Clinica Chimico Acta, 193 (1990) 103-112 Elsevier


CCA 04861

Polymorphonuclear Theodossia Ludwig Boltzmann

elastase in neonatal sepsis Tsaka and Kurt R. Herkner

Institutefor Paediatric Endokrinology,

Vienna (Austria)

(Received 12 February 1990; revision received 24 August 1990; accepted 30 August 1990) Key wora!x PMN-elastase; Neonatal sepsis; Infection diagnosis; Newborn; Neonatal disorder


Quantitative analysis of polymorphonuclear elastase was carried out in 135 newborn infants during the first 28 days of life. In 15 neonates with septicaemia, significantly increased PMN-elastase activity (range 75 to 700 pg/l; P < 0.0001) was observed at the time of recognition of infection. Cut-off level 65 pg/l. Normalisation of the elastase activities was observed only under real recovery of the patients. It is concluded that plasma elastase determination serves as a sensitive indicator of systemic infection in neonatology, and as a good parameter for monitoring the course of the disease, because PMN-elastase correlates with the clinical condition of the patient.


Septicaemia in the newborn is associated with high morbidity and mortality and still causes diagnostic and therapeutic problems. There is no doubt, that the early detection of sepsis is critical for effective treatment and for the survival of the infected neonate. In order to facilitate the early diagnosis of septicaemia a number of laboratory tests have been used: measurement of C-reactive protein (CRP) [l-3], total neutrophil and immature form counts [4-61, serum orosomucoid (a,-acid glycoprotein) [2,7], serum immunoglobulin M [8,9], serum haptoglobin [lo], fibrinogen [ll], erythrocyte sedimentation rate [12,13], buffy-coat smear [14], immature/ total neutrophil ratio [15], and elastase-a,-proteinase inhibitor (E-al-PI) [16]. However, there is no reliable test capable of rapid diagnosis of infection in the neonatal period.

Correspondence to: Dipl.-Ing. Th. Tsaka, Ludwig Boltzrnann Institut fir Khnische Endokrinologie. Arbeitsgruppe fidiatrische Endokrinologie und Immunologie, Universitiits Rinderkhnik, W&ringer Giirtel 18-20. A-1090 Wien. Austria. 0009-8981/90/%03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)


Further it is rarely if ever possible to wait for the results of culture before starting treatment. The difficulties of making an early diagnosis of neonatal sepsis are reflected in various reports [17-191. The most frequently used laboratory investigations for the diagnosis of sepsis during the first 28 days of life are CRP, total neutrophil count and the immature/ total neutrophil ratio, though their reliability to serve as early indicators is controversial [20,21]. A combination of some of these tests may increase the accuracy of the diagnosis [22]. However some of these tests reflect the immunological response of the infant, which might fail in cases of severe diseases. Additionally, neonates who are extremely stressed may also present hemato~o~cal abnorma~ties of a high degree but without infection [23]. In order to overcome these disadvantages and with the aim of evaluating a rapid responding analytical parameter, which is more related to the presence of bacteria, polymorphonuclear elastase (PMN-elastase) was tested for its clinical relevance in neonatology. Methods and probands Sampie collection

For the measurements of the PMN-elastase activity fresh EDTA containing plasma was used, which was obtained as a part of the daily routine laboratory analyses. 1 ml blood drawn by v&puncture in K,EDTA Monovette (Fa. Sarstedt), or in K,EDTA Microvette CB 1000 tubes (Fa. Sarstedt) in cases of heel prick, was centrifuged at 1500 x g for 10 mm at 18°C within 1 h after collection. The first specimen was obtained on admission. Further specimens were collected during the hospitalisation of the babies, as often as blood for routine diagnostics was drawn. Assay procedure

Analyses of PMN-elastase were carried out using a homogenous enzyme-linked immunoassay (ELISA), which is commercially available. The test kit was from Diagnostica Forschung, Merck, Darmstadt, DDR. The standard curves for the elastase assay were obtained using 5 standards (0, 50,100,200, 395 pg,/l). Standards and plasma samples (50 ~1) were incubated with 100 ~1 of monoclonal sheep anti-elastase Fab’antibody fragment conjugated with horseradish peroxidase (reconstituted in 20 mmol/l tris buffer pH 7.5 ) for 10 mm at 37% Dilution medium (20 mmol/l, tris buffered gelatine solution, pH 7.5, 50 ~1) used as a standard blank and an additional plasma sample (50 ~1) were parallel incubated with blank conjugate solution (sheep Fab’antibody fragment conjugate). Subsequently, samples and standard blanks were incubated with 500 ~1 substrate (80 mmo1/l H,O,; 50 mmol/l phenol; 0.10 mol/l phosphate buffer pH 7.0; 1.5 mmol/l cl-aminophenazone) for 10 mm at 37°C. After this fixed incubation time, substrate hy~olysis was stopped by adding 2000 ~1 of stop solution (0.6 mmol/l for~ldehyde). The absorbance values (AA; A A = Asampie- Asamptebtank ) obtained at 500 nm were plotted against the standard concentrations.


Probands 135 preterm and term newborns, being admitted to the neonatal intensive care unit, were included in the study. They were assigned to one of 4 groups. Group I included six preterm and five full-term newborn infants with sepsis, confirmed by positive blood culture. Group 2 consisted of one full-term and three preterm neonates with suspected sepsis (negative blood culture) and who were given antibiotics. At least two of the following predisposing risk factors were the criteria for admission to this group: (a) premature rupture of the membranes (> 24 h), maternal fever or leukocytosis, and premature or prolonged labor without any good reason; (b) overt clinical signs of sepsis; (c) serum CRP concentrations above the reference interval. Table I shows the clinical data of the affected infants, and the microorganisms which grew in the blood cultures.



Clinical data of affected infants Case no.


Gestation (wk)

Proved sepsis (group 1): 1 M 38 2 M 31

Birth wt.

Age at onset of sepsis



Organism identified

3 4” 5 6


40 36 40 38

3.920 2.520 2.550 2.100

8 7 8 6











9 10 11


31 41 30

1.450 3.800 1.040

28 6 10

Group B str. Cand. albicans S. haemolyticus S. epidermidis Ps. aeruginosa Sir. faecalis Kleb. oxytoca Kleb. pneumoniae Cand. albicans S. epidermidis Srr. faecalis Cand. albicans Cand. albicans Cand. albicans Ps. aeruginosa


2.800 1.460

1 23

Suspected sepsis (group 2): ;:


40 36

2.200 3.650

birth 3


30 33

1.430 1.700

6 2

a Infant died during course of illness. b Infants died. All of them had overt clinical signs of sepsis but blood cultures were negative.


Group 3 consisted of 40 non-infected preterm and 34 full-term newborn infants whose clinical course was without signs of infection. This group served as a reference population. Group 4 included 46 neonates who underwent surgery (n = 5) or had cephalhematomas (n = 5), Rh-incompatibility (n = 3) local infections such as enteritis, pyelonephritis, urinary tract infections, etc. (n = 14 confirmed: n = 4 suspected), pneumonia (n = ll), or acidosis (n = 4). These neonates were included in order to determine the sensitivity, specificity, and the predictive value (positive and negative) of the test.

Results Assay precision

The within-run and between-run imprecision of the method was tested with 3 different plasma pools (see Table II). Reference interval

The reference interval (Table III) for the concentration of PMN-elastase in plasma during the first 28 postnatal days was calculated from 223 single values,

TABLE II Precision of the assay Mean @g/I)



Exp.per series

cv (%I

Within-run precision: Plasma pool 1 Ptasma pool 2 Plasma pool 3

50 130 462

1 1 1

15 15 15

6.5 4.9 2.1

Between-run Plasma pool Plasma pool Plasma pool

47 135 455

15 15 I5

2 2 2

7.2 6.8 5.2

precision: 1 2 3

TABLE III Reference interval Age (days) l- 3 4-28

Single values 46 177

a 95% Confidential range.

Mean&l SD bg/l) 42.28 + 15.76 28.19 i 13.74

95% CR’ &g/I) 37.61-46.95 26.16-30.22


measured at approximately clinical signs of infection. 33.54 f 2.9 weeks (mean + gestational age and elastase

10 days intervals in 74 neonates (group 3) without any The gestational age of the 40 preterm infants was 1 SD; range 28 to 37 wk). No correlation between concentration was found (r = 0.11).

Patient results As shown in Fig. 1 and Fig. la all neonates with proven sepsis (group 1) had significantly raised plasma PMN-Elastase concentrations at the time of diagnosis with a mean of 231 f 137 pg/l (mean + 1 SD; range 75-700; P < 0.0001). (The upper limit of the reference interval as shown in this figure was calculated by excluding the highest 2.5% of the values measured in the plasma samples of the healthy neonates.) In seven of these neonates, much increased elastase concentrations were associated with thrombocytopenia (platelet counts of < lOOOOO/Cu mm) as defined by

PMN-Elastase =.-..-I_





Neonatal PMN-Elastase


600 -







400 -

\ )/


300 t




,l-s:;E. ._;: i,; y






Age [days)







p ‘:





0 0


1 10

_-A_ 15 Neonatal

.z. 20 Age (days)

-_i_______ ~_.. 25 30


Fig. 1. a. Thin lines, course of PMN-Elastase concentration in plasma of 7 neonates with proved sepsis; thick line, upper limit of reference interval; numbers, case no; 2, age at diagnosis; * neonate died during course of illness. b. Thin lines, course of PMN-Elastase concentration in plasma of 4 neonates with proved sepsis; thick line, upper limit of reference interval; numbers, case no; -), age at diagnosis.

108 PMN-Elastase 300 -~--~-

[tig/ll ~--- -.

250 ’!m


-- --












4 5 Neonatal



~. -



7 [days)








Fig. 2. Thin lines, course of PMN-Elastase concentration in plasma of 4 neonates with suspected sepsis: thick line, upper limit of reference interval; numbers, case no; _‘, age at onset of clinical signs of sepsis; * neonates who died during course of illness.

Zipursky et al. [24]. An increase in the absolute neutrophil count above the normal range or a decrease below it, as defined by Manroe et al. [25], could not be observed. By contrast, eight of them showed a ratio of i~ature/tot~ neutrophil > 0.3. Recovery of the patients was indicated by norma~sation of the PMN-elastase concentration. Two patients (see Fig. la; cases nr. 10 and 111, who developed persistent septicaemia (caused by Candida a&cans and Pseudomonas aeruginosa, respectively) showed a recurrent increasing of the PMN-elastase concentration in plasma, which reflected their clinical condition. Other laboratory parameters (CRP, leukocytes, thrombocytes, immature/ total neutrophil ratio) gradually normalised inspite of persistent infection, after having started the antibiotic therapy, whereas the normalisation of the PMN-elastase occurred only under definite recovery of the patients. The infants in group 2 (see Fig. 2) with suspected systemic bacterial infection, also showed significantly increased PMN-elastase levels at the onset of signs of sepsis, as well as on the day of death. Values ranged from 90 to 275,/l (P < 0.001). The decrease of the elastase con~ntration in the plasma samples of the patients nr. 2 and nr. 4 as indicated in this figure, was due to a total blood exchange. In 41 patients of group 4 (18 with local infections; 11 with neonatal pneumonia and 12 with aseptic problems) PMN-elastase levels were also shown to be increased (above the cut-off level of 65 pg/l). Sensitivity and specijity of PMN-Elastase in systemic bacterial infection When differentiating patients with systemic bacterial infections from those with local bacterial infections or other noninfectious conditions, the sensitivity of an elevated PMN-elastase test result was 100% (E/15), the specificity was 65.8% (79/120), the predictive value of a positive test result was 26.8% (H/56), and the predictive value of a negative test result was 100% (79/79). When neonates with


systemic and local bacterial infections or pneumonia were ~ffer~ntiated from those with nonbacterial infections, the sensitivity of an elevated PMN-elastase test result remained 100% (44/44) and the specificity increased to 86.8% (79/91); the predictive value of an elevated test result increased to 78.6% (44/56), and the predictive value of a negative test result remained 100% (79/79). Discussion

Polymorphonuclear leukocytes (PMNs) and monocytes (MNLs) play an essential role in the mechanism of early cellular defense against bacterial and fungal infections. In newborn infants (premature as well as mature) with increased susceptibility to systemic infections, i.e. subnormal phagocyte functions, have been described [26]. Most authors reported normal bactericidal activities in PMNs from newborn infants, and their investigations yielded data suggesting that the neonatal PMN is equivalent to the adult PMN in phagocytic ability [27-291. During phagocytosis, intracellular granules fuse with the newly formed phagocytic vacuole, and subsequent degranulation and exocytosis occur [30]. Elastase, a neutral protease in the azurophilic granules of neutrophils, is extracellularly rapidly deactivated, i.e. binding to a,-proteinase inhibitor (90%) and a,-macroglobulin (lo%), respectively [31]. The formed complex is highly stable and can easily be identified by enzyme-linked immunoassay [ 32J. Plasma PMN-elastase concentrations in healthy neonates proved to be higher during the first three days of life (Table III), probably as a result of the increased number of neutrophils [25], or the resorption of necrotic tissues of the umbilical cord blunt 1331. All neonates studied with systemic bacterial infection, and those who were suspected to suffer from septicaemia, had significantly increased elastase concentrations at the time of diagnosis. After effective therapy, normalisation of PMN-elastase levels was observed in patients who recovered from septicaemia. Our data suggest that PMN-elastase is a sensitive and rapidly responding indicator of the neonatal bacterial infection, because it is directly related to the presence of bacteria and is independent from the immunological system of the newborn infant. In addition it seems to be a good parameter for reflecting the clinical situation of the patient. In that sense sequential measurements of the PMN-elastase is recommended for the mo~to~ng of patients with septicaemia, in order to evaluate the adequacy of therapy, or for the better recognition of complications. Elastase results can be returned to the ward within one hour, which is a very important factor in the treatment of neonatal sepsis. The increased levels of the PMN-elastase in plasma of neonates with local infections and pneumonia may also help to identify the infected neonate in order to start appropriate therapy early and to prevent sepsis. The low specificity of the elastase test may be compensated by combining it with the analysis of CRP, and the immature/ total neutrophil ratio, thus in order to quarantee the early diagnosis of neonatal septicaemia. Our results are similar to those reported by Speer et al. [16], but this group used a coated-tube ELBA for the elastase determination which was time consuming


(results obtained neonate.

after 5 h), and therefore

of reduced clinical relevance

in the

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Polymorphonuclear elastase in neonatal sepsis.

Quantitative analysis of polymorphonuclear elastase was carried out in 135 newborn infants during the first 28 days of life. In 15 neonates with septi...
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