigated the reliability of this assay. 1. Clinical Pathologist. 2. Registrar. 3. Medical Technologist. *Present Address: Perth Pathology Service, 7 Burgay Court, Osborne Park, Western Australia.
PATIENTS AND METHODS
The specimen was collected by placing a strip of Whatman's no. 1 filter paper measuring 8.0 X 1.0 cm in the vagina for approximately 30 seconds. A speculum was not required for this procedure, but it was important that the whole strip was moistened. The strip was eluted in 6 ml of 1/15M phosphate buffer, pH 7.4 and DAO activity in vaginal fluid was measured using C14 putrescine as the substrate according to the method of Tufvesson and Tryding (1969). Permablend 5 g/1 toluene was the schtillant used and radioactivity was measured using a Packard Tri-Carb Liquid Scintillation Spectrometer 2450. Activity was calculated according to the definition that one unit of enzyme is that amount of diamine oxidase that catalyses the degradation of a pmole of substrate per minute under defined conditions. It was not possible to express activity per ml of secretion as the amount of fluid absorbed by the filter paper strip was unknown.
24
AUST. AND
N.Z.
JOURNAL OF OBSTETRICS AND
The enzyme is not present in normal vaginal secretions or urine. The specificity of the assay is high and in the absence of contamination with blood, the presence of any DAO activity in vaginal secretions is an indication of ruptured membranes. The sensitivity of the test is also high, measuring down to 1 p per 100 p l of amniotic fluid. Initially 146 patients were studied to assess accuracy of the method; in 76, artificial rupture of the membranes had been performed (DAO activity was measured within 2 hours of this procedure), and in the remaining 70 the membranes were known to be intact. Following this the assay was performed on 149 patients delivered at this Hospital with suspected premature rupture of the membranes. All patients were assessed clinically by one of us (M.M.W. or D.T.J.) and the case histories reviewed for any subsequent clinical evidence of ruptured membranes. If delivery did not occur within 72 hours and there was no further suggestion of drainage of liquor, the membranes were considered to be intact. A result was regarded as a false negative when the assay indicated intact membranes but clinical assessment strongly suggested that the membranes were ruptured and a repeat assay confirmed that clinical suspicion. RESULTS
Diamine oxidase activity was present in all 76 patients whose membranes had been ruptured artificially (range 1-780 p units). No activity was detected in any of the 70 patients with known intact membranes. These results give an accuracy rate of 100%. There was positive DAO activity in the vaginal fluid (range 1-513 p units) in 48 of the 149 patients admitted with suspected premature rupture of the membranes: 3'7 of these delivered within 72 hours of admission, 1 I delivered more than 72 hours after admission and in 8 of these 11 labour was pharmacologically inhibited but clinical evidence of ruptured membranes persisted, giving a presumed accuracy of 94%. Three patients initially gave false negative results; a positive result was obtained in 2 on single repeat assay and the remaining patient after 3 further assays. There was no DAO activity in the vaginal fluid in the remaining 101 patients in the group with suspected membrane rupture; 90 of these delivered more than 72 hours after the assay was performed and 11 delivered within 72 hours ( 8 following artificial rupture of the membranes for complications of pregnancy and 3 after the spontaneous onset of labour). The presumed accuracy in this group was 97%. DISCUSSION
Diamine oxidase is an enzyme present in the plasma of normal adults and increases during preg-
GYNAECOLOGY
nancy. It is also present in amniotic fluid, although its activity is variable and levels are lower than in maternal plasma (Tornqvist et al., 1971). I t is produced, at least partly, by decidual cells (Swanberg, 1950). Correlation of clinical outcome with assay results can be difficult. It has been shown that 95% of pregnancies at or near term will deliver within 72 hours of rupture of the membranes and that although the latent period between rupture of the membranes and delivery increases with increasing prematurity, 81% of premature infants will still deliver within 72 hours (Eastman and Hillman, 1961). The 100% accuracy claimed by Elmfors et al. (1974) for this method was not reproduced in the 149 patients in the present study where the overall accuracy obtained was 96%. The 3 patients with initial false negative results all drained liquor intermittently and all the specimens were collected at an interval greater than 2 hours after liquor was seen clinically. It became obvious that DAO activity in vaginal fluid was present for not more than 2 hours after liquor was discharged into the vagina. This limits the usefulness of the method. Since many patients drain liquor intermittently and as the assay takes 4-5 hours to complete, the specimen, which has to be processed immediately, must be collected at a time suitable to the laboratory in order for the assay to be completed within a normal working day. As sophisticated equipment is required, this test cannot be performed by many small laboratories. Although not included in the present study, there were 2 patients in whom fetal death had occurred and in whom there was no DAO activity in the presence of obvious ruptured membranes. It seems, therefore, that a functioning fetal-placental unit is required if DAO activity is to be measured. We consider that measurement of DAO activity in vaginal fluid is a useful diagnostic aid (with an overall accuracy of 96%) in cases of suspected premature rupture of the membranes and a positive result is an absolute indication that the membranes are ruptured. References Eastman, N. J., and Hellman, L. M. (1961), Williams Obstetrics, 12 Edn., Appleton-Century-Crofts, New York, p. 432. Elmfors, B., Tryding, N., and Tufvesson, G. (19741, J . Obstet. Gynaec. Brit. Cwlth, 81: 301. Friedman, M. L., and McElin, T. W. (1969), Amer. J. Obstet. Gynec., 104: 544. Swanberg, H. (1950), A d a Physiol. scand., 23: Supp. 19. Tornqvist, A., Jonassen, F., Johnson, P., Fredholm, A. M. (1971), Acta obstet. gynec. scand., 50: 79. Tufvesson, G., and Tryding, N. (1969), Scund. 1. din. Lab. Invest., 24: 163.
