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Instruction Manual Cellometrics Inc. PO Box 89053

991 King Street West Hamilton ON Canada

L8S 4R5289-828-0488

www.cellometrics.com

ACCUFILTER® Sputum Processing Kit

The ACCUFILTER® is a sputum filtration device for in -vitro use to diagnose and guide treatment of airway diseases. It is presented in a kit with a detailed procedure which, if followed, standardizes the processing of freshly expectorated spontaneous or induced sputum, for measurement of inflammatory cells or other components. Interpretation must be made within the clinical context.

Portions of sputum free of saliva are selected and mixed with a solution of dithiothreitol to disperse the mucus. This is then filtered to obtain a homogeneous suspension of cells from which reliable measurements of quantitative non-squamous total and differential cell counts, cell viability or other cell or supernatant components can also be made.

• Dithiothreitol stock solution (DTT)

• Trypan Blue Stain 0.4%

• Distilled Water (D-H2O)

• Phosphate Buffered Saline (PBS)

• 10 - ACCUFILTER® devices (including ACCUFILTER® Collection Vial, Housing and Receiving Vial)

• Sputum Processing and Examination Worksheet

ACCUFILTER® Sputum Processing Kit includes reagents and materials including:

Manufactured by Southmedic Inc. for Cellometrics Inc.

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1. This procedure is to be done on the freshly expectorated sample. If the sample is to be examined for quantitative cell counts it can be processed within 8 hours providing it is stored in the fridge at 4ºC.

Note: If supernatant collection is required, sputum should be processed within 2 hours of collection

2. Pour entire expectorated sample into a tissue culture dish, and record its macroscopic characteristics (colour) on the provided Sputum Processing and Examination Worksheet .

3. Record the weight of the Receiving Vial (Figure 2) including the lid and set aside. This weight will be required to calculate the amount of filtrate recovered.

4. Using fine end forceps, macroscopically select portions of sputum, which appear denser then saliva, opaque or colored, and place these on the lid of a tissue culture dish for inspection under an inverted microscope.

5. Under an inverted microscope (100 x magnification), spread thin the selected portion and examine the chosen portion to ensure that there are none to very few squamous cells.

6. Zero the weight of the empty ACCUFILTER® Collection Vial (Figure 2), remove from the scale and use forceps to insert 0.100-0.300 g of the selected squamous cell-free sputum, into the vial. Greater quantities of sputum will be incompletely dispersed and will give inaccurate cell counts.

7. Obtain the weight of selected sputum portion and record in (a) on the Sputum Processing and Examination Worksheet.

8. Remove the dithiothreitol (DTT) (amber glass vial) from the kit and prepare a sufficient amount of working solution for the weight of selected sputum to be processed (a).

9. The working solution is prepared by adding 9 parts distilled water to one part of DTT into a tube.

10. Vortex the working solution for 15 sec and set aside.

11. Add a volume of DTT working solution equal to four times the weight of selected sputum into the ACCUFILTER® Collection Vial. Example: 0.08 ml DTT + 720 ml distilled water will provide 0.8ml of DTT working solution which is a sufficient amount for processing .200g of selected sputum.

12. Vortex for 30 sec.

13. Mix solution and sputum gently up and down several times with a disposable transfer pipette, to ensure adequate homogeneity.

14. Place the ACCUFILTER® Collection Vial on a tube rocker and rock for 15 min at room temperature to allow dispersion of mucus.

15. Add a volume of PBS equal to four times that of the selected sputum weight and cap the tube tightly. Example: 0.200 g of sputum add, 0.8 ml PBS. Note: Cap PBS vial immediately after use to limit possible bacterial contamination.

16. Vortex 15 sec and set aside.

17. Obtain an ACCUFILTER® Housing unit and pre-wet the filter with 0.5 ml of PBS. Dispense PBS directly into the conical filter and shake off the excess into a discard container.

Note: Pre wet filter immediately prior to filtration.

18. Remove the cap from the pre-weighed Receiving Vial, and screw the Receiving Vial to the pre-wet ACCUFILTER® Housing, and place it securely in a tube rack.

19. To filter the sample, insert the ACCUFILTER® Collection Vial into the ACCUFILTER® Housing and press firmly to break the diaphragm located on the bottom of the collection vial.

20. Allow the cell suspension to filter through into the Receiving Vial for 3-5 minutes, then tap on bench

The process has not been established in other fluids although it is likely to be effective.

The process can interfere with measurements of certain proteins.

Common Lab Materials not provided in kit include:

• Single-channel precision pipettes with disposable tips: 20uL, 200uL, 1000uL

• Fine end forceps

• Tissue culture dishes

• Disposable transfer pipettes

• Micro tubes - 1.5 ml

• Test tube racks

• Timer

• Tube rocker

• Inverted microscope

• Light microscope

• Haemocytometer and cover glass

• Precision balance

• Differential cell counter

• Glass slides

• Cytocentrifuge

• Wright’s or Geimsa stains

Storage and Shelf Life

Store kit at room temperature 15–25 0C.

All performance claims are void after expiration date of reagents.

SPUTUM PROCESSING PROCEDURE (page 1)Please Note

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to receive any remaining drops of suspension.

21. Unscrew the Receiving Vial containing the filtered cell suspension, cap tightly and label.

22. Discard the ACCUFILTER® Collection Vial and attached filter Housing into a biological hazard waste bin.

23. Obtain the total weight of the filtered cell suspension and Receiving Vial and subtract the weight of the empty Receiving Vial to obtain the weight of the filtered cell suspension. Record in (b) on the Sputum Processing and Examination Worksheet.

24. Mix 10 µl of trypan blue stain with 10 µl of the filtered cell suspension in a micro tube and vortex.

25. Flood a haemocytometer with 10 µl of the trypan-blue suspension and count the non-squamous, viable and non-viable cells separately on the entire counting chamber 9 sq mm ruled grid area labelled 1-9 (Figure 3). Note: All non-viable cells stain blue and viable cells do not.

26. Record the total number of viable and non-viable non-squamous cells in chamber as (c) on the Sputum Processing and Examination Worksheet.

27. Calculate the absolute number of non-squamous cells retrieved by multiplying (b)x(c) and record as (d) on the Sputum Processing and Examination Worksheet (Figure 4).

28. Calculate total non-squamous cell count per sputum weight = (d)÷(a).

29. Determine the non-squamous cell viability, express in percent and record. Example: Cell viability % = viable leukocytes x 100 ÷ ( viable + non-viable leukocytes)

Note: If supernatant collection is not needed, skip 30-34 and go to step 35

30. Centrifuge the cell suspension at 3000 rpm (circa 600 G) for 4 min.

31. Remove supernatant with a disposable transfer pipette leaving behind about 50 µl of fluid and the undisturbed pellet.

32. Aliquot equal volumes of supernatant into micro tubes as required and store at -700C until needed.

33. Re-suspend cell pellet with PBS to a concentration of 1.0 x 106

cells/ml.

34. Vortex 15 sec.

35. Adjust cell suspension with PBS if count in (c) exceeds 1.0 x 106, to achieve the desired concentration of 1.0 x 106 cells/ml.

36. Prepare cytospins by adding 60 – 70 µl of the 1.0 x 106 cells/ml suspension, into a cytocentrifuge and spin at 450 rpm (22 G) for 6 min.

37. Allow cytospins to air-dry and examine slide preparation for minimum to no overlap of cells under a light microscope using 400 x magnification, prior to staining.

Note: If cytospin staining is not being done locally, stop at step 37.

38. Stain with Wright’s or Giemsa, allow to air dry and cover slip.

39. Perform a differential on 400 consecutive stained cells to determine proportion of neutrophils, eosinophils, macrophages, lymphocytes and bronchial epithelial cells.

40. Note presence of free eosinophil granules which occur with sputum eosinophilia.

41. Note presence of macrophage inclusions most commonly seen in current or former smokers.

42. Standard laboratory practices for handling and disposing of biohazardous materials, specimens and specimen contacting disposables should be followed.

1. Process the freshly expectorated sample for cell or fluid phase measurements. If there is delay, quantitative cell counts are unchanged if the sample is stored at 4ºC and processed within 8 hours.

2. Select sputum carefully to eliminate squamous cells as much as possible. This will ensure the accuracy of the cell counts.

3. Do not exceed the 0.30 g, of selected sputum for processing. This may result in inadequate cell dispersion and decreased cell yield.

4. All reagent vials should be capped immediately after use.

5. DTT working solution must be made up fresh daily. Stability is 24 h.

6. Stock DTT should be clear and colourless. Colour or clarity changes are indicative of reagent instability.

7. Differential cell counts need to be done by technologists trained in cell morphology. This is to ensure accuracy of cell staining, morphological assessment of the cells, and recognition of other technical errors.

8. Wright’s or Giemsa stains are used according to manufacturer specifications, and the pH is closely monitored to ensure correct balance of color.

SPUTUM PROCESSING PROCEDURE (page 2) Please Note

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A device used for determining the number of cells per unit volume of a suspension is called a counting chamber. The most widely used type of chamber is called a haemocytometer, since it was originally designed for performing blood cell counts.

To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid.

The coverslip is placed over the counting surface prior to putting on the cell suspension. The suspension is introduced into one of the V-shaped wells with a Pasteur or other type of pipet. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the microscope stage and the counting

Figure 3Improved Neubauer Haemacytometer

Interpretation Of ResultsQuantitative sputum non-squamous cell counts carried out by the method described here are valid, reproducible (reliable) and responsive, the qualities of good measurements. They provide the most comprehensive and specific information currently clinically available of the presence, type and severity of airway bronchitis. It is applicable to all chronic airway diseases including the bronchitis of chronic cough and the bronchitis component of asthma, chronic obstructive pulmonary disease (COPD), emphysema, cystic fibrosis, and bronchiectasis. Normal reference values are well described.

The different types of inflammation include eosinophilic, neutrophilic, both of these together and paucigranulocytic. The first three types precede clinical exacerbations and guide corticosteroid or antibiotic treatment. When sputum eosinophils are used to identify minimum maintenance corticosteroid treatment, eosinophilic and perhaps neutrophilic exacerbations are reduced in patients with asthma or COPD.

Sputum cell counts are useful in clinical practice in asthma, COPD, cough and bronchiectasis especially when disease is moderate-severe or difficult to control,

• to predict benefit from steroid treatment,

• to establish minimum steroid dose and reduce exacerbations,

• to identify infective bronchitis,

• to guide treatment of eosinophilic or infective exacerbations, and

• to diagnose occupational eosinophilic bronchitis with or without asthma.

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Bibliography

Methods

1. Efthimiadis A, Pizzichini E, Pizzichini MMM, Hargreave FE. Sputum examination for indices of airway

inflammation: laboratory procedures Astra Draco AB, Lund, Sweden 1997

2. Pavord ID, Pizzichini MMM, Pizzichini E, Hargreave FE. The use of induced sputum to investigate airway inflammation. Thorax 1997; 52:498-501

3. Kelly MM, Efthimiadis A, Hargreave FE. Induced Sputum: Selection method. In: DF Rogers & LE Donnelly, eds. Methods in molecular medicine. Human airway inflammation: sampling techniques and

analytical protocols. Humana Press, London; 2001:77-92

4. Efthimiadis A, Spanevello A, Hamid Q, Kelly MM, Linden M, Louis R, Pizzichini M, Pizzichini E, Ronchi C, Overveld FV, Djukanovic R, Methods of sputum processing for cell counts, immunocytochemistry and in situ hybridisation, Eur Respir J 2002; 20:1-5

Normal Values

5. Cai Y, Carty K, Henry RL, Gibson PG. Persistences of sputum eosinophilia in children with controlled asthma when compared with healthy children. Eur Respir J 1998; 11: 848-53

6. Belda J, Leigh R, Parameswaran K, O=Byrne PM, Sears MR, Hargreave FE Induced sputum cell counts in healthy adults. Am J Respir Crit Care Med 2000; 161:475-8

7. Spanevello A. Confalonieri M, Sulotto F, Romano F, Balzano G, Migliori GB, Bianchi A, Michetti G. Induced sputum cellularity. Reference values and distribution in normal volunteers. Am J Respir Crit Care Med 2000; 162: 1172-4

Clinical Uses

8. Jayaram L, Parameswaran K, Sears MR, Hargreave FE. Induced sputum cell counts: their usefulness in clinical practice. Eur Respir J 2000; 16:150-8

9. Brightling CE, Chronic Cough due to asthmatic eosinophilic bronchitis; ACCP evidence-based clinical practice guidelines. Chest 2006; 129:116S-21S

10. Siddiqui S, Brightling CE, Airway disease: phenotyping heterogeneity using measures of airway inflammation. J Allergy, Asthma, and Clinical Immunol, 2007; 3: 60-9

11. Hargreave FE, Nair P. The definition and diagnosis of asthma. Clin Exp Allergy. 2009; 39: 1652-8

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12. Gibson PG, Inflammatory phenotypes in adult asthma: clinical applications. Clin Respir J 2009; 3:

198-206

13. Nair P, Hargreave FE. Measuring bronchitis in airway diseases: clinical implementation and application. Chest 2010; 138 (suppl): 385-435.

Research Application

14. Jayaram L, Pizzichini MM, Cook RJ, et al. Determining asthma treatment by monitoring sputum cell

counts; effect on exacerbations. Eur Respir J 2006; 27:483-94

15. Kelly MM, Leigh R, Jayram L, Goldsmith CH, Parameswaran K, Hargreave FE. Eosinophilic bronchitis in asthma: a model for establishing does-response and relative potency of inhaled corticosteroids. J Allergy Clin Immunol 2006; 117:989-94

16. Pavord ID, Brightling CE, Woltmann G, Wardlaw AJ. Non-eosinophilic corticosteroid unresponsive

asthma. Lancet 1999; 353: 2213-14

17. Jayaram L, Pizzichini E, Lemiere C, Man SFP, Cartier A, Hagreave FE, Pizzichini MMM. Steroid naïve eosinophilic asthma: anti-inflammatory effects of fluticasone and montelukast. Thorax 2005; 60: 100 – 5

18. Boulet LP, Gauvreau G, Boulay ME, O’Byrne P, Cockcroft DW, Clinical Investigative Collabration, Canadian Network of Centers of Excellence AllerGen, The allergen bronchoprovocation model: an

important tool for the investigation of new asthma anti-inflammatory therapies, Allergy 2007: 62: 1101-10

19. Leigh R, Pizzichini MMM, Morris MM, Maltais F, Hargreave FE, Pizzichini E. Stable COPD: predicting benefit from high-dose corticosteroid treatment. Eur Respir J 2006; 27: 964-71

20. Girard F, Chaboillez S, Cartier A, Cote J, Hargreave FE, Labrecque M, Malo JL, Tarlo SM, Lemiere C.

An effective strategy for diagnosing occupational asthma: Use of induced sputum. Am J Respir Crit Care Med 2004; 169:845-50

21. Nair P, Pizzichini MM, Kjarsgaard M, Inman MD, Efthimiadis A, Pizzichini E, Hargreave FE, O'Byrne PM. Mepolizumab for prednisone-dependent asthma with sputum eosinophilia. N Engl J Med 2009; 360: 985-93

22. Haldar P, Brightling CE, Hargadon B et al. Mepolizumab and exacerbations of refractory eosinophilic asthma. N Engl J Med 2009; 360: 973-84.

23.

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Diagnostic Use of Sputum Cell Counts

Normal Values

Total cell count < 9.7 x 106/g (ml)Eosinophils < 2%, Neutrophils < 64.4%

Eosinophilic Bronchitis Identified by eosinophils > 3%. This can be associated with symptoms and is a predictor for exacerbations.

Infective BronchitisA raised total cell count is suggestive of an infective bronchitis. The count tends to be higher in bacterial than viral causes.

In bacterial infections, the neutrophils tend to be >80%. In viral infections they tend to be lower.

Combined Infective and Eosinophilic BronchitisThis is indicated by a raised total cell count and proportion of neutrophils and eosinophils.

Trivial NeutrophiliaThis is indicated by a normal total cell count and raised proportion of neutrophils. It can result from many stimuli (e.g. smoking and atmospheric pollutants) and is of uncertain clinical significance.

Treatment Options When Using Sputum Cell Counts

Normal Counts If the cell counts are normal and the patient is not on steroid treatment, current symptoms may be due to variable airflow limitation (and require treatment with long-acting beta-agonist or leukotriene antagonists) or to other conditions.

Eosinophilic BronchitisAn eosinophilia > 3% predicts improvement with adequate steroid treatment in asthma, COPD and chronic cough. If eosinophils are zero (or <1%) and the patient is being treated with corticosteroid, consideration should be given to a trial of reducing the dose.

Infective BronchitisConsideration needs to be given for a course of antibiotic. An infective neutrophilia can mask an associated eosinophilia; the presence or absence of this can be investigated when the neutrophilia has resolved.

Combined Infective and Eosinophilic BronchitisTreatment with an antibiotic and the addition (or increase) in steroid treatment is indicated.

Trivial NeutrophiliaIf the patient is a smoker, encouragement should be given to quit.

Accufilter® Clinical Use and Interpretation of Sputum Cell Counts

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ACCUFILTER ® SPUTUM PROCESSING AND EXAMINATION WORKSHEETACCUFILTER ® SPUTUM PROCESSING AND EXAMINATION WORKSHEETACCUFILTER ® SPUTUM PROCESSING AND EXAMINATION WORKSHEETACCUFILTER ® SPUTUM PROCESSING AND EXAMINATION WORKSHEETACCUFILTER ® SPUTUM PROCESSING AND EXAMINATION WORKSHEETACCUFILTER ® SPUTUM PROCESSING AND EXAMINATION WORKSHEETACCUFILTER ® SPUTUM PROCESSING AND EXAMINATION WORKSHEETACCUFILTER ® SPUTUM PROCESSING AND EXAMINATION WORKSHEET

Date:

Type: SpontaneousSpontaneous InducedInducedInducedInduced

Colour: Colourless White Green Yellow Brown Red

Sputum ProcessingSputum ProcessingSputum ProcessingSputum ProcessingSputum ProcessingSputum ProcessingSputum Processing

Used Inverted Microscope : Yes / NoUsed Inverted Microscope : Yes / NoUsed Inverted Microscope : Yes / NoUsed Inverted Microscope : Yes / No

a = Weight of portion to be processed = 0. _____________ gramsa = Weight of portion to be processed = 0. _____________ gramsa = Weight of portion to be processed = 0. _____________ gramsa = Weight of portion to be processed = 0. _____________ gramsa = Weight of portion to be processed = 0. _____________ gramsa = Weight of portion to be processed = 0. _____________ gramsa = Weight of portion to be processed = 0. _____________ grams

b = Weight suspension after filtration = ________________ ml b = Weight suspension after filtration = ________________ ml b = Weight suspension after filtration = ________________ ml b = Weight suspension after filtration = ________________ ml b = Weight suspension after filtration = ________________ ml b = Weight suspension after filtration = ________________ ml b = Weight suspension after filtration = ________________ ml

c = Total cell count in chamber x2.22 / 1000 = _______________ x 10^6 cells / mlc = Total cell count in chamber x2.22 / 1000 = _______________ x 10^6 cells / mlc = Total cell count in chamber x2.22 / 1000 = _______________ x 10^6 cells / mlc = Total cell count in chamber x2.22 / 1000 = _______________ x 10^6 cells / mlc = Total cell count in chamber x2.22 / 1000 = _______________ x 10^6 cells / mlc = Total cell count in chamber x2.22 / 1000 = _______________ x 10^6 cells / mlc = Total cell count in chamber x2.22 / 1000 = _______________ x 10^6 cells / ml

d = Absolute number of retrieved cells = ( b x c ) = _______________ x 10^6 cellsd = Absolute number of retrieved cells = ( b x c ) = _______________ x 10^6 cellsd = Absolute number of retrieved cells = ( b x c ) = _______________ x 10^6 cellsd = Absolute number of retrieved cells = ( b x c ) = _______________ x 10^6 cellsd = Absolute number of retrieved cells = ( b x c ) = _______________ x 10^6 cellsd = Absolute number of retrieved cells = ( b x c ) = _______________ x 10^6 cellsd = Absolute number of retrieved cells = ( b x c ) = _______________ x 10^6 cells

Processing Time :Processing Time :Processing Time :Processing Time : Observer:Observer:Observer:

Examination ResultsExamination ResultsExamination ResultsExamination ResultsExamination ResultsExamination ResultsExamination Results Reference Values

Viability : _____________ %Viability : _____________ %Viability : _____________ %Viability : _____________ %Viability : _____________ %Viability : _____________ %Viability : _____________ % > 40

Total leukocyte count per selected sputum weight = ( d / a ) : __________ x10^6 gramsTotal leukocyte count per selected sputum weight = ( d / a ) : __________ x10^6 gramsTotal leukocyte count per selected sputum weight = ( d / a ) : __________ x10^6 gramsTotal leukocyte count per selected sputum weight = ( d / a ) : __________ x10^6 gramsTotal leukocyte count per selected sputum weight = ( d / a ) : __________ x10^6 gramsTotal leukocyte count per selected sputum weight = ( d / a ) : __________ x10^6 gramsTotal leukocyte count per selected sputum weight = ( d / a ) : __________ x10^6 grams < 9.7

Squamous Cell: __________________ %Squamous Cell: __________________ %Squamous Cell: __________________ %Squamous Cell: __________________ %Squamous Cell: __________________ %Squamous Cell: __________________ %Squamous Cell: __________________ %Squamous Cell: __________________ %

Neutrophils : __________ %Neutrophils : __________ %Neutrophils : __________ %Neutrophils : __________ %Neutrophils : __________ %Neutrophils : __________ %Neutrophils : __________ % < 64.4

Eosinophils : ___________ % Free granules None Few Moderate ManyEosinophils : ___________ % Free granules None Few Moderate ManyEosinophils : ___________ % Free granules None Few Moderate ManyEosinophils : ___________ % Free granules None Few Moderate ManyEosinophils : ___________ % Free granules None Few Moderate ManyEosinophils : ___________ % Free granules None Few Moderate ManyEosinophils : ___________ % Free granules None Few Moderate Many < 2.0

Macrophages : __________ % Smokers inclusions None Few Moderate ManyMacrophages : __________ % Smokers inclusions None Few Moderate ManyMacrophages : __________ % Smokers inclusions None Few Moderate ManyMacrophages : __________ % Smokers inclusions None Few Moderate ManyMacrophages : __________ % Smokers inclusions None Few Moderate ManyMacrophages : __________ % Smokers inclusions None Few Moderate ManyMacrophages : __________ % Smokers inclusions None Few Moderate Many < 86.1

Lymphocytes : ___________ %Lymphocytes : ___________ %Lymphocytes : ___________ %Lymphocytes : ___________ %Lymphocytes : ___________ %Lymphocytes : ___________ %Lymphocytes : ___________ % < 2.6

Bronchial epithelial : ___________ %Bronchial epithelial : ___________ %Bronchial epithelial : ___________ %Bronchial epithelial : ___________ %Bronchial epithelial : ___________ %Bronchial epithelial : ___________ %Bronchial epithelial : ___________ % < 4.4

Observer :Observer :Observer :Observer : Date : Date : Date : Date :

< 100%

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