Impact of lymphocyte differential count > 15% in BALF on the mortality of patients with acute exacerbation of chronic fibrosing idiopathic interstitial pneumonia
© The Author(s). 2017
Received: 27 September 2016
Accepted: 13 April 2017
Published: 20 April 2017
Chronic fibrosing idiopathic interstitial pneumonia (CFIIP) has a potential risk of acute exacerbation (AE). However, the usefulness of cellular analysis of bronchoalveolar lavage fluid (BALF) has never been evaluated. This study aimed to evaluate the impact of the lymphocyte differential count > 15% in BALF on the mortality of patients with AE of CFIIP.
We retrospectively analysed 37 patients with AE of CFIIP who underwent BAL on admission. Patients were divided into two groups: one group consisting of those with a lymphocyte differential count > 15% and the other consisting of those with a lymphocyte differential count ≤ 15%. We compared the 90-day mortality between the two groups as the primary outcome, using the two-tailed log-rank test.
The median follow-up duration was 6.9 months. Twenty-four patients had a lymphocyte differential count > 15%. The 90-day mortality was significantly higher in the group with a lymphocyte differential count ≤ 15% than in the group with a lymphocyte differential count > 15% (long rank test, p = 0.003). In the multivariate analysis a lymphocyte differential count > 15% was shown to be an independent favourable prognostic factor for 90-day mortality (HR: 0.125; 95% CI: 0.0247–0.589; p = 0.009).
A lymphocyte differential count > 15% in BALF may be associated with favourable outcomes in patients with AE of CFIIP.
KeywordsInterstitial Lung Disease Acute exacerbation Lymphocyte differential count in BALF
Acute exacerbation (AE) in patients with idiopathic pulmonary fibrosis (IPF), characterised by clinically progressive respiratory failure and worsening pulmonary fibrosis, was first reported in 1993 and has been recognised as a relatively common and highly critical event in the clinical course of IPF [1, 2]. AE has also been described in other cases of fibrosing interstitial pneumonia [3–6], suggesting the potential risk of AE in chronic fibrosing idiopathic interstitial pneumonia (CFIIP).
The diagnostic consensus criteria for AE were suggested by Collard et al. in 2007  and include a lack of evidence of pulmonary infection by endotracheal aspirate or bronchoalveolar lavage (BAL). However, there have been no studies of the specificity of the cellular analysis of BAL fluid (BALF) and appropriate treatment for AE has not been established, and so the mortality rate of AE remains high .
Although corticosteroids were weakly recommended in the American Thoracic Society/European Respiratory Society/Japanese Respiratory Society/Latin American Thoracic Association (ATS/ERS/JRS/ALAT) guidelines for patients with AE of IPF , no controlled trials on the effectiveness of corticosteroids have been conducted. According to the ATS/ERS guidelines, BAL cell differential counts > 15% lymphocytes represent a lymphocytic cellular pattern such as cryptogenic organizing pneumonia , which has been shown to have a good recovery with corticosteroid treatment . So patients with BAL cell differential counts > 15% lymphocytes may be good responders to corticosteroids. However, the effect of corticosteroids and other immunosuppressants in AE of CFIIP with > 15% lymphocytes in BALF has never been evaluated.
In this study, we retrospectively analysed patients with AE of CFIIP who underwent bronchoscopy with BAL in order to evaluate the impact of the lymphocyte differential count > 15% in BALF on outcomes.
Patients with AE of CFIIP who were admitted to our department and underwent BAL examination between August 2006 and February 2015 were retrospectively studied. This study protocol was approved by the ethics committee of Kurashiki Central Hospital in accordance with the Declaration of Helsinki (approved number 1972). The categorization of major idiopathic interstitial pneumonias in ATS/ERS guidelines was used to define CFIIP .
Criteria for AE
AE was defined using the following features based on the defined criteria : (1) previous or concurrent diagnosis of CFIIP, (2) acute worsening or development of dyspnea within 1 month duration, (3) computed tomography with new bilateral ground-glass opacity and/or consolidation superimposed on a background pattern, (4) deterioration not fully explained by cardiac failure, fluid overload, or an identifiable cause of acute lung injury. A significant decline in oxygenation was confirmed by previous PaO2 or SpO2 in every patient. Drugs such as anticancer agents, herbal medicines, health foods, and other suspicious drugs were considered as the cause of acute lung injury when they were used within 30 days before admission, and these patients were excluded.
BAL was performed with a fiber-optic bronchoscope in a wedge position within the selected bronchopulmonary segment [10, 12]. Sterile saline (0.9% NaCl) at room temperature was instilled through the bronchoscope. The total instilled volume was 100 or 150 ml and was divided into two or three 50-ml aliquots. After instillation of each aliquot, the instilled sterile saline was retrieved using negative suction pressure adjusted to avoid visible airway collapse. The recovered BALF was separated from the cellular components by centrifugation (1 min, 1300 rpm). Preparations of cell suspensions were made in a cytocentrifuge (Shandon, Woburn, MASS). Cytospin slides of BALF cells were stained with May-Grünwald-Giemsa for cell differentiation.
Clinical characteristics and laboratory findings
The patient’s clinical characteristics were retrieved from the available clinical record. A respiratory function test was performed within 1 year before AE. Two pulmonologists, who had no knowledge of any prior clinicopathological reports relevant to these cases, reviewed the computed tomography and classified the patients into two groups according to the ATS/ERS criteria as those with usual interstitial pneumonia (UIP) and those with not UIP .
We divided the patients with AE of CFIIP into two groups; one group had a lymphocyte differential count > 15%, and the other group had a lymphocyte differential count ≤ 15%. We set 90-day mortality as the primary outcome and overall survival (OS) as the secondary outcomes. The 90-day mortality was measured at 90 days after the BAL procedures. The OS was also measured from the date of the BAL procedure until the date of death from any cause or until the date on which the patient was last known to be alive. The cause of death was regarded as AE when the patient died within 90 days from the date of performing BAL. We estimated the 90-day mortality and OS using Kaplan-Meier analysis . Differences between survival curves were tested for statistical significance using the two-tailed log-rank test. Univariate and multivariate prognostic analyses were performed for 90-day mortality using logistic regression model. For multivariate analysis, a stepwise backward procedure to derive a final model of the variables that had a significant independent relationship with survival was employed. All variables with a P value of less than 0.10 in the univariate analyses were entered into the multivariate analysis. Categorical variables were compared using the Fisher test, and continuous variables were compared using the t-test or the Mann-Whitney U test. All statistical analyses were performed using R (The R Foundation for Statistical Computing V.3.0.3). Statistical significance was defined as a p value of <0.05. Data were expressed as the median (interquartile range) or mean ± standard deviation.
Clinical characteristics of patients
Lymphocyte differential count in BALF
>15% (n = 24)
≤15% (n = 13)
Smoking habit (current/former/never/unknown)
25 ± 8
23 ± 7
7.1 ± 5.5
8.0 ± 5.2
3.2 ± 0.5
3.0 ± 0.4
323 ± 93
270 ± 63
1.09 ± 1.48
0.80 ± 0.22
1543 ± 922
1163 ± 874
387 ± 363
215 ± 140
96.4 ± 45.6
84.5 ± 39.0
7.44 ± 0.03
7.43 ± 0.07
261 ± 73
210 ± 89
8.7 ± 23.2
4.3 ± 6.9
CT pattern (UIP/not UIP)
possible UIP pattern
Inconsistent with UIP pattern
FVC, % predicted
80.1 ± 15.4
72.0 ± 19.8
FEV1, % predicted
81.7 ± 17.4
82.1 ± 18.5
DLco, % predicted
53.9 ± 23.6
54.5 ± 23.8
total volume of retrieved BALF, %
Total cell count, /ml
5.0 × 105 (4.0 × 105–5.3 × 105)
3.0 × 105 (2.0 × 105–4.0 × 105)
Days from admission until BAL, day
Treatment and outcome
Treatment and outcome of patients
Lymphocyte differential count in BALF
>15% (n = 24)
≤15% (n = 13)
Treatment for CFIIP before admission
corticosteroid + CyA
Treatment for AE of CFIIP
corticosteroid + CyA
corticosteroid + CY
corticosteroid + CyA + CY
Overall survival, months
Death during observation period
Of the nine patients who were mechanically ventilated, five died without withdrawal from the respirator. A total of 22 patients died during the observation period and 12 patients died from AE. Autopsies were performed in two patients who died during their hospitalization. Histopathological examination from these two patients showed the exudative phase of diffuse alveolar damage (DAD) in one patient and the exudative to fibrotic phase of DAD in the other patient.
In the two patients who underwent autopsy, the patient (case 1) with the exudative phase of DAD showed 6.0% lymphocytes and 67% neutrophils in the BALF, and the patient with the exudative to fibrotic phase of DAD (case 2) showed 36% lymphocytes and 17% neutrophils in the BALF. In case 1, the patient received corticosteroid pulse therapy and was mechanically ventilated, but died on the fifth day. In case 2, the patient received corticosteroid pulse and cyclosporine A therapy and was also mechanically ventilated; this patient died on the 40th day.
Evaluation of the impact of the lymphocyte differential count in BALF on mortality
Univariate and multivariate analyses of factors associated with 90-day mortality
Prognostic impacts on the 90-day mortality
BAL >15% lymphocytes
FVC, % predicted
DLco, % predicted
In this study, we demonstrated that the lymphocyte differential count > 15% in BALF was associated with favourable outcomes in patients with AE of CFIIP. To the best our knowledge, this is the first study to demonstrate the impact of the lymphocyte differential count > 15% in BALF on the 90-day mortality and OS in patients with AE of CFIIP.
The pathogenesis of AE of CFIIP remains unclear, but the pathological findings of AE of CFIIP were reported as DAD or organizing pneumonia (OP), without other evidence of the organizing phase of DAD [1, 4, 7, 11, 15]. The ATS guidelines indicate that the presence of > 15% lymphocytes in BALF represents a lymphocytic cellular pattern such as OP, nonspecific interstitial pneumonia, etc. . Some of these conditions may respond to corticosteroids. In this study, corticosteroids and other immunosuppressants were more effective in the group with a lymphocyte differential count > 15% than in the group with a lymphocyte differential count ≤ 15%. These results suggest that the 15% lymphocytes in BALF in AE of CFIIP patients is a helpful factor for identifying the therapeutic response and prognosis in these patients. BAL cell differential counts with > 3% neutrophils represent a neutrophilic cellular pattern such as DAD  and, in previous reports, patients with DAD were more resistant to corticosteroid and other immunosuppressants and showed a worse prognosis than patients with OP [1, 4, 16, 17]. We analysed association the degree of neutrophil differential count in BALF and OS. The area under the receiver operating characteristic curve for the neutrophil differential count in BALF for predicting the OS in AE of CFIIP was 0.78 [95% CI: 0.59–0.92] (Additional file 1: Figure S1). The univariate analysis identified neutrophil differential count in BALF was associated with OS (HR 1.02, 95% CI 1.01–1.04, p < 0.01). In this study, however, 35 patients (94.6%) showed > 3% neutrophils in BALF and a > 3% neutrophil differential count in BALF was not useful in predicting prognosis.
Cause of death after AE of CFIIP
Lymphocyte differential count in BALF
>15% (n = 20)
≤15% (n = 5)
Reccurence of AE
In the present study, we analysed the prognosis of AE in all CFIIP patients including both UIP and not UIP patients. Usui et al. reported that the computed tomography classification (UIP pattern and not UIP pattern) had no significant effect on survival in AE patients , and we considered it valid to place all patients in the CFIIP category. However, although many reports have described the prognosis of AE in IPF patients, few reports have thoroughly investigated the prognosis of AE in not UIP patients [1–3, 7]. Therefore, we classified all patients into the two groups (UIP and not UIP) and analysed the 90-day mortality and OS. Using a cut point of 15% lymphocytes in BALF appeared to be a helpful factor in identifying patients with AE-CFIP who may respond to immunosuppressive therapy, and this appeared to be the case for patients with or without apparent UIP. These findings suggest that >15% lymphocytes in the BALF differential cell count may be a stronger prognostic indicator than whether the underlying histopathologic lesion is UIP or a non-UIP form of CFIIP.
In this study, most of the patients underwent BAL on the first day and received treatment at an early phase. The average total volume of retrieved BALF was 43.3% of the total instilled volume, and this amount was adequate to estimate the BALF cell differential counts . BAL is a safe and well-tolerated procedure , and no patient in this study had any serious complications, such as severe worsening of AE. Video-assisted thoracoscopic surgery was too risky to perform during AE. BAL was not only helpful distinguishing AE from infection , but it was also useful and safe for evaluating the impact of the lymphocyte differential count in BALF on the mortality of patients with AE of CFIIP.
There are several limitations to the present study. The first is its retrospective design. The second is the fact that it included a relatively small number of patients with AE of CFIIP. The third is that the diagnosis of CFIIP and the classifications were somewhat dependent on the HRCT scanning. Especially, the diagnosis of nonspecific interstitial pneumonia was not adequately accurate because most of the patients were difficult to perform surgical lung biopsies. So, we classified CFIIP into UIP and not UIP and showed “UIP/not UIP” in Table 1. Needless to say, we excluded, in reference to the 2013 ATS guideline, known entities associated with the development of pulmonary fibrosis such as collagen vascular disease and chronic hypersensitivity pneumonitis. The fourth is that we have not compared between BAL in stable CFIIP and in AE of CFIIP. BAL differential counts in stable CFIIP may influence that in AE of CFIIP. However, BAL was performed at the new abnormal infiltration and may represent the acute phase of AE of CFIIP. Finally, because some patients were treated with corticosteroids or other immunosuppressants for CFIIP before AE, the BALF cell differential counts may have been affected. Prospective studies with larger patient cohorts are required to overcome these limitations.
In conclusion, our findings indicate that the lymphocyte differential count > 15% in BALF may be associated with good outcomes in patients with AE of CFIIP. Further studies are needed to confirm our findings, to elucidate the mechanism of AE of CFIIP, and to develop the optimal treatment strategies for AE of CFIIP.
- 95% CI:
95% confidence interval
American Thoracic Society/European Respiratory Society/Japanese Respiratory Society/Latin American Thoracic Association
Bronchoalveolar lavage fluid
Chronic fibrosing idiopathic interstitial pneumonia
Diffuse alveolar damage
Idiopathic pulmonary fibrosis
Usual interstitial pneumonia
Availability of data and materials
The database used for the study can be available from the corresponding author under demand if needed.
RT, MA, SK, YI, MN, FT and TI designed this study and were involved in the acquisition of the data. RT, MA and SK analysed the data. RT wrote the manuscript. All authors approved the final version of the manuscript.
Tadashi Ishida has received honoraria from Pfizer Japan Inc. The other authors have no conflict of interest.
Consent for publication
Ethics approval and consent to participate
This study protocol was approved by the ethics committee of Kurashiki Central Hospital in accordance with the Declaration of Helsinki (approved number 1972). Due to retrospective nature of this study, informed consent was waived.
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- Kondoh Y, Taniguchi H, Kawabata Y, Yokoi T, Suzuki K, Takagi K. Acute exacerbation in idiopathic pulmonary fibrosis. Analysis of clinical and pathologic findings in three cases. Chest. 1993;103:1808–12.View ArticlePubMedGoogle Scholar
- Kim DS, Park JH, Park BK, Lee JS, Nicholson AG, Colby T. Acute exacerbation of idiopathic pulmonary fibrosis: frequency and clinical features. Eur Respir J. 2006;27:143–50.View ArticlePubMedGoogle Scholar
- Park IN, Kim DS, Shim TS, Lim CM, Lee SD, Koh Y, Kim WS, Kim WD, Jang SJ, Colby TV. Acute exacerbation of interstitial pneumonia other than idiopathic pulmonary fibrosis. Chest. 2007;132:214–20.View ArticlePubMedGoogle Scholar
- Churg A, Muller NL, Silvia CIS, Wright JL. Acute exacerbation (acute lung injury of unknown cause) in UIP and other forms of fibrotic interstitial pneumonias. Am J Surg Pathol. 2007;31:277–84.View ArticlePubMedGoogle Scholar
- Suda T, Kaida Y, Nakamura Y, Enomoto N, Fujisawa T, Imokawa S, Hashizume H, Naito T, Hashimoto D, Takehara Y, Inui N, Nakamura H, Colby TV, Chida K. Acute exacerbation of interstitial pneumonia associated with collagen vascular diseases. Respir Med. 2009;103:846–53.View ArticlePubMedGoogle Scholar
- Miyazaki Y, Tateishi T, Akashi T, Ohtani Y, Inase N, Yoshizawa Y. Clinical predictors and histologic appearance of acute exacerbations in chronic hypersensitivity pneumonitis. Chest. 2008;134:1265–70.View ArticlePubMedGoogle Scholar
- Collard HR, Ryerson CJ, Corte TJ, Jenkins G, Kondoh Y, Lederer DJ, Lee JS, Maher TM, Wells AU, Antoniou KM, Behr J, Brown KK, Cottin V, Flaherty KR, Fukuoka J, Hansell DM, Johkoh T, Kaminski N, Kim DS, Kolb M, Lynch DA, Myers JL, Raghu G, Richeldi L, Taniguchi H, Martinez FJ. Acute exacerbations of idiopathic pulmonary fibrosis. An international working group report. Am J Respir Crit Care Med. 2016;194:265–75.View ArticlePubMedGoogle Scholar
- Ley B, Collard HR, King TE. Clinical course and prediction of survival in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2011;183:431–40.View ArticlePubMedGoogle Scholar
- Raghe G, Rochwerg B, Zhang Y, Garcia CA, Azuma A, Behr J, Brozek JL, Collard HR, Cunningham W, Homma S, Johkoh T, Martinez FJ, Myers J, Protzko SL, Richeldi L, Rind D, Selman M, Theodore A, Wells AU, Hoogsteden H, Schünemann HJ. American Thoracic Society; European Respiratory society; Japanese Respiratory Society; Latin American Thoracic Association. An official ATS/ERS/JRS/ALAT clinical practice guideline: treatment of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2015;192:e3–19.View ArticleGoogle Scholar
- Meyer KC, Raghu G, Baughman RP, Brown KK, Costabel U, du Bois RM, Drent M, Haslam PL, Kim DS, Nagai S, Rottoli P, Saltini C, Selman M, Strange C, Wood B. American Thoracic Society Committee on BAL in Interstitial Lung Disease. An official American Thoracic Society clinical practice guideline: the clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease. Am J Respir Crit Care Med. 2012;185:1004–14.View ArticlePubMedGoogle Scholar
- Travis WD, Costabel U, Hansell DM, King Jr TE, Lynch DA, Nicholson AG, Ryerson CJ, Ryu JH, Selman M, Wells AU, Behr J, Bouros D, Brown KK, Colby TV, Collard HR, Cordeiro CR, Cottin V, Crestani B, Drent M, Dudden RF, Egan J, Flaherty K, Hogaboam C, Inoue Y, Johkoh T, Kim DS, Kitaichi M, Loyd J, Martinez FJ, Myers J, Protzko S, Raghu G, Richeldi L, Sverzellati N, Swigris J, Valeyre D. ATS/ERS Committee on Idiopathic Interstitial Pneumonias. An official American Thoracic Society/European Respiratory Society statement: update of the international multidisciplinary classification of the idiopathic interstitial pneumonias. Am J Respir Crit Care Med. 2013;188:733–48.View ArticlePubMedGoogle Scholar
- Drent M, Mulder PG, Wagenaar SS, Hoogsteden HC, van Velzen-Blad H, van den Bosch J. Differences in BAL fluid variables in interstitial lung diseases evaluated by discriminant analysis. Eur Respir J. 1993;6:803–10.PubMedGoogle Scholar
- Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown KK, Colby TV, Cordier JF, Flaherty KR, Lasky JA, Lynch DA, Ryu JH, Swigris JJ, Wells AU, Ancochea J, Bouros D, Carvalho C, Costabel U, Ebina M, Hansell DM, Johkoh T, Kim DS, King Jr TE, Kondoh Y, Myers J, Müller NL, Nicholson AG, Richeldi L, Selman M, Dudden RF, Griss BS, Protzko SL, Schünemann HJ. ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary Fibrosis. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med. 2011;183:788–24.View ArticlePubMedGoogle Scholar
- Kaplan E, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc. 1958;53:457–81.View ArticleGoogle Scholar
- Mallick S. Outcome of patients with idiopathic pulmonary fibrosis (IPF) ventilated in intensive care unit. Respir Med. 2008;102:1355–9.View ArticlePubMedGoogle Scholar
- Homma S, Sakamoto S, Kawabata M, Kishi K, Tsuboi E, Motoi N, Yoshimura K. Cyclosporin treatment in steroid-resistant and acutely exacerbated interstitial pneumonia. Intern Med. 2005;44:1144–50.View ArticlePubMedGoogle Scholar
- Akira M, Hamada H, Sakatani M, Kobayashi C, Nishioka M, Yamamoto S. CT findings during phase of accelerated deterioration in patients with idiopathic pulmonary fibrosis. Am J Roentgenol. 1997;168:79–83.View ArticleGoogle Scholar
- Maher TM, Whyte MK, B Hoyles RK, Parfrey H, Ochiai Y, Mathieson N, Turnbull A, Williamson N, Bennett BM. Development of a consensus statement for the definition, diagnosis, and treatment of acute exacerbations of idiopathic pulmonary fibrosis using the delphi technique. Adv Ther. 2015;32:929–43.View ArticlePubMedPubMed CentralGoogle Scholar
- Kataoka K, Taniguchi H, Kondoh Y, Nishiyama O, Kimura T, Matsuda T, Yokoyama T, Sakamoto K, Ando M. Recombinant human thrombomodulin in acute exacerbation of idiopathic pulmonary fibrosis. Chest. 2015;148:436–43.View ArticlePubMedGoogle Scholar
- Iwata T, Yoshida S, Nagato K, Nakajima T, Suzuki H, Tagawa T, Mizobuchi T, Ota S, Nakatani Y, Yoshino I. Experience with perioperative pirfenidone for lung cancer surgery in patients with idiopathic pulmonary fibrosis. Surg Today. 2015;45:1263–70.View ArticlePubMedGoogle Scholar
- Abe S, Azuma A, Mukae H, Ogura T, Taniguchi H, Bando M, Sugiyama Y. Polymyxin B-immobilized fiber column (PMX) treatment for idiopathic pulmonary fibrosis with acute exacerbation: a multicenter retrospective analysis. Intern Med. 2012;51:1487–91.View ArticlePubMedGoogle Scholar
- Usui Y, Kaga A, Sakai F, Shiono A, Komiyama K, Hagiwara K, Kanazawa M. A cohort study of mortality predictors in patients with AE of CFIP. BMJ Open. 2013;3:e002971.View ArticlePubMedPubMed CentralGoogle Scholar
- Drent M, Jacobs JA, Wagenaar SS. Bronchoalveolar lavage. Eur Respir J. 2000;2000:1–8.Google Scholar