Laboratory Methods

2.3.1 Serum 25-hydroxyvitamin D

Serum 25-hydroxyvitamin D level was measured in all subjects using a 2 dimensional high performance liquid chromatography system - tandem mass spectrometry (2D LC-MS-MS), by the biochemistry department at the RBH.

Serum 25-hydroxyvitamin D levels are considered the best circulating biomarker of vitamin D metabolic status because this form has a longer half-life (2-3 weeks) than 1α,25VitD3 (6-12 hours) and reflect contributions from all sources of vitamin D (i.e. diet and sun exposure).³⁵¹ Serum 25-hydroxyvitamin D levels were measured in duplicate and the lowest limit of detection was 3 nmol/L and upper limit of detection was 100nmol/L.

Vitamin D deficiency was defined as serum 25-hydroxyvitamin D <50nmol/L (20ng per milliliter)^{185, 352} and serum levels between 50-75 nmol/L (20-30 ng per millilitre) were defined as vitamin D insufficiency.^{185, 352} Based on changes in parathyroid hormone levels and intestinal calcium transport values of less than 75nmol/L have been suggested as insufficient.¹⁸⁵ The date the 25-hydroxyvitamin D level was measured was recorded to assess seasonal variation between subjects. Subjects were grouped as follows; winter (December to February), spring (March to May), summer (June to August) or autumn (September to November).

2.3.2 Serum total and allergen specific IgE

Serum IgE was analyzed by Beckman Access 2 immunoassay analyser and allergen-specific IgE levels (normal <0.35 IU/ml) to cat, dog, tree pollen, Dermatophagoides pteronyssinus, Aspergillus fumigatus, grass, egg, milk and peanut were analyzed by Phadia Immunocap 250 analyser in the biochemistry department at the RBH. Recently it has been postulated that it is

useful to quantify atopy rather than just reporting it as present or absent. This was done by summing the results of specific IgE for food and aeroallergen.³⁵³

2.3.3 Sputum induction

For subjects with a post-bronchodilator FEV1 > 65% predicted, sputum induction was performed using 3.5% saline inhalation for four 5 min periods as part of clinical investigations. For subjects with a post-bronchodilator FEV1

< 65% predicted, sputum induction was performed with 0.9% saline. I performed sputum induction and processing in 10 patients and in the remaining 12 patients was performed by Ms Adesimbo Sogbesan, sputum technician at RBH.

Children were asked to blow their nose and rinse their mouth to reduce squamous cell contamination before sputum induction was started. The children were instructed how to cough and clear their throat in order to obtain the best sample. The recorded baseline FEV1 was the highest manoeuvre from 3 satisfactory spirometric curves. Children were seated comfortably and asked to inhale the saline solution for periods of increasing duration from an ultrasonic nebuliser (De Vilbliss 2000, Somerset). FEV1 was measured after each inhalation period and the higher of 2 readings was selected. After each 5 minute nebulisation period children were encouraged to cough and expectorate sputum into a falcon tube. If the FEV1 had not fallen below 10-15% of the baseline value, further aliquots of 3.5% saline were nebulised.

Symptoms such as cough or wheeze were recorded at each stage. If an inadequate sample of sputum was produced the process was continued for up to a maximum of 15 minutes.

Children whose FEV1 had decreased by more than 15% were given salbutamol (1000µg) through a large volume spacer and spirometry was repeated 10 minutes later to ensure return to baseline values before discharge.

Sputum processing

Sputum samples were stored on ice until ready for processing. All samples were processed in less than 4 hours. The sample was poured into a petri dish and sputum was selected by identifying mucoid plugs and gelatinous portions.

A selected rather than whole sample processing method was used to reduce salivary contamination because this improves intra-observer repeatability of differential cell counts.¹¹⁹ The selected sputum was returned to a clean pre-weighed tube and pre-weighed using a microbalance. A stock dithiothreitol (DTT) (0.1%) was then added to the sputum in the ratio of 4 ml DTT to 1g sputum.

The mixture was gently aspirated with a 3ml disposable wide bore plastic pipette and then gently agitated on a rolling mixer (DenleySpiramix 5, Denley Instruments, Colchester, UK) for 15 minutes. The sample was filtered using a 48µm pore nylon mesh filter. Phosphate buffered saline (PBS) (in the ratio of 4 ml PBS to 1g original sputum weight) was used to wash out the tube and added to the filtrate (through the filter). The filtrate was centrifuged at 4°C for 10 minutes at 400g (1500rpm). The remaining pellet was re-suspended in 1ml PBS and mixed by gentle vortexing. 20µl of sputum cell suspension was added to 20µl trypan blue (1 in 2 dilution factor). 10µl of the mixture was placed on a haemocytometer and examined using a light microscope (Leitz, Wetzlar, Germany) (improved Neubauer, BDH, Leicestershire, UK). All leukocytes (viable stained yellow and dead stained blue) and squamous cells were counted in the bottom left middle and top right quadrants of the haemocytometer grid. The total leukocyte count per ml was calculated using the following formula:

Total count = no of viable & dead leukocytes X trypan blue dilution factor X10⁴

After calculation of the total leukocyte count the sputum cell suspension was adjusted to 200,000 cells/ml by diluting with PBS. The mixture was centrifuged (Shandon Cytospin Preparation System, Thermo Shandon, Cheshire, UK) at 450rpm for 3 min. If the total leukocyte count was less than 2 x 10⁵ /ml aliquots of 200µl or 400µl of sputum cell suspension were loaded. 1- 4 slides were left to air dry for 30 minutes and then fixed with methanol. The slides were then stained with a modified Wright Giemsa stain Reastain®

Quick-Diff staining kit (Reagena Ltd. Toivala, Finland). Characteristic images of cells are shown in Figure 2.1

Figure 2.1 Characteristic appearances of sputum cytology shown under light microscopy (magnification x400) (a) normal count showing macrophages (b) eosinophilic (c) neutrophilic.

A" " " " B" " " " C"

Photograph taken by Dr Cara Bossley.

Cell counts

The 400 cells were counted and expressed as % total inflammatory cells and total cell counts. Eosinophilia was defined as ≥2.5% eosinophils; neutrophilia as ≥54% neutrophils.³⁵⁴

The intra-observer sputum eosinophil variability, percentage of eosinophils counted in the same slides on 3 separate occasions from 3 different subjects (blinded to previous results) is shown in Table 2.1

Table 2.1. Intra-observer sputum eosinophils repeatability. This show acceptable coefficient of variation (CoV) of less than 10%

Slide Count 1 Count 2 Count 3 Average CoV

1 4 3.7 4.3 4 7.5

2 11 9.8 10.4 10.4 5.8

3 2 2 2.2 2.1 5.6

Mean CoV 6.2 CoV-Coefficient of variation