Abstract
One hundred normal pregnant women in labour with full term pregnancy were included in the study. The study involved examination of amniotic fluid for testing for foetal maturity by performing shake test, tap test and Nile blue sulphate test. For the tap test specificity 92%; whereas that for shake test was 87%. Nile blue sulphate test had a specificity of 69%. The specificity of both shake test and tap test is good and form a useful bedside rapid, simple, cheap tests which is easy to perform and with high predictive value for maturity even in rural India.

Introduction
Amniotic fluid analyses have been used in the correct timing of delivery prior to elective termination of pregnancy.

The ideal test for foetal lung maturity should be rapid, inexpensive, capable for being done at the patient’s bed side, with a high predictive value for both mature and immature results.

Lecithin/Sphingomyelin ratio and amniotic fluid lecithin concentration are accepted as best means of surfactant assessment. However these tests are costly and are not readily available outside normal working hours.

Looking for an alternative simple, quick, cheap bed side tests were decided to investigate the reliability of shake test, tap test and nile blue sulphate test singly or in combination for prediction of foetal maturity.

Material and Methods
One hundred patients (normal pregnant women) in labour with full term pregnancy (37 completed weeks or more) with the below mentioned inclusion and exclusion criteria being applied were included in the study.

All patients included in the study were counselled and provided with patient information sheet. Written valid informed consent was taken of all patients. The study was approved by the institutional Ethics Committee.

Inclusion criteria
Only full term normal pregnancy women in labour selected.

Term women: 37 completed or more weeks of pregnancy in a woman sure of her dates or otherwise with a gestational age of 37 completed weeks or more according to mid second trimester obstetric ultrasound.

Normal pregnant woman: No maternal or foetal high risk factor e.g. Chronic Hypertension, Diabetes mellitus, IUGR.

Exclusion criteria

1. Gestational age less than 37 completed weeks.
2. Any medical maternal high risk factor (e.g. Diabetes mellitus, Chronic Hypertension, PIH, Rheumatic heart disease, other endocrine disturbances, renal disease).
3. History of receiving steroid therapy or taking Injection Dexamethasone or Betamethasone for foetal lung maturity.
4. History of premature rupture of membranes.
5. IUGR or macrosomic foetuses.
6. Patient not sure of her dates and no early obstetric ultrasonography for gestational age dating available.
7. Antepartum haemorrhage.
8. Presence of meconium stained amniotic fluid.
9. Foetal malformations.
10. Polyhydramnios or Oligohydramnios.

The study involved examination of amniotic fluid for testing for foetal maturity. A complete case record form of every patient was maintained which included a detailed history, clinical and Obstetric examination and results of all investigations. Amniotic fluid was collected in a sterile test tube per vaginum, when there was spontaneous rupture of membranes or following low artificial rupture of membranes. To avoid contamination of amniotic fluid with blood and vaginal secretions, the initial amniotic fluid was not collected (as it is likely to be contaminated), the amniotic fluid that comes out after the few initial ml was collected escaping from the cervix. Low rupture of membranes was done only in those patients in labour who required augmentation of labour. Patients with meconium and/or blood stained amniotic fluid were excluded from the study.

Once the amniotic fluid is collected in a sterile test tube, all the tests were done immediately by a single investigator in all the patients, to assess foetal maturity.

The quality control in the study was ideal as all the tests were performed by a single investigator.

Techniques of the tests

1. Shake test
Take 1 ml of amniotic fluid; add 1 ml 95% ethanol in a small test tube. Shake vigorously for 15 seconds. Place in upright rack for 15 min. Persistence of intact ring of bubbles at the air liquid interface after 15 minutes is to be considered as foetal lung maturity. The shake test was performed as by Clements et al,¹ but without amniotic fluid dilution.

2. Tap test
The tap test was performed as described by Socol et al 1984. Take 1 ml of amniotic fluid, add one drop of 6N HCl, and to this add 1.5 ml of diethyl ether from the side in a small test tube. Tap briskly three to four times creating bubbles. In amniotic fluid from a mature foetus the bubbles rise to the surface and break down, in amniotic fluid from an immature foetus the bubbles are stable or break down slowly. The cut off for maturity was arbitrarily set as five bubbles as recommended by Socol et al. If no more than five bubbles persisted in the ether layer, the test was considered as positive. Occasional bubbles confined to the amniotic fluid layer or not dispersed in the ether layer were ignored.²

3. Nile blue sulphate test
Place on a slide, one drop of amniotic fluid and add one drop of 0.1% Nile blue sulphate. Place on slide with a cover slip and observe under high power microscope. More than 50% orange foetal squame cells is considered as an indicator of foetal lung maturity.

The specificity, and predictive values for shake test, tap test and Nile blue sulphate test were determined by comparing the results of these tests with the actual incidence of neonatal respiratory distress syndrome.

As all our babies were term and none had respiratory distress syndrome, we could not assess sensitivity and predictive value immature. The gold standard for correlation of maturity was assessment of gestational age at birth by New Ballard’s scoring system.

Results
As all the women in labour were with term pregnancy and all the babies did not have respiratory distress syndrome, there were no false mature and no true immature results. So, it was not possible to assess sensitivity and predictive value immature of these tests

True mature : Predicted mature, no respiratory distress syndrome.

False mature : Predicted mature, respiratory distress syndrome present.

True immature : Predicted immature, respiratory distress syndrome present.

False immature : Predicted, no respiratory distress syndrome.²

Sensitivity = Correctly predicted immature all immature

Specificity = Correctly predicted mature all mature

Predictive = Correctly predicted mature value, mature all predicted mature

Predictive = Correctly predicted immature value, immature all predicted immature²

For the tap test specificity 92%; and predictive values for maturity 100% and whereas that for shake test was 87%, 100% respectively. Nile blue sulphate test had a specificity 69%, predictive value for maturity 100% which were significantly lower than that with the shake test and tap test. The predictive value for maturity was 100% in all the tests as all the babies were term deliveries and no true immature babies delivered (Table 2).

Discussion
Accurate prediction of foetal lung maturity is important to the management of high risk obstetric patients. No single test of amniotic fluid has yet been found completely reliable, easily performed and universally applicable in the prediction of foetal pulmonary maturity.

Lecithin/sphingomyelin (L/S) ratio and PG tests are used more frequently but have the major limitations. First, they are time consuming and costly and require extensive laboratory technician training and experience, and rigorous quality control.

Consequently, the affability of the tests is often limited to regional laboratories and to daytime and weekday hours. Second immature values are associated with absence of the RDS in up to 50% of the cases, although each is reliable when mature values are found. The test of Lecithin and sphingomyelin takes 3-4 hours or more to perform. Because of these limitations, the test is generally not available on a 24 hours, 7 days a week basis. PG results are reliable when blood or meconium contaminates samples of amniotic fluid, whereas lecithin and sphingomyelin ratio and tap test may be altered. PG may be reliably determined from amniotic fluid obtained from the vaginal pool in cases of premature rupture of the membranes.³

Socol et al^2,4 found that the ability of the tap test to predict foetal pulmonary maturity was comparable, if not superior, to that of the phospholipid profile. In their analysis of clear amniotic fluid samples, the sensitivity, specificity, mature and immature results at 5 minutes were 90.9%, 87.4%, 97.5% and 63.5% respectively² whereas 95.5%, 74.2%, 98.5% and 47.2% respectively at 2 minutes.

Guiddozi and Gobetz found the tap test to be more reliable than the shake test, with a positive predictive value of 63%, whereas the shake test had a positive predictive value of 79% and a negative predictive value of 31%. Repeating the tap test and reading the result at 5 and 10 minutes did not improve the outcome.⁵

Sepulveda and co-workers also demonstrated that the tap test is a reliable indicator for foetal pulmonary maturity and probably with a higher specificity than the shake test.⁶ In normal pregnancies, L/S ratios correlate closely with gestational age. In many high risk pregnancies with maternal disease, however, there is not a good correlation. Biochemical maturation of the foetal lung may be either accelerated or delayed depending upon the maternal disease. The L/S ratio is particularly useful in the management of high risk pregnancies. Maternal condition associated with acceleration of maturation usually are associated with pathologic placentas.^3,7

A potential disadvantage of the tap test is that it is semiquantitative, with an arbitrary cut off for maturity. Evaluation of the tap test at 5 minutes gives the highest predictive value for a mature test in the study by M Kucuk. The tap test had a sensitivity (80%), specificity (91.3%) and predictive value for maturity (94.96%) and immaturity (68.29%) and was comparable to those of the PG, which had a sensitivity of 82.85%, specificity of 92.41% and predictive value for maturity of 95.71% and immaturity of 72.50%.⁸

The quality control in our study was ideal as all the tests were performed by a single investigator.

For obstetricians working in a peripheral hospital with limited access to a central biochemical laboratory the tap test, shake test and Nile blue sulphate tests appears to be a valuable procedure for predicting foetal pulmonary maturity.

Rome RM et al analyzed the efficiencies of the L/S ratio, creatinine concentration and the Nile blue sulphate tests used singly and in combination, the assessment of foetal lung maturity. They concluded when the results of all three tests are high, there is virtually no risk of the infant developing RDS, but when all three results are low the foetus is immature and runs a high risk of developing RDS. When one or two results were low, 2 of 140 infants (1.4%) developed RDS and when all three test results were low, 3 of 5 infants (60%) developed RDS.⁹

The rationale of the shake (surfactant) test is based on the ability of pulmonary surfactant to form highly stable surface films that can support the structure of a foam for relatively long periods. Since other substances in amniotic fluid, such as proteins, bile salts or salts of free fatty acids, can also form a stable foam, these are excluded from the surface films by the non foaming competitive surfactant ethanol. It is expected therefore that mixing amniotic fluid with an equal volume of 95% ethanol should poise the system so as to reveal the pulmonary surfactant in it when it is shaken with air to generate a foam. To make the test semiquantitative, tube dilutions can be done.¹

A number of factors require careful considerations for interpreting this test¹ -

1. The final concentration of alcohol in the system is critical and the volumes used must be carefully measured.
2. The glass tubes must be clean, without remnants of soap, serum or biologic fluids, which might produce foam. The diameter of the tube might affect foam stability by changing the surface area of glass in contact with the foam.
3. Evaporation from the foam could decrease the stability, especially in hot dry rooms. Clean rubber stoppers on the tubes minimize evaporation.
4. Movement of the glass tubes after the foam is produced may break the foam or distribute it over the sides of the tube so that it is difficult to detect. It is essential not to move the tubes after the foam is produced.
5. Do not attempt to retrieve results by shaking the tubes a second time, if reading are missed. Instead start over with a new sample of amniotic fluid.
6. A precipitate occasionally forms on addition of ethanol to amniotic fluid. When this happens the meniscus must be observed carefully to avoid confusion between the precipitate and foam.
7. The test should not be used when meconium is seen in the amniotic fluid or when there is blood in the sample.
8. The shake test requires test tubes, pipettes and alcohol, and is easily taught to an unskilled person. It does not require centrifugation unless red blood cells are present in the sample. It takes less than half an hour to complete.

Masood Shahla analyzed amniotic fluid for foetal lung maturity by measuring the lecithin-sphingomyelin ratio and performing the shake test and Nile blue sulphate staining. The shake test proved to be a valuable screening tool in assessment of foetal pulmonary maturity when the foetus is immature. The estimation of foetal fat cells seemed to be of lesser value in predicting foetal maturity. In their experience, a Nile blue sulfate test result of more than 10% (the percentage of orange staining anuclear cells under low power) has been matched by a lecithin-sphingomyelin ratio indicating maturity. The Nile blue sulphate test was seen to give twice as many falsely immature values as the shake tst.10 In our study, the Nile blue sulphate test had a false immature of 31%.

A comparison of the shake test, optical density, L/S ratio and PG for the assessment of foetal lung maturity was done by Anceschi MM et al. Their aim was to determine the diagnostic accuracy and reliability of these tests for the assessment of foetal lung maturity. The diagnostic accuracy of each test was as follows: the shake test and the OD650 had a sensitivity of 50%, while the steicometric L/S had a sensitivity of 75%, the planimetric L/S and the presence of PG were 100%. All four tests demonstrated a specificity greater than 64%, the highest for the PG presence being 83% and the shake test 86%. Predictive negative values for lung maturity were > 93% for all tests, with the highest for the planimetric L/S and presence of PG being 100%.¹¹

Kuchenbecker WK et al concluded that the tap test is a rapid, easy and accurate predictor of the need for neonatal ventilation.¹²

Rodriguez-Macais KA found that the tap test is a good alternative to other diagnostic methods of foetal pulmonary maturity. The sensitivity of the shake test was 92% for foetal pulmonary maturity as was the sensitivity of phospholipid profile while for the tap test at 2, 5 and 10 minutes it was between 96 and 100%. In terms of specificity the 5 and 10 minutes tap test diagnoses were 100% correct for immaturity while all other tests were 60-78% correct. The predictive value for maturity was 100% for the tap test (5 and 10 minutes), 67% for the 2 minutes tap test, 64% for PG, 60% for L/S and 56% for the shake test.¹³

Our results show that the tap test has a specificity of 92%; and predictive values for maturity 100%, whereas that for shake test was 87%, 100%, respectively. Nile blue sulphate test had a specificity 69%, predictive value for maturity 100% which were significantly lower than that with the shake test and tap test. The predictive value for maturity was 100% in all the tests as all the babies were term deliveries and no true immature babies delivered. Thus our results show that both shake test and tap test have a good specificity and a 100% predictive value for maturity which is comparable with the results of shake test, tap test, L/S ratio and PG test.^8,10,11,13

To conclude, the prime importance to us is assessment of foetal lung maturity. It is important to remember that 80% of our population lives in rural India. The benefits of shake test and tap test are:

1. Cutting down of cost, against L/S ratio and PG test.
2. Tests are simple, but efficacious and can be easily performed in rural India as against L/S ratio and PG test which are complicated.
3. No microscope facility may be available in rural India. Nile blue sulphate test requires microscope.
4. These tests can be done easily by any technician with minimal training. Nile blue sulphate test requires training of microscope handling and expertise in recognising and counting orange cells. Hence one may easily eliminate performing Nile blue sulphate test.
5. If a single test is to be performed then tap test should be performed for assessing foetal maturity.
6. If two tests are to be performed then one should perform both shake test and tap test.
7. However, if finances permit, all three tests may be done as there are no false positives in any of these tests. Hence if baby is found to be mature the pregnancy can be safely terminated.

Conclusion
Shake test and tap test in combination provide a useful guide whether neonatal ventilation will be required postnatally or not. The specificity of both shake test and tap test is good and form a useful bedside rapid, simple, cheap tests which is easy to perform and with high predictive value for maturity even in rural India.

References

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