Prevalence of Different Haptoglobin Phenotypes in Patients With COPD- Frequent Exacerbators Versus Non Exacerbators

Laboratory Procedures:

Overview:

3 ml of Serum will be collected and stored at 4 degrees Celsius (up to one week) or minus 20 degrees Celsius until delivery to the laboratory.

Haptoglobin phenotype will be determined at the Department of Anatomy and Cell Biology, the B. Rappaport Faculty of Medicine, 1 Efron st. Haifa, Israel. The method for phenotypin Hp is protein gel electrophoresis.

Serum remaining will be kept frozen in minus 70 degrees Celsius for 5 years in a freezer shelf dedicated for the Pulmonology Institute at the Serology Laboratory in Carmel Medical Center for future analysis of proteins.

Haptoglobin Electrophoresis:

The Haptoglobin protein is separated based on size. Hp 2-2 is the largest therefore was expected to travel the shortest distance in the gel and Hp 1-1 is the smallest and therefore would travel the farthest down the gel. The gel that is used for the electrophoresis was a Polyacrylamide gel. Two concentrations are used in order to provide loading and separating conditions.

The lower gel is a Polyacrilamide gel that contains 10.8 ml 1M Tris pH 8.8, 3.55 ml of Acrylamide 40%, 11.5 ml of dH2O, 225 μl of Ammonium persulfate (APS) 10%, and 18 μl of tetramethylethylenediamine (TEMED). The upper gel is a Polyacrylamide gel that contains 937.5 μl of 1M Tris pH 6.8, 787.5 μl of Acrylamide 40%, 5.7 ml of dH2O, 75 μl of APS 10%, and 7.5 μl of TEMED.

The lower gel is poured in between two glass plates and left for 30 minutes to solidify. Once lower gel is solidified upper gel is poured on top of it, and left for 30 min to solidify. Wells comb placed superiorly to upper gel in order to create wells for sample placement.

The sample for each patient is prepared using 10 μl of the serum, 2 μl of Hemoglobin, and 12 μl of a commercial loading buffer. The sample is then carefully placed in the prepared wells using a pipette. 4 wells are reserved for 3 controls of Hp 2-2, Hp 2-1, and Hp 1-1 followed medially by an empty well.

The western blot analysis is run in a Protean II xi cell by BIO-RAD. The apparatus is assembled and an electrophoresis running buffer is added. The running buffer contains 15.1 g of Tris, 72 g of Glycine, and 1000 ml of DDW. The running buffer was then placed in the upper and lower reservoir of the Protean II. The electrophoresis was run at 240 Volts for 3 hrs.

Once the samples are finished running, the gel is removed from between the glass plates. The gel was then stained using a color staining solution. The Solution was made using 40 mg of 3,3'-5,5' tetramethyl benzidine and 20 ml of methanol. This was left to mix for 15 minutes. Next 2 ml of dimethyl sulfoxide is added and left to mix for 5 min. This was followed by the addition of 40 ml of acetic acid 5% which is made of 2 ml AA and 38 ml of DDW. Next 40 mg of potassium fericyanide dissolved in 4 ml of DDW are added. Finally 600 μl of H2O2 is added to the staining solution and left to mix for 10 min.

Once the staining solution is ready, the gel is placed in a 22 cm by 32 cm tray and the staining solution is poured over the gel. The tray is then placed on a shaker for 25 minutes. Once the Gel is finished being stained it is legible. Nevertheless, the gel is photographed for image enhancement and data storage. The gel is removed from the tray and placed in an Image Analyzer (Imagequant LAS 4000) for imaging, or Canon powershot sx40 hs digital camera.

Data Tabulation:

The data - the Haptoglobin phenotype with corresponding IDs - is gathered and tabulated into a Microsoft excel table.

Inclusion criteria

• Patients diagnosed with COPD according to GOLD guidelines1
• Age 40-100 years
• Smoking history of > 10 pack - years
• Spirometry consistent with airflow obstruction (FEV1/FVC ratio < 70%)
• Moderate to severe airflow obstruction (FEV1<60%)
• At least two episodes of acute exacerbations in the former 12 months, or no exacerbations in the former 24 months. (Acute exacerbations are defined as worsening symptoms requiring treatment with systemic steroids (oral or parenteral) or antibiotics, a visit to the emergency room, and/or admission to a hospital. Events separated by at least 21 days are considered as separate events of exacerbation.)

Exclusion criteria

Patients with a history of any of the following conditions will be excluded from the study:

• Active Tuberculosis
• Pulmonary fibrosis or Asbestosis
• Organ transplantation
• Lung volume reduction surgery
• Previous lung or lobe resection.