Practical Biomedical Engineering 2023/2024 PDF

Practical Biomedical Engineering 2023/2024 Week 43 Determination of Diagnostic Blood Parameters Persons in charge of this practical course: Prof. Dr. Thomas Lemmin, [email protected] and Prof. Dr. Christine Peinelt, [email protected] Institute of Biochemistry and Molecular Medicine T...

Practical Biomedical Engineering 2023/2024 Week 43 Determination of Diagnostic Blood Parameters Persons in charge of this practical course: Prof. Dr. Thomas Lemmin, [email protected] and Prof. Dr. Christine Peinelt, [email protected] Institute of Biochemistry and Molecular Medicine Technical assistance: Monika Rauch and Jennifer Jelk Overview Biochemistry practicals: General information ..............................................................................2 A. Practical work in groups ...........................................................................................................2 B. General working instructions....................................................................................................2 C. Protocol....................................................................................................................................2 1. Protein determination in human serum (Biuret method)...........................................................3 1.1 Practical work ...................................................................................................................3 1.2 Objectives of this practical work .......................................................................................3 1.3 Background.......................................................................................................................3 1.4 Protocol .............................................................................................................................3 2. Determination of hemoglobin concentration in human blood ...................................................6 2.1 Practical work ...................................................................................................................6 2.2 Objectives of this practical work .......................................................................................6 2.3 Background.......................................................................................................................6 2.4 Protocol .............................................................................................................................6 3. Serum electrophoresis .............................................................................................................8 3.1 Practical work ...................................................................................................................8 3.2 Objectives of this practical work .......................................................................................8 3.3 Background.......................................................................................................................8 3.4 Protocol ...........................................................................................................................10 4. Questions ...............................................................................................................................12 Practical Biomedical Engineering, 2023/2024 Week 43 Page 1 Biochemistry practicals: General information A. Practical work in groups Each student performs the experiment by herself/himself. Location: Course rooms Biochemistry, Anatomy / Cell Biology, 2nd floor, Course rooms 1-4, Baltzerstrasse 2, 3012 Bern B. General working instructions The equipment used in the practicals is costly and must be handled with proper care. Incorrect operation can result in expensive repairs. Pipettes must be handled with care. These pipettes have 2 pressure points (from top to bottom): 1) nominal suction volume, 2) additional piston strokes to completely pipet out the absorbed liquid. A lateral release will eject the pipette tip. Never pipet without a pipette tip! Begin by pipetting large volumes (1-2 ml) first, followed by smaller volumes. Safety: Experimental work in the laboratory is associated with certain health risks, even when exercising the utmost caution. It is mandatory to wear safety glasses and gloves at all times. Protective gloves and glasses are available in the laboratories. You have to pick up your lab coat prior to the first practical session. C. Protocol Each student is required to maintain a record of her/his laboratory work (protocol) throughout the practical session. The protocol must include at a minimum: - measurements - calculations - results - conclusions Practical Biomedical Engineering, 2023/2024 Week 43 Page 2 1. Protein determination in human serum (Biuret method) 1.1 Practical work Determination of protein concentrations in biological samples using a photometer. 1.2 Objectives of this practical work • Get used to accurately pipet small volumes • Gain knowledge in operating a photometer and understand the basics of photometry • Determine unknown sample protein concentrations using standards with known concentrations 1.3 Background Under alkaline conditions and in the presence of divalent cupper ions, proteins and oligopeptides, but not dipeptides and free amino acids, form purple-colored chelate complexes (Biuret method). Using a dilution series containing protein (albumin) standards of known concentrations, a relationship between absorbance (indicative of color intensity in the sample) and protein concentration will be established. A linear relationship is expected at absorbance values below 1.0, and the empirical correlation can be established either through graphical analysis or curve fitting. 1.4 Protocol Wavelength: Cuvette thickness: Assay temperature: Measurement: Practical Biomedical Engineering, 2023/2024 550 nm 1 cm Room temperature Determine sample concentrations against a reagent blank (used to set the photometer reading to extinction ZERO). Week 43 Page 3 Pipetting scheme and measurements: No. Probe 0.9% NaCl Standard µl µl Serum Biuret blue/red reagent mg protein / µl µl cuvette 1 Blank 500 0 - 2500 0 2 Standard 1 400 100 - 2500 1 3 Standard 1 400 100 - 2500 1 4 Standard 2 300 200 - 2500 2 5 Standard 2 300 200 - 2500 2 6 Standard 3 200 300 - 2500 3 7 Standard 3 200 300 - 2500 3 8 Standard 4 100 400 - 2500 4 9 Standard 4 100 400 - 2500 4 10 Serum blue 450 - 50 2500 ? 11 Serum blue 450 - 50 2500 ? 12 Serum red 450 - 50 2500 ? 13 Serum red 450 - 50 2500 ? Mixing: Cover cuvette with parafilm, turn upside down 3-4 times; do not shake. Color reaction: Keep the samples at room temperature for 15 min. Photometer: Please ask the staff for technical assistance if you have never used a photometer before! Measurements: Measure extinctions at a wavelength of 550 nm against a reagent blank. Calibration curve: Create a plot with extinctions on the y-axis and protein concentrations on the x-axis using the provided technical paper or a suitable calculation program. Data points are expected to form a linear trend. Practical Biomedical Engineering, 2023/2024 Week 43 Page 4 Calculations: Using the empirical calibration, the protein concentrations (P) of the unknown samples are determined in mg / 50 µl volume. Calculate the protein concentration in serum samples (S) as follows: !""" S = PŸ #" = P Ÿ 20 (mg/ml) = P Ÿ 20 (g/l) Range of protein concentrations in healthy individuals: 66-82 g/l No. mg protein / cuvette 1 0 2/3 1 4/5 2 6/7 3 8/9 4 10 / 11 Serum blue 12 / 13 Serum red Absorbance at 550 nm (A550) Serum blue: ............... mg protein / cuvette equivalent to ............... g/l in serum Serum red: equivalent to ............... g/l in serum ............... mg protein / cuvette Interpretation: Discuss your results. Practical Biomedical Engineering, 2023/2024 Week 43 Page 5 2. Determination of hemoglobin concentration in human blood 2.1 Practical work Hemoglobin concentration is measured using a device commonly employed in medical practices and laboratories. 2.2 Objectives of this practical work • Determine the hemoglobin concentration in your own (!) blood • Understand the importance of hemoglobin in human physiology 2.3 Background The test strips consist of several layers of dry reagents to lyse the red blood cells (erythrocytes) and convert the hemoglobin from its Fe2+ state to methemoglobin (Fe3+ state), through oxidation by potassium hexacyanoferrate. The hemoglobin concentration of the sample is directly proportional to the extinction (color change) and is measured at 567 nm. 2.4 Protocol General remark: The technical staff will guide you through the process of drawing blood and operating the instrument. Proceed as follows: 1) Wash and dry your hands. 2) Take out a test strip from the container and promptly recap the container to prevent moisture from damaging the remaining unused strips. 3) Prepare and insert the test strip. 4) Rub your fingertip to enhance blood flow through the capillaries. 5) Disinfect the tip of your finger. 6) Using the lancet, puncture the tip of your finger on one side. 7) Dry off the first drop of blood; collect the next drop(s) in the capillary; fill the capillary till the ring mark; tilt slightly to facilitate blood flow; ensure that no air bubbles enter the capillary. 8) Place the capillary perpendicular above the test strip; slowly empty the capillary by gently pressing the air balloon; do not touch the test strip with the tip of the capillary. 9) Following a short incubation period, record the value displayed; the hemoglobin concentration is indicated in mg/dl. 10) Remove the test strip. Practical Biomedical Engineering, 2023/2024 Week 43 Page 6 Interpretation: Discuss the obtained result in the context of the hemoglobin values typically observed in healthy individuals, as indicated on the provided chart. Can you name conditions under which the hemoglobin concentration is below – or above – the normal range? Practical Biomedical Engineering, 2023/2024 Week 43 Page 7 3. Serum electrophoresis 3.1 Practical work Human plasma and serum will be separated electrophoretically on agarose gel. 3.2 Objectives of this practical work • Understand the principles of electrophoresis • Comprehend the individual steps of the method • Recognize the differences between human plasma and serum 3.3 Background Many analytical and preparative methods in biochemical laboratories are aimed at isolating and purifying biologically active molecules. Frequently, the initial sample is comprised of a mixture of hundreds or thousands of different components, from which the molecules of interest must be isolated. The most important separation techniques are based on the different chemical characteristics of the compounds in the mixture, encompassing characteristics like molecule size, electrical charge, density or polarity. Techniques for separating molecules based on their electrical charge, such as electrophoresis and isoelectric focusing, are of central importance and are widely used. Electrophoresis is a technique based on the ability of charged molecules to migrate in an electrical field. The velocity of migration depends on the electric field strength, the net charge, size and threedimensional structure of the molecule (Fig. 1), as well as on the composition of the surrounding medium (surface, buffer). Fig. 1: Influence of charge and molecule size on the electrophoretic separation of proteins. Agarose gels will serve as support in this experiment. The individual protein bands will be made visible using a non-specific protein stain (Fig. 2); the relative abundance of an individual protein band will be determined by scanning the color intensity of each band (Fig. 3). Practical Biomedical Engineering, 2023/2024 Week 43 Page 8 Fig. 2: Electrophoresis of human serum and purified albumin (the major protein in human serum); 1, normal human serum; 2, serum of a patient with agammaglobulinemia (lacking immunoglobulins); 3, purified serum albumin. Fig. 3: Electrophoretic separation of human serum. The photometric quantification reveals the following 5 fractions: albumin (65.5±4.3%), a1-globulins (4.1±1.1%), a2-globulins (6.7±1.3%), b-globulins (9.8±1.6%), g-globulins (14.1±2.9%). Practical Biomedical Engineering, 2023/2024 Week 43 Page 9 3.4 Protocol Samples: The following samples are provided: • Human serum • Human plasma • Purified human albumin • Purified human γ-globulin fraction Materials and reagents: • • • • • • • • • • Electrophoresis apparatus Agarose gel Drying paper for Agarose gel and sample template Pipette: 50 µl Timer Working buffer: ATX buffer (Tris-hippuric acid, pH 8.6) Fixing solution: Ethanol : Distilled water : Acetic acid (in a ration of 50:40:10) Staining solution: Amido Black solution Destaining solution: Aqueous acetic acid Oven (100 °C) Practical Biomedical Engineering, 2023/2024 Week 43 Page 10 Procedure: 1. The electrophoresis chamber already contains the working buffer. 2. Carefully remove the gel from the gel envelope. Position the gel on a piece of filter paper in front of you with the gel side upwards. Carefully remove the blue protection foil. 3. Gently place a filter paper on the gel to remove excess buffer from the gel surface. Make sure that the gel is not damaged or lifted off the carrier material when removing the filter paper. 4. Position the sample template with small marking holes between the markers (> and <) 5. Place a small filter paper on the slots of the application template and gently swipe your finger over it to remove possible air bubbles between the template and gel. Then remove the filter paper again. 6. Add 5 µl of the sample to each slot, placing it in the center of the slot. 7. Wait 5 min. for the samples to enter the gel. 8. Place the small filter paper on the template again to absorb the rest of the sample material and then remove the application template and filter paper from the gel. 9. Put the gel onto the gel bridge with the gel side facing down. Pay attention to the correct polarity. Place the gel bridge in the electrophoresis tank. 10. Close the tank lid and start the electrophoresis. Run electrophoresis at 100 V for 25 min. 11. Remove the gel and place it in the fixing solution for 4 min. 12. Place the gel on a filter paper, label it and dry for 20 min. at 100 °C in the oven. 13. Cut the gel in half. 14. Incubate for 3 min. in staining solution. 15. Place sequentially the gel in the three destaining baths (3 min per bath). Gently agitate the trays occasionally. The gel should become fully destained, revealing clearly visible bands. 16. Dry the gel again in the oven for 10 min. Interpretation: Discuss your results. Practical Biomedical Engineering, 2023/2024 Week 43 Page 11 4. Questions Explain how the following parameters influence the photometer reading: A) Cuvette volume B) Cuvette size C) Concentration of a solubilized compound D) Wavelength E) Which compounds are suitable for photometric measurements? F) What is the definition of ‘anemia’? What physiological consequences do you expect in an anemic patient? G) Under which circumstances could YOUR hemoglobin concentration be below the normal range? H) Under which circumstances could YOUR hemoglobin concentration be above the normal range? I) Which biomolecules can be separated electrophoretically? J) Which factors determine the migration velocity/distance of molecules in a constant electric field? K) Explain which chemical groups contribute to the net charge of a protein. L) Determine the net charges of the following amino acids at the indicated pH values: - glutamic acid at pH 8.6 - glutamic acid at pH 1.0 - lysine at pH 12.0 - lysine at pH 8.6 - lysine at pH 1.0 M) What is the difference between ‘serum’ and ‘plasma’? N) How could you quantify the bands of the obtained electrophoretic pattern? Practical Biomedical Engineering, 2023/2024 Week 43 Page 12