Autor : Rodríguez-Martín Isabel1 *, Galán-Paéz Juan2, Sánchez Margalet Víctor3
1 Clinical Biochemistry Laboratory. Hospital Universitario Virgen Macarena. Sevilla. Spain 2Universidad de Sevilla Escuela Técnica Superior de Ingeniería Informática. Universidad de Sevilla. Sevilla 3Laboratorio de Bioquímica Clínica. Hospital Universitario Virgen Macarena. Sevilla. España
Correspondencia :Isabel Rodríguez Martín - c/ Jauregui, Nª 4, 3.3 Sevilla. Spain - Telephone number: +34 646639397 - E-mail: isarodm@gmail.com
Abstract
Introduction: Diffuse interstitial lung diseases are a hard-to-diagnose heterogeneous
group of respiratory diseases. The study of bronchoalveolar lavage through flow
cytometry may define typical cell patterns in different diseases and so help
confirm the differential diagnosis. The purpose of this study was to
retrospectively analyze the clinical utility of cell and lymphocyte
subpopulations detected in the bronchoalveolar lavage by flow cytometry in
order to define typical cell patterns that allow for making a differential
diagnosis of granulomatous lung diseases.
Materials and methods: The retrospective study included 44 patients. The subjects were
diagnosed with sarcoidosis or hypersensitivity pneumonitis during a period of
3 years. We performed the cellular analysis of bronchoalveolar lavage through
flow cytometry and histological and imaging testing (HRCAT, High Resolution
Computed Axial Tomography) as part of the diagnosis. The percentages of T
cells, B cells, NK cells, CD4, CD8 and CD4/CD8 were analyzed by flow cytometry
for the following markers: CD3 +, CD19 + CD4 +, CD8 +, CD3 + CD4-CD8- and CD3 +
CD16-CD56-.
Results: We conclude that the most important parameters were lymphocytosis and
especially the CD4/CD8 quotient. This quotient was high for diseases such as
sarcoidosis and low for hypersensitivity pneumonitis, in comparison with the
values found in the peripheral blood.
Conclusions: The BAL (Bronchoalveolar Lavage) study is useful for differentiating
between granulomatous interstitial lung diseases and other DILDs (diffuse
interstitial lung diseases).
Key words: Interstitial lung diseases; Flow
cytometry; Bronchoalveolar lavage
Received: 08/06/2020
Accepted: 03/01/2021
Introduction
Diffuse interstitial lung
diseases (DILDs) are a very heterogeneous group of entities that mainly affect
the lung interstitium.
The diagnosis of such a
heterogeneous group of diseases frequently poses a challenge to the clinical
staff, for multiple reasons.
First, there are more than 150
causes of DILDs (but it is only possible to establish the etiologic diagnosis
in 40-50% of the cases). Many factors are involved in the pathogenesis of the
disease, both exogenous (metals, organic substances, wood, drugs, virus) and
endogenous (autoimmune diseases, gastroesophageal reflux). Secondly, for many
years there wasn’t uniformity in the classification of these diseases. Finally,
the clinical context in which these diseases are developed is frequently common
and non-specific; on many occasions the symptoms can’t be distinguished from
other neoplastic or autoimmune diseases1,
2.
The most frequent clinical
symptoms presented by patients with these diseases are dyspnea and cough that
worsens after exercise.
Extrapulmonary symptoms are also
common and sometimes help make a diagnosis. Clinical manifestations may be
present, at the neurological, ocular, dermatologic, digestive or cardiac level.
Sarcoidosis and hypersensitivity
pneumonitis (HN) are the most important of those diseases. Both diseases share
their granulomatous nature (60% of HPs are manifested by granulomas) and the existence
of common pulmonary and extrapulmonary manifestations, which extremely
complicate the etiologic diagnosis.
The diagnostic process begins
with a clinical evaluation (including the thorough knowledge of the medical
record, physical examination, patient’s biochemical and immunological analysis,
etc.), chest x-ray, pulmonary functional tests and image-based testing such as
HRCAT (High Resolution Computed Axial Tomography). If that weren’t enough, the
study could also be completed with other more invasive tests such as surgical
lung biopsy or the study of bronchoalveolar lavage (BAL).
Bronchoalveolar lavage is a
method that allows for the study of the lower airways through the analysis of
cellular and biochemical components.
This technique consists in the
instillation of a saline solution in boluses of about 20-50 ml (until getting
120-200 ml) at the lung segment level. After each instillation, the content is
aspirated, obtaining a first aliquot (bronchial sample) that is representative
of the cellularity of the airway.
It is a simple, safe (less than
3% complication rate), well-tolerated technique that provides a lot of clinical
information for the study of lung diseases.
However, the usefulness of this
technique is still controversial, since it appears to show high diagnostic
value for certain lung diseases (alveolar proteinosis, eosinophilic pneumonia,
histiocytosis X, alveolar infections or hemorrhage) but is just a guideline for
others (pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis,
pneumoconiosis or drug toxicity)3, 4.
Thus, we set forth the usefulness
and diagnostic value of bronchoalveolar lavage cytological study, which seems
to define certain cellular patterns typical of the disease that could be useful
for the etiologic diagnosis of diffuse interstitial lung diseases.
The purpose of this study was to
carry out a retrospective descriptive analysis of the cytological study and
lymphocyte subpopulations in the BAL performed in patients with sarcoidosis and
hypersensitivity pneumonitis in our hospital area.
We pretend to show that
bronchoalveolar lavage can present cellular patterns typical of the disease,
allowing us to differentiate the DILDs and greatly supporting the clinical
diagnosis.
Materials and methods
Retrospective, observational
study including all the patients diagnosed with sarcoidosis and hypersensitivity
pneumonitis who underwent a bronchoalveolar lavage as part of the diagnosis
between January 2015 and June 2018 at the H.U.V. Macarena de Sevilla.
All the patients underwent a
bronchoalveolar lavage unless otherwise contraindicated, in order to identify
and determine percentages and absolute lymphocyte counts as well as lymphocyte
subpopulations. The same parameters were measured in peripheral blood, for the
purpose of doing a comparative study as part of the diagnosis. Both samples,
the BAL and peripheral blood were obtained and processed at the same time.
The patients: The study includes all the patients diagnosed with sarcoidosis and
hypersensitivity pneumonitis (N = 44), from January 2015 to June 2018.
Bronchoalveolar lavage was performed in all the patients as part of the
diagnosis.
The final diagnosis was
established in two ways: a) through histological confirmation, and b)
clinical diagnosis without histological confirmation based on clinical and
analytical data, functional study, HRCAT, cellular and immune parameters in the
BAL and patient’s follow-up over time.
The final diagnosis was
established by the Pulmonology Service of our hospital center and included in
the medical record of each patient (with the Diraya system of the Andalusian
Health Service). Clinical data for this study (clinical diagnosis of each
patient) were taken from the Diraya, an electronic system of medical records,
and were directly compared with the data provided by the personnel of the
clinical biochemistry laboratory.
The study was approved by the
Research Ethics Committee of our center.
Bronchoalveolar lavage: Bronchoalveolar lavage was performed through a fibrobronchoscope (Olympus).
The instillation volume was 180 ml. At least 3 aliquots were recovered and sent
to different laboratories (general biochemistry, microbiology and pathologic
anatomy) to be studied fully.
Together with the collection of
the BAL, a peripheral blood sample was taken from each patient through
venipuncture.
Despite variability (smoking or
age, mostly), it is an accepted fact that the cellularity present in the BAL
fluid of normal subjects consists of: mainly macrophages (80-95%), lymphocytes
(< 15%) and neutrophils (2-5%). Eosinophils, basophils and plasma cells
represent the minority group (1-3%).
Processing of bronchoalveolar
lavage: The antiserum used for identifying and determining
percentages and absolute counts of lymphocytes was the Becton Dickinson BD
Multitest 6-color TBNK reagent.
The immunological study, done by
flow cytometry (BD FACSCanto II cytometer), included in both samples the
identification and count of T, B and NK lymphocytes, monocytes and
polymorphonuclear cells, as well as CD4+ and CD8+ lymphocyte T subpopulations
and the existing relationship between them (CD4+/CD8+ quotient).
Results
The study includes 44 patients:
32 patients with sarcoidosis and 12 patients who showed hypersensitivity
pneumonitis.
The mean age (± standard
deviation) of the sample was 60.80 ± 15.46 years.
As regards gender, 62% were male
(N = 27) and 38% were female (N = 17).
As for patients with sarcoidosis,
there were 22 females (70%) and 10 males (30%); and the mean age of this
population was 56.1 ± 11.07 years. Differences regarding age and gender were
demonstrated in comparison with the rest of the diffuse respiratory diseases:
in sarcoidosis, females predominated and the mean age was lower (56.1 ± 11.07).
In order to estimate the
diagnostic utility of bronchoalveolar lavage, we will now describe the results
obtained from each one of the respiratory diseases:
Firstly, in the study of patients
with sarcoidosis, our results concluded that sarcoidosis is presented as
lymphocyte alveolitis with a predominance of CD4+ lymphocytes: 60.94% (± 20.20)
lymphocytes, 8.94% (±19.00) monocytes and 30.12% (± 16.19) polymorphonuclear
cells. A clear increase in the lymphocyte population is observed compared to the
rest of the cell populations of the BAL.
On the other hand, with respect
to the quotient of CD4+/CD8+ lymphocyte subpopulations, we can say that there
is a clear increase of such quotient in the bronchoalveolar lavage in
comparison with its blood values. The mean CD4+/CD8+ quotient in blood was 1.09
(± 0.59), whereas in bronchoalveolar lavage it was 5.35 (± 3.75). Thus, we can
see that most patients (68.8% of patients) show a CD4+/ CD8+ quotient of more
than 3.5.
Secondly, in patients with hypersensitivity
pneumonitis, the BAL study showed an increase in lymphocytes, just like in the
previous case: 63.33% (±10.40) lymphocytes, 5.00 (± 0.00) monocytes, 31.67 (±
10.40) polymorphonuclear cells.
But the BAL quotient in these
patients had noticeably decreased in comparison with the values found in
peripheral blood: the mean CD4+/CD8+ quotient in blood was 2.07 (± 1.47),
whereas in the BAL it was 0.2 (± 0.119).
Discussion
The diagnostic utility of BAL has
been the object of numerous studies. Most studies agree on the diagnostic
value of BAL for certain diseases such as alveolar proteinosis, histiocytosis X
or alveolar hemorrhage. But we should also add the fact that there are many
studies still discussing its use for many other DILDs (sarcoidosis, pneumonias
associated with drugs or connective tissue diseases), where it seems to have a
mostly illustrative and not diagnostic value. For that reason, in most
respiratory disorders the study of BAL doesn’t seem enough for making the final
diagnosis.
With respect to our work, we
agree with the literature that was consulted about the diagnostic value of BAL
for certain DILDs, including sarcoidosis and hypersensitivity pneumonitis,
where BAL shows a high value5-7.
Sarcoidosis is a multisystemic
disease of unknown origin, characterized by the presence of non-caseating
granulomas. Due to this multisystemic characteristic, the diagnostic approach
may be complex (there isn’t an individual diagnostic test for sarcoidosis).
Despite the fact that sarcoidosis
may affect multiple organs, the lung is involved in most cases (80- 95% of
cases). Lung symptoms include dyspnea, cough, distress and wheezing. Other
frequently affected organs include: eyes, skin and lymph nodes8.
Sarcoidosis predominantly affects
young adults. Most of them are 20-40 years old and it is clearly most common in
women than in men (ratio 3:1). This fact was confirmed in our patients, and we
concluded there is a higher incidence in young women.
Even though its etiology is
unknown, we suggest a cause of genetic origin together with certain
environmental factors. As regards the genetic factors, the individuals more
susceptible to the disease are those with HLA DR11, 12, 14,15 and 17; on the
other hand, the studies seem to conclude that having HLA DR1 and DR2 provides
a protection capacity. As for the exogenous factors, it is important to
mention: microbial agents (mycobacteria), organic agents (pine pollen),
inorganic agents (beryllium, aluminum) or drugs (methotrexate).
So, the characteristic granulomas
that are formed are caused by a persistent immune response to antigens acting
in a continuous way, capable of inducing an exaggerated response in genetically
predisposed individuals.
As a consequence of this immune
response, patients diagnosed with sarcoidosis showed lymphocyte alveolitis with
noticeable increase in the BAL CD4+/CD8+ quotient. Thus, 68.8% of patients
showed a CD4+/CD8+ quotient of more than 3.5. An increase in said quotient is
very specific of sarcoidosis (whereas an increase in lymphocytes is more
sensitive). So, according to the consulted literature, those patients with a
CD4+/CD8+ quotient > 4 have a high probability of developing sarcoidosis
(predictive value of 94%), whereas CD4+/CD8+ quotients < 1 allow for the
exclusion of the disease9-11.
This fact seems to be related to
the nature of the disease itself, characterized by the presence of
non-caseating granulomas which consist mainly of macrophages and CD4+
lymphocytes that appear as a consequence of an exaggerated immune response.
Also, hypersensitivity
pneumonitis is a diffuse interstitial disease normally characterized by the
presence of granulomas (60% of the cases) caused by the chronic inhalation of a
wide variety of organic products. In this group we include a lot of substances:
soy, hay, wood, coffee grains, sugar cane, cured meats. In almost every case
its origin is occupational. The most frequent of these diseases are: the
“farmer’s lung disease” (moldy hay, Ag. T. vulgaris) and the “bird fancier’ s
lung disease” (serum, bird proteins and droppings, Ag. T. vulgaris).
The pathogenesis is based on an
immune response to these inhaled particles giving place to type III
hypersensitivity reactions (mediated by immune complexes and CD8+ lymphocytes)
and type IV hypersensitivity reactions (mostly mediated by CD8+ lymphocytes
responsible for the formation of granulomas)12.
In patients with hypersensitivity
pneumonitis, the immune study through BAL cytometry yielded a high value. HP has
a complex diagnosis, due to its granulomatous nature and extrapulmonary
clinical manifestation (cough, dyspnea or weight loss); it can be clinically
similar to other diseases, such as sarcoidosis.
All the patients with HP show
alterations in the BAL in such a way that an unaltered BAL excludes the HP
diagnosis. It is the most sensitive method. Lymphocytosis is predominant, with
CD8+ lymphocytes that appear as a consequence of type III and type IV
hypersensitivity reactions to inhaled particles, thus, the quotients in these
patients are lower than one. An increase in CD4+ lymphocytes suggest a
bad prognosis, given that an increase in CD4+ (as well as neutrophils) has been
related to a higher possibility of suffering from fibrosis13.
As can be observed, these
diseases can show common clinical symptoms that greatly complicate their
diagnosis; however, there are clear differences regarding the cellular
populations present in the bronchoalveolar lavage. Thus, under certain
circumstances where it is difficult to distinguish between HP and sarcoidosis,
the cytological study of BAL has allowed us to guide the diagnosis: CD4/CD8
quotients are reduced in BAL for HP, and those quotients increase considerably
for sarcoidosis.
Conclusions
Diffuse interstitial lung
diseases (DILDs) entail in most cases a diagnostic challenge for the clinician.
But, in the adequate clinical and
diagnostic context, the analysis of bronchoalveolar lavage by flow cytometry is
useful to study lung diseases, since it shows high diagnostic value for certain
diseases such as sarcoidosis and hypersensitivity pneumonitis.
Likewise, after the review that
was conducted, we believe more studies about bronchoalveolar lavage are
necessary to discover new BAL markers of any nature (immune, biochemical,
cellular), more specific, that contribute to a better understanding of diffuse
interstitial lung diseases.
Conflict of interests: The authors declare that there is no conflict of interests.
Funding source: None.
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