(planimetric and stechiometric) and

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A comparison of the shake test, optical density, L/S ratio (planimetric and stechiometric) and PG for the assessment of fetal lung maturity Maurizio M. Anceschi, Juan J. Piazze Garnica, Vittorio Unfer, Maria R. Di Benedetto, and Ermelando V. Cosmi 2nd Institute for Gynecology and Obstetrics, University "La Sapienza", Rome, Italy

1 Introduction The tests currently available for the determination of fetal lung maturation (FLM) are based on the biochemical or biophysical analysis of the pulmonary surfactant found in amniotic fluid (AF) (see for review [4]). The biochemical approach is the most widely accepted and is based on the determination of Lecithin to Sphingomyelin Ratio (L/S) by thin-layer chromatography (TLC) [8] and the determination of Phosphatidylglycerol (PG) [13, 18]. Among the biophysical methods are the bubble formation tests (shake test, FSI test, the tap test) [3, 4, 9], optical density at 650 nm [2, 17, 19], the fluorescence polarization [10, 15] and the lamellar body concentration [5, 6]. Because these methods enjoy distinct specificities and greatly differ in terms of the technical skills and the time required to be performed, we have undertaken a prospective study to compare four commonly used AF methods for the assessment of FLM, i. e., shake test, OD650, L/S (planimetric and stechiometric) and PG. 2 Material and methods 2.1

Cases

A total of 88 amniotic fluid specimens were obtained from 74 pregnant women by transabdominal amniocentesis and in nine cases from the vaginal pool at the time of delivery. The mean age of our study group was 30.4 (± 4.8) years and

Curriculum vitae MAURIZIO M. ANCESCHI was born in 1955 in Scandiano, Italy, and graduated with honors at the Medical School of Modena in 1980. He specialized in Obstetrics and Gynecology in 1984 and in 1988 became PhD in Perinatology in Perugia, Italy. He was a research fellow in Dallas (University of Texas), in Heidelberg (EMBL), in New York (Stony Brook) and in London (Hammersmith Hospital). He is now Assistant Professor in the 2nd Dept of Obstetrics and Gynecology in the University "La Sapienza ", Rome. His main research interests are fetal lung maturation, preterm delivery, hypertension in pregnancy and management of pregnancies at risks.

the week of delivery was 35.9 (±2.7) weeks. Amniotic fluid was obtained from more than one amniotic sac in six multiple pregnancies. Patients' characteristics are shown in table I. In order to determine the respiratory outcome of each neonate, we reviewed hospital records of the 76 infants delivered in the study group (table II). The presence or absence of neonatal respiratory distress syndrome (RDS) was diagnosed by the neonatology staff by means of objective criteria, including clinical signs and chest radiograms and blood gas analysis when appropriate. Neonatal

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outcome was considered for statistical analysis only in those neonates delivered within 48 hours of AF collection (41 cases). For studies on developmental changes of the AF indexes studied (i. e., value for each test as dependent variable vs. gestational age as the independent one) mothers with hypertension, diabetes and alloimmunization were excluded (table III). 2.2 Amniotic fluid tests Amniotic fluid specimens were discarded if heavily contaminated with blood or meconium. Following collection, AF specimens were stored Table I. Characteristics of patient population* Pregnancies

No. of patients (n = 74)

Normal pregnancies Preterm delivery Hypertension Diabetes Intrauterine growth retardation Multiple gestation Alloimmunization Premature rupture of membranes Nephrotic syndrome Other characteristics

26 35 7 5 5 6 3 3 1 15

* Some patients had multiple characteristics

Table II. Neonatal outcome in the study group*

No.of

Neonatal outcome

neonates (n = 76)

47 14 12 4 4 2 2 1 1 2

Uneventful Hypoglicemia Jaundice

RDS

Transitory apnea Cardiopathy Wet lung Hydronefrosis Electrolyte alterations Perinatal death Some neonates had multiple complications

at 4 °C and analyzed within 24 hours from collection. Shake test. The Clements test (shake test) was performed according to the original method [3], with minor modifications; in brief, immediatly before analysis, uncentrifuged specimens were gently inverted several times to obtain a uniform solution; AF was made to 0.5 ml with 0.9% saline in order to obtain dilutions of 1 : 1, 1:2, 1 : 3 and 1 : 4, respectively, and pipetted into 10 X 60 mm glass tubes. Half millimeter of 95% ethanol was then added to each tube. The tubes were capped with parafilm and shaked vigorously for 15 seconds and placed in a rack. Fifteen minutes later the air-liquid interface was examined against a black background; the presence of bubbles in at least 3/4 of the upper ring was recorded as a positive result. We considered negative the test in which bubbles were present in tubes with dilutions > 1 : 2. OD650. The OD650 was performed according to the published method [16]. Amniotic fluid (2 ml) were centrifuged at 2,000 X g for 8 minutes at room temperature. The supernatant was placed in a 1 cm path cuvette and the absorbance read at 650 nm in a Pharmacia LKB Ultrospec spectrophotometer against a control of deionized water at room temperature. A value > 0.150 was considered as mature. L/S ratio. The L/S ratio was determined by twodimensional TLC [8]. Lipid extraction of AF samples (4ml) was performed according to FOLCH [7]; the dried lipid extract was resuspended in chloroform/rnethanol (2:1) and applied with a Hamilton syringe to silica gel plates (10 X 10 cm) (Silica Gel 60, Merck, Germany) preheated at 100 °C for one hour. Following chromatography, the lipid spots were visualized by blowing iodine vapors with a pasteur glass pipette onto the plate. The planimetric L/S ratio was estimated semiquantitatively by assessment of the area of the individual spots. A L/S ratio ^ 2 was considered a negative test (mature test). The stechiometric L/S was performed by determination of the organic phosphorus of the lecithin and sphyngomyelin spots in the silica plate; briefly, following visualization, the spots were J. Perinat Med. 24 (1996)


357


Table III. Diagnostic accuracy of the methods for FLM determination in specimens tested 48 hours before delivery Clinical outcome (n = 41) Methods

RDS

No RDS

Shake test 2 : 1

2 2

5 32

OD650 < 0.150 > 0.150

2 2

13 24

Planimetrie L/S L/S < 2 L/S > 2

4 0

7 30

Stechiometric L/S L/S < 3 L/S > 3

3 1

10 27

PG PG absence PG presence

4 0

7 30

§

Reliability parameters Specificity

PV+§

50

86

28

95

83

50

64

15

93

60

81%

36%

100%

83%

73%

23%

96%

73%

83%

36%

100%

83%

Sensitivity

100%

75%

100%

PV~§

Efficiency

PV : Predictive negative value, PV+: Predictive positive value

scraped into individual tubes and phosphorus determined by the method of AMES-UUBIN [1], The ratio of organic phosphorus content were calculated after subtraction of blank, consisting of the phosphorus content of the silica gel scraped from a comparable area of the plate known to be lipid free after the two solvent runs. Because Stechiometric L/S is not frequently used, we had to calculate the cutoff by using the best percentual agreement with respect to the other known methods (figure 1), which resulted to be > 3. The presence of phosphatidilglycerol (PG) was evaluated by visualization of the corresponding spot in the TLC plate following exposure to iodine vapours. 3 Statistics Statistical analysis was performed using the SigmaStat statistical package (Jandel Co., Germany). We performed a Spearman rank order correlation for assessing FLM test values against gestational

Full agreement of FLM tests and US Stechiometric cutoff

Shake test ι L/S planimetric > PG

US cutoff

Figure 1. Full agreement of FLM tests and L/S Stechiometric cutoff as % agreement of L/S Stechiometric with respect to other known methods. As seen in figure above, a cutoff of ^ 3 was accepted.

J. Perinat. Med. 24 (1996)


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age in all tests except for presence of PG where we performed a point-serial correlation. Considering a True positive test = Immature test (RDS) and a True negative test = Mature test (No RDS), we analyzed data for sensibility, specificity, predictive positive value, predictive negative value and efficience for every single FLM test (4). 4 Results The values of each test were plotted and correlated against gestational age at the time of amniocentesis. Considering the gestational age at the time of amniocentesis as an independent variable, we found a significant correlation between the shake test and gestational age (r = 0.46, p < 0.005) (figure 2). Positive correlations coefficients and values of the tests tending to increase together with gestational age were also found for: OD650 (figure 3) (r - 0.31, p < 0.005), planimetric L/S (figure 4) (r = 0.77, p < 0.005), stechiometric L/S (figure 5) (r = 0.52, p < 0.005). With the visualization technique used, i. e., exposure of the plateo to iodine vapours, PG was not detectable before 34 weeks of gestation (figure 6) (r = 0.54, p < 0.005). When we analyzed the accuracy of each test (table III), the shake test and the OD650 had a sensitivity of 50%, while the planimetric L/S and the

presence of PG were 100%. All four tests demonstrated a specificity greater than 64%, with the highest for the planimetric L/S being 83% and the shake test 86%. Predictive negative values for lung maturity were > 93% for all tests, the highest being for the planimetric L/S and presence of PG (100%). In the prediction of RDS none of the tests fulfilled the requirements of an ideal test, considering a 36% predictive value for planimetric L/S, and PG presence. For the overall efficiency, the shake test, planimetric L/S and PG presence shared the same percentual (83%); the lowest was for OD650 (60%). 5 Discussion Since GLUCK et al. [8] reported the use of the L/S ratio as a useful parameter for the prediction of FLM many methods have been developed. Yet, any of these tests is far from fullfilling the requirements of an ideal test. In the present work we sought to determine the accuracy and usefulness of four different tests, i. e. space shake test, OD650, L/S (planimetric and stechiometric) and PG, for the assessment of FLM, by performing them in the same amniotic fluid specimen. The shake test has proven to have a reduced falsepositive rate (specificity 86%), a high predictive value for a mature test (PV~ 95%), and can be used as a screening test as suggested by others (r=0.31,P< 0.005)

(n=0.56, P0.005)

Negative-

25

30

35

Gestational age

20 22 24 26 28

30 32 34 36 38 40 42

Gestational age

Figure 2. Shake test dilutional factor vs. gestational age. (r = 0.46, p < 0.005; No. of cases used for normality curve =41).

Figure 3. OD650 vs. gestational age (r = 0.31, ρ = 0.005; No. of cases used for normality curve = 41). J. Perinat. Med. 24 (1996)



359

(r=0.71,P< 0.005)

(r=0,52, P 93% for all tests, with the highest for the planimetric L/S and presence of PG being (100%). The study confirms that the determination of L/S ratio is still superior to other tests in terms of overall diagnostic accuracy. In addition, it was found that presence of PG was highly associated with the absence of respiratory complications in the newborn.

Keywords: Fetal lung maturity (FLM), Lecithin/Sphyngomyelin ratio, OD650, L/S, Phosphatidylglycerol, shake test. Zusammenfassung Ein Vergleich des Schütteltestes, der optischen Dichte, des L/S-Verhältnisses (planimetrisch und stöchiometrisch) und des PG für die Bewertung der fetalen Lungenreife Die Studie wurde durchgeführt, um die diagnostische Genauigkeit und Zuverlässigkeit von vier Tests für die Bewertung der fetalen Lungenreife (FLM) zu bestimmen: Schütteltest, optische Dichte bei 650 nm (OD650), Lecithin zu Sphingomyelin-Verhältnis durch Dünnschichtchromatographie (L/S) durch Planimetrie und Stöchiometrie, und das Vorhandensein von PG. Insgesamt 88 Fruchtwasserproben wurden von 74 schwangeren Frauen durch transabdominale Amniocen-

tese erhalten. Das respiratorische Ergebnis, d.h. das Vorhandensein oder das Nichtvorhandensein des Respiratorischen Insuffizienz-Syndroms der 76 in der Untersuchungsgruppe entbundenen Säuglinge wurde vom Neonatologie-Personal diagnostiziert und für die statistische Analyse berücksichtigt, wenn die Geburt innerhalb von 48 Stunden der Fruchtwasserprobenentnahme stattgefunden hatte (41 Fälle). Positive Korrelationskoeffizienten und Werte der Tests, die zur Zunahme zusammen mit dem Schwangerschaftsalter tendierten, wurden festgestellt für: Schütteltest (r = 0,46, p < 0,005), OD650 (r = 0,31, p < 0,005), planimetrisch L/S (r = 0,77, p < 0,005), J. Perinat Med. 24 (1996)


Anceschi et al, Correlation of different FLM tests stöchiometrisch L/S (r = 0,52, p < 0,0052). Als wir die Genauigkeit jedes Tests analysierten, hatten der Schütteltest, die OD650 und das stöchiometrische L/S eine Empfindlichkeit von 50%, während das planimetrische L/S und das Vorhandensein von PG 100% betragen. Alle vier Tests zeigten eine Spezifizität größer als 64%, mit der höchsten für das planimetrische L/S (83%) und den Schütteltest (86%). Prognostische negative Werte für Lungenreife waren > 93% für alle Tests, mit den höchsten für das planimetrische L/S und das Vorhandensein von PG (100%). Bei der Prognose des Respiratorischen InsuffizienzSyndroms erfüllte keiner der Tests die Anforderungen Schlüsselwörter:

361

an einen Idealtest, in Anbetracht eines 36% prognostischen positiven Wertes für das planimetrische L/S und das Vorhandensein von PG. Für die Gesamteffizienz teilten sich der Schütteltest, das planimetrische L/S und das Vorhandensein von PG den gleichen Prozentsatz (83%), und der niedrigste war der für die OD650 (60%). Selbst obwohl die Bestimmung des L/S-Verhältnisses arbeitsintensiv ist, ist das L/S-Verhältnis hinsichtlich der diagnostischen Gesamtgenauigkeit noch allen anderen Tests überlegen. Darüberhinaus wurde festgestellt, daß das Vorhandensein von PG in hohem Maße mit dem Nichtvorhandensein von respiratorsichen Komplikationen im Neugeborenen verbunden war.

Fetale Lungenreife (FLM), Lecithin/Sphyngomyelin (L/S)-Verhältnis, optische Dichte (OD650), Phosphatidylglycerol (PG), Schütteltest.

Resume Comparaison du test par vibration, la densite optique, la taux L/S (planimetrique et stoechiometrique) et PG pour la mesure de la maturite pulmonaire du foetus La presence etude a ete effectuee pour determiner la pertinence diagnostique et la confiance de cinq tests destines ä mesurer la maturite pulmonaire foetale (FLM): le test par vibration, la densite optique a 650 nm (OD650), le rapport lecithine/sphingomyeline par TLC (L/S) par planimetrie et stoechiometrie et la presence de PG. Nous avons obtenu de 74 femmes enceintes 88 prelevements de liquide amniotique par amniocentese transabdominale. L'etat respiratoire, c'est-a-dire la presence ou absence de syndrome de detresse respiratoire (RDS) sur les 76 enfants du groupe etudie, a ete diagnostique par l'equipe de neonatalogie et, pour analyse statistique, nous avons retenu si accouchement a eu lieu dans les 48 heures de la collection AF (41 cas). Nous avons trouve des coefficients de correlation et des valeurs positives des tests avec une tendance ä Faccroissement en function de l'äge gestionnel pour: test par vibration (r = 0,46, p < 0,005), OD650 (r = 0,31, p < 0,005), L/S planimetrique (r = 0,77, p < 0,00(), L/S stcechiometrique (r = 0,52,

p < 0,0052). Du point de vue de la pertinence des tests, le test par vibration, OD650 et le L/S stoechiometrique ont une sensibilite de 50% contre 100% pour le L/S planimetrique et identification de PG. Les cinq tests ont une specificite superieure a 64%, les taux les plus eleves etant enregistres pour le L/S planimetrique (83%) et le test par vibration (86%). Les valeurs previsionnelles negatives de la maturite pulmonaire sont > 93% pour tous les tests, les valeurs les plus elevees etant le L/S planimetrique et identification de PG (100%). Pour la prevision du syndrome de detresse respiratoire, aucun des tests ne satisfait aux specifications de test ideal avec 36% de valeur previsionnelle positive pour le L/S planimetrique et identification de PG. Du point de vue de l'efficacite globale, le test par vibration, le L/S planimetrique et identification de PG s'etablissent au meme taux (83%), le niveau le plus bas etant celui du OD650 (60%). Bien que le taux L/S represente une intervention tres forte dans le travail, le rapport L/S est superieur a tous les autres tests en termes de pertinence diagnostique. Nous avons aussi trouve que 1'identification de PG presentait une correlation tres forte avec absence de complications respiratoires chez le nouveau-ne.

Mots-cles: FLM, L/S taux de lecithine/sphyngomyeline, maturite pulmonaire foetale, OD650, phosphatidylglycerol, PG, test par vibration. Acknowledgements:

This work was supported by grants from CNR (FATMA, SP7).

References [1] AMES BN, DT DUBIN: The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem 235 (I960) 769

[2] CETRULLO CL, AJ SBARRA, RJ SELVARAJ: Amniotic fluid optical density and neonatal respiratory outcome. Obstet Gynecol 55 (1980) 262



362


[3] CLEMENTS JA, AC PLATZKER, DF THERNEY, CJ HOBEL, RK CREASY, AJ MARGOLLS, W ΉΙΕΒEAULF, WH TOOLEY, W OH: Assessment of the risk of the respiratory distress syndrome by a rapid test for surfactant in amniotic fluid. N Engl J Med 286 (1972) 20 [4] DUBIN SB: The laboratory assessment of fetal lung maturity. Clin Chem 97 (1992) 6 [5] DUBIN SB: Determination of lamellar body size, number density, and concentration by differential light scattering from amniotic fluid: Physical significance of A650. Clin Chem 34 (1988) 938 [6] FAKHOURY G, NH DAIKOKU, J BENSER, N DUBIN: Lamellar body concentrations and prediction of fetal pulmonary maturity. Am J Obstet Gynecol 170 (1993)1 [7] FOLCH J, M LEES, AH SLOANE-STANLEY: A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226 (1957) 497 [8] GLUCK L, MV KULOVICH, RC BORER JR: Diagnosis of the respiratory distress syndrome by amniocentesis. Am J Obstet Gynecol 109 (1971) 440 [9] GUIDOZZI F, L GOBETZ: The tap-test a rapid bedside indicator of fetal lung maturity. Br J Obstet Gynaecol 98 (1991) 479 [10] HAGEN E, J LINK, F ARIAS: A comparison of the accuracy of the TDx-FLM assay, Lecithin-Sphingomyelin ratio, and Phosphatidylglycerol in the prediction of neonatal respiratory distress syndrome. Obstet Gynecol 82 (1993) 6 [11] HALLMAN M, M KULOVICH, E KIRKPATRICK, RG SUGARMAN, L GLUCK: Phosphatidylinositol and phosphatidylglycerol in amniotic fluid: indeces of lung maturity. Am J Obstet Gynecol 125 (1976) 613 [12] HERBERT WNP, JF CHAPMAN: Clinical and economic considerations associated with testing for

fetal lung maturity. Am J Obstet Gynecol 155 (1986) 820 KOGON DP, M OULTON, JH GRAY, RM LISTON, ER LUTHER, LJ PEDDLE, DC YOH: Amniotic fluid phosphatidylglycerol and phosphatidylcoline phosphorus as predictors of fetal lung maturity. Am J Obstet Gynecol 154 (1986) 226 KULOVIC M, M HALLMAN, L GLUCK: The lung profile. I Normal pregnancy. Am J Obstet Gynecol 135 (1979) 57 RUCH AT, R LENKE, ER ASHWOOD: Assessment of fetal lung maturity by fluorescence polarization in high risk pregnancies. J Reprod Med 38 (1993)2 SALMONA M, L DIOMEDE, G MORO, I MINOLI, S BERNINI, S AGOSTI: Fetal lung maturity evaluation with fluorescence polarization of the amniotic fluid. J Perinat Med 21 (1993) 349 SBARRA AJ, H MICHLEWITZ, RJ SELVARAJ: Correlation between amniotic fluid optical density and L/S ratio. Obstet Gynecol 48 (1976) 613 TSAO FH, RD ZACHMAN: Use of quantitative amniotic fluid phosphatidylglycerol as a criterion for fetal lung maturation. Am J Perinatol 9 (1992) 34 TURNER RJ, JA READ: Practical use and efficiency of amniotic fluid OD 650 as a predictor of fetal pulmonary maturity. Obstet Gynecol 61 (1983) 5

[13]

[14] [15]

[16]

[17] [18] [19]

Received August 29, 1994. Revised May 22,1995. Accepted May 25, 1995. Maurizio M. Anceschi II Istituto di Clinica Ginecologica ed Ostetrica Policlinico Umberto I Viale regina Elena 324 1-00161 Rome Italy

