KL-6, a Human MUC1 Mucin, as a prognostic marker for diffuse alveolar hemorrhage syndrome

Consecutive patients who were admitted to the ICU and diagnosed as DAH between 2004 and 2011 were retrospectively studied. Patients’ characteristics including age, sex, laboratory findings, radiological findings, ventilatory modes or therapeutic regimens were extracted from the medical records. The vital status of the patients was determined by reviewing medical records, death certificates, as well as by phone interviews.

Serum KL-6 and lactate dehydrogenase (LDH), prothrombin time-international normalized ratio (PT-INR), activated partial thromboplastin time, D-dimer and hemoglobin levels and platelet counts were included as variables for analysis when taking routine blood tests and performing BAL.

Baseline KL-6 was defined as serum KL-6 level at admission, and peak KL-6 was defined as the highest serum KL-6 level during the follow-up. KL-6 was measured once-weekly. The etiologies of DAH were grouped into 5 categories: pulmonary infection, excessive anticoagulation, vasculitis, interstitial pneumonia or idiopathic based on the different pathogenesis in our patients. The diagnosis of vasculitis was defined by histological evidence, high titers of anti-neutrophil cytoplasmic antibodies, anti- deoxyribonucleic acid antibodies or anti-basement membrane antibodies [10, 11]. Correlations between survival and clinical variables were evaluated.

Clinical diagnosis of DAH was based on the findings of diffuse ground glass or airspace-filling opacities on chest radiograph and computed tomography, and BAL fluid showing progressively bloodier returns and the presence of 20% or more hemosiderin-laden macrophages [12]. Exclusion criteria were apparent deterioration of left heart failure and contact bleeding with bronchoscope.

The procedure of BAL was done as previously described [13]. In brief, a flexible bronchoscope was wedged into a segmental bronchus of the middle lobe or the lingula. Sterile isotonic saline at 37°C was instilled in three 50 ml aliquots up to a total volume of 150 ml, with immediate aspiration by gentle suction after each aliquot. The total of recovered BAL fluid fractions were mixed and immediately filtered through two layers of gauze, and centrifuged at 500 g for 10 min at room temperature. Slides were stained with Prussian blue to detect haemosiderin-laden macrophages [12]. After incubation of the slides with 1% hydrochloric acid and 2% potassium ferricyanide (Katayama Medical Co. Ltd., Osaka, Japan) for 20 minutes, slides were counterstained with Kernechtrot stain (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan). A total of 200 or more macrophages were examined for calculating a percentage of Prussian blue positive cells. The BAL fluid samples also underwent routine microbiologic testing for detecting bacteria and viruses.

Data are expressed as mean ± standard deviation. Comparison of non-normally distributed variables between groups was done with the Mann-Whitney’s U test or Fisher’s PLSD test. Analysis of changes in non-normally distributed variables between groups was done with the Wilcoxon’s rank test or repeated measures of analysis of variance. Comparison of categorical variables between two groups was done with the χ² test. The probability of survival was estimated with the Kaplan-Meier method, and the differences in survival rates were evaluated by log-rank test. Multivariate analysis of prognostic factors was done using the Cox regression model. All statistical analyses were done using SPSS version 13.0 for Windows (SPSS Inc., Chicago, IL). Differences were considered statistically significant when the p value was <0.05.

There were 25 males and 16 females with a median age of 69 (range, 16–83) years. There were 27 never smokers, 12 former smokers, 2 current smokers, respectively. The etiologies of DAH were pulmonary infection (n=19), excessive anticoagulation (n=9), vasculitis (n=6), interstitial pneumonia (n=2) and idiopathic (n=5). Thirteen patients survived, and 28 died. The ICU mortality was 54% (22/41), the in-hospital mortality was 68% (28/41), and the 28-day mortality was 32% (13/41), respectively. No significant differences were observed in age, sex, smoking status, radiological findings, use of mechanical ventilation, baseline P/F ratio, and laboratory markers at admission between the groups (Table 1). Use of methylprednisolone pulse after admission was more frequent, prothrombin time-international normalized ratio and activated partial thromboplastin time were higher, and duration of ICU stay was significantly longer in non-survivors than survivors (p=0.038, 0.043, 0.029, 0.005, respectively). Meanwhile, P/F ratio 48 hrs after admission was significantly lower in non-survivors than survivors (p=0.023). There was no difference in the frequency of etiologies of DAH between survivors and non-survivors.

The baseline and peak serum KL-6 levels in 41 patients are shown in Figure 1. The median duration before serum KL-6 level reached the peak level was 11±10 days. The median duration from the time of peak serum KL-6 level to death was 23±23 days. Baseline and peak serum KL-6 levels were significantly higher in non-survivors than in survivors (baseline levels, 845±831 vs 492±606 U/mL, p=0.01; peak levels, 1471±1648 vs 659±813 U/mL, p=0.004, respectively).

In our cohort, 34 patients were intubated, whereas the remaining 7 patients were treated without mechanical ventilation. Mean serum KL-6 levels increased during the follow-up in both groups (p=0.01; Ventilated patients, baseline 703±799 U/mL, peak 1,189±1,567 U/mL; Non-ventilated patients, baseline 881±694 U/mL, peak 1,330±995 U/mL; Figure 2). There was no difference in serum KL-6 levels according to the use of mechanical ventilation (p=0.72).

Serial changes in serum KL-6 in patients with DAH are shown in Figure 3. Patients who showed a decrease in P/F ratio (n=13, Figure 3A) or an increase in oxygenation index (n=16, Figure 3B) during the initial week showed a significant increase in serum KL-6 levels (p=0.002, p=0.003, respectively). In Figure 3A, two subjects appear to have extremely high levels of KL-6 one week after admission. When they were excluded to avoid statistical error, the increase in serum KL-6 levels during the initial week was still significant (p=0.003).

Receiver operating characteristic curve analysis was used to evaluate the sensitivity, specificity and accuracy of baseline and peak serum KL-6 levels in relation to non-survival (Figure 4). The larger area under the curve which is associated with non-survival was found for peak KL-6 with 0.78 (95% confidence interval (CI), 0.61 to 0.96) compared with baseline KL-6 with 0.74 (95% CI, 0.56 to 0.93). When the cut-off levels were set at the closest point to 100% sensitivity and 100% specificity, the levels in the prediction of non-survival were 700 U/mL for peak KL-6 (sensitivity, 75%; specificity, 85%; accuracy, 78%, respectively) and 240 U/mL for baseline KL-6 (sensitivity, 86%; specificity, 69%; accuracy, 81%, respectively).

The Kaplan-Meier analysis showed that higher baseline KL-6 levels were associated with a shorter median survival period (p=0.002) (Figure 5A). The same was true for higher peak KL-6 levels (p=0.0006) (Figure 5B).

In the univariate survival analysis, P/F ratios <200 48 hours after admission (Hazard ratio, 2.58, 95% CI, 1.17-5.68; p=0.018), baseline serum KL-6 levels ≥240 U/mL (Hazard ratio, 4.72, 95% CI, 1.63-13.7; p=0.004) and peak serum KL-6 levels ≥700 U/mL (Hazard ratio, 4.43, 95% CI, 1.76-11.1; p=0.002) were associated with non-survival, whereas no correlations were found between survival and age, sex, smoking status, radiological findings, baseline P/F ratio prothrombin time-international normalized ratio levels and activated partial thromboplastin time (Table 2). In the multivariate survival analysis only peak serum KL-6 levels ≥700 U/mL were associated with non-survival (Hazard ratio, 3.95, 95% CI, 1.14-13.7; p=0.031), after adjustment for age, sex, smoking status, extent of pulmonary involvement and P/F ratio 48 hrs after admission.

The experiments in this study comply with the current laws of Japan and Germany.