Differential sputum cell counting is not widely available despite proven clinical utility in the management of asthma. We compared eosinophil counts obtained using liquid-based cytology (LBC), a routine histopathological processing method, and the current standard method. Eosinophil counts obtained using LBC were a strong predictor of sputum eosinophilia (≥3%) determined by the standard method suggesting LBC could be used in the management of asthma

A Green

Bafadhel

C Reynolds

D E Shaw

Efthimiadis

G Meakin

Gibson

Green

H Lee

I Soomro

Korevaar

M J Martin

Pavord

Petsky

R L Simms

S Winters

T W Harrison

Ward

Martin, M.J.

Lee, H.

Meakin, G.

Green, A.

Simms, R.L.

Reynolds, C.

Winters, S.

Shaw, Dominick E.

Soomro, Irshad N.

Harrison, Timothy W.

Name not available

Assessment of a rapid liquid-based cytology method for measuring sputum cell counts

Nottingham eTheses

Nottingham ePrints

Martin, M.J. and Lee, H. and Meakin, G. and Green, A. and Simms, R.L. and Reynolds, C. and Winters, S. and Shaw, Dominick E. and Soomro, Irshad N. and Harrison, Timothy W. (2016) Assessment of a rapid liquid-based cytology method for measuring sputum cell counts. Thorax . ISSN 1468-3296 Access from the University of Nottingham repository: http://eprints.nottingham.ac.uk/38383/1/Cell%20Count%20Paper%20Accepted%20Manuscript.pdfCopyright and reuse: The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions.This article is made available under the University of Nottingham End User licence and may be reused according to the conditions of the licence.  For more details see: http://eprints.nottingham.ac.uk/end_user_agreement.pdfA note on versions: The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher’s version. Please see the repository url above for details on accessing the published version and note that access may require a subscription.For more information, please contact eprints@nottingham.ac.ukAssessment of a rapid liquid based cytology method formeasuring sputum cell countsMartin MJ, Lee H, Meakin G, Green A, Simms RL, Reynolds C, Winters S*,Shaw DE, Soomro I*, Harrison TWThe Asthma CentreNottingham Respiratory Research Unit,University of Nottingham,Nottingham City Hospital,Nottingham,UK*Department of Cellular Pathology,Nottingham University Hospitals NHS Trust,Nottingham City HospitalNottingham,UKCorresponding author:Professor Tim HarrisonRoom B23Clinical Sciences BuildingNottingham City HospitalHucknall RdNottingham NG5 1PBE-mail: tim.harrison@nottingham.ac.ukPhone: +44 0115 8231714Keywords:Word Count: 938AbstractDifferential sputum cell counting is not widely available despite proven clinical utility inthe management of asthma. We compared eosinophil counts obtained using liquid basedcytology (LBC), a routine histopathological processing method, and the current standardmethod. Eosinophil counts obtained using LBC were a strong predictor of sputumeosinophilia (≥3%) determined by the standard method suggesting LBC could be used in the management of asthma.BackgroundDifferential cell counts (DCC) of induced sputum samples have been shown to be usefulin the management of patients with moderate–to-severe asthma(1) and in the diagnosisof eosinophilic bronchitis(2). Unfortunately, sputum processing is time consuming(3)taking approximately 4 hours and a meta-analysis concluded that its routine use couldnot be justified due to the technical expertise required(4). We aimed to establish if asimplified method using routine liquid based cytology (LBC), used in histopathologylaboratories throughout the NHS, could provide similar results.MethodSubjects with asthma or chronic cough presenting to an outpatient respiratory clinic whowere willing to take part in the study were consented (REC 08/H0407/2) and spirometryperformed. Sputum samples were induced using hypertonic saline as previouslydescribed(3), following which sputum plugs were isolated, weighed and divided into twoequal portions.The first sample was processed as described previously(3) and the resultant slidecounted by a trained research scientist. Cell counts were later reviewed by a secondscientist blinded to the results and any discrepancies in counts were resolved byreassessment by both scientists.For the LBC method, sputum samples were manually mixed in 10ml CytoRich Red (CRR)solution with a pipette. The mixture was shaken on an Ika-Vibrax-VXR shaker at1000rpm for 30 mins to homogenise the sample and then centrifuged at 780g for 5minutes. The resultant pellets were re-suspended in 1.5ml CRR and 0.75ml aliquots weredeposited into settling chambers positioned on coated slides for a minimum of 10minutes. Any excess fluid was extracted from the chambers using a pipette. Thechambers were then removed and the slides fixed and stained using the Papanicolaoumethod. Eosinophils in 100 high power fields (x400) were counted and averaged foreach slide by a single consultant histopathologist (IS) blinded to the DCC results. Eachslide was graded on an ordinal scale of 0-4 based on the approximate number ofeosinophils per high power field (eos/hpf) where: 0=0 eos/hpf, 1=1-2 eos/hpf, 2=3-4eos/hpf, 3=5-10 eos/hpf and 4=>10 eos/hpf.Data were entered into GraphPad Prism. Empirical receiver operator characteristics(ROC) curves and sensitivity, specificity, positive predictive value (PPV) and negativepredictive values (NPV) for each LBC ‘grade’ to identify a sputum eosinophil count of≥3%(1) were generated. This analysis was repeated with exclusion of samples with ≤50% viability and/or ≥20% squamous cell contamination for comparative purposes with previous studies investigating the inter-observer agreement of DCCs(5).ResultsDemographic and lung function data are available in the online supplement. Of 55patients, 6 produced samples of insufficient volume to process by either technique, and4 samples were uncountable by both methods, leaving 45 pairs of slides available forcounting. Four slides were identified by DCC with ≤50% viability and 2 slides had ≥20% squamous contamination leaving 39 pairs of slides for counting in the ‘exclusion set’. Theempirical ROC curve to assess the utility of the LBC based method to identify a sputumeosinophil count of ≥3% had an area under the curve (ROC AUC) of 0.90 (p<0.001) (Fig 1) which increased to 0.95 (p<0.001) upon excluding slides with low viability orsignificant squamous contamination. The cut-point with the highest combined set ofvalues for sensitivity, specificity, PPV and NPV for identifying a sputum eosinophil countof ≥3% was >10 eos/hpf, as shown in table 1. Processing of sputum took approximately 1 hour with LBC compared with approximately 4 hours using the current cytospin andDCC technique. Examples of slides produced by both methods are shown in the onlinesupplement.LBC grade(eos/hpf)Sensitivity% 95% CI (%) Specificity% 95% CI (%) PPV% NPV%1 (1-2) 100 78.2 - 100 20 7.7 - 38.6 37.5 1002 (3-4) 93.3 68.1 - 99.8 56.7 37.4 - 74.5 51.9 95.43 (5-10) 93.3 68.1 - 99.8 70 50.6 - 85.3 60.9 95.54 (>10) 66.7 (83.3*) 38.4 - 88.2 96.7 82.8 - 99.9 90.9 85.3 (93.1*)Table 1: Sensitivity and specificity values for each LBC grade to identify a DCCsputum eosinophil count of ≥3% for all slides. *Values for sensitivity and NPV afterexcluding slides with low viability/squamous contamination (n=6)Figure 1: Empirical ROC curve showing the sensitivity (%) and 100-specificity (%) of LBCderived eosinophil count (eos/hpf) to identify a sputum differential eosinophil count of ≥3% for all  samples and after exclusion of samples with ≤50% viability and/or ≥20% squamous contamination*DiscussionOur results indicate that a cut-off point of >10 eos/hpf from the LBC technique has ahigh PPV and NPV to identify patients with a sputum differential eosinophil count of ≥ or <3%. This is important in asthma management because this value is associated withincreased risk of exacerbations and can be modified with additional oral or inhaledcorticosteroids(1).Sputum differential cell counts have demonstrated utility in guiding treatment decisionsin patients with more severe asthma(1) but the DCC technique has not been widelyadopted into clinical practice because of the time and technical expertise required toperform it. Any novel method of assessing sputum eosinophilia must therefore becompared with DCC. However, differences in results do not necessarily mean the DCCmethod is superior to the LBC approach. Firstly, it cannot be excluded that differences ineosinophil counts produced by the two methods were due to heterogenous distribution ofthe cellular portion of the sample, resulting in a higher eosinophil concentration in onepart of the sample than the other. Secondly, the DCC technique is not subject to theformal quality control procedures in place for laboratory investigations performed in theNHS. It remains possible that LBC with an eosinophil count performed by a consultanthistopathologist is more accurate than research scientists performing DCC. LBC also hassome practical advantages: 1) samples do not have to be processed on the day ofcollection as for DCC(6) because CRR acts as a mucolytic and a fixative and 2) the LBCtechnique is less time intensive making it more suitable for use in NHS histopathologydepartments.The next step is to demonstrate that these results can be reproduced at other centresand that treatment decisions based on LBC are beneficial in terms of asthma outcomesas has been shown for DCC’s(1). These studies should include blood eosinophil countswhich are more easily obtained and have been demonstrated to be a good predictor ofsputum eosinophilia in subjects undergoing COPD exacerbations(7) and a moderatepredictor of sputum eosinophilia in asthma(8). The reliability of LBC eosinophil countsfrom mucopurulent sputum samples from subjects with COPD also needs to be assessed.References:1. Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P, et al.Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial.Lancet. 2002;360(9347):1715-21.2. Gibson PG, Dolovich J, Denburg J, Ramsdale EH, Hargreave FE. Chronic cough:eosinophilic bronchitis without asthma. Lancet. 1989;1(8651):1346-8.3. Pavord ID, Pizzichini MM, Pizzichini E, Hargreave FE. The use of induced sputumto investigate airway inflammation. Thorax. 1997;52(6):498-501.4. Petsky HL, Cates CJ, Lasserson TJ, Li AM, Turner C, Kynaston JA, et al. Asystematic review and meta-analysis: tailoring asthma treatment on eosinophilicmarkers (exhaled nitric oxide or sputum eosinophils). Thorax. 2012;67(3):199-208.5. Ward R, Woltmann G, Wardlaw AJ, Pavord ID. Between-observer repeatability ofsputum differential cell counts. Influence of cell viability and squamous cellcontamination. Clin Exp Allergy. 1999;29(2):248-52.6. Efthimiadis A, Jayaram L, Weston S, Carruthers S, Hargreave FE. Inducedsputum: time from expectoration to processing. Eur Respir J. 2002;19(4):706-8.7. Bafadhel M, McKenna S, Terry S, Mistry V, Reid C, Haldar P, et al. AcuteExacerbations of Chronic Obstructive Pulmonary Disease Identification of BiologicClusters and Their Biomarkers. Am J Respir Crit Care Med. 2011;184(6):662-71.8. Korevaar DA, Westerhof GA, Wang J, Cohen JF, Spijker R, Sterk PJ, et al.Diagnostic accuracy of minimally invasive markers for detection of airway eosinophilia inasthma: a systematic review and meta-analysis. Lancet Respir Med. 2015;3(4):290-300.Subjects with asthma Subjects with chroniccoughTotal number recruited to study 43 12Total number who producedadequate samples forprocessing41 8Total number who produced‘countable’ samples38 7Mean age (range) a 55 (26-76) 59 (41-79)Sex: male (%) 27 (62.8) 5 (41.7)Ethnic group:White Or White British (%) 43 (100) 12 (100)Mean FEV1 % predicted (SD) 63 (15) 73 (12)Mean FEV1/FVC % (SD) 67 (23) 107 (25)Median blood eosinophil countx109/L (IQR)0.75 (3.25) 1.9 (2.8)Table S1: Demographics and clinical measures of study subjects with asthmaand chronic coughABFigure S1: Cytological analysis of sputum samples from the study. Eosinophilsare indicated with arrows. (A) A cytocentrifuge slide prepared by the standard(DCC) technique (Giemsa stain, original magnification x400) (B) A slideprepared by the LBC technique (Papanicolaou stain, original magnificationx400)

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Assessment of a rapid liquid based cytology method formeasuring sputum cell countsMartin MJ, Lee H, Meakin G, Green A, Simms RL, Reynolds C, Winters S*,Shaw DE, Soomro I*, Harrison TWThe Asthma CentreNottingham Respiratory Research Unit,University of Nottingham,Nottingham City Hospital,Nottingham,UK*Department of Cellular Pathology,Nottingham University Hospitals NHS Trust,Nottingham City HospitalNottingham,UKCorresponding author:Professor Tim HarrisonRoom B23Clinical Sciences BuildingNottingham City HospitalHucknall RdNottingham NG5 1PBE-mail: tim.harrison@nottingham.ac.ukPhone: +44 0115 8231714Keywords:Word Count: 938AbstractDifferential sputum cell counting is not widely available despite proven clinical utility inthe management of asthma. We compared eosinophil counts obtained using liquid basedcytology (LBC), a routine histopathological processing method, and the current standardmethod. Eosinophil counts obtained using LBC were a strong predictor of sputumeosinophilia (≥3%) determined by the standard method suggesting LBC could be used in the management of asthma.BackgroundDifferential cell counts (DCC) of induced sputum samples have been shown to be usefulin the management of patients with moderate–to-severe asthma(1) and in the diagnosisof eosinophilic bronchitis(2). Unfortunately, sputum processing is time consuming(3)taking approximately 4 hours and a meta-analysis concluded that its routine use couldnot be justified due to the technical expertise required(4). We aimed to establish if asimplified method using routine liquid based cytology (LBC), used in histopathologylaboratories throughout the NHS, could provide similar results.MethodSubjects with asthma or chronic cough presenting to an outpatient respiratory clinic whowere willing to take part in the study were consented (REC 08/H0407/2) and spirometryperformed. Sputum samples were induced using hypertonic saline as previouslydescribed(3), following which sputum plugs were isolated, weighed and divided into twoequal portions.The first sample was processed as described previously(3) and the resultant slidecounted by a trained research scientist. Cell counts were later reviewed by a secondscientist blinded to the results and any discrepancies in counts were resolved byreassessment by both scientists.For the LBC method, sputum samples were manually mixed in 10ml CytoRich Red (CRR)solution with a pipette. The mixture was shaken on an Ika-Vibrax-VXR shaker at1000rpm for 30 mins to homogenise the sample and then centrifuged at 780g for 5minutes. The resultant pellets were re-suspended in 1.5ml CRR and 0.75ml aliquots weredeposited into settling chambers positioned on coated slides for a minimum of 10minutes. Any excess fluid was extracted from the chambers using a pipette. Thechambers were then removed and the slides fixed and stained using the Papanicolaoumethod. Eosinophils in 100 high power fields (x400) were counted and averaged foreach slide by a single consultant histopathologist (IS) blinded to the DCC results. Eachslide was graded on an ordinal scale of 0-4 based on the approximate number ofeosinophils per high power field (eos/hpf) where: 0=0 eos/hpf, 1=1-2 eos/hpf, 2=3-4eos/hpf, 3=5-10 eos/hpf and 4=>10 eos/hpf.Data were entered into GraphPad Prism. Empirical receiver operator characteristics(ROC) curves and sensitivity, specificity, positive predictive value (PPV) and negativepredictive values (NPV) for each LBC ‘grade’ to identify a sputum eosinophil count of≥3%(1) were generated. This analysis was repeated with exclusion of samples with ≤50% viability and/or ≥20% squamous cell contamination for comparative purposes with previous studies investigating the inter-observer agreement of DCCs(5).ResultsDemographic and lung function data are available in the online supplement. Of 55patients, 6 produced samples of insufficient volume to process by either technique, and4 samples were uncountable by both methods, leaving 45 pairs of slides available forcounting. Four slides were identified by DCC with ≤50% viability and 2 slides had ≥20% squamous contamination leaving 39 pairs of slides for counting in the ‘exclusion set’. Theempirical ROC curve to assess the utility of the LBC based method to identify a sputumeosinophil count of ≥3% had an area under the curve (ROC AUC) of 0.90 (p<0.001) (Fig 1) which increased to 0.95 (p<0.001) upon excluding slides with low viability orsignificant squamous contamination. The cut-point with the highest combined set ofvalues for sensitivity, specificity, PPV and NPV for identifying a sputum eosinophil countof ≥3% was >10 eos/hpf, as shown in table 1. Processing of sputum took approximately 1 hour with LBC compared with approximately 4 hours using the current cytospin andDCC technique. Examples of slides produced by both methods are shown in the onlinesupplement.LBC grade(eos/hpf)Sensitivity% 95% CI (%) Specificity% 95% CI (%) PPV% NPV%1 (1-2) 100 78.2 - 100 20 7.7 - 38.6 37.5 1002 (3-4) 93.3 68.1 - 99.8 56.7 37.4 - 74.5 51.9 95.43 (5-10) 93.3 68.1 - 99.8 70 50.6 - 85.3 60.9 95.54 (>10) 66.7 (83.3*) 38.4 - 88.2 96.7 82.8 - 99.9 90.9 85.3 (93.1*)Table 1: Sensitivity and specificity values for each LBC grade to identify a DCCsputum eosinophil count of ≥3% for all slides. *Values for sensitivity and NPV afterexcluding slides with low viability/squamous contamination (n=6)Figure 1: Empirical ROC curve showing the sensitivity (%) and 100-specificity (%) of LBCderived eosinophil count (eos/hpf) to identify a sputum differential eosinophil count of ≥3% for all  samples and after exclusion of samples with ≤50% viability and/or ≥20% squamous contamination*DiscussionOur results indicate that a cut-off point of >10 eos/hpf from the LBC technique has ahigh PPV and NPV to identify patients with a sputum differential eosinophil count of ≥ or <3%. This is important in asthma management because this value is associated withincreased risk of exacerbations and can be modified with additional oral or inhaledcorticosteroids(1).Sputum differential cell counts have demonstrated utility in guiding treatment decisionsin patients with more severe asthma(1) but the DCC technique has not been widelyadopted into clinical practice because of the time and technical expertise required toperform it. Any novel method of assessing sputum eosinophilia must therefore becompared with DCC. However, differences in results do not necessarily mean the DCCmethod is superior to the LBC approach. Firstly, it cannot be excluded that differences ineosinophil counts produced by the two methods were due to heterogenous distribution ofthe cellular portion of the sample, resulting in a higher eosinophil concentration in onepart of the sample than the other. Secondly, the DCC technique is not subject to theformal quality control procedures in place for laboratory investigations performed in theNHS. It remains possible that LBC with an eosinophil count performed by a consultanthistopathologist is more accurate than research scientists performing DCC. LBC also hassome practical advantages: 1) samples do not have to be processed on the day ofcollection as for DCC(6) because CRR acts as a mucolytic and a fixative and 2) the LBCtechnique is less time intensive making it more suitable for use in NHS histopathologydepartments.The next step is to demonstrate that these results can be reproduced at other centresand that treatment decisions based on LBC are beneficial in terms of asthma outcomesas has been shown for DCC’s(1). These studies should include blood eosinophil countswhich are more easily obtained and have been demonstrated to be a good predictor ofsputum eosinophilia in subjects undergoing COPD exacerbations(7) and a moderatepredictor of sputum eosinophilia in asthma(8). The reliability of LBC eosinophil countsfrom mucopurulent sputum samples from subjects with COPD also needs to be assessed.References:1. Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P, et al.Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial.Lancet. 2002;360(9347):1715-21.2. Gibson PG, Dolovich J, Denburg J, Ramsdale EH, Hargreave FE. Chronic cough:eosinophilic bronchitis without asthma. Lancet. 1989;1(8651):1346-8.3. Pavord ID, Pizzichini MM, Pizzichini E, Hargreave FE. The use of induced sputumto investigate airway inflammation. Thorax. 1997;52(6):498-501.4. Petsky HL, Cates CJ, Lasserson TJ, Li AM, Turner C, Kynaston JA, et al. Asystematic review and meta-analysis: tailoring asthma treatment on eosinophilicmarkers (exhaled nitric oxide or sputum eosinophils). Thorax. 2012;67(3):199-208.5. Ward R, Woltmann G, Wardlaw AJ, Pavord ID. Between-observer repeatability ofsputum differential cell counts. Influence of cell viability and squamous cellcontamination. Clin Exp Allergy. 1999;29(2):248-52.6. Efthimiadis A, Jayaram L, Weston S, Carruthers S, Hargreave FE. Inducedsputum: time from expectoration to processing. Eur Respir J. 2002;19(4):706-8.7. Bafadhel M, McKenna S, Terry S, Mistry V, Reid C, Haldar P, et al. AcuteExacerbations of Chronic Obstructive Pulmonary Disease Identification of BiologicClusters and Their Biomarkers. Am J Respir Crit Care Med. 2011;184(6):662-71.8. Korevaar DA, Westerhof GA, Wang J, Cohen JF, Spijker R, Sterk PJ, et al.Diagnostic accuracy of minimally invasive markers for detection of airway eosinophilia inasthma: a systematic review and meta-analysis. Lancet Respir Med. 2015;3(4):290-300.Subjects with asthma Subjects with chroniccoughTotal number recruited to study 43 12Total number who producedadequate samples forprocessing41 8Total number who produced‘countable’ samples38 7Mean age (range) a 55 (26-76) 59 (41-79)Sex: male (%) 27 (62.8) 5 (41.7)Ethnic group:White Or White British (%) 43 (100) 12 (100)Mean FEV1 % predicted (SD) 63 (15) 73 (12)Mean FEV1/FVC % (SD) 67 (23) 107 (25)Median blood eosinophil countx109/L (IQR)0.75 (3.25) 1.9 (2.8)Table S1: Demographics and clinical measures of study subjects with asthmaand chronic coughABFigure S1: Cytological analysis of sputum samples from the study. Eosinophilsare indicated with arrows. (A) A cytocentrifuge slide prepared by the standard(DCC) technique (Giemsa stain, original magnification x400) (B) A slideprepared by the LBC technique (Papanicolaou stain, original magnificationx400)

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Assessment of a rapid liquid-based cytology method for measuring sputum cell counts

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