OF WORKING GROUP 3 Methods of sputum processing for cell counts , immunocytochemistry and in situ hybridisation Leader of the Working Group :

Since the first attempts to use standardised methods for sampling induced airways sputum, two methods for processing the expectorate have evolved. The first involves selecting all viscid or denser portions from the expectorated sample with the aid of an inverted microscope [1, 2]. This method has been extensively evaluated and reported in detail [2–4]. The second approach involves processing the entire expectorate, comprising sputum plus variable amounts of saliva [5]. Recent modifications to this method include collecting saliva and sputum separately in order to reduce salivary contamination [6–8]. Both methods have advantages and disadvantages. The advantages of using selected sputum are: squamous cell contamination is v5%, making cell counting easier and quicker to perform, the total cell count (TCC) can be expressed per gram of lower airway secretions, and concentrations of chemicals in the fluid phase are unaffected by the confounding influence of saliva, and can be accurately corrected for dilution. The disadvantage is that selection takes a few minutes longer to perform and requires an inverted microscope. The advantage of using the entire expectorate is that the technique is quicker to perform, but there are some disadvantages that require consideration. The expectorate contains a variable mixture of sputum plus saliva which may dilute the sputum and confound its analysis. The reproducibility of cell counts has been reported to be lower if squamous cell contamination represents w20% of all recovered cells [4]. There is conflicting data as to whether or not differential cell counts (DCCs) differ between the two methods. One study reported a higher percentage of eosinophils in sputum processed by the selection method compared to the entire expectorate [9] but this has not been confirmed in other studies [2, 6, 10]. Although, both the selected sputum and the entire expectorate methods have the same ability to distinguish asthmatics or bronchitics from healthy subjects, they are not interchangeable, and, once a technique has been adopted for a given study, it should always be applied. When evaluating the most appropriate method for processing sputum for cellular analysis, a number of issues need to be considered: 1) sputum sample homogenisation; 2) duration and temperature of homogenisation; 3) volume and concentration of added mucolytic; 4) sample filtration; 5) TCC and viability; 6) centrifugation and storage of supernatant; 7) cytospin centrifugation, staining and counting; 8) metachromatic cell staining and counting; 9) slide reading quality control; 10) immunocytochemical staining; and 11) in situ hybridisation. Figure 1 is representative of the two methods of processing to yield accurate cell counts.