Differential cell analysis in ovine bronchoalveolar lavage fluid: Effect of different sampling methods and seasons

Abstract

Background:

Bronchoalveolar lavage (BAL) is a method for collecting the cellular and fluid components of the airway surface in the lungs. The assessment of differential cell profiles is potentially valuable in the diagnosis of pulmonary diseases, but there is no information about the normal BAL profiles in the Gezel breed.

Aim:

This study aimed to characterize the normal cryptologic findings of BAL with Gezel sheep.

Methods:

Twenty healthy sheep (15 females: 5 males, bodyweight: 55–65 kg) were sedated with xylazine (0.02–0.04 mg/kg IV). Two methods; the transtracheal bronchoalveolar lavage technique and the bronchoscopic bronchoalveolar lavage using a scope were evaluated. Sampling was performed in the summer and winter seasons.

Results:

Normal value (Mean ± SEM) for total cell, macrophage, lymphocyte, neutrophil, epithelial, and bronchial cells in BAL sampled in summer were (343.75 ± 30.23), ) 24.50 ± 2.62), (2.81 ± 0.51), (1.43 ± 0.88), and (3.12 ± 0.32), respectively. The normal values for the total cell, macrophage, lymphocyte, neutrophil, epithelial, and bronchial cells in BAL sampled in winter were (355.55 ± 37.67), (59.11 ± 4.30), (21.33 ± 3.10), (3.88 ± 1.07), (8.88 ± 3.78), and (6.33 ± 1.44), respectively.

Conclusion:

No significant change in the percentage of neutrophils was detected between seasons, although the percentages of bronchial and epithelial cells in winter were significantly high (p < 0.05). Except for the mentioned cases, neither the total cell number nor the percentage differential cell populations of BAL changed significantly (p < 0.05) in different sampling methods and seasons. Normal BAL profiles in the Gezel breed were determined and could be used in result interpretations. Also, both sampling methods can be used without significantly affecting the results.

Introduction

Bronchoalveolar lavage (BAL) is a diagnostic, useful, and safe method for sampling the cellular elements of the lungs, which is used to accurately diagnose various infections and a wide range of inflammatory mechanisms involving the respiratory airways (Klech, 1992). BAL analysis provides valuable information in the diagnosis and management of lower respiratory tract disease in various domestic animals including horses, cattle, sheep, and goats (Couëtil et al., 2007; Rola-Pleszczynski et al., 2007).

Respiratory diseases are one of the most common causes of poor weight gain and mortality that occur in sheep individually and mainly in outbreaks in the herds and lead to considerable financial losses (Scott, 2011). The incidence of respiratory diseases in small ruminants depends on many factors; therefore, there are several methods for identifying respiratory diseases (Lacasta, 2008).

An increase in the prevalence of respiratory diseases in sheep and goats often results from adverse climatic conditions and physiologic stress combined with infectious agents including viruses and bacteria (Scott, 2011). Ruminant barns are overcrowded, dark, and damp in the winter months and hot and poorly ventilated during the winter months. It is proposed these weather conditions in different seasons could act as a stress factor and affect lung defense mechanisms consequently predisposing animals to respiratory tract diseases. For these reasons, it is important to identify and introduce normal amounts of BAL fluid parameters in different seasons.

The aim of this study was to determine the normal cytological values of BAL fluid in healthy Ghezel sheep and to declare possible changes in BAL fluid cytology between two sampling methods, bronchoscopic bronchoalveolar lavage (BBAL) and transtracheal bronchoalveolar lavage (TBAL).

Materials and Methods

This study is designed and performed in the veterinary teaching and educational hospital of the Faculty of Veterinary Medicine of Urmia University. Twenty healthy Ghezel sheep were 2–3 years old and weighed in the range of 55–65 kg of both males and females were used. Animals were fed twice a day with an alfalfa-based ration fed as TMR with ground corn and vitamins premix and had free access to the water. BAL samples were taken once in two different seasons, summer as the hot season and winter as the cold season. Clinical examination (vital signs) and laboratory (differential count of blood cells on a venous blood sample) were conducted before sampling to confirm healthy status.

The animal was selected as a healthy sheep based on the following criteria:

The absence of clinical signs of respiratory disorders and total white blood cell count in the peripheral blood sample was in the normal range accepted for sheep (Burrells, 1985).

Grouping the animals:

Group (1): Sheep that were prepared for TBAL in the summer.

Group (2): Sheep that were sampled by bronchoscopy BBAL in summer.

Group (3): Sheep that were prepared by bronchoscopy BBAL in winter.

Method of TBAL

To perform this method, sheep were pretreated with xylazine (0.05 mg/kg IM) as a sedative, and the animal was restrained in a standing position with its neck stretched. An area over the trachea was shaved approximately 5 cm away and below the larynx and extended slightly (5–7 cm around the proposed entry site). The site was first disinfected with 4% chlorhexidine and then with 70% alcohol, then anesthetized with lidocaine hydrochloride using a 21-gauge needle. An intravenous catheter 12 G was inserted between the two tracheal rings and a 570 mm polyethylene catheter was inserted through the catheter into the trachea and pushed forward until it resists (Sheehan, et al., 2005). At this stage, the animals usually coughed, then 50 ml of 0.9% sterile isotonic saline at 37°C was injected continuously through a 60 ml syringe; based on available references, this amount is well tolerated by sheep. The liquid will be removed after 5 seconds by suction using a syringe. In this method, an average of 10 ml of liquid was recovered.

Method of bronchial-alveolar lavage sampling with bronchoscopy (BBAL)

In this procedure, deep sedation with intravenous xylazine (0.2 mg/kg) was applied to sheep. A flexible bronchoscope (Vet Video Endoscope PV-G 34-325; KARL STORZ Germany GMBH) is inserted through the nostril and conducted to the bifurcation of the main bronchi. At this stage, the animals usually cough due to the presence of a bronchoscope inside the trachea. 50 ml of 0.9% sterile isotonic saline at 37°C was injected continuously through the suction port of the bronchoscope. This amount was well tolerated by the sheep in the present study. The lavage process was completed after 5 seconds using the suction of installed fluid (Collie, et al., 1999). On average, more than 30 ml of liquid is recycled by this method. Bronchial-alveolar lavage was performed only once to ensure the welfare of the animals, although not enough fluid was collected (Katsoulos, et al., 2009). The collected fluid was placed on ice and then sent to the Laboratory for further evaluation.

Laboratory evaluation of BAL sample

All samples were centrifuged at room temperature at 800 g for 10 minutes. The supernatant was removed and the remaining sediments were suspended in 2 ml of 0.9% normal saline solution and washed again. Total cell count and differential count were performed by two methods; automatically obtained by Celltacα Hematology Analyzer (NIHON KOHDEN CORPORATION, Tokyo, Japan) and manually with Neubauer hemocytometer and values expressed as cell per milliliter BALF. The prepared slides were dried and then fixed in methanol and finally stained with Giemsa stain. The percentage of macrophages, mast cells, and leukocytes, including lymphocytes, neutrophils, and eosinophils, was calculated by counting 200 cells per sample. Samples with an identifiable cell count of fewer than 200 cells were considered undetectable and excluded from the study. Smear counting analysis was performed by two persons separately and manually.

Statistical analysis

SPSS software was used to analyze the statistical parameters of the data. The normality of the distribution and the homogeneity of the variances were checked and a one-way analysis of variance parametric tests was used to compare the data. p < 0.05 was considered as the level of statistical significance differences.

Ethical approval

Ethical approval for the study on sheep was certified by the Animal Research Ethics Committee of Urmia University NO: IR-UU-ACU 320.

Results

To ensure the health of the animals, venous blood samples were taken. Blood parameters were in the normal range in both sampling seasons (Table 1). There was no significant difference in blood cell indices between the groups. Total cell count (per microliter), and percentage of macrophage, lymphocyte, neutrophil, epithelial cell, and bronchial cell of BAL samples by two different sampling methods in summer (groups 1–2) and winter (group 3) are displayed in the Table 2.

Table 1. Hematological parameters of animals in the first and second groups (in the summer season) and group 3 (in the winter season).

Parameter	Unit	Group (1 and 2)		Group (3)
		Statistic		Statistic
		Mean	Std. error	Mean	Std. error
Total cells	103/ Ml	8.87	0.68	6.68	0.81
Nutrophils	%	36.95	4.81	50.88	3.92
Lymphocytes	%	58.71	5.50	41.81	4.52
Eosinophils	%	2.50	0.48	5.06	1.44
Monocytes	%	1.93	0.86	1.31	0.37
RBC	106/ Μl	11.87	0.71	9.99	0.68
HGB	g/dl	11.43	0.64	9.62	0.65
HCT	%	34.15	2.25	30.66	2.16
MCV	Fi	28.75	0.71	30.65	0.59
MCH	Pg	9.65	0.16	9.63	0.08
MCHC	g/dl	33.67	0.56	31.50	0.46
PLT	103/ μl	290.33	49.93	267.62	43.55
RDW	%	14.41	0.15	14.57	0.39
PCT	%	0.10	0.01	0.09	0.03
MPV	Fl	3.36	0.14	3.10	0.74
PDW	%	14.18	0.34	12.12	1.88
Total Protein	g/ dl	9.63	0.16	8.70	0.20
Fibrinogen	mg/ dl	622.22	86.24	250.00	92.19

RBC, red blood cells; HGB,hemoglobin; HCT,hematocrit; MCV,mean corpuscular volume; MCH,mean corpuscular hemoglobin; MCHC,mean corpuscular hemoglobin concentration; PLT,platelets; RDW,red cell distribution width; PCT,platelets-crit; MPV,mean platelets volume; PDW,platelets distribution width.

Table 2. Total and differential cell counts of BAL fluid in the groups1–3 sampled in summer and winter seasons.

Parameter	Unit	Group 1 (TBAL)		Group 2 (BBAL)		Group 3 (BBAL)
		statistic		statistic		statistic
		Mean	Std. error	Mean	Std. error	Mean	Std. error
Total cells	103/ ml	355.55	29.39	343.75	30.23	355.55	37.67
Macrophages	%	65.66	1.33	68.18	3.544	59.11	4.30
Lymphocytes	%	23.22	0.86	24.50	2.62	21.33	3.10
Neutrophils	%	5.44	0.85	2.81†	0.51	3.88	1.07
Epithelial cells	%	0.00†	0.00	1.43†	0.88	8.88†	3.78
Bronchial cells	%	0.00†	0.00	3.12†	0.32	6.33†	1.44
CONT1	103/ ml	337.25	0.80	381.25	0.54	290.00	0.33
CONT2	103/ ml	366.00	0.38	475.00	0.64	276.00	0.77

The statistics describe the all data (N = 20 for each of groups).†One-way analysis of variance results indicates significant diffrences; p = 0.05. BBAL,Bronchoscopic bronchoalveolar lavage; TBAL,transtracheal bronchoalveolar lavage; Smear manually counted and analyzed by two persons separately and therefore their results have presented as CONT 1 and 2.

The results of manual counting of the total cell number in the BAL sample (381.25 ± 54.17) were not significantly different from counting by Hematology Analyzer apparatus (343.75 ± 30.23). In the manual method, two experts performed total cell count and there was no significant difference between the report of the first expert (381.25 ± 17.54) and the second one (475.00 ± 64.87).

In the BAL fluid prepared from healthy Ghezel sheep in summer by transtracheal method the normal value of total cell count was 355.55 ± 29.39, which in the differential count the highest value was declared for macrophages 60.66 ± 1.33, and any epithelial and bronchial cells were detected.

In the BAL fluid samples prepared from healthy Ghezel sheep in winter with bronchoscopy the normal amount of total cells was 355.55 ± 37.67, which in the differential count the highest value for macrophages and the lowest values for bronchial cells were 59.11 ± 4.30 and 6.38 ± 1.44, respectively.

There was no significant difference in the number of total cells, and the percentage of macrophages and lymphocytes between the methods and seasons, although at first glance the low number of total cells in winter is noticeable. The percentage of neutrophils in the bronchoscopy method was not significantly different between summer and winter. In summer, sampling was done with two different methods, there was a significant change (p <0.05) in the count of neutrophils. The increase in the epithelial and bronchial cells was significant in samples obtained in the winter compared to the summer (p <0.05).

In BBAL prepared from healthy animals, the percentage of neutrophils was between 2.8 and 3.8; however, TBAL samples in summer, have large amounts (5.44) of neutrophils. Interestingly, eosinophils were not observed in healthy sheep studied in this study, or in the case of mast cells, only a sample prepared from one of the studied animals in both seasons showed a small number.

Discussion

The BAL method can provide useful and valuable microbiological and cytological data in the study of respiratory diseases of small ruminants, but little information is available on the interpretation of abnormal cell profiles in small ruminants. Recent articles show that advanced bronchoscopy is now widely used in animal lung research (Dawson, et al., 2005). Of course, its clinical tools are quite limited and require the implementation and introduction of easier methods. In the present study two sampling methods, BAL and, TBAL have been evaluated in different seasons, and side effects such as bleeding were not observed in sheep during or after the sampling. Acute bleeding rarely occurs during BAL unless the iatrogenic bleeding is caused by trauma. The presence of erythrophagocytosis or hemosiderin in cytology can be used to differentiate iatrogenic trauma from actual pulmonary hemorrhage (Woods, et al., 2013). Methods and seasons do have not any significant effects on the cell profiles of samples.

This study was designed to evaluate the clinical performance of the TBAL technique compared with BAL obtained via a bronchoscope BBAL. Both methods can be performed successfully and quickly. Although we think a likely significant fall in PaO₂ occurs post-lavage in animals subjected to bronchoscopic BAL due to scope diameter and airway resistance especially if respiratory distress is present, unfortunately, we were not able to measure oxygen saturation and this was a limitation in the present study.

The TBAL technique did not differ from that used with bronchoscopy, and samples obtained with both procedures had excellent quality. There was no statistical difference between the cytological parameters of BBAL compared with TBAL. Therefore, the TBAL technique for collecting BAL can be performed by clinicians in field conditions without the need for advanced sophisticated apparatus, but in cases where there is a suspicion of the presence of intrabronchial lesions, it will be possible to observe them only by using bronchoscopy, and in such cases, it is a preferred tool. As previously mentioned (Rola-Pleszczynski, et al., 2007), this method TBAL of pulmonary lavage is well tolerated by the animal. The simplicity of this method is a good advantage and can be used in the clinic as a practical diagnostic method for the respiratory diseases of sheep (Sheehan, et al., 2005).

Comparative results of samples taken by TBAL and BBAL did not show a significant difference between two the performed techniques. The results showed that the differential counting of fluid cells by an automatic analyzer device is not authentic, because the device is not able to identify all the different cells in the normal and abnormal BAL sample, therefore could not be a suitable option for this case, but could be recommended for total cell counting.

There was no significant difference in cell number and percentage of macrophages and lymphocytes between applied procedures and seasons. The percentage of neutrophils in BBAL was not significantly different between the summer and winter seasons. There was a significant increase in the percentage of epithelial and bronchial cells in winter compared to summer. Large numbers of epithelial cells (more than 5% of nucleated cells) in BALF may be observed due to excessive suction, abnormal sampling, trauma, or viral infections (Woods, et al., 2013). A similar study on the horse has shown a significant difference between the summer and winter seasons in the cytology profiles of the BAL samples. It is assumed that these seasonal differences are responsible for the high incidence of some respiratory diseases in horses (Davis And Sheats, 2019).

If the neutrophil percentage is higher than 10, it indicates pneumonia (Taniuchi, et al., 2009). In this study, in BALF prepared from healthy animals, the percentage of neutrophils was less than 10% although a different level was declared with TBAL samples in summer. This increase in BALF neutrophil percentage has been linked to an increase in organic dust particles in dry and hot weather. Reciprocally, it was demonstrated that weather humidity significantly increases during autumn and winter; furthermore, decreases the concentration of airborne endotoxins and explains the low BAL neutrophil cell count (Hansen et al., 2018).

The total number of cells in BAL specimens in healthy sheep was reported as 31× 10⁴ per milliliter (with a range of 6−110× 10⁴) and the mean percentage of macrophages, neutrophils, and lymphocytes determined 79.8, 2.6, and 4.4, respectively (Dawson, et al., 2005). In comparison to the findings of the present study, the percentage of macrophages (68%–59%) and neutrophils(15.2–8.2) was higher, also lymphocytes (23–21) were less (Dawson, et al., 2005).

Interestingly a very low number (6.01×10⁴ per milliliter) of total cells in sheep normal BALF, with a predominance of macrophages 93.4%, has been reported by Collie et al., (1999) in the same geographical area that was done previously by Dawson et al. (2005) and show a noticeable difference. Regarding neutrophil and lymphocyte counts, there is a clear difference between the two mentioned studies and the findings of the present study. Breed differences and various husbandry and climatic conditions may be described in these findings. Slight differences between normal samples could be explained by closed barn environments during the wintertime, in such situations, where ventilation is not enough as well as during warm conditions (Hansen et al., 2018).

In a bronchoscopic study of sheep lungs in Japan, the normal range of cell count values in the BAL sample was 44.6—29.7× 10⁴ per ml, which is compared with this study data (35.5 25.5× 10⁴) has a close overlap, although higher values are considered as normal (Burrells and Sutherland, 1994). The difference in the reported values of the sample does not seem to be a new topic, as the reference books on the horse field indicate that even the normal number of sample cells varied significantly between studies and they have reported a difference range of 1,000–4,000 cells per micro-liter (Cowell and Tyler, 1992).

BAL findings have been compared in Bighorn and domestic sheep (Silflow et al., 1989). The total and differential cell count obtained from domestic sheep was similar in number and composition to the values reported by Woodside et al. (1983). Because no specific unit is mentioned for total cell count; therefore, they are not comparable with the present study data, but in the case of differential cell counts, the macrophage values of Bighorn sheep and domestic sheep in the United States, California (73.70% and 81.50%, respectively), which is more similar to the data of this study, while the neutrophil count in domestic and Bighorn sheep (4.7%–11%) was higher compared to the data obtained in Gezel sheep. The eosinophils were not observed in the normal BAL in the Ghezel breed while it was reported by others (Silflow et al., 1989; Woodside et al., 1983). Alveolar macrophages are the major cells in BALF; other cells commonly detected in normal BALF include lymphocytes, neutrophils, eosinophils, and mast cells (Woods, et al., 2013).

Almost, the same value was revealed in the number of lymphocytes in Ghezel at 21%, and Bighorn sheep at 19.53%. The increase in the number of lymphocytes observed in the Bighorn sheep may be the result of several mechanisms. It could be a result of a subclinical viral infection or non-infectious particles.

Rola-Pleszczynski et al. (2007) vaguely have stated the total number of cells and, therefore, can not be discussed, but about the differential count of immune cells in the BALs, the more common cells type in adult sheep reported as macrophages (70%) and lymphocytes (10%–15%). Accordingly, there are similarities in macrophage rates, but a higher rate of lymphocytes in the Ghezel breed has been counted in the present study (Rola-Pleszczynski, et al., 2007).

The fact that macrophages normally have the highest number of inflammatory cells indicates the importance of their presence in lung clearance (Jarikre et al., 2016). In this study, the range of macrophage cells in normal BAL is reported between 59% and 68%. Therefore, changes in this ratio can help in the diagnosis process. It has been demonstrated that in the BAL fluid of the lungs infected with the Maedi virus compared to healthy sheep, a higher percentage of lymphocytes and a lower percentage of macrophages were present (Katsoulos et al., 2009).

The results of many studies on normal BAL in sheep also show that macrophages and neutrophils have a very important role in the prevention of specific respiratory diseases (Katsoulos et al., 2009; Silflow et al.,1989). In the Ghezel breed, a smaller number of macrophages have been identified compared to the other breeds, which could be a turning point for further studies to identify it, as the predisposing factor to respiratory diseases.

The neutrophilia and lymphocytosis indicate lung response to stress, and bacterial or viral damage (Jarikre et al., 2016), as expected no abnormal increases were observed in the normal samples. BAL cell differential count is used in describing the stage of the inflammatory process. In the early stages of pneumonia, giant multinucleated cells were frequently observed, and in the advanced stages, neutrophils predominated. When the result is eosinophilia, indicates that the animal may have been exposed to lungworm (Jarikre et al., 2016). Therefore, the cytological examination can be used to evaluate the stages and the agent of the pneumonia process.

Considering the issues discussed above, it seems that the parameters of the normal sample among various breeds of the same species may also have very obvious and significant differences, therefore, using the criteria of other species and breeds is most likely associated with error and will lead to wrong decision making, these subjects further highlights the importance of the present study because this study for the first time reports the criteria for normal BAL characteristics in the Ghezel sheep.

Conclusion

It was concluded that TBAL has equivalent efficacy to bronchoscopic BAL. The TBAL is easy to perform and does not require bronchoscopy and extensive training to use. Even though it has limitations compared with bronchoscopic BAL, it is accessible and less expensive. The normal parameters for evaluating BAL samples in Ghezel sheep were obtained. There was no significant difference in the total and differential cell count between the procedures and the seasons. In other words, normal BAL fluid retains its properties in different seasons. The automatic analyzer device is not suitable for the differential counting of BAL fluid cells but is recommended for total cell counting.

Conflict of interest

The authors declare that they have no competing interests.

Funding

This work was funded as a dissertation of the first author, Fatemeh Ghorbani submitted as a Partial Fulfillment of the Degree of Doctor of Veterinary Medicine (DVM) at Urmia University (Code—2020/1702).

Authors contributions

G. Jalilzadeh-Amin and B. Dalir-Naghade designed and directed the project; F. Ghorbani and S. Asri Rezaei performed the experiments; G. Jalilzadeh-Amin analyzed data and also wrote the article.

Data availability

All data are provided in the manuscript.