**2. Automated detection of immature granulocytes- Clinical applicability**

Automated measurement of IGC could represent a reliable and utile method in the prediction of bacterial infection in neonates. In a study evaluating 106 samples from patients with an absolute neutrophil count (ANC) less than 2.0 × 109/L measured with an automated 5-part differential hematology instrument the IGC showed a very good precision and accuracy when compared with a flow cytometric neutrophil count using monoclonal antibodies for cell classification (Amundsen et al., 2012). In another investigation of 200 febrile patients suspected to have infection the performance characteristics of automated IGCs in predicting blood culture results and their clinical utility were assessed. The study population included adults, children, infants, and neonates. Measurements were performed using the Coulter Act Diff 5 counter which can perform a 5-part differential leucocyte count and can also numerate the percentage and absolute number of IG using a technology that combines cytochemistry, focused flow impedance, and light absorbance. The means of IGC and the percentage of IG (IG%) between culture positive and negative groups were statistically significant suggesting that they are potential markers for bacteremia. Among the 51 culture positive cases, 49 had an IT-ratio > 0.65% giving a sensitivity of 96.1%. IGC of 0.03×103/µL and IG% of 0.5% offered a sensitivity of 86.3% and 92.2%, respectively. Higher values of IGC > 0.3 and IG% > 3 had a specificity greater than 90%, although the values were infrequent. Receiver operating characteristic (ROC) curves showed that IGC was a better predictor of infection than WBC and ANC in adults and the ratios IGC/WBC and IGC/ANC did not improve the prediction outcome (Senthilnayagam et al., 2012). Another study reported an in parallel increase of IG values to an increase of the ANC and an inverse relation to the lymphocyte count (Bernstein & Rucinski, 2011).

In an adult study population including patients suspected for sepsis higher percentages of IGs have been observed in infected than in non-infected patients and in patients with positive than patients with negative blood cultures (Ansari-Lari et al., 2003). Also in preterm

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

The Role of Immature Granulocyte Count

and Immature Myeloid Information in the Diagnosis of Neonatal Sepsis 65

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

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

newborn infants (Raimondi et al., 2010; Raimondi et al., 2011).

**2.2. The Sysmex XE-2100** 

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

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 sepsis (Bernstein & Rucinski, 2011).
