Red cell fragility

The McGill Physiology Virtual Lab

Blood Laboratory

Red cell fragility > Procedure

Six labelled centrifuge tubes are prepared containing 10 ml of 0.9%, 0.6%, 0.5%, 0.4%, 0.3% and 0.0% NaCl solutions.

The blood sample is mixed, to obtain a homogeneous suspension of blood cells;
0.1 ml of blood is delivered into each of the 6 centrifuge tubes with an adjustable pipette on which a disposable tip is inserted.

The tubes are capped, inverted several times, and allowed to stand for 20 minutes. After 20 minutes, all six tubes are centrifuged for 10 minutes at maximal speed.

The supernatant and the pellet are now visible. Pasteur pipettes are used to transfer enough of the supernatant fluid (up to the mark on the spectrophotometer cuvette) from each tube into square spectrophotometer cuvettes.

The use of adjustable pipettes and proper pipetting

These expensive instruments are continuously adjustable pipettes designed to dispense precise volumes of liquid safely. Each model (see above) cover specific volume ranges. If these volume ranges are not respected, the fragile volumeter breaks!

A pipette is equipped with a volumeter which is used to set the volume. The volume is continuously adjustable within the volume range for the pipette. The maximum volume for the pipette is shown on the push-button and corresponds to the model.

The volumeter consists of three number dials (which are used to set the volume of liquid to be transferred). They are read from top (most significant digit) to bottom (least significant digit).

There is also a locking mechanism (black ring) which prevents accidental change in volume.

NEVER USE A PIPETTE OUTSIDE ITS DESIGNATED RANGE!
Set the desired volume by turning the knurled volumeter adjustment knob until the correct volume shows on the volumeter read-out.
Attach the appropriate tip to the end of the pipette by pushing down until a "click" occurs.
Depress the plunger to the first positive stop.
Holding the pipette as near to vertical as possible, immerse the tip ONLY 2-4 mm below the surface of the sample.

Allow the volumeter knob to return slowly to the up position. Never permit it to snap up.
Wait one to two seconds to ensure that the full volume has been drawn into the tip (longer for viscous solutions such as blood).
Withdraw the tip from the blood sample and carefully remove any excess blood on the outside tip with a "kimwipe" (blood being a viscous liquid, extra cells can be carried over...)
Dispense the sample by placing the outlet of the tip against the wall of the receiving tube, with the pipette as close to vertical as possible, and depressing the plunger to the first stop.
Wait to allow blood to drain to the end of the tip. Then, depress the knob to the second stop to fully expel the measured volume.
With the knob still fully depressed, withdraw the tip from the receiving tube. Then allow the knob to return slowly to the top (uppermost) position.
Discard used tips into the proper receptacle using the tip ejector.

Pipette tip sizes used in the laboratories

Note the different sizes available for specific pipette models, you will be using in this laboratory exercise as well as others requiring precise pipetting. Use the proper tip with the proper pipette!

Examples of volumeter adjustments for different pipettes

For pipettes with a specified range of: 2-20 microL (verify on top of the knob):

This reading indicates a volume setting of: 5 microL (the red indicates a decimal, so a volume of 5.x microLl is possible with this pipette)

For pipettes with a specified range of: 20-200 microL (verify on top of the knob):

This reading indicates a volume setting of: 175 microL or 0.175 ml

For pipettes with a specified range of: 100-1000 microL (verify on top of the knob):

This reading indicates a volume setting of: 1000 microL or 1 ml. Notice that there are only 3 dials on this particular pipette. The topmost digit in red should only read 0 (for volumes under 1 ml) or 1 (for a volume of exactly 1 ml; in such a case, the bottom two dials should all read zero)

Pipette specifications, accuracy and precision

It is also important to use the proper pipette, for the amount of liquid handled because accuracy and precision must be taken into account. Accuracy is the degree of closeness of a measured or calculated quantity to its actual (true) value. Precision is the degree to which further measurements or calculations show the same or similar results.

Look at the following table showing the specifications of different adjustable pipette models. For a given volume, for different models, notice how accuracy and precision vary. Which pipette would you choose to dispense a 10 microL volume with best accuracy and precision? and to dispense a 20 microL or a 100 microL volume?

Adjustable range Model	Volume microL	Accuracy		Precision
Adjustable range Model	Volume microL	microL +/-	%	microL <=	%
1-10 microL	10	0.1	1.0	0.04	0.4
2-20 microL	10	0.15	1.5	0.05	0.5
2-20 microL	20	0.2	1.0	0.06	0.3
20-200 microL	20	0.5	2.5	0.2	1.0
20-200 microL	100	0.8	0.8	0.25	0.25
100-1000 microL	100	3.0	3.0	0.6	0.6

To deliver a volume of 100 microL : when choosing a 20-200 microL range pipette model, the error is +/- 0.8 microL which represents 0.8% of the volume (0.8 % deviation from the desired value). However, if the 100-1000 microL model is chosen, then the error becomes +/- 3 microL , that is 3% deviation from the desired value.

Spectrophotometry readings

The spectrophotometer is prepared. The wavelength is selected for optimum light absorption to read 540 mu.

How does it work?

Zero OD is set with a cuvette containing distilled water only. Then, the cuvettes containing the different supernatants are read sequentially.

A spectrophotometer cuvette holding a maximum of about 3 ml.

To continue with the measurement of erythrocyte fragility, click here