Multiple leucocyte activation markers to detect neonatal infection

Abstract

SUMMARYDiagnosis of congenital or neonatal infection is often based on clinical signs. However, clinical symptoms of infections may not be specific, and for this reason early diagnosis is often determined on results of laboratory tests, which may not currently be adequate. A more reliable method of detection of infection may be the demonstration of activated lymphocytes, which can be conducted rapidly and before the isolation of the infected organism. We have shown that detection of up-regulation of CD45RO, an activated/memory isoform of CD45 present on T cells, provides a reasonably sensitive screening test for neonatal infection. We also showed that dual expression of CD45RA/CD45RO was up-regulated early during the infective process in neonates with documented infection. However, other leucocytes are also activated during the infective process. To improve the sensitivity of the neonatal infection screening test and to identify the types of leucocytes involved in the immune response to the infective organism, we studied further the up-regulation of a comprehensive range of surface activation markers on T cells, monocytes and natural killer (NK) cells from a group of 17 newborn patients with positive culture, a group of 40 possibly infected patients based on clinical signs and a control group. 'Normal' ranges were established for each activation marker for each leucocyte subset from 1 to 7 and 7-14-day-old newborns < 35 weeks' gestation and 35-40 weeks' gestation. There was a significant increase in the percentage of T cells expressing CD25 in the peripheral blood from infants at 2 weeks of age. Expression of HLA-DR on T cells, CD25 and CD69 on monocytes and HLA-DR on NK cells was also increased significantly in the peripheral blood from infants at 2 weeks of age and may reflect a maturation of these functional surface molecules. Up-regulation of CD69 on NK cells was the most sensitive marker for neonatal sepsis (positive in 13/16 patients). CD69 and CD25 expression was increased significantly on T cells in 11/17 and 10/17 patients, respectively. A combination of CD45RA/CD45RO and CD45RO identified 11/16 infected patients. Measurement of CD69 expression on NK cells with CD45RA, CD45RO, CD25 and CD69 expression on T cells resulted in a significant increase in at least two leucocyte activation markers from infected patients. In conclusion, this is the first report of the up-regulation of CD69 on NK cells as a sensitive marker of neonatal infection. A combination of this marker with CD45RA, CD45RO, CD25 and CD69 expression on peripheral blood derived T cells is the most sensitive and specific for neonatal infection.

Flow Cytometric Methods in the Detection of Neonatal Infection

“…In this study, CD11b expression gradually increased in preterm infants during the 3 days prior to sampling of blood cultures. The sensitivity and specificity of CD11b was 100 and 56% for neutrophils and 86 and 94% for monocytes, respectively [37], but other study results show a variability [35][36][37][38][39]. This discrepancy is related to pre-analytic differences, evaluation of different populations, criteria for sepsis, time points of test per-forming, and standardization of the flow cytometric technique.…”

“…Blood specimens for cell surface markers should be transported to the laboratory on ice and processed immediately: Studies showed that a substantial proportion of neutrophils undergoes apoptosis after 24 h and down-regulates surface receptor antigens during the apoptotic process [36,37]. Therefore, analysis of neutrophil surface antigens should be performed immediately so that pre-analytic conditions (temperature, time, continuous activation of inflammatory cells in vitro, pH) should not affect receptor expression levels [21].…”

“…As it can be expressed without de novo protein synthesis, receptor densities increase considerably within minutes after contact between inflammatory cell and bacteria, bacterial products and/or endotoxins [36]. These unique characteristics enable CD11b to be used as an early marker of bacterial infection.…”

Neutrophil CD64 Is a Sensitive Diagnostic Marker for Early-Onset Neonatal Infection

“…Recent reports suggested that the expression of other cell surface antigens such as CD11b on neutrophils (5,6), CD45RO and CD25 on lymphocytes (19 -21), and CD69 on NK cells (22) were potential valuable markers for predicting neonatal sepsis. However, not all have been validated to be suitable for clinical use.…”

Detection of the CD64 on Neutrophils and CD69 on Lymphocytes by Flowcytometry as a Marker for Early Diagnosis of Neonatal Sepsis

“…Since diagnosis of neonatal sepsis is one of the most difficult tasks in clinical practice, as the disease progress more rapidly than adult and the mortality rate is higher in neonates (3) , several different laboratory determinations are helpful in diagnosis of neonatal sepsis for instances; numerous cell surface antigens have been studied as potentially promising biomarkers of infection, including CD69 and CD64 (4) . Flow cytometric analysis of cell surface antigens (CD11b, CD64, CD32 CD16, CD69, CD25 and CD45) has been performed to detect neonatal sepsis (5) . Other surface markers that have been investigated in different studies include CD69 on peripheral T and B lymphocytes may also have a role (6) .…”