HUMAN IMMUNOLOGY doi: 10.1111/sji.12181 ..................................................................................................................................................................
Reduced NK Cell Percentage at Birth is Associated with Late Onset Infection in Very Preterm Neonates L. Ma*†1, R. Chen†1, F. Liu*†, Y. Li†, Z. Wu†, W. Zhong‡, G. Lu† & B. Wang§
Abstract *Southern Medical University, Guangzhou, Guangdong Province, China; †Department of Neonatology, Shenzhen Bao’an Maternal and Child Health Hospital, Guangzhou, Guangdong Province, China; ‡Department of Medical Information, Shenzhen Bao’an Maternal and Child Health Hospital, Guangzhou, Guangdong Province, China; and §Department of Paediatrics, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong Province, China
Received 15 February 2014; Accepted in revised form 2 April 2014 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 protected] 1
The two authors contribute equally to this paper.
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.
Introduction 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 [1]. Over half would develop one or more episodes of infection in preterm infants with gestational age 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 [5]. Diagnosis was made by the Table 1 Demographic information of study populations.. Groups
37w–42w
33w–36w
≤32w
n GA (w)
49 39.5 0 (38.60–40.25) 3350 (3090–3665) 61.22
59 34.00 (32.86–35.14) 1950 (1700–2200) 66.10
84 30.29 (29.29–31.14) 1355 (1252–1600) 60.71
Birth wt (g) Gender male (%) Vaginal delivery (%)
51.02
64.41
66.67
GA, gestational age. The median and interquartile ranges for both gestational age and birth weight were shown.
Ó 2014 John Wiley & Sons Ltd
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 ruleout 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 [10]. 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 ll) were incubated for 15 min in the dark at room temperature with TBNK reagents. The erythrocytes were then lysed with 450 ll of 1 9 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). Statistical analysis. 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 chisquare test was used for testing the association of variables in a 2 by 2 table format. P value 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.
Figure 2 Absolute counts of lymphocyte subsets on D0 in preterm and term neonates. Results were shown as median with interquartile range. *compared with term neonates, P < 0.05. #compared with preterm neonates of 33w–36w, P < 0.05.
Figure 3 Percentages of lymphocyte subsets on D0 in preterm and term neonates. Results were shown as median with interquartile range. *compared with term neonates, P < 0.05. #compared with preterm neonates of 33w–36w, P < 0.05.
Scandinavian Journal of Immunology, 2014, 80, 50–56
L. Ma et al. Immune Status and Late Onset Infection 53 ..................................................................................................................................................................
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) /ll and 3.5 (2.7–4.5)%, respectively] and D7[137 (89–209) /ll and 4.3 (2.9–5.5)%, respectively] were significantly reduced compared with those on D0 [263 (145–403) /ll and 8.7 (4.9–14.8)%, respectively] (P all
Reduced NK Cell Percentage at Birth is Associated with Late Onset Infection in Very Preterm Neonates L. Ma*†1, R. Chen†1, F. Liu*†, Y. Li†, Z. Wu†, W. Zhong‡, G. Lu† & B. Wang§
Abstract *Southern Medical University, Guangzhou, Guangdong Province, China; †Department of Neonatology, Shenzhen Bao’an Maternal and Child Health Hospital, Guangzhou, Guangdong Province, China; ‡Department of Medical Information, Shenzhen Bao’an Maternal and Child Health Hospital, Guangzhou, Guangdong Province, China; and §Department of Paediatrics, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong Province, China
Received 15 February 2014; Accepted in revised form 2 April 2014 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 protected] 1
The two authors contribute equally to this paper.
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.
Introduction 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 [1]. Over half would develop one or more episodes of infection in preterm infants with gestational age 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 [5]. Diagnosis was made by the Table 1 Demographic information of study populations.. Groups
37w–42w
33w–36w
≤32w
n GA (w)
49 39.5 0 (38.60–40.25) 3350 (3090–3665) 61.22
59 34.00 (32.86–35.14) 1950 (1700–2200) 66.10
84 30.29 (29.29–31.14) 1355 (1252–1600) 60.71
Birth wt (g) Gender male (%) Vaginal delivery (%)
51.02
64.41
66.67
GA, gestational age. The median and interquartile ranges for both gestational age and birth weight were shown.
Ó 2014 John Wiley & Sons Ltd
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 ruleout 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 [10]. 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 ll) were incubated for 15 min in the dark at room temperature with TBNK reagents. The erythrocytes were then lysed with 450 ll of 1 9 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). Statistical analysis. 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 chisquare test was used for testing the association of variables in a 2 by 2 table format. P value 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.
Figure 2 Absolute counts of lymphocyte subsets on D0 in preterm and term neonates. Results were shown as median with interquartile range. *compared with term neonates, P < 0.05. #compared with preterm neonates of 33w–36w, P < 0.05.
Figure 3 Percentages of lymphocyte subsets on D0 in preterm and term neonates. Results were shown as median with interquartile range. *compared with term neonates, P < 0.05. #compared with preterm neonates of 33w–36w, P < 0.05.
Scandinavian Journal of Immunology, 2014, 80, 50–56
L. Ma et al. Immune Status and Late Onset Infection 53 ..................................................................................................................................................................
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) /ll and 3.5 (2.7–4.5)%, respectively] and D7[137 (89–209) /ll and 4.3 (2.9–5.5)%, respectively] were significantly reduced compared with those on D0 [263 (145–403) /ll and 8.7 (4.9–14.8)%, respectively] (P all
