**3. Reference values in an adult and pediatric population**

Generally, normal values of laboratory parameters in a neonatal population are difficult to define, because removal of blood is usually not performed in healthy neonates and reference ranges are composed by assessing patients with minor illness (Christensen et al., 2009). To our knowledge, the first published reference values for neutrophil cells in neonates including the total neutrophil count, the absolute number of immature neutrophils, and the IT-ratio during the first 28 days of life refers to a study by Manroe in 1979 (Manroe et al., 1979). About 15 years later the same study group found that in VLBW these reference values are of limited applicability because a wider range of distribution was found in this subgroup of patients compared to larger or older counterparts (Mouzinho et al., 1994). These new data comprised a wider range of the absolute total neutrophil count and a considerable decreased lower limit in the first 60 hours after birth, whereas reference ranges for the immature neutrophil count and IT values remain unchanged. It has been assumed that low neutrophil counts soon after birth might be caused by a placental factor inhibiting neutrophil production. Clearance of this factor within the first week could lead to the observed increase in immature neutrophil cells. Anyhow, the capability of the bone marrow to rapidly produce immature as well as mature neutrophil forms by the second week is well documented. Neutropenia occurred rarely in infants at an age of more than 7 days, but neutrophilia occurred frequently in association with stress conditions inducing an adrenergic increase in cyclic adenosine monophosphate (cAMP) leading to a release of neutrophil cells (Mouzinho et al., 1994). Hence, neutropenia has been described as a better predictor for neonatal sepsis than an elevated neutrophil count because besides accelerated utilization in case of infection there a fewer factors (i.e. hemolytic disease, asphyxia, maternal hypertension) causing a decrease of neutrophil granulocytes. Lower levels of normal for neutrophil values have been set at 1800/mm3 at birth and < 7800/mm3 12-14 hours after birth in term and late preterm infants (Manroe et al., 1979).

A large trial evaluating more than 30000 samples from infants born at 23 to 42 weeks of gestational age reinvestigated the previously published reference ranges using an automated blood cell counter (Schmutz et al., 2008). In this study lower limits of normal for the neutrophil count were determined as follows (Table 1):

#### The Role of Immature Granulocyte Count and Immature Myeloid Information in the Diagnosis of Neonatal Sepsis 67


**Table 1.** Neutrophil count at birth and 6-8 hours postnatally (pn) comparing groups of different gestational age (Polin, 2012; Schmutz et al., 2008).

66 Neonatal Bacterial Infection

nucleus size (Sysmex Corporation, 2005).

al., 2003; Fernandes & Hamaguchi, 2007).

composition of the cell (nucleus, granules). Differences in RF resistance detected as electrical pulses are plotted in a two dimensional scattergram reflecting the distribution of cell and

The IMI-channel determines the total number of myeloid precursor cells by distinguishing selectively immature myeloid cells from mature leukocytes. The reaction principle of the IMI-channel is based on differences in membrane composition between mature and immature cells. It has been shown that the flow cytometric IGC performed by the Sysmex XE-2100 is superior to the manual morphology count as a reference method for IG counting and that the percentage of IGs is a better predictor of infection than the WBC (Ansari-Lari et

Generally, normal values of laboratory parameters in a neonatal population are difficult to define, because removal of blood is usually not performed in healthy neonates and reference ranges are composed by assessing patients with minor illness (Christensen et al., 2009). To our knowledge, the first published reference values for neutrophil cells in neonates including the total neutrophil count, the absolute number of immature neutrophils, and the IT-ratio during the first 28 days of life refers to a study by Manroe in 1979 (Manroe et al., 1979). About 15 years later the same study group found that in VLBW these reference values are of limited applicability because a wider range of distribution was found in this subgroup of patients compared to larger or older counterparts (Mouzinho et al., 1994). These new data comprised a wider range of the absolute total neutrophil count and a considerable decreased lower limit in the first 60 hours after birth, whereas reference ranges for the immature neutrophil count and IT values remain unchanged. It has been assumed that low neutrophil counts soon after birth might be caused by a placental factor inhibiting neutrophil production. Clearance of this factor within the first week could lead to the observed increase in immature neutrophil cells. Anyhow, the capability of the bone marrow to rapidly produce immature as well as mature neutrophil forms by the second week is well documented. Neutropenia occurred rarely in infants at an age of more than 7 days, but neutrophilia occurred frequently in association with stress conditions inducing an adrenergic increase in cyclic adenosine monophosphate (cAMP) leading to a release of neutrophil cells (Mouzinho et al., 1994). Hence, neutropenia has been described as a better predictor for neonatal sepsis than an elevated neutrophil count because besides accelerated utilization in case of infection there a fewer factors (i.e. hemolytic disease, asphyxia, maternal hypertension) causing a decrease of neutrophil granulocytes. Lower levels of normal for neutrophil values have been set at 1800/mm3 at birth and < 7800/mm3 12-14 hours

**3. Reference values in an adult and pediatric population** 

after birth in term and late preterm infants (Manroe et al., 1979).

the neutrophil count were determined as follows (Table 1):

A large trial evaluating more than 30000 samples from infants born at 23 to 42 weeks of gestational age reinvestigated the previously published reference ranges using an automated blood cell counter (Schmutz et al., 2008). In this study lower limits of normal for The notable difference in altitude between the two studies might have influenced the results. The dynamic process of granulopoesis after birth is reflected by a rapid increase of neutrophil cells reaching peak levels at 6 to 8 hours postnatally (Polin, 2012; Schmutz et al., 2008). Allowing sufficient reaction time to inflammatory stimuli alterations in mature and immature granulocytes are more likely to occur between 6 to 12 hours after birth. This should be taken into account when planning blood sampling (Polin, 2012).

A quite similar time course has been shown for the absolute immature neutrophil count: Maximal values increase from 1100/µL soon after birth to a peak of 1500/µL at 12 hours postnatally. In contrast to that, maximum normal values for the IT-ratio have been observed directly after birth followed by a decline with increasing age (Polin, 2012; Schmutz et al., 2008). In the most immature infants between 24 and 26 weeks of gestational age, an elevation of ANC has been shown during the first month of life. In the first three weeks of life a weekly decrease of ANC to values between 2000/µL and 4000/µL has been observed. As the prevalence of both neutropenia as well as neutrophilia decreased with maturity, it can be concluded that granulopoetic function stabilizes with higher gestational age enabling adequate reactions to infectious or stress stimuli. Deviations from the normal range of neutrophil granulocytes without additional signs of clinical symptoms or conditions occurred frequently even in a hospitalized population (Juul et al., 2004). In the face of these data more interest should be attracted on considering the gestational age as well as the time point of blood sampling when interpreting CBC results (Polin, 2012). The influence of birth weight on CBC in healthy term infants was examined in a study performed by Ozyürek and co-workers. Their data revealed a clear difference in several CBC parameters comparing healthy, term infants with intrauterine growth retardation to appropriate for gestational age (AGA) counterparts showing neutropenia in 21% as well as higher IT-ratios in small for gestational age (SGA) newborns. Beyond these findings, a higher rate in immature neutrophil cells, namely in the absolute number of metamyeolcytes, was observed in the SGA babies. The authors suggested that this elevation might be interpreted as a reaction of the bone marrow to compensate for the initially frequent low neutrophil count (Ozyurek et al., 2006).

Some authors have considered the method of automated measurement of IGC as not sensitive enough to be used as a sole screening assay for the prediction of infection. However, it has been demonstrated that a high percentage of IG (> 3%) is a very specific predictor (> 90%) of sepsis (Ansari-Lari et al., 2003) and that IG values less than 0.5% are associated with a high negative predictive value. These findings might be of use in a clinical context (Nigro et al., 2005). Recently published reference values have defined a median of 0.63x103/µL (0.1–2.4; 2.5%–97.5% confidence interval) for IG number (IG#) and a cut-off value of 3.2% for IG% as optimal for a normal adult population. Using a cut-off in a range between 4% and 5% of total WBC would result in a too high rate of missed cases (Bernstein & Rucinski, 2011). In a large outpatient pediatric population comprising more than 2400 samples, age dependent upper limits for reference ranges for the automated enumeration of IG were defined as 0.30% and 40/µL for IG% and IG#, respectively for children aged below 10 years (Roehrl et al., 2011). Above the age of 10 years, an upper limit of 0.90% and 70.0/µL for relative and absolute IG count was recommended (Roehrl et al., 2011). In this study blood samples were analyzed using the Sysmex XT-1800i instrument (Sysmex, Kobe, Japan). The defined upper limits showed no differences dependent on the patient sex. As expected the cause of elevated IGC differed between both groups. While respiratory or gastrointestinal infections were common associations with elevated IGC in the group < 10 years, the older children showed hematologic malignancies, drug therapy (glucocorticoids, chemotherapy), severe infections, and pregnancy (young females). In a subgroup analysis of patients < 1 year this study revealed age-stratified nonparametric estimates of upper limits of normal (95th percentiles) and associated 90% confidence intervals (CI) for IG# and IG% of 40/µL (30.0–50.0) and 0.30% (0.20–0.40). In addition, this study described an important observation: Even the most abnormal IGCs in the younger age group were quite low compared with abnormal IGCs in the older individuals. This fact highlights the importance of particular reference values appropriate for different age groups. Otherwise especially younger children with associated disease and with only small elevations of IGCs could be overlooked (Roehrl et al., 2011). As neonates represent a highly particular and often vulnerable patient population we aimed at investigate a possible correlation between IGC and sepsis.

The Role of Immature Granulocyte Count

and Immature Myeloid Information in the Diagnosis of Neonatal Sepsis 69

infection, blood samples were always taken before the initiation of antibiotic therapy. ROC curves were used for comparison of infectious indices by plotting the test sensitivity (equivalent to the true positive rate) on the y-axis and 1-specifity (equivalent to the false positive rate) on the x-axis for all possible cut off values of the diagnostic test (see Figure 1).

**Figure 1.** a and b: Diff- and IMI scattergram showing graphic output of WBC differential results performed with the Sysmex XE 2100. By courtesy of © Sysmex Europe GmbH, Norderstedt, Germany.
