Lab 9

Approximate Time: 3 hours

“This material is adapted from iWorx Systems Inc with permission.” 

Equipment Required

IXTA, and power supply for IXTA

A-FH-300L Spirometer flow head and plastic tubes

A-SRK student respiratory kit (blue filter, disposable cardboard mouthpiece, nose clip) – SINGLE STUDENT USE!!!

It is important that the volunteer is healthy and has no history of respiratory or cardiovascular problems.

Setup

Note: Student respiratory kit (Fig. 1A) – this is a single-student test/session use.  When done with lab, please discard them away.

1. Locate the flow head and the airflow tubing (Fig. 1B). You will notice that the two air flow tubes onto the are firmly push onto the two outlets on the flow head as it demonstrated in Figure 1b.

Figure 1. A. The student respiratory kit. B. the A-FH flow head and the airflow tubing

1. The other ends of the two air flow tubes are connected into Channel A1 input (Fig. 2).
2. Attach the blue filter to the flow head (Fig. 2)

Figure 2. The flow head connected to the internal spirometer of the IXTA unit.  Blue filter and disposable cardboard piece attach to the flow head.

Before Starting

1. Please read the procedures for each exercise completely before beginning the experiment. You should have a good understanding of how to perform these lab exercises before making recordings.
2. The spirometer will monitor breathing form a subject. It is important that the subject is HEALTHY and has NO HISTORY OF RESPIRATORY OR CARDIOVASCULAR PROBLEMS.
3. On the flow head, the outlets connected to the airflow tubing should always be pointed up to avoid problems with condensation developing within the tubing.
4. To reduce turbulence within the flow head, place a disposable cardboard mouthpiece over the opening of the flow head.
5. Use a nose-clip to prevent air from entering or leaving the nose as the subject is breathing. Air passes through the nose causes errors in the lung volume values.

Start the Software

5. Turn onthe iWorx TA-ROAM box.
6. Clickthe Labscribe23 icon on the computer screen.
7. A default mode box will appear, clickOK. A pop-up box should appear indication that the iWorx IX-TA ROAM (hardware) has been found, click
8. When the program opens, Clickon Settings on the toolbar on top. Select Human Spirometry and then Breathing-Rest Exercise
9. LabScribe will appear on the computer screen as configured by the Human Spirometry settings.
10. Check the calibration of your spirometer on the Lung Volume channel:

• Clickon the words Human (Air Flow), that are next to the title of the Lung Volumes channel, to open the computed function pull-down menu.
• SelectSetup Function from this pull-down menu to open the Spirometer Calibration Dialog window.
• Check that the internal spirometer is being used making sure the Type of Spirometer is the IXTA and the flow head is 300 L.
• Make sure the reset time is set to “No Reset”, and the first 10 seconds of the recording are used to zero the baseline of the Lung Volumes channel.
• Enter37 for temperature of exhaled air.
• Click OK.

7. Allow the IXTA to warm up for 10 minutes before recording for the first time.
8. Make sure the subject breathes through the red port side of the flow head.

Procedure

1. Instruct the subject to:

• Sit quietly and become accustomed to breathing through the spirometer flow head.
• Make sure the subject is breathing through the red port side of the flow head.
• Breathe normally before any recordings are made.
• Hold the flow head so that its outlets are pointed up.
• Remove the flow head from his or her mouth and hold it at the mouth level in a position that prevents a breath from moving through the flow head.

Note: The LabScribe software will zero the Lung Volumes channel during the first ten seconds of recording.  No air should be moving through the flow head during this time.

2. TypeResting in the Mark box.
3. ClickRecord. After waiting at least 10 seconds for the Lung Volumes channel to zero, have the subject place the flow head in his o her mouth and begin breathing.  Click the mark button to mark the recording.
4. Clickthe AutoScale All Notice the slowly moving wave on the Lung Volumes channel.  Record five-six breaths, which normally takes about forty-five seconds to record.
5. Type Forced in the Mark box. Click the mark button as the subject inhales as deeply as possible.  After reaching his or her maximum inhalation volume, the subject should exhale as quickly and as completely as possible.  Blow all the air out.
6. After the forced exhalation is complete, the subject should continue to breathe normally through the spirometer for five-six breath cycles.
7. ClickStop to halt recording. Your data may look like the image below (Fig 4).

Fig. 4. Air flow and lung volumes of the normal and forced breathing of a subject at rest.

Data Analysis-Normal Breathing at Rest

1. Scroll through the recording and find the section of data recorded when the subject was breathing while resting.
2. Use the Display Time icons to adjust the Display Time of the Main Window to show the complete breathing cycles on the Main Window. The adjacent breathing cycles can also be selected by:

• Placing the cursors on either side a group of complete breathing cycles; and
• Clicking the Zoon between Cursors button on the LabScribe toolbar to expand the selected breathing cycles to the width of the Main Window.

Fig.5.LabScribe_Toolbar

3. Once the cursors are placed in the correct positions for determining the volumes of each breath cycle, the values of the parameters can be recorded.
4. On the Lung Volumes channel, use the mouse to click on and drag the cursors to specific points on the recording to measure the following volumes:

• Tidal Volume (TV),which is the volume of air inhaled during a normal breathing cycle. Place one cursor in the trough prior to inhalation, and the second cursor on the peak of the cycle.  The value for the V2-V1 function on the Lung Volumes channel is the tidal volume (Fig. 5).

Fig. 5. Breathing pattern of a subject at rest, displayed on the Lung Volumes Channel in the Analysis window.  The cursors are positioned on the trough and the peak of the breath cycle to measure the tidal volume (TV) with V2-V1 function and the maximum inspiratory flow rate with the Max-dv/dt function.

1. Use the slider or the arrows on the scroll bar, at the bottom of the Analysis window, to position data recorded when the subject exhaled with maximum force in the window.
2. Use the Display Time icons to adjust the Display Time of the Analysis window to show the forced expiration curve and the two normal breaths, that occur before the force expiration curve, on the same window.
3. Use the same techniques used earlier to record volumes in Table 2.
4. Place the cursors on the forced expiration data displayed on the Lung Volumes channel to measure the following volumes using the V2-V1 functions. Check the labels to identify the volumes and rates that you will measure:

• Inspiratory Reserve Volume (IRV), by placing one cursor on the peak of the normal breath prior to the maximum inhalation and the second cursor on the peak of the forced breath cycle. The value for the V2-V1 function on the Lung Volumes channel is the forced inspiratory flow rate (Fig. 8).
• Expiratory Reserve Volume (ERV), by placing one cursor in the trough before maximal inhalation and the second cursor on the flat line after subject has expelled all the air from his or her lungs. The value for V2-V1 function on the Lung Volumes channel is the expiratory reserve volume (Fig. 8).

Fig. 8. Recording of normal and forced lung volumes taken from a subject at rest and displayed on the Lung Volumes channel in the Analysis window.  The normal breathing cycles are to the left of the forced inspiration and expiration.  Lines and labels were added to figure to indicate the volumes that should be measured for each subject: Tidal Volume (TV), Inspiratory Reserve Volume (IRV), and Expiratory Reserve Volume (ERV).

Table 1. Lung volumes for an Average Sized Human Male (70Kg).

Procedure

1. In this exercise, use the same healthy subject whose breathing parameters at rest were measured in Activity 1.
2. TypeAfterExercise in the the Mark Box.
3. The subject should exercise to sufficiently elevate breathing rate, but with minimal class disruption. Use the treadmills or exercise bikes are a good method.  The subject should sit down immediately after the exercise period.
4. Click Record. After waiting ten seconds have the subject place the flow head in his or her mouth and begin breathing.  Click the mark button to mark the recording.
5. Click the AutoScale All button. Record at least five-six breaths as the subject is recovering from exercise.  Remember the baseline of the Lung Volume channel automatically resets every sixty seconds.
6. Type Forced in the Mark box. Click the mark button as the subject inhales as deeply as possible.  After reaching his or maximum inhalation volume, the subject should exhale as quickly and as completely as possible.  Blow out as much as possible.
7. The subject should return to breathing as normally as possible through the spirometer.
8. Click Stop to halt recording.

Data Analysis-Forced Expiration at Rest

1. Perform the same types of measurements on the data recorded in Activity 1. Record the measurements in the table.

TURN OFF THE IXTA UNIT WHEN DONE!!!!!!!

Students will perform the ELISA to detect the presence of COVID-19 antibodies in simulated patient samples (wear gloves and safety goggles)

1. LABELthe wells of the microtiter plate.
2. LABELthe transfer pipets as follows.  These pipets will be used to add and remove liquid from the wells.

(-) Negative Control                         (CAP) used to add/remove the capture antibody

(+) Positive Control                           (WASH) used to add wash buffer to each well

(P1) Patient 1                                    (SUB) used to add substrate to each well

(P2) Patient 2                                    (DET) used to add detection antibody to each well

(P3) Patient 3

3. Using the transfer pipet for CAP, ADD50 ul of the capture antibody solution (CAP) to all of the wells.  (if using transfer pipets, three drops is approximately 50 ul).  INCUBATEthe plate at room temperature for 5 minutes.
4. INVERTthe strip over the sink or a stack of paper towels to remove the samples.  Gently TAP the strip 4-5 times onto a fresh paper towel.  DISCARD the wet paper towels.
5. Using the WASH transfer pipet, ADDwash buffer to fill each well, being careful not to overfill.

NOTE: To minimize cross-contamination it is important that you avoid spilling buffer into neighboring wells.  

6. REPEATstep 4 to REMOVE the wash buffer.
7. Using the same transfer pipet,REPEAT the wash a second time.  INVERT strip onto fresh paper towels and TAP.
8. Using the appropriately labeled transfer pipet, ADD50 ul of each of the controls and patient samples to the appropriate well.  INCUBATE the plate for 5 minutes at room temperature.
9. INVERTonto paper towels and TAP.  WASH the wells twice as in steps 4-7.
10. ADD50 ul of the detection antibody solution (DET) to each well.  INCUBATE the plate for 5 minutes at room temperature.
11. INVERTonto paper towels and TAP.  WASH the wells twice as in steps 4-7.
12. ADD50 ul of the substrate solution (SUB) to each well.  INCUBATE the plate for 5 minutes at room temperature.
13. EXAMINEyour results:

Negative COVID Test – Clear

Positive COVID Test – Dark

14. RECORD your results in the table, below.

Fill out your results for the COVID ELISA test in the following chart: