**2.1. Neutrophil positional parameters as a new tool in the prediction of sepsis**

Morphologic and physical properties of cells including cell volume, internal composition, and cytoplasmatic granularity and nuclear structure assessed via cell conductivity and cell scatter are described as positional parameters. In contrast to more mature cells IGs and band cells for instance are known to be larger, whereas nuclear composition is less complex. Based upon these facts positional parameters are used to classify different types of white blood cells, but recently this method has also been applied as a screening tool for neonatal sepsis to detect morphologic changes within the same blood cell population (i.e. in reactive neutrophil cells occurring during acute bacterial infection). Chaves et al. tried to assess and quantify these parameters as indicators of acute infection. In retrospective studies of adult septic patients and controls, the mean neutrophil volume (MNV) and its standard deviation, the neutrophil volume distribution width (NDW), reflecting the neutrophil size variability, showed high specifities (Chaves et al., 2005; 2006). In the control group the neutrophil population presented more homogenous than in bacteremic patients and the individual cell size varied less. Furthermore, a correlation of NDW respectively MNV and positive blood culture results, higher percentages of neutrophils and higher WBCs has been shown, whereas increased values were also present in patients without leukocytosis or neutrophilia possibly representing an important early diagnostic parameter in this subgroup of patients (Chaves et al., 2006). Another study using this technology showed good performance characteristics of MNV in detecting LOS in VLBW neonates with a NPV of 98.9%. Because of a considerably lower PPV the authors emphasize the possible combination with CRP-values in the prediction of sepsis. Interestingly, in contrast to an adult population the NDW did not reveal any clinical significance in a neonatal population. The authors suggested that this might be due to an originally more heterogeneous morphology of neutrophil cells in newborn infants (Raimondi et al., 2010; Raimondi et al., 2011).

#### **2.2. The Sysmex XE-2100**

64 Neonatal Bacterial Infection

positive blood culture (Nigro et al., 2005).

sepsis (Bernstein & Rucinski, 2011).

hospitalized infants elevations of IGs were associated with positive blood culture results. In this study, values exceeding 0.5% showed a more than three-fold increased likelihood of a

In several studies comparing the manual microscopic method and the automated method for IG% and IGC significant correlation coefficients between 83% and 87% have been demonstrated (Field et al., 2006; Senthilnayagam et al., 2012). Compared with a flow cytometric reference count with monoclonal antibodies the correlation coefficient was even higher and amounted to 96% (Briggs et al., 2003). It has been shown that an increased percentage of more than 2% of IGs can be useful in identifying infection even when the neutrophil count is within the normal range and infection is not suspected. Conversely, in patients with a high IGC sample rates were positive for CRP and the erythrocyte sedimentation rate in 84% and 95%, respectively. Furthermore, elevated IGC showed a correlation with other inflammation markers such as CD 64 expression on

The detection of IGs using automated hematology analyzers represents a fast, accurate, and less-labor intensive method and could improve screening and monitoring for neonatal septicaemia (Briggs et al., 2000; Fernandes & Hamaguchi, 2007; Nigro et al., 2005). The detection limit of IGs has been described to be 0.1% which is considerably lower than in a manual smear. The automated simultaneous enumeration of IGs in the course of performing a routine CBC provides additional information without the need of further costs and blood sampling, which might be of special importance in preterm babies. This new technology of automated measurement of IGs offers additional information reflecting the increase in bone marrow activity as an indicator of a left-shift of neutrophil cells in a more sensitive and specific way than the manual examination of a peripheral blood smear differential count.

Detection of IGs comprises the amount of metamyelocytes and myelocytes, but not band neutrophils and therefore reflects early stages of maturation of granulocytes. As the band cell is defined as a cell in the transitional state of granulopoetic maturation after the differentiation of metamyelocytes and myelocytes, the band count itself has been described as nonspecific, imprecise, and inaccurate as laboratory marker for the early detection of sepsis (Bernstein & Rucinski, 2011; Cornbleet, 2002). Hence, determination of IGs in contrast to the more mature band neutrophils, which arise later on, could be advantageous at the onset of moderate to severe inflammation (Cornbleet, 2002). Moreover, it has already been shown that the measurement of granulocyte maturation correlates to the identification of

**2.1. Neutrophil positional parameters as a new tool in the prediction of sepsis** 

Morphologic and physical properties of cells including cell volume, internal composition, and cytoplasmatic granularity and nuclear structure assessed via cell conductivity and cell scatter are described as positional parameters. In contrast to more mature cells IGs and band cells for instance are known to be larger, whereas nuclear composition is less complex. Based upon these facts positional parameters are used to classify different types of white

polymorphonuclear cells and interleukin 6 concentration (Briggs et al., 2003).

The Sysmex XE-2100 (Sysmex Corporation, 2005), a multiparameter automated hematology analyzer offers the possibility to detect IGs including metamyelocytes, myelocytes, and promyelocytes by the measurement of white blood cell differential counts by flow cytometry in the DIFF-channel. Besides the quantification of IGs, physical properties of immature cells and reactivated neutrophils are provided. Therefore blood samples are incubated with Stromatolyser-IM, a fluorescent dye and a proprietary reagent, selectively leaking the membrane of mature leukocytes. Immature myeloid cells are not modified in their size, structure and integrity, because the IG has a lower cholesterol content than the mature granulocyte, and its phospholipid composition has a relatively higher ratio of phosphatidylcholine and a lower ratio of sphingomyelin (Gottfried, 1967). Depending on the dispersion angle when the cell passes the beam of a semiconductor laser, information about the volume, inner structure and complexity, and DNA/RNA content of each cell is obtained by a combination of forward-scattered light, lateral-scattered light, and lateral fluorescent light. The light is received by a photodiode respectively a photomultiplier tube and is then converted into electrical pulses. The higher content of RNA and DNA in IGs compared with segmented neutrophils is reflected in an increased fluorescence emission after excitation with the laser beam. The XE-2100 is equipped with an additional immature information (IMI) channel, where not only IGs, but also bands, blasts, and hematopoietic progenitor cells are detected. Detection of cell size, information about the nuclei and composition of cytoplasm is generated by direct current and radio frequency resistance when cells pass an aperture in the IMI-channel. The direct current (DC) pulse height is equivalent to cell volume. The radio frequency (RF) measurement provides information on the internal

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 nucleus size (Sysmex Corporation, 2005).

The Role of Immature Granulocyte Count

and Immature Myeloid Information in the Diagnosis of Neonatal Sepsis 67

Gestational age Neutrophil count at birth Neutrophil count 6-8h pn

> 36 weeks 3500/µL 7500/µL 28-36 weeks 1000/µL 3500/µL < 28 weeks 500/µL 1500/µL

**Table 1.** Neutrophil count at birth and 6-8 hours postnatally (pn) comparing groups of different

should be taken into account when planning blood sampling (Polin, 2012).

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

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

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

gestational age (Polin, 2012; Schmutz et al., 2008).

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