The detection of NRBC in the blood was not associated with one particular cause of death (Fig. 2). The mean NRBC concentration in patients who died due to malignant tumors (55 ± 14/μL) was significantly lower than in those patients who died due to cardiac failure (338 ± 101/μL, P < 0.001) or multiorgan failure (399 ± 108/μL, P < 0.05). However, during the detection of NRBC in blood, none of the deceased NRBC-positive patients with malignant tumors were provided with chemotherapy or irradiation. Concerning the other causes of death, none was apparently associated with a significantly lower or higher NRBC concentration than others, although overall the number is rather low.
Association between NRBC in blood and erythropoietin, IL-3, IL-6, and IL-12p70, respectively
The concentrations of erythropoietin, IL-3, IL-6, and IL12p70 in the blood of NRBC-negative and NRBC-positive patients is summarized in Figure 3. Concerning the concentrations of those cytokines, no differences were found between male and female patients. In patients with renal deficiency, the erythropoietin concentration was not significantly lower than in patients without renal deficiency.
Correlation analysis of NRBC concentration revealed significant correlations with erythropoietin (r = 0.180, P < 0.001, n = 551), IL-3 (r = 0.348, P < 0.001, n = 551), IL-6 (r = 0.180, P < 0.001, n = 551), and IL-12p70 (r = 0.084, P < 0.05, n = 551), respectively.
As summarized in Table 2, the multiple logistic regression showed a significant association between the appearance of NRBC in the blood and age, erythropoietin, IL-3, and IL-6, respectively.
Moreover, concerning the fatal outcome (death), the odds ratios of age, gender, NRBC, erythropoietin, IL-3, IL-6, and IL-12p70 were calculated (Table 3). Significant odds ratios concerning death were found for age, the appearance of NRBC in blood, increased erythropoietin, increased IL-6, and decreased IL-12p70, respectively.
Erythropoiesis of the bone marrow
Erythropoietic function of the bone marrow was estimated by the measurement of the reticulocyte concentration in blood. The reticulocyte concentration in NRBC-positive patients on the day of the highest NRBC concentration of the respective patients was considered as a basic value. On that day, the reticulocyte concentration was 69 ± 2/nL (n = 301; reference interval, 26-78/nL) and thus significantly higher than in NRBC-negative patients (60 ± 2/nL; n = 250; P < 0.01). During the additional course of hospitalization, reticulocytes increased to 94 ± 3/nL (NRBC-positive patients; n = 224) and 70 ± 3/nL (NRBC-negative patients; n = 125; P < 0.001), respectively. Therefore, as shown in Figure 4, in NRBC-positive patients, the increase in the reticulocyte concentration was significantly higher (24 ± 3/nL; n = 224) than in NRBC-negative patients (11 ± 3/nL; n = 125; P < 0.01).
Furthermore, we analyzed the increase in the reticulocytes in deceased and surviving NRBC-positive patients. In deceased NRBC-positive patients, the increase in the reticulocyte concentration was 23 ± 6/nL (n = 41), whereas in NRBC-positive survivors, the increase was 24 ± 3/nL (n = 183; not significant). Therefore, as shown by the reticulocytes in NRBC-positive patients, disturbed bone marrow function seems not to be the reason for the high in-hospital mortality of NRBC-positive patients.
NRBC and other laboratory parameters
Concerning the C-reactive protein concentration, a significant difference was found between NRBC-positive patients and NRBC-negative patients (80.9 vs. 22.9 mg/L). Correlation of the NRBC concentration and other laboratory parameters was assessed by Spearman correlation analysis. NRBC significantly correlated with leukocytes (r = 0.3805; P < 0.001; n = 301), thrombocytes (r = −0.2133; P < 0.001; n = 301), C-reactive protein (r = 0.2218; P < 0.001; n = 266), alanine aminotransferase (r = 0.3405; P < 0.001; n = 131), and creatinine (r = 0.3194; P < 0.001; n = 277), respectively. On the other side, reticulocytes or hemoglobin showed no correlation with the NRBC concentration.
In earlier studies, we and others (1, 4, 11-15) have shown that NRBCs were detected particularly in the blood of patients suffering from severe diseases. If detectable, those patients have an extremely poor prognosis (1-4, 9, 10, 31). In association with various diseases, our present data revealed significant differences in the mean NRBC concentration. The lowest concentration was found in deceased patients who died of malignant tumors. However, at that period of time, none of the deceased NRBC-positive patients with malignant tumors were provided with chemotherapy or irradiation. Therefore, the reason remains unclear.
In general, the pathophysiology concerning the appearance of NRBC in blood of patients with high in-hospital mortality remains to be clarified (9). In particular, it is widely unknown whether a malfunction of the bone marrow by a disturbed cytokine environment could lead to the appearance of NRBC in blood. Therefore, in this study, we have analyzed erythropoietin together with IL-3, IL-6, and IL-12p70 in patients with NRBC in the peripheral blood. Moreover, the reticulocytes in blood as a marker of the efficiency of the erythropoiesis were analyzed as well.
Univariate analysis revealed that erythropoietin, IL-3, IL-6, and IL-12p70 were significantly higher in NRBC-positive patients than in NRBC-negative patients. Compared with the reference interval of 6 to 25 mU/mL (32), the average erythropoietin concentration was increased in NRBC-positive patients. Furthermore, increased IL-3 concentrations (concentration in healthy adults is <8 pg/mL) (33) were found in NRBC-negative and NRBC-positive patients. Moreover, IL-3 concentration in NRBC-positive patients was significantly higher than in NRBC-negative patients. Because erythropoietin and IL-3 are stimulators of the early phases of the erythropoiesis (17, 21), the appearance of NRBC under distinct conditions could be due to a stimulation by erythropoietin and other cytokines.
Furthermore, we found increased IL-12p70 concentrations (concentration in the plasma of healthy adults is <238 pg/mL) (34) in NRBC-negative patients and even significantly higher concentrations in NRBC-positive patients. IL-12p70 is the biologically active polypeptide heterodimer that binds to the IL-12 receptor (35, 36). Although lymphocytes are generally considered to be the main target cells of IL-12p70 (37, 38), several additional effects on the erythropoiesis have been observed (29, 30, 39). In turn, based on our study, these effects of IL-12p70 on the erythropoiesis could strengthen the appearance of NRBC in blood of critically ill patients.
Increased IL-6 concentrations (reference range of <10 pg/mL) (40) were found in NRBC-negative and NRBC-positive patients. However, the IL-6 concentrations in NRBC-positive patients were significantly higher than in NRBC-negative patients. IL-6 plays a pivotal role in the development of inflammation. Concerning such association between IL-6 and NRBC, it seems attractive to speculate whether an anti-inflammatory therapy could improve the poor prognosis of NRBC-positive patients.
Furthermore, a direct impact of IL-6 on the erythropoiesis has been reported (19, 24, 25). Thus, the relatively high IL-6 concentrations in NRBC-positive patients may indicate not only a strong inflammatory process in those patients, but also a stimulation of the erythropoiesis leading to the, in part, excessive appearance of NRBC in blood.
However, erythropoietin, IL-3, IL-6, and IL-12p70 may be members of a functional network in which they influence each others' concentration. Such interdependency of the parameters would not be elucidated by a univariate analysis, as shown in this study. Therefore, in addition, we performed a multivariate logistic regression that circumvents such interdependency. This analysis revealed a significant association between the appearance of NRBC in blood and age, erythropoietin, IL-3, and IL-6, respectively. In contrast, the odds ratios of gender and IL-12p70 were not significant.
Moreover, the multivariate logistic regression analysis showed that the detection of NRBC is significantly associated with an older age. However, with regard to in-hospital mortality, the detection of NRBC itself had a significant odds ratio. Therefore, the poor prognosis of NRBC-positive patients is not simply a result of a higher rate of NRBC in older patients. On the contrary, in a recent study we have shown that the prognostic impact of NRBC is even higher in younger patients (9).
Erythropoietin production occurs nearly completely in the kidney, stimulated by the oxygen tension registered in the kidney (41, 42). Therefore, taken together, our results about erythropoietin and other cytokines, NRBC in blood may be considered as a parameter that sums up hypoxic (erythropoietin) and inflammatory (IL-3 and IL-6) injuries as supposed by us elsewhere (43). This may be the reason why the appearance of NRBC is a strong predictor of in-hospital mortality. Moreover, under consideration of age, gender, erythropoietin, IL-3, IL-6 and IL-12p70 NRBCs had a significant odds ratio of 15.2. Thus, for the identification of patients at high risk, the measurement of NRBC cannot be replaced by the measurement of erythropoietin, IL-3, IL-6, or IL-12p70.
Finally, NRBC significantly correlated with laboratory parameters of liver (alanine aminotransferase) and renal injuries (creatinine). Furthermore, in a recent study on trauma patients, some authors reported on a release of NRBC into the circulation that was associated with bone marrow failure (44). However, in the present study, the reticulocyte concentration strongly increased in NRBC-positive patients. This increase in the reticulocyte concentration is likely to be induced by the high erythropoietin as well as high IL-3 concentrations in NRBC-positive patients. Furthermore, in deceased and surviving NRBC-positive patients, no difference was found regarding the increase in the reticulocyte concentration. Therefore, it seems unlikely that the appearance of NRBC is associated with a disturbed bone marrow function as far as the erythropoiesis is concerned.
In summary, our study revealed that the detection of NRBC in blood was associated with increased erythropoietin, IL-3, and IL-6 concentrations. Therefore, the detection of NRBC may be considered a parameter that sums up life-threatening hypoxic and inflammatory injuries. However, due to the reticulocyte data in NRBC-positive patients, the respective bone marrow function is obviously not disturbed under those conditions.
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Nucleated red blood cells; erythropoietin; IL-3; IL-6; reticulocytes