Department of Paediatrics, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong Province, China
Correspondence to: B. Wang, Department of Pediatrics, ZhuJiang Hospital of Southern Medical University, No. 253 Industrial Avenue, Guangzhou City, 510280 Guangdong Province, China. E-mail: email@example.com
Immune status in the early life of preterm infants and its association with late onset infection has not been fully described. To investigate immune status of lymphocyte subsets in the first week in preterm neonates and its association with late onset infection, 143 preterm neonates (84 neonates ≤32 weeks, 59 neonates of 33–36 weeks) and 49 term neonates were recruited. Absolute counts and percentages of lymphocyte subsets were measured by flow cytometry in umbilical cord or venous blood at birth (in all neonates), on day 3 and 7 (in preterm neonates). The presence of late onset infection was recorded in very preterm neonates ≤32 weeks. At birth, absolute counts of most lymphocyte subsets in all preterm neonates and percentages of B cell and NK cell in those ≤32 weeks were reduced compared with term neonates. Absolute counts of all the subsets in preterm neonates showed decline after birth then beginning to rise after day 3. Late onset infections were documented in 33 of 84 very preterm infants ≤32 weeks and 27 of 45 very preterm infants ≤30 weeks. Percentages of NK cell at birth in very preterm neonates ≤30 weeks with late onset infection were significantly reduced compared with those without infection (P < 0.01). In conclusion, immune status of lymphocyte subsets in preterm neonates at birth is less developmental than in term neonates, in spite of the ability of getting improvement in the first week. Reduced NK cell percentage at birth would increase the risk of subsequent infection in very preterm infants.
Infectious diseases are major causes of morbidity and mortality in neonates especially those born prematurely. Throughout their hospitalization, preterm infants are at increased risk of infection. It is reported the incidence of late onset sepsis varies from 21% to 36% in preterm infants depending on their gestational age or birth weight and increases with decreasing gestational age . Over half would develop one or more episodes of infection in preterm infants with gestational age <25 weeks . In our neonatal intensive care unit (NICU), the incidence of late onset bacterial infection is around 40% in preterm neonates with gestational age (GA) <32 weeks during the hospital stay.
Despite the extensive researches, the mechanism underlying the heightened susceptibility is only partly understood . Preterm neonates usually experience more frequent invasive procedures than term neonates do, which is believed to be one contribution for their vulnerability to infection. The immature immune system is thought to be another main reason [3, 4]. It is known that the defence of neonates against infection relies on their innate immunity as adaptive immunity develops later in life. Different from full-term neonates who can benefit from maternal antibodies transferred through the placenta as supplemental protection, the preterm infants can only depend on their innate immunity against infections . Several authors have tried to investigate the innate immune status of lymphocyte subsets in preterm infants and found that preterm neonates have different immune components compared with term neonates and children [6-9]. However, detailed information related to immune status in the early life of preterm infants has not been fully described, let alone its relationship with infections in later life.
This study was designed to investigate the lymphocyte subsets and other immune parameters in preterm neonates in the first week and follow up their subsequent clinical situations to document whether the high incidence of late onset infections in preterm neonates could be due to their immunodeficiency at birth.
Study subjects and sample preparation
One hundred and ninety-two newborn infants were recruited in this prospective study conducted at Shenzhen Baoan Maternal and Child Health Hospital from May 2009 to April 2011. The ethics committee of the hospital approved this study. Written informed consents for participation were obtained from the neonates' parents.
To demonstrate the immune status of lymphocyte subsets at birth (D0) in preterm infants, infants were classified into three main groups according to GA. Absolute counts and percentages of lymphocyte subsets were compared among healthy full-term neonates (37w–42w) (n = 49) who would stay with their mother after birth in obstetric department and preterm neonates with GA ≤32 weeks (n = 84) and >32 weeks (33w–36w) (n = 59). All the preterm neonates were admitted to the department of neonatology after birth, and those with GA ≤32 weeks would be routinely under intensive care in NICU. All the neonates with known congenital anomalies and maternal immunosuppressive disorders were excluded. Demographic details among three groups were shown in Table 1.
Table 1. Demographic information of study populations
GA, gestational age.
The median and interquartile ranges for both gestational age and birth weight were shown.
39.5 0 (38.60–40.25)
Birth wt (g)
Gender male (%)
Vaginal delivery (%)
Lymphocyte subsets in all preterm infants on day 3 (D3) and day 7 (D7) were also examined to observe the developmental trends of immune function in preterm neonates in the first week.
To investigate whether there was a relationship between the lymphocyte subsets at birth and the risk of infection later in preterm infants, we also followed up the very preterm infants with GA ≤32 weeks who would stay in hospital for a longer period than those >32 weeks. These infants were re-classified into two subgroups based on the presence or absence of late onset infection during their hospitalization. Late onset infections comprise culture proven or suspected late onset sepsis, hospital-acquired pneumonia or other infectious diseases presenting later than 72 h after birth . Diagnosis was made by the attending doctors based on clinical syndrome, positive findings on examination, laboratory test, imaging and bacteria culture.
Cord blood samples in full-term neonates were collected on D0. Surplus venous blood samples from those of rule-out sepsis evaluation in preterm neonates were collected on D0, D3 and D 7.
Analysis of lymphocyte subsets
Percentages and absolute counts of lymphocyte subsets were determined in ethylene di-aminetra-acetic acid (EDTA) blood samples within 6 h of sampling at Flow Cytometry Laboratory in Department of Neonatology, Shenzhen Baoan Maternal and Child Health Hospital by six-colour flow cytometry using a BD FACSCanto flow cytometer and bd facscanto clinical software (Becton Dickinson, San Jose, CA, USA) as before . Expression of cell surface markers was determined by staining the cells using the BD Multitest 6-colour TBNK kit combined CD3 Fluorescein isothiocyanate (FITC),CD16 phycoerythrin (PE)+CD56PE,CD45 PerCP-Cy5.5, CD4 PE-Cy7, CD19 allophycocyanin (APC), and CD8 APC-Cy7, resulting in percentages of total lymphocyte (CD45+), T cells(CD3+), helper (Th) cells (CD3+CD4+), suppressor (Ts) cells (CD3+CD8+), natural killer (NK) cells (CD16+CD56+) and B cells (CD19+). Absolute counts of subsets were then calculated by the software using the internal reference beads. Fig. 1 showed the representative flow cytometry stainings for identification of different immune cells in a very preterm neonate of 30w on D0.
Procedures were performed according to the instructions of BD Company. Briefly, quality control was performed daily after startup using the BD FACS 7-colour setup beads. Aliquots of whole blood (50 μl) were incubated for 15 min in the dark at room temperature with TBNK reagents. The erythrocytes were then lysed with 450 μl of 1 × BDFACS Lysing Solution. Subsequently, the cells were mixed by vortexing and incubated for 15 min in the dark at room temperature and analysed by flow cytometer with bd facscanto clinical software. To exclude operator variability, all the analyses were performed by the same operator.
Analysis of white blood cells and plasma immunoglobulin
White blood cell (WBC) counts were examined using a Sysmex XE 1000 haematology analyser (Kobe, Japan). Immunoglobulin (Ig) G, M and A were measured on Olympus AU640 by immunoturbidimetry using Randox reagents (Randox Laboratory Ltd., London, UK).
All measurement data are given as median (25th–75th percentiles). Statistical analysis was performed using spss 17.0 software (SPSS Inc., Chicago, IL, USA). Differences between groups were analysed using nonparametric tests (Mann–Whitney U test). Pearson chi-square test was used for testing the association of variables in a 2 by 2 table format. P value <0.05 by two-sided test was considered significant.
Absolute counts and percentages of lymphocyte subset on D0
As shown in Fig. 2, compared with those in full-term neonates, absolute counts of all the subsets except Ts cell in preterm infants were significantly reduced (P < 0.05). Absolute counts of Ts cell in very preterm infants ≤32 weeks were lower than in full-term and preterm neonates >32 weeks. Differences in subset values except Ts cell between the two preterm groups were not found.
There were also developmental differences in terms of percentage of subsets as shown in Fig. 3. Contrary to the trend in absolute count, percentages of total lymphocyte, T cell, Th and Ts cell in very preterm ≤32 weeks were higher than in full-term neonates. Accordingly, percentages of B cell and NK cell in very preterm neonates ≤32 weeks were significantly lower than in term neonates. Percentages of lymphocyte, B cell and NK cell in very preterm neonates ≤32 weeks were different from those >32 weeks.
Developmental trend of lymphocyte subsets in preterm neonates in the first week
As shown in Fig. 4, absolute counts of all the subsets in preterm neonates showed decline after birth then beginning to rise after D3. On D7, most subsets except NK cell rose to the levels higher than those at birth.
Percentages of B cell and Ts cell continued to drop in the first week, while percentages of T cell and Th cell showed trends to increase (Fig. 5). Thus, percentage of total lymphocyte demonstrated a trend of drop first on D3 then increasing.
Absolute counts and percentages of NK cell on D3 [63 (45–120) /μl and 3.5 (2.7–4.5)%, respectively] and D7[137 (89–209) /μl and 4.3 (2.9–5.5)%, respectively] were significantly reduced compared with those on D0 [263 (145–403) /μl and 8.7 (4.9–14.8)%, respectively] (P all <0.05). They both rose after day 3, but still did not reach the level at birth on D7.
Clinical characteristics of very preterm infant ≤32 weeks with or without late onset infection
Late onset infections were documented in 33 of 84 very preterm infants of GA ≤32 weeks at the age of 10.5 (7.25–13.0) days during hospitalization, including 15 cases of culture proven late onset sepsis (four cases of Pseudomonas aeruginosa, three cases of Klebsiella pneumoniae, three of Staphylococcus haemolyticus, three case of Escherichia coli, two cases of coagulase-negative Staphylococcus), 12 cases of suspected sepsis and six cases of pneumonia. The most serious episode would be recorded in those experienced more than once infectious diseases. All the cases of sepsis and some cases of suspected sepsis were performed lumbar puncture for analysing cerebrospinal fluid, and no meningitis was proved. Gestational age and birth weight were significantly lower in infants with late onset infection than in those without infection (P < 0.01). There were more infants receiving mechanical ventilation in late onset infection group than in non-infection group (P all <0.05). Apgar scores, incidence of respiratory distress syndrome (RDS) and early onset infection in two groups were not different. (Table 2).
Table 2. Clinical data of preterm infants ≤32w with or without late onset infection during hospitalization
Late onset infection group
RDS, respiratory distress syndrome.
The median and interquartile ranges for gestational age, birth weight and Apgar score were shown.
P value <0.05 by two-sided test was considered significant.
Birth weight (g)
Gender male (%)
Vaginal delivery (%)
Antenatal corticosteroid (%)
1 min Apgar score
5 min Apgar score
Early onset infection (%)
Mechanical ventilation (%)
Immune status of lymphocyte subset at birth was associated with late onset infection in very preterm infants
Immune status including lymphocyte subsets, WBC and blood immunoglobulin levels was compared between very preterm neonates ≤32 weeks in the presence or absence of late onset infection. As shown in Table 3, percentages and absolute counts of NK cell showed significant reduction in infants with late onset infection, whereas both GA and birth weight were also lower than in control group. Percentages of T cell were slightly higher in infection group than in control group. Blood immunoglobulin levels and WBC counts were not different between the two groups. To avoid the confounding factors of GA and birth weight since they have profound impacts on lymphocyte subsets [6-8], we further analysed those significant factors in the very preterm neonates with GA ≤30 weeks. There were 45 very preterm infants with GA ≤30 weeks in total, with 27 infants in presence of late onset infection. As shown in Table 4, GA and birth weight showed no significant differences any more between two groups after re-analysis, while percentages of NK cell in the late onset infection group still showed significant reduction compared with control group (P < 0.01).
Table 3. Immune status of lymphocyte subset on D0 in preterm neonates ≤32w with or without late onset infection
Non-infection group (n = 51)
Late onset infection group (n = 33)
The absolute counts and percentages of immune cell were shown as median and interquartile ranges.
P value <0.05 was considered significant.
NK cell counts (/μl)
Total WBC (/μl)
Lymphocyte count (/μl)
Th (CD4+) cell counts (/μl)
Ts (CD8+) cell counts (/μl)
B cells count (/μl)
T cells count (/μl)
%Th (CD4+) cell
%Ts (CD8+) cell
Table 4. Comparison of selected factors of lymphocyte subset on D0 in preterm neonates ≤30w with or without late onset infection
GA ≤30w and non-infection (n = 18)
GA ≤30w and infection (n = 27)
GA, gestational age.
Results were shown as median and interquartile ranges.
P value <0.05 was considered significant.
Birth weight (g)
NK cell counts (/μl)
In this study, we found that immune status of lymphocyte subsets in preterm neonates at birth was different from those in term neonates, manifested as reduced absolute counts of almost all the subsets and reduced percentages of B and NK cells in preterm neonates. Our results are similar with previous reports despite of slight discrepancies [7, 8, 11]. Pérez A et al.  found a correlation between gestational age and both absolute counts and percentages of NK cell, which was different from the work of Walker TC et al. showing no differences in NK cell absolute counts between preterm neonates with GA ≤32 weeks, 33–36 weeks and term neonates . Although our study did not show a difference in NK cell counts between the two preterm groups, NK cell numbers in both premature infant groups were lower than in term infants, and the very preterm neonates ≤32 weeks had a lowest percentage of NK cells. Differences in sample size and characteristics of study population, varied types of flow cytometer and other factors may contribute to the discrepancies of results.
When looking at the pattern of lymphocyte subsets development after birth in the first week, we found that absolute counts of all the subsets in preterm neonates demonstrated decline after birth then starting to rise after day 3. These results and others  demonstrated that the preterm immune system has the ability to improve after birth although their immune system is less developed than term neonates. Furthermore, it is also worth noting that although absolute counts and percentages of NK cell both began to rise after day 3, they still did not reach the basal level on day 7. The weakness of this study is that we did not check the lymphocyte subset levels week by week until infection occurred. However, according to another report , the low levels would continue beyond 12 weeks, which might be one potential factor for their vulnerability to infection.
As expected, preterm infants with less gestational age and birth weight or with mechanical ventilation had higher incidence of late onset infection. In addition to these risk factors, it has long been thought that susceptibility to infection in preterm infants is due to their immature innate immunity, which was supported by our findings that very preterm infants ≤32 weeks with late onset infection had reduced percentages of NK cell, T cell and absolute counts of NK cell compared with those without infection. However, the fact that some preterm infants are more susceptible to infection than the others even if they have similar gestation or birth weight should not be neglected either. Although the reasons might be various, differences in immune status at birth may be an important factor, which is supported by our further findings in the very preterm neonates with GA ≤30 weeks when confounding factors of GA and birth weight had been excluded. Preterm neonates ≤30 weeks who developed late onset infection had reduced NK cell percentages at birth compared with those who did not, suggesting reduced NK cell percentages at birth make the very preterm infants more susceptible to infection later in hospital. Azizia M and colleagues found that infants with evidence of sepsis during the first week of life already had lower expression of major histocompatibility complex class antigen in their cord blood monocytes than infants without sepsis , which also supports the concept that immune status at birth is associated with subsequent infection in preterm infants.
Reduced proportion or count of NK cell, which is an important part of innate immune system, could play a crucial role in the increased occurrence of infection in preterm neonates. Although the role of NK cells has been mainly to provide antiviral and antitumor effects, recent researches demonstrate that NK cells may also play a role in antibacterial and antifungal by crosstalk with other immune cells, which interact with NK cells through the production of cytokines such as interleukin (IL)-2, IL-12, IL-15 and IL-18, which boost NK cell activities . NK cells can be directly involved in the antibacterial response through expressing pattern recognition receptors, or through producing interferon (IFN)-γ, which is a key contributor to antibacterial immune defence . Reduced NK cell cytotoxicity has been shown to be associated with neonatal sepsis, pneumonia, and recurrent infection [13, 14]. Activating signals by way of massage could improve the overall outcomes in preterm infants through enhanced NK cell cyotoxicity in these infants . Deficiency in NK cells and/or their function due to genetically defects in an increasing number of patients has been implicated recently , suggesting genetic deficiency of NK cells might be one possible reason for those preterm infants prone to infection. Additionally, Ivarsson et al. showed that expression of killer-cell immunoglobulin-like receptors (KIRs) on NK cell that normally educated adult NK cells did not educate foetal NK cells but rendered them hyporesponsive to target cells lacking HLA class I. Besides, foetal NK cells were highly susceptible to TGF-β-mediated suppression . Their data implicate that the hyporesponsiveness of NK cells in prematurely born neonates might be a reason for susceptibility to infection later in the very preterm neonates. Further researches are required for exploring the detailed mechanisms of reduced NK cell percentage responsible for late onset infection in very preterm neonates.
In conclusion, in this study, we found that immune status of lymphocyte subsets in preterm neonates at birth is less developmental than in term neonates, in spite of the ability of getting improvement in the first week. Very preterm infants with reduced NK cell percentages at birth had higher incidence of late onset infection, suggesting that measurement of NK cells at birth could be used to identify a group of preterm neonates who might be particularly at risk of infection and maybe suitable for potential immune modulation or prophylactic antibiotics.
The authors would like to thank the staff of delivery room and NICU in Shenzhen Baoan Maternal and Child Health Hospital for their assistance in collection of blood samples. This work was supported by the Shenzhen Key Medical Subject Program.
Liya Ma and Rui Chen collected data, drafted the manuscript and participated in laboratory testing. Fang Liu, Wenming Zhong, Yuefeng Li and Zhijun Wun participated in patients recruiting and collection, analysis and interpretation of data. Guangjin Lu and Bin Wang designed and coordinated the study and made the decision to submit. All authors read and approved the final manuscript.