KAAACI Standardization Committee report on the procedure and application of induced sputum examination

Min-Hye Kim; Mi-Yeong Kim; Kyung-Hwan Lim; Min-Suk Yang; Woo-Jung Song; Jeongmin Lee; Dong In Suh; Yoo Seob Shin; Jae-Woo Kwon; Sae-Hoon Kim; Sang-Heon Kim; Byung-Jae Lee; Sang-Heon Cho; Jae-Woo Jung

doi:10.4168/aard.2017.5.6.307

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Kim, Kim, Lim, Yang, Song, Lee, Suh, Shin, Kwon, Kim, Kim, Lee, Cho, Jung, and Korean Academy of Asthma, Allergy and Clinical Immunology Standardization Committee: KAAACI Standardization Committee report on the procedure and application of induced sputum examination

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Allergy Asthma Respir Dis 2017;5(6):307-311.

Published online: 5 November 2017

DOI: https://doi.org/10.4168/aard.2017.5.6.307

KAAACI Standardization Committee report on the procedure and application of induced sputum examination

Min-Hye Kim¹, Mi-Yeong Kim², Kyung-Hwan Lim³, Min-Suk Yang⁴, Woo-Jung Song⁵, Jeongmin Lee⁶, Dong In Suh⁷, Yoo Seob Shin⁸, Jae-Woo Kwon⁹, Sae-Hoon Kim¹⁰, Sang-Heon Kim¹¹, Byung-Jae Lee¹², Sang-Heon Cho⁵, Jae-Woo Jung¹³; Korean Academy of Asthma, Allergy and Clinical Immunology Standardization Committee

¹Department of Internal Medicine, Ewha Womans University School of Medicine, Seoul, Korea

²Department of Internal Medicine, Inje University Busan Paik Hospital, Inje University College of Medicine, Busan, Korea

³Department of Internal Medicine, Armed Forces Capital Hospital, Seongnam, Korea

⁴Department of Internal Medicine, Seoul Metropolitan Government - Seoul National University Boramae Medical Center, Seoul, Korea

⁵Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea

⁶Department of Pediatrics, Yonsei University Wonju College of Medicine, Wonju, Korea

⁷Department of Pediatrics, Seoul National University College of Medicine, Seoul, Korea

⁸Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, Korea

⁹Department of Internal Medicine, Kangwon National University School of Medicine, Chuncheon, Korea

¹⁰Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea

¹¹Department of Internal Medicine, Hanyang University College of Medicine, Seoul, Korea

¹²Division of Allergy, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

¹³Department of Internal Medicine, Chung-Ang University College of Medicine, Seoul, Korea

Correspondence to: Jae-Woo Jung https://orcid.org/0000-0002-3411-735X Department of Internal Medicine, Chung-Ang University Hospital, Chung-Ang University College of Medicine, 102 Heukseok-ro, Dongjak-gu, Seoul 06973, Korea Tel: +82-2- 6299-1437, Fax: +82-2-825-7571, E-mail: jwjung@cau.ac.kr

• This research was supported by a fund (A092076) by Research of Korea Centers for Disease Control and Prevention.

Received 17 March 20176 September 2017 Accepted 8 September 2017

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Induced sputum and sputum cell count analysis is a test for the diagnosis of various respiratory diseases. In particular, it has long been used as an important biomarker in the diagnosis or characterization of asthma or eosinophilic bronchitis. Despite a relatively long history of this test, there has been no consensus report for conducting and interpreting the analyses in Korea. Based on this awareness and necessity, the Korean Academy of Asthma, Allergy and Clinical Immunology launched the Standardization Committee to review the international guidelines and the literature and to develop a consensus report on the diagnostic procedure and interpretation of the sputum induction test.

Keywords: Sputum, Induced sputum, Methods, Analysis

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Fig. 1.

Image of the sputum cells (×200). M, macrophage; N, neutrophil; E, eo-sinophil; EP, epithelial cell.

Table 1.

Equipment for sputum induction

Oxygen saturation monitor, oxygen supply device
Salbutamol inhaler (200–400 μg)
Spirometry or peak expiratory flow meter, nose clip
Ultrasonic nebulizer, tube, lids
Sterile hypertonic (3.0% or 4.5%) or isotonic (0.9%) saline
Glass of water, petri dish for sample collection
Worksheet, calculator
Clock

Table 2.

Process of sputum induction

① Explain the test method to the patient in detail (rinsing the mouth before the test, inhalation with tidal breathing, and collecting sputum by coughing every 5 minutes)
② Set the nebulizer output (≥1 mL/min) and sterile saline preparation (usually hypertonic saline, 4.5%)
③ Measure baseline FEV₁ (or PEF)
④ FEV1 measurement after 10 minutes of inhalation of pre-treatment Salbutamol (200 μg)
⑤ Start inhalation of saline with tidal breathing through a nebulizer. The sputum is collected every 5 minutes by coughing, during 15 to 20 minutes in total. (The time required for cough and sputum excretion was excluded from the total examination time and the total sputum induction time is recorded). However, if the patient wants to spit out, they can collect sputum at any time.
⑥ Measure FEV₁ (or PEF) every 5 minutes after the start of the test. Examination should be stopped when symptoms occur or FEV₁ is reduced by more than 20% of postbron chodilator FEV₁.

FEV₁, forced expiratory volume in 1 second; PEF, peak expiratory flow.

Table 3.

Process of induced sputum preparation

When using whole sputum samples
① Put total sputum sample in a polystyrene tube and record volume and weight.
② Mix PBS with 0.1% DTT or DTE in a 1:1 volume ratio with the sputum sample.
③ Mix the sample with pipet and mix more with vortex.
④ Mix the sample using rocker or water-bath at room temperature (22° C).
⑤ The mixture is filtered and the weight is measured.
⑥ Cell viability and total cell count is measured.
⑦ Calculate as total number of cells per mL.
⑧ Cells and supernatant are separated by centrifugation (300–1,500×g, 5–10 minutes; usually 400×g, 10 minutes).
⑨ The cell precipitate is resuspended in a salt solution (or buffer) (concentration 1.0×10⁶ cells/mL). Use approximately 40–65 μL of sample for each cytospin.
⑩ Cytospin is centrifugated at 10–51×g for 6 minutes.
⑪ Giemsa or Wright staining of cytospin slides.
⑫ Differential cell count: Count 400–500 nonsquamous cells and record the percentage of eosinophils, neutrophils, macrophages, lymphocytes, and bronchial epithelial cells. Record the fraction of squamous cells.
When using only sputum mucus plug samples
① Only a thick sputum mucus plug without saliva is selected and put into a polystyrene tube. Record volume and weight.
② Dilute sample with PBS with 0.1% DTT or DTE in a 1:4 volume ratio.
③ The following procedure is the same as the treatment step for the whole sputum sample.

PBS, phosphate-buffered saline; DTT, dithiotreitol; DTE, dithioerythritol.

Table 4.

Report form of induced sputum processing

	Cell counts	%
Bronchial epithelial cells
Macrophages
Neutrophils
Eosinophils
Lymphocytes
Total cell counts	400 (500)∗	100

^∗ For differential cell count, count 400–500 nonsquamous cells, and then record percentage of eosinophils, neutrophils, macrophages, lymphocytes, and bronchial epithelial cells.

Table 5.

Causes of increase in specific cell type of induced sputum examination

Cell type	Causes of increased number of cells in sputum
Increased	Uncontrolled asthma, eosinophilic bronchitis, allergen or chemical
eosinophils	sensitizer exposure, steroid-reactive chronic airway obstruction
Increased neutrophils	resistant asthma Smoking, air pollutions (ozone, etc.), infection, endotoxin, steroid-
Increased lymphocytes	Sarcoidosis, Chlamydia pneumonia infection

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