Lab 6 - Turbidimetry and reflectance photometry PDF

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BTI class in Biomed Lab Tech program...

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LABORATORY MANUAL

140-115-DW (F2020)

LABORATORY #6 TURBIDIMETRY AND REFLECTANCE PHOTOMETRY

PART A: Total protein determination using the SSA method (turbidimetry) PART B: Chemical urinalysis (reflectance photometry)

Safety precautions Students must adhere to all safety precautions as described in the Biomedical Laboratory Technology Program General Laboratory Procedures and Safety Manual. WHMIS principles must be followed. Gloves and safety glasses must be worn.

OBJECTIVES After completing this laboratory the student will be able to: • Accurately determine total protein concentration using a manual turbidimetric technique • Accurately perform manual and automated chemical urinalysis • Demonstrate an understanding of the analytical techniques used in this lab: turbidimetry and reflectance photometry

How to prepare for this laboratory 1. Read the entire laboratory exercise very carefully 2. Complete all pre-lab exercises. See “Pre-lab exercises” section.

Report: To be submitted (in order): 1. Professional technical competency sheet 2. Pages 2-5 and 8-9 of this lab 3. Printout from the automated urine analyzer (stapled to page 9) 4. Standard curve (done by hand) 5. Flow chart 1

LABORATORY MANUAL

140-115-DW (F2020)

PRE-LAB EXERCISES (LABORATORY #6) Answer in the space provided, unless otherwise indicated. PART A: Total protein determination using the SSA method (turbidimetry) 1. What is the purpose of adding SSA in this assay?

2. In this assay the analyte of interest _______________ light. a. Absorbs b. Reflects c. Scatters d. Transmits Circle the correct answer. 3. In this assay light ________________ is measured by the detector. a. Absorbed b. Reflected c. Scattered d. Transmitted Circle the correct answer. 4. What type(s) of pipette will you use to transfer the protein solution? (be specific: name and size)

5. What type(s) of pipette will you use to transfer the saline solution? (be specific: name and size) 6. What type of pipette will you use to transfer the patient specimen? (be specific: name and size)

7. What is the total amount of 3% SSA required?

8. What type of pipette will you use to transfer the 3% SSA? (be specific: name and size)

9. Fill in the “Protein concentration” row in the table on page 4. Show calculations in the space provided below.

10. Prepare a flow chart for this assay (on a separate page). (continued on the next page) 2

LABORATORY MANUAL

140-115-DW (F2020)

PRE-LAB EXERCISES (LABORATORY #6) CONT. PART B: Chemical urinalysis (reflectance photometry) 1. Use the Chemstrip 10 product insert (page 7) and instructions provided for manual chemical urinalysis (page 8) to answer the following questions: a. After dipping the test strip in the urine specimen, how long should you wait before comparing the test pads with the colour scale?

b. If the colour of a test pad changes 5 minutes after it has been dipped in the specimen, how should the result be interpreted?

c. Can a specimen be stored overnight before testing? If not, what is the maximum storage time?

d. Why is it necessary to close the container immediately after removing a test strip?

2. With respect to the automated urinalysis: concentration is directly proportional to ________. a. Absorbance b. Reflected light c. Scattered light d. Transmitted light Circle the correct answer. 3. In this assay light ________________ is measured by the detector. a. Absorbed b. Reflected c. Scattered d. Transmitted Circle the correct answer. 4. What type of (a) light source and (b) detector does the urinalysis instrument use?

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LABORATORY MANUAL

140-115-DW (F2020)

PART A: Total protein determination using the SSA method (turbidimetry) Introduction Turbidimetric measurements are based on the amount of light blocked by particulate matter in a sample as light passes through the cuvette. The SSA method is used to determine total protein concentration. SSA precipitates protein, and the precipitate scatters light. The amount of light scattered (i.e. “blocked”) is directly proportional to protein concentration. The relationship between %T and light scatter is analogous to that between %T and absorbance: absorbance readings obtained using a spectrophotometer can therefore be considered equivalent to amount of light scattered (remember: the spectrophotometer measures %T, and then mathematically converts the readings to Abs). Procedure Materials:

3% sulfosalicylic acid (SSA) Protein solution (1.00 g/L) 0.85% NaCl (w/v) Pipettes Cuvettes Spectrophotometer Patient specimen (CSF)

1. Label 7 cuvettes and add the appropriate solutions, as shown in the following table: 7 Patient ID:

Cuvette #

1

2

3

4

5

6

0.85% NaCl (w/v)

0.5 mL

0.4 mL

0.3 mL

0.2 mL

0.1 mL

0.0 mL

-------

Protein solution

0.0 mL

0.1 mL

0.2 mL

0.3 mL

0.4 mL

0.5 mL

-------

Patient specimen

-------

-------

-------

-------

-------

-------

0.5 ml

OD @10 min Protein conc. (mg/L)

-------------

2. Pipette 2.0 mL of 3% sulfosalicylic acid into each cuvette 3. Mix, then wait 10 min. 4. Mix well again and then read the absorbance of each cuvette at 620 nm using cuvette #1 as the blank.

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LABORATORY MANUAL

140-115-DW (F2020)

Calculations, final results and questions Use the space provided, unless otherwise indicated. 1. Use the concentration and absorbance values for your blank and all of your standards to prepare a standard curve. The standard curve must be prepared by hand. 2. Use the standard curve from Question 1 to determine the concentration of protein in the CSF specimen. Record the protein concentration in the table provided at the bottom of this page. 3. Determine the concentration of protein in the CSF specimen using a calibration factor (follow the procedure described in the pre-class question set 5). Show all calculations in the space provided below. Record the calibration factor and the protein concentration in the table provided below.

CSF protein concentration Patient ID: __________________________________________________________ Standard curve Calibration factor: ______________ QUESTION: What effect do you think that not mixing before reading the absorbance would have on the results of the SSA assay? Explain your answer.

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LABORATORY MANUAL

140-115-DW (F2020)

PART B: Chemical urinalysis (reflectance photometry) Introduction Chemical urinalysis is usually performed using a reagent strip. The reagent strip consists of single or multiple reagent pads on a solid support, usually plastic. Each pad contains a reagent that reacts with a particular analyte to produce a coloured product. When immersed in a urine specimen the reagent pads will change color in proportion to analyte concentration. Results can be either qualitative (i.e. positive or negative), or semi-quantitative (i.e. give an approximate concentration), depending on the analyte and reagent strip. Today you will be using Chemstrip reagent strips to perform manual chemical urinalysis, and Multistix 10 SG reagent strips to perform automated chemical urinalysis. Additional information about the Chemstrip reagent strips (including reactions occurring in each test pad), is provided in the Chemstrip product insert on the following page. Reactions occurring on the Multistix 10 SG reagent strips are very similar to those that occur on the Chemstrip reagent strips. For both manual and automated chemical urinalysis: (1) a test strip is briefly dipped into the urine specimen, (2) in each pad on the test strip a reagent reacts with a particular analyte to produce a colored product, (3) results are obtained for each test pad through visual examination (manual method) or using an automated urine analyzer. In general: more analyte = more coloured product formed. The automated urine analyzer that we will be using (the Siemens Clinitek Advantus) is shown below: Printer Display

Waste bin

Strip loading station

Push bar Incubation/read station

Most automated urine analyzers, including the Clinitek Advantus, use reflectance photometry to determine analyte concentration: light is directed onto each test pad, and reflected light is measured. For most of the analytes: higher concentration = more colored product = more light absorbed = less light reflected. The Clinitek Advantus measures reflected light as follows: • In the read station the reagent strip is scanned by a read head, which contains an incandescent (tungsten) lamp and a photodiode pack. The incandescent lamp serves as the light source, and the photodiode pack as the detector. • The photodiode pack contains four filters, for blue (400-510 nm), green (510-586 nm), red (586-660 nm), and IR (825-855 nm) light. As the reagent strip is scanned each reagent pad is illuminated by the incandescent lamp, and a portion of the reflected light is detected by the photodiode pack. For each reagent pad the amount of light reflected at a specific wavelength (i.e. blue, green, red or IR) is dependent on the degree of color change in the pad, and is related to the concentration of the particular constituent in the urine. • The light intensity detected by the photodiode pack for each reagent pad is converted to an electrical signal, which is processed by a microprocessor into a clinically meaningful result. 6

LABORATORY MANUAL

140-115-DW (F2020)

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LABORATORY MANUAL

140-115-DW (F2020)

Manual procedure Materials:

Chemstrip 10 Urine specimen

1. Mix the urine specimen well. 2. Pour 10 ml of the urine specimen into a conical tube. 3. Take a test strip out of the container. Close the container again immediately after removal of the strip. 4. Briefly (about 1 second) dip the test strip into the urine specimen, making sure that all test areas are moistened. 5. When withdrawing the test strip, wipe the edge (or back) against the rim of the vessel to remove excess urine. 6. Place the strip on a kimwipe with the reagent pads facing up. 7. After 60 seconds (60-120 seconds for the leukocyte test area) compare the reaction colors of the test areas with the colors on the label. Compare the 10th (blood) test area with both color scales as separate color scales are given for erythrocytes and hemoglobin. NOTE: • Reaction colors should be observed under a good light source • Visual interpretation of these results can be very subjective • Any color changes appearing only along the edges of the test areas, or developing after more than two minutes, do not have any diagnostic significance 8. Record your results in the following table: Fill in the “parameters” column before dipping the Chemstrip into the specimen, and make sure that you know how the strip should be oriented when reading the results.

Patient ID: ___________________________________________________________________ Parameter

Result

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LABORATORY MANUAL

140-115-DW (F2020)

Automated procedure NOTE: The instructor will demonstrate proper use of the urine analyzer Materials:

Automated urine analyzer: Clinitek Advantus Multistix 10 SG reagent strip (NOTE: this is not the same type of test strip that you used for the manual procedure) Urine specimen

Procedure: 1. 2. 3. 4. 5. 6. 7. 8.

9.

Mix urine specimen well. Enter specimen ID (Unit # ONLY). Enter clarity using the cycle key (the sample you have is CLEAR). Press Enter (Push Bar will retract to loading position). Take a Multistix 10SG strip out of the container and immediately close the container. Completely immerse all of the reagent pads of the test strip in the urine specimen. Immediately remove the test strip. Ø While removing the strip, run the edge against the side of the container to remove excess urine . Place the test strip onto the strip loading station with the reagent pads facing up. Ø Place to the right of, and parallel to, the push bar. Ø Ensure that the end of the strip is against the back wall of the platform, and not touching the bottom of the strip loading station. Wait while specimen is being processed.

RESULTS: Results will print automatically, and the instrument will return to the Home Screen Submit the printout along with your lab report: staple the report to this page

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LABORATORY MANUAL

140-115-DW (F2020)

Course: _140-115-DW_____________ Laboratory: __6__________________

Student Name: _____________________________________

The following professional technical competencies will be assessed during each of the laboratories given in this course during the semester. Assessment will be made according to the criteria listed below on the basis of pass (P), fail (F), or improvement required (I). Professional Technical Competency 1. Dependability with respect to time.

2. Ability to follow written and oral instructions. 3. Ability to show initiative

4. Ability to use and maintain instruments and lab materials 5. Ability to organize work

6. Ability to produce proper reports

7. Ability to meet the objectives of the laboratory

Criteria for Assessment -

8. Ability to interact constructively with instructors and peers

9. Ability to show professional judgement, honesty and maturity

10. Ability to work safely

Overall Assessment:

-

comes to laboratories on time. works in a timely fashion. completes laboratory work within the lab period. meets stated deadlines. follows written protocol given in laboratory manual. responds to and follows oral directions given by the instructor. comes to the laboratory prepared. works without being prompted (i.e., self-motivated). works autonomously as required. uses instruments and lab materials properly. cleans instruments after use if necessary. restocks work area if necessary pays attention to detail. manages time effectively able to multi-task (when appropriate) organizes her/his work area for safe and efficient work. leaves work area clean without being reminded. produces reports which are neat and clear. produces reports which are complete, according to the established protocol for the laboratory. produces the required quantity of work. produces work to an acceptable level of accuracy or within an acceptable standard as appropriate to the competency. demonstrates understanding of the tests and protocols being carried out interprets results correctly interacts with instructional staff and peers in a professional manner. communicates effectively both orally and in writing works as a team member when required accepts constructive criticism with respect to her/his work. seeks help when needed. carries out work in a logical sequence. admits to and corrects mistakes reports results honestly responds to questionable results appropriately handles stressful situations calmly and efficiently adheres to all safety regulations of the laboratory

Assessment P I

F

P I F P I F

P I F

P I F

P I F

P I F

P I F

P I F

P I F

Satisfactory _____ Improvement required _____Unsatisfactory _____

Instructor’s initials: _____________ Comments: ____________________________________________________________________________________________ ______________________________________________________________________________________________________ ______________________________________________________________________________________________________

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