Page 57 - Škrgat, Sabina, ed. 2023. Severe Asthma Forum - Monitoring and Treatable Traits in Severe Asthma. Koper: University of Primorska Press. Severe Asthma Forum, 2
P. 57
It is necessary to weight sputum plugs be- – mixed granulocytic asthma: >61% 57
fore the homogenisation. Homogenisation is sputum neutrophils and >3% eosino-
performed by the use of fresh solution of 0,1% phils induced sputum role in severe asthma phenotyping
dithiothreitol (DTT) that breaks disulphide
bonds in mucin molecules and preserves cells – paucigranulocitic asthma: <61% spu-
morphology. The added DTT volume is 2-4 tum neutrophils and <3% eosinophils
times of recorded weight of the plugs, tubes
with mixture are placed in the shaking water Asthma inflammatory phenotypes
bath on 37°C for 15 minutes to ensure com-
plete homogenisation. It is necessary to stop Asthma inflammatory subtypes are charac-
the effect of DTT and preserve cells morpho- terized by some important clinical differenc-
logy by adding buffer solution (PBS) in a vo- es. Eosinophilic asthma is the most common
lume equal to the sputum volume plus DTT phenotype. It is very prevalent in individuals
volume.36 with nonsevere disease, and also accounts for
approximately 50% to 60% of the total severe
Filtration of the fluid mixture through asthma population11. It has classically been
48-52 μm nylon gauze can remove remaining associated with allergic sensitization and a
debris and mucus. Next step is recording of T2-dominant inflammatory response. Eosin-
the filtrate volume, accessing cell viability and ophilic group is characterised by the highest
measuring total cell count/millilitre using ha- degree of airway hyperresponsiveness. Wood-
emocytometer. ruff et al demonstrated that the percentage
of eosinophils in induced sputum was inde-
Dissolved induced sputum should be pro- pendently associated with more severe airflow
cessed as any other liquid cytology sample in obstruction and methacholine reactivity12.
cytocentrifuge and centrifuge. Cytospin slides Eosinophilic phenotype of asthma mainly re-
are usually May Grunwald-Giemsa (MGG) sponds well to corticosteroid treatment. Strat-
stained and used for cell counting. Remaining egies that are based on sputum examination
supernatant can be stored on -80°C for addi- to guide treatment decisions have been effec-
tional analyses4. Cytological examination of tive in improving lung function and decreas-
the slides should be performed under light mi- ing asthma symptoms and exacerbations.
croscope high power magnifications (400x, Normalization of induced sputum eosino-
1000x). Adequate are samples with less than phil counts has been shown to be an effective
20% of squamous cells counting on 300-500 strategy for preventing severe asthma exacer-
of all cells. Differential cell count (%) sho- bations and hospitalizations. Sputum exami-
uld be calculated on 300-500 non-squamous nation can detect an increase in airway eosin-
cells: eosinophils, neutrophils, macrophages, ophils up to 3 months before the development
lymphocytes, columnar epithelial cells and of a clinical exacerbation13. This approach re-
mastocytes, if any. Both counts (%) should be quires frequent sputum analyses and is im-
recorded in final report9,10. On the basis of cell practical for routine clinical use in most cen-
differential counts in induced sputum diffe- tres. However, the ability to analyze sputum
rent inflammatory phenotypes of acute asth- is necessary in centers dealing with more se-
ma can be divided in four types4: vere forms of asthma. The effectiveness of a
treatment strategy based on assessment of air-
– eosinophilic asthma (EA): >3% spu- way inflammation was not as clear in patients
tum eosinophils with mild asthma, suggests that sputum anal-
ysis is not necessary in those with milder asth-
– neutrophilic asthma (NA): >61% spu- ma that responds well to initial therapy14.
tum neutrophils and <3% eosinophils
fore the homogenisation. Homogenisation is sputum neutrophils and >3% eosino-
performed by the use of fresh solution of 0,1% phils induced sputum role in severe asthma phenotyping
dithiothreitol (DTT) that breaks disulphide
bonds in mucin molecules and preserves cells – paucigranulocitic asthma: <61% spu-
morphology. The added DTT volume is 2-4 tum neutrophils and <3% eosinophils
times of recorded weight of the plugs, tubes
with mixture are placed in the shaking water Asthma inflammatory phenotypes
bath on 37°C for 15 minutes to ensure com-
plete homogenisation. It is necessary to stop Asthma inflammatory subtypes are charac-
the effect of DTT and preserve cells morpho- terized by some important clinical differenc-
logy by adding buffer solution (PBS) in a vo- es. Eosinophilic asthma is the most common
lume equal to the sputum volume plus DTT phenotype. It is very prevalent in individuals
volume.36 with nonsevere disease, and also accounts for
approximately 50% to 60% of the total severe
Filtration of the fluid mixture through asthma population11. It has classically been
48-52 μm nylon gauze can remove remaining associated with allergic sensitization and a
debris and mucus. Next step is recording of T2-dominant inflammatory response. Eosin-
the filtrate volume, accessing cell viability and ophilic group is characterised by the highest
measuring total cell count/millilitre using ha- degree of airway hyperresponsiveness. Wood-
emocytometer. ruff et al demonstrated that the percentage
of eosinophils in induced sputum was inde-
Dissolved induced sputum should be pro- pendently associated with more severe airflow
cessed as any other liquid cytology sample in obstruction and methacholine reactivity12.
cytocentrifuge and centrifuge. Cytospin slides Eosinophilic phenotype of asthma mainly re-
are usually May Grunwald-Giemsa (MGG) sponds well to corticosteroid treatment. Strat-
stained and used for cell counting. Remaining egies that are based on sputum examination
supernatant can be stored on -80°C for addi- to guide treatment decisions have been effec-
tional analyses4. Cytological examination of tive in improving lung function and decreas-
the slides should be performed under light mi- ing asthma symptoms and exacerbations.
croscope high power magnifications (400x, Normalization of induced sputum eosino-
1000x). Adequate are samples with less than phil counts has been shown to be an effective
20% of squamous cells counting on 300-500 strategy for preventing severe asthma exacer-
of all cells. Differential cell count (%) sho- bations and hospitalizations. Sputum exami-
uld be calculated on 300-500 non-squamous nation can detect an increase in airway eosin-
cells: eosinophils, neutrophils, macrophages, ophils up to 3 months before the development
lymphocytes, columnar epithelial cells and of a clinical exacerbation13. This approach re-
mastocytes, if any. Both counts (%) should be quires frequent sputum analyses and is im-
recorded in final report9,10. On the basis of cell practical for routine clinical use in most cen-
differential counts in induced sputum diffe- tres. However, the ability to analyze sputum
rent inflammatory phenotypes of acute asth- is necessary in centers dealing with more se-
ma can be divided in four types4: vere forms of asthma. The effectiveness of a
treatment strategy based on assessment of air-
– eosinophilic asthma (EA): >3% spu- way inflammation was not as clear in patients
tum eosinophils with mild asthma, suggests that sputum anal-
ysis is not necessary in those with milder asth-
– neutrophilic asthma (NA): >61% spu- ma that responds well to initial therapy14.
tum neutrophils and <3% eosinophils
