Category Archives: LABORATORY PROCEDURES

COLORIMETRIC GLUCOSE METHOD

COLORIMETRIC GLUCOSE METHOD
The glucose-oxidase enzymatic method is recommended because it is specific for glucose. A protein precipitation stage is included because this removes substances such as urate which may be present in blood samples in sufficient concentration
to interfere in the final stage of the reaction.

Principle of glucose oxidase-peroxidase
method

Glucose oxidase (GOD) catalyzes the oxidation of glucose to give hydrogen peroxide (H2O2) and gluconic acid. In the presence of the enzyme peroxidase (POD), the hydrogen peroxide is broken
down and the oxygen released reacts with 4- aminophenazone (4-aminoantipyrine) and phenol to give a pink colour.
The absorbance of the colour produced is measured in a colorimeter using a green filter 520 nm or in a spectrophotometer at
515 nm.

See also:

ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

The APTT is a screening test of the intrinsic clotting system. It will detect the inhibition or deficiency of one or more of the following factors: prothrombin, V, VIII (antihaemophilic factor), IX, X, XI, XII and fibrinogen. The APTT is also used to monitor patients being treated with heparin.

Principle of test

Kaolin (surface activator) and platelet substitute (phospholipid) are incubated with citrated plasma at 37°C for the time
specified in the test method. Calcium chloride is added and the time taken for the mixture to clot is measured.

Reagents

Kaolin/platelet substitute mixture
Purchase from a reliable manufacturer in lyophilized form and reconstitute as instructed.

Calcium chloride, 0.025 mol/l (25 mM)
This is best obtained ready made unless the laboratory has facilities to make the reagent accurately and standardize it.

See also:

APTT TEST PROCEDURE

SOURCES OF ERROR WHEN PERFORMING APTT, PT & TT TESTS


● Difficulty in obtaining a venous sample, resulting in haemolysis or small clots in the sample.

● Delay in testing the plasma and leaving the sample at room temperature.

● Using tubes or pipettes which are not clean and dry or are contaminated with detergent. Whenever possible use disposable tubes.

● Not timing accurately the different stages of the test (a stop-watch must be used).

● Using unsatisfactory reagents (will be detected when testing control plasma).

● Not pipetting correct volumes of plasma and reagents.

● Reconstituting reagents and control plasma with contaminated deionized water.

APTT MEASUREMENT USING PLATELET SUBSTITUTE MIXTURE

Test the control plasma and patient’s plasma in duplicate.

1. Pipette 0.2 ml of well-mixed kaolin/platelet substitute in a small glass tube.

2. Add 0.1 ml of plasma, mix, and incubate at 37° C for exactly 2 minutes (tilting the tube at intervals).

3. Add 0.1 ml 0.025 mol/l calcium chloride, mix and start the stop-watch. Hold the tube in the water bath and tilt the mixture back and forth, looking for clot formation. When a clot forms, stop the stop-watch and record the time.

4. Report the patient’s APTT (average of the duplicate tests) providing the APTT of the normal control plasma is satisfactory.

Reference APTT range

36–50 seconds

What causes prolonged APTT?

  • DIC (involving several clotting factors)
  • Deficiency of clotting factors: prothrombin, V, VIII, IX, X, XI or XII due to vitamin K deficiency, liver disease, heparin or warfarin anticoagulation, or less commonly an inherited coagulation disorder.

See also:

The Principle of APTT test

BLOOD COLLECTION TUBES

Red-top tube: Contains no anticoagulant or preservative.
Use: Serum or clotted whole blood. Serum must be separated from cells within 45 minutes to two hours depending on the test

Lavender-top tube: Contains K2 EDTA.
Use: EDTA whole blood or plasma. Separate and send plasma in a plastic transport tube labeled “Plasma, EDTA.” Send whole blood in a lavender-top tube.

Gray-top tube: Contains sodium fluoride (a preservative) and potassium oxalate (an anticoagulant).
Use: Sodium fluoride whole blood or plasma. Send plasma in a plastic transport tube labeled “Plasma, Sodium Fluoride.” Send whole blood in a gray-top tube.

Blue-top tube (also light blue-top tube): Contains sodium citrate. Be sure to use only tubes with a 3.2% sodium citrate concentration. These are easily identified by the yellow diagonal stripes on the label.
Use: Sodium citrate plasma. Send plasma in a plastic transport tube labeled “Plasma, Sodium Citrate.” Send whole blood in a blue-top tube.

Green-top tube: Contains sodium heparin or lithium heparin.
Use: Heparinized whole blood or plasma. Send plasma in a plastic transport tube labeled “Plasma, Sodium Heparin” or “Plasma, Lithium Heparin.” Send whole blood in a green-top tube.

Yellow-top tube: Contains acid citrate dextrose (ACD) solution.
Use: ACD whole blood. Send whole blood in a yellow-top tube.

Royal blue-top tube: Contains sodium EDTA for trace metal studies. Some royal blue-top tubes do not contain EDTA.
Use: EDTA whole blood or plasma. Send whole blood in a royal blue-top tube.

CAUSES OF ERRORS OF INACCURACY (BIASE)

The causes of these can be summarized as follows:

  • Incorrect calibration of a test method.
  • Tests being read at the incorrect wavelength or An automatic pipettor is used that has been set to measure incorrect amount
  • The wrong filter being used.
  • Consistent calculation error e.g. incorrect factor is used
  • Use of unsatisfactory reagents, standards, or controls

CAUSES OF ERRORS OF IMPRECISION (SCATTER)

  • Incorrect and variable pipetting and dispensing.
  • Inadequate mixing of sample with reagents.
  • Samples are not incubated consistently at the correct temperature (when incubation of tests is required).
  • Individual items of glassware or plastic-ware are not clean or dry before reuse.
  • Equipment malfunctioning (erratic performance).
  • Incomplete removal of interfering substances, e.g. red cells when measuring analytes in serum or plasma.

WHAT MAKES A SPECIMEN TO BE REJECTED

It is unlabelled or the identity on the form does not match that on the specimen.

It is not correct for the test being requested or it has been collected in the wrong container, e.g. containing the incorrect anticoagulant.

The specimen container is leaking.

There is evidence of contamination.

A blood sample appears haemolyzed or an anticoagulated specimen contains clots.

There is too long delay in the specimen reaching the laboratory or it has not been transported

LEISHMAN STAINING TECHNIQUE


1. Cover the blood film (preferably methanol pre- fixed, ) with undiluted stain but do not flood the slide. If using a dropper bottle count the number of drops required to cover the film.

The undiluted stain not only acts as a fixative but also partially stains the smear. This stage is required to obtain the best possible staining results.

2. Add twice the volume of pH 6.8 buffered water (i.e. twice the number of drops as stain). The stain should not overflow. Ensure the water is well mixed with the stain by blowing on the diluted stain or mixing the stain and water using a plastic bulb pipette. Allow to stain for 10 minutes (time may require adjusting).

Diluting the stain in buffered water brings about full staining of the blood cells. The exact staining time to use should be decided when a new batch of stain is prepared.

3. Wash off the stain with tap water (filtered if not clean). Do not tip off the stain, because this will leave a fine deposit covering the film. Wipe the back of the slide clean and stand it in a draining rack for the smear to dry. The blood film should
appear neither too pink nor too blue (check
results microscopically).

HOW TO MAKE A THIN BLOOD FILM


1. Make a blood spreader from a slide which has ground glass polished sides

2. Place a drop of blood on the end of a clean dry slide too large (if too large, use a drop from the excess blood to make the film).

3. Using a clean smooth edged spreader, draw the spreader back to touch the drop of blood and allow the blood to extend along the edge of the spreader. Holding the spreader at an angle of about 30, spread the drop of blood to make a film about 40–50 mm in length (two thirds of the slide)

4. Wipe clean the end of the spreader.

5. Immediately air dry the film by waving the slide back and forth. Protect the dried film from dust and insects.

When not using a frosted ended slide,
write the patient’s name and number on the dried blood at the top or along the side of the film using a lead pencil.

6. When completely dry and within a few minutes of making the blood film, fix it in absolute methanol