METHODS
Subjects
The study group comprised 38 high-risk
new-borns admitted to North Shore University
Hospi-Polymorphonuclear
Leukocyte
Function
in the
Preterm
Neonate:
Effect
of Chronologic
Age
Shaista
S.
Usmani, MD; Jerrold S. Schlessel, MD; Concepcion G. Sia,MD; Shahid
Kamran,
MD; and Shahnaz
D. Orner,
MD
From the Department of Pediatrics, Division of Perinata! Medicine, North Shore University Hospital-Cornell University Medical College, Manhasset, New York
ABSTRACT. In this study, effect of chronologic age on
polymorphonuclear leukocyte (PMN)
chemilumines-cence and random and chemotactic motility was evalu-ated in 38 stable preterm neonates of less than 32 weeks’ gestation during the first month of life.
Chemilumines-cence and random and chemotactic motility of PMNs
from preterm neonates were first evaluated at mean
postnatal age of 9.8 days and then weekly for an ensuing
21-day period. For comparison, one blood sample was
obtained for PMN functions from 14 healthy term
neo-nates younger than 72 hours of age and seven normal
adults. On day 1 PMN chemiluminescence and random
and chemotactic motility values in preterm neonates were
significantly lower (P < .001) compared with those in
term neonates and PMN function values of term
neo-nates were significantly lower (P < .001) than those of
adults. Although initial PMN chemiluminescence and
random and chemotactic motility values in preterm
neo-nates were depressed, subsequent values on days 7, 14,
and 21 increased significantly (P < .002). On day 21 (mean postnatal age of 30.8 days) no differences existed in chemiluminescent activity and random motility be-tween preterm and term neonates; chemotactic motility
in preterm neonates, however, remained impaired. Mean
cumulative age (gestational age at birth plus postnatal age) of preterm neonates on day 21 of study was 32.5 weeks, suggesting that chronobogic age has more effect
on maturational changes in PMN functions than
gesta-tional age. Pediatrics 1991;87:675-679;
polymorphonu-clear leukocyte functions, chemotaxis, chemiluminescence, bactericidal activity, preterm neonates.
Bacterial infections are a major cause of morbid-ity and mortality in newborn infants, particularly
preterm infants.’3 The high incidence of sepsis in
the neonate appears in part to be secondary to the
immaturity of the immune system.48 The two
ma-jor deficits proposed to increase the risks of bacte-rial infection are abnormal .function of
polymor-phonuclear leukocytes (PMNs) and defects in the
antibody-mediated immunity.4 The rate of
septi-cemia is highest in the first week of life, with onset
after the first week comprising only 10% of the
cases during the neonatal period.’3 This decline in
frequency of sepsis during the first month of life
occurs despite the falling level of maternally
trans-mitted IgG as well as inadequate numbers of bone
marrow granulocyte progenitor cells and a limited
PMN storage pool.9’2 Many investigators have
sug-gested that neonatal PMNs have decreased
chemo-taxis, impaired adherence, and defective
chemilu-minescence.1321 These subtle defects in function
are further amplified in stressed or infected
neo-nates.’321 To our knowledge, PMN
chemilumines-cence and random and chemotactic motility have
not been sequentially assessed in preterm neonates
during the first month of life. Driscoll et a!22
eval-uated PMN chemiluminescence in preterm infants
longitudinally during a 2-month period and
re-ported that chemiluminescence during the first
week of life was equal to that of healthy adults;
however, through the remainder of the first month
of life preterm neonates had significantly decreased
chemiluminescence compared with that of adults.
In the present study we evaluated PMN
chemilu-minescence and random and chemotactic motility
in preterm neonates of less than 32 weeks’ gestation
sequentially during the first month of life and
as-sessed the effect of chronologic age on PMN
func-tions.
Received for publication Mar 26, 1990; accepted May 15, 1990.
Reprint requests to (S.S.U.) North Shore University
Hospital-Cornell University Medical College, 300 Community Dr,
Man-hasset, NY 11030.
PEDIATRICS (ISSN 0031 4005). Copyright © 1991 by the
tal, Neonatal Intensive Care Unit. The neonates
were of 25 to 32 weeks’ gestation (mean ± SD
gestational age 28.1 ± 2.5 weeks, birth weight 1.0 ±
0.4 kg, age at entry into the study 9.8 ± 4.8 days
[range 3 to 27 days]). Nineteen neonates had mild
to moderate and 18 had severe respiratory distress
syndrome. One neonate had transient tachypnea of
the newborn. Criteria for entry into the study
in-cluded hemodynamic stability, normal acid-base
balance, no proven or suspected sepsis, a fraction
of inspired oxygen requirement of less than 0.3,
peak inspiratory pressure of less than 20 cm of
water, and respiratory rate of fewer than 20 breaths
per minute. All neonates studied remained stable
and showed no evidence of proven or suspected
sepsis throughout the study.
After a baseline sample for complete blood cell
count and PMN functions, a blood sample was
obtained for PMN functions on study days 7, 14,
and 21, mean postnatal age of 16.8 (range 10 to 34
days), 23.8 (17 to 41 days), and 30.8 (24 to 48 days),
respectively. To compare the PMN functions of
preterm neonates with those of term neonates and
adults, one blood sample was obtained for complete
blood cell count and PMN functions from 14
healthy term neonates (mean ± SD birth weight
3.3 ± 0.4 kg, gestational age 39.6 ± 1.1 weeks)
between 12 and 72 hours of age and seven normal
adult volunteers. These data were collected as a
part of a larger study.23 The study was approved by
the Institutional Research and Publications Review
Committee on human subjects.
PMN Functional
Studies
Two milliliters (10 U/mL) of heparinized blood
was obtained and tested immediately after
collec-tion. Total and differential leukocyte counts were
performed manually on each whole blood sample.
Polymorphonuclear leukocytes were separated
ac-cording to the method of Boyum24 and washed twice
in Hanks’ balanced salt solution. After isolation,
cell suspension contained more than 85% PMNs
and trypan blue dye showed more than 95% PMN
viability. The cells were adjusted to a concentration of 1.0 x 106 PMNs per milliliter in Hanks’ balanced
salt solution for chemiluminescence, and 2.0
x
106PMNs per milliliter in 2% bovine serum albumin
for random and chemotactic motility.
Luminol-enhanced chemiluminescence (Sigma Diagnostics,
St Louis, MO), an assay of oxidative and metabolic
function ofphagocytizing PMNs,2527 was measured
in a liquid scintillation system (Beckman model
LS-230, Beckman Instruments, Inc, Fullerton, CA)
using latex particles of 0.80 m in diameter (Seragen Diagnostics, Indianapolis, IN) as a stimulant.
Sam-ples were tested in duplicate and peak levels in
counts per minute (cpm x iO) for 1.0 x 106 PMNs
were recorded.
Chemotactic and random motility were tested by
modified Boyden technique with the use of
blind-well chambers (Neuro-Probe Inc, Bethesda, MD)
and 5-jm pore cellulose nitrate filters (Millipore
Corp, Bedford, MA).28 Pooled human serum
acti-vated by zymosan (Sigma) was used for chemotaxis
and tissue culture medium-199 (Difco Laboratories, Inc, Detroit, MI) for random motility. Samples were
tested in duplicate after incubation of cells for 2
hours at 37#{176}C.Chemotactic and random motility
were expressed as the mean depth in micrometers
at which the leading front of PMNs was in focus
(x40 objectives). Mean depth represents the
aver-age of reading in 10 randomly chosen microscopic
fields in each filter.
Statistical Analysis
Repeated measures analysis of covariance was
used to evaluate all data obtained in preterm
neo-nates, with age of the neonate at the start of the
study as the covariate. A multivariate repeated
measures analysis of covariance was performed to
detect any differences in chemiluminescence and
random and chemotactic motility between the time
periods while adjusting for the neonate’s age at the
start of the study.
For the comparison of preterm neonates with
full-term neonates and adults, an analysis of
van-ance was performed with the type of patient
(pne-term, full-term, and adult) as the grouping variable.
Because preterm neonates had multiple measures
over time, the analysis was first done using the first
values of the preterm neonates and a second time
using the last (fourth) values of the preterm
neo-nates. When a difference was detected between at
least two types of patients, ie, the analysis of van-ance was significant (P < .05),
Student-Newman-Keuls multiple comparisons were performed (at the
5% level) to determine which of the types of
pa-tients were different. Where the P value for the
analysis of variance was not significant (ie, P value
above .05), multiple comparisons were not
per-formed.
RESULTS
In stable pnetenm neonates of less than 32 weeks’
gestation, mean PMN chemiluminescence and
ran-dom and chemotactic motility values on study day
1 (mean postnatal age of 9.8 days) were significantly
lower compared with those in healthy term
neo-nates (P < .001, Table 1). Term neonates likewise
and random and chemotactic motility values corn-pared with those of adults (P < .001, Table 1).
Although day 1 PMN chemiluminescence and
random and chemotactic motility values in preterm
neonates were depressed, subsequent values on days
7, 14, and 21 increased significantly (P < .02, Table
2). There were no significant differences in PMN
chemiluminescence and random and chemotactic
motility values in preterm neonates at entry into
the study and through study period when compared
for gestational age, birth weight, and postnatal age. Neonates who were still receiving assisted
ventila-tion or supplemental oxygen at initiation of study
tended to have depressed PMN functions compared
with those not receiving respiratory support;
how-ever, these differences were not statistically signif-icant.
On day 21 of the study (mean postnatal age 30.8
days) no differences existed in chemiluminescence
activity and random motility between preterm and
term neonates; chemotactic motility in preterm
neonates, however, remained impaired (P < .001,
Table 3). The PMN functions of both preterm and
term neonates were depressed when compared with
PMN functions of healthy adults (P < .001, Table 3).
DISCUSSION
Our study of PMN functions confirms and
ex-tends previous reports which indicated
abnormali-ties in the bactericidal and chemotactic activities
of PMNs from preterm and term neonates.4”#{176}2’
We noted that within the first 10 days oflife, PMNs
from stable preterm neonates demonstrated
signif-icantly decreased chemiluminescence and random
and chemotactic motility when compared to PMN
functions from healthy term neonates and adults.
The PMN functions of the preterm neonates,
how-ever, improved over time so that on day 21 of study (mean postnatal age 30.8 days) chemiluminescence
and random motility of PMNs were similar to those
of term neonates. At this time, the cumulative age
(gestational age at birth + postnatal age) of preterm
neonates was 32.5 weeks vs 39.6 weeks for term
neonates. These improvements in
oxidative-meta-bolic activity and motility of PMNs in preterm
neonates correlated positively with chronologic age
as opposed to gestational age. These findings
sup-port the hypothesis of a maturational defect in
structure and/or metabolism of PMNs in
new-borns.8”#{176}’2 Analogous to pulmonary maturation,
development of the neutrophil system may be
un-necessary during fetal life, but critical to postnatal
survival. The enhancement of PMN functions in
preterm neonates may be an adaptive mechanism
to postnatal existence.
Driscoll et a122 recently evaluated
chemilumines-cence activity responses of PMNs from preterm
infants of between 24 and 35.5 weeks’ gestation
during the first 2 months of life. The mean peak
TABLE 1. Study Day 1: Polymorphonuclear Leukocyte Fun Compared With Full-Term Neonates and Adults*
ctions in Preter m Neonates
Test Variables Patient Groups Pt
Preterm (n = 38) Term (n = 14) Adult (n = 7)
Chemiluminescence, 186 ± 86.7 302 ± 66.1
cpm X iO
Random motility, depth 34.6 ± 12.0 52.1 ± 12.8
436 ± 40.9
68.3 ± 10.3
<.001
<.001 m
Chemotaxis, depth zm 68.5 ± 14.9 97.8 ± 17.3 111.4 ± 9.0 <.001
* Values represent mean ± SD.
t Values in preterm and term neonates were significantly different from each other and
adults on multiple comparisons. Preterm < term (P < .001), term < adults (P < .001).
Analysis of variance.
TABLE 2. Effect of Chronologic Age on Polymorphonuclear Leukocyte Functions in
Preterm Neonates*
Test Variables Day of Study Pt
1 7 14 21
Chemiluminescence, 186 ± 86.7 223 ± 90.6 241 ± 95.6 256 ± 82.0 <.02
cpm X iO
Random motility, 34.6 ± 12.0 41.9 ± 9.9 48.0 ± 9.4 47.2 ± 9.0 <.02
depth m
Chemotaxis, depth j.m 68.5 ± 14.9 75.4 ± 12.6 82.4 ± 13.6 82.5 ± 14.4 <.01
* Values represent mean ± SD.
TABLE 3. Study Day 21: Polymorphonuclear Leukocyte Functions in Preterm
Neonates Compared With Full-Term Neonates and Adults*
Test Variables Patient Groups P
Preterm (n = 38) Term (n
=
14) Adult (n = 7)Chemiluminescence, 256.4 ± 82.0 302 ± 66.1 436 ± 40.9t <.001
cpm X i0
Random motility, depth 47.2 ± 9.0 52.1 ± 12.8 68.3 ± 10.3t <.001
m
Chemotaxis, depth m 82.5 ± 14.4 97.8 ± 17.3t 111.4 ± 9.Ot <.001
* Values represent mean ± SD.
t Values in adults were significantly greater than in preterm and term neonates (P <
.001); no difference was detected between preterm and term neonates except in chemotaxis
by multiple comparisons (P < .001). Analysis of variance.
chemiluminescence activity for preterm neonates
during the first week of life was not significantly
different from that of healthy adults. However,
mean peak chemiluminescence activity at postnatal
ages of 12, 26, 40, and 54 days was significantly
lower than that of adults. They did not study
che-motactic or random motility of PMNs. The
differ-ences in chemiluminescence noted by Driscoll et a!
and our group are not clear and may lie in the
dissimilarities in the study populations or factors
intrinsic to the neonatal intensive care unit
envi-ronment (eg, invasive procedures, nosocomial
path-ogens, drugs, etc). Thirty-three “serious infections” occurred in 23 of the 70 infants studied by Driscoll
et a!22 and no episode of suspected or proven
infec-tion was noted in our study population. Defective
oxidative metabolic responses are more common in
stressed or infected neonates.14”8’19’2’ This may be
a reason for the differences in chemiluminescence between our study and that of Driscoll et al.22
Our study indicates that hemodynamically stable,
growing preterm neonates show improvement in
PMN functions with advancing chronologic age.
Although the cellular mechanisms responsible for
the enhancement in PMN function is as yet
Un-clear, there are several possibilities to be consid-ered. These may include structural and functional differences such as cell surface receptors, metabolic
activity, and cell adherence, all of which appear
necessary to mediate and sustain
chemilumines-cence and directed migration. Investigators have
reported a correlation between the presence of Fc
receptors and certain functional capabilities of
PMNs, eg, adherence, chernotaxis, and microbicidal
activity, and have shown decreased chemotactic
function by cells showing fewer Fc receptors.8”2’29’3#{176}
These receptors appear at different times during
PMN differentiation.3#{176} It is possible that the Fc
receptors on PMNs show maturational
develop-ment with advancing chronologic age. Also,
im-proved PMN function may derive from
matura-tional changes in cell metabolism; and the enhanced
chemiluminescence with advancing postnatal age
as found in our study is indicative of an improved
ability of PMNs to sustain oxidative metabolism.
Further studies are necessary to delineate the
molecular basis of maturational aspects of PMN
function and to define the environmental or
extrin-sic factors influencing cell function.
ACKNOWLEDGMENT
We thank Claudia Bock for her contribution in the
preparation of the manuscript.
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BIRTH WEIGHT/HEART DISEASE
Men who were born as low birth weight infants-less than or equal to 5.5
pounds-are more likely to die from heart disease than men of normal birth
weight, a new British study in Lancet (Vol. 2, No. 8663; pp. 577-580) concludes.
The death rate caused by heart disease among low birth weight men is 104
when 100 is used as the national average. The study involved health records of
5,654 men, and researchers concluded that factors influencing birth weight and
early growth may affect cardiovascular health in later life. Promoting early
growth may also reduce the risk of heart disease, the study found.
Birth Weight/Heart Disease. Nutrition Week (CNI). No. 17, April 26, 1990.
