HEMATOLOGY – Page 4

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.

THROMBOCYTOPENIA

It occurs when platelets fall below the normal range of 150–400×10^9 per litre of blood

REDUCED PRODUCTION OF PLATELETS

● Infections, e.g. typhoid, brucellosis
● Deficiency of folate or vitamin B12
● Aplastic anaemia
● Drugs (e.g. cytotoxic, quinine, aspirin), chemicals
(e.g. benzene), some herbal remedies, alcoholism
● Leukemias, lymphoma, myeloma, myelofibrosis, carcinoma
● Hereditary thrombocytopenia (rare condition).

INCREASED DESTRUCTION OR CONSUMPTION OF
PLATELETS

● Infections, e.g. acute falciparum malaria, dengue, trypanosomiasis, visceral leishmaniasis
● Disseminated intravascular coagulation (DIC)
● Hypersplenism
● Immune destruction of platelets, e.g. idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), other connective tissue disorders, chronic lymphatic leukaemia, lymphomas and HIV/AIDS. Also, exposure to drugs, e.g. quinine, mefloquine, penicillin, and some herbal remedies.

RETICULOCYTE COUNT

Value of test:

Reticulocytes are immature red cells
normally present in small numbers in the blood (up to 2%). Reticulocyte numbers increase when there is an increase in erythropoietic activity. A reticulocyte count assesses bone marrow activity, e.g. whether there is an effective erythropoietic response when there is a reduction in the number of red cells due to haemolysis or haemorrhage. A reticulocyte count is also of value in monitoring the erythropoietic response of an anaemic patient following treatment.

Principle of test
An isotonic solution of a supravital stain (i.e. one that stains living material) such as New methylene blue or brilliant cresyl blue is incubated with a few drops of blood. To detect ribosomal RNA in reticulocytes, the red cells must be stained while they are still living (not fixed). A thin preparation is made and the reticulocytes counted microscopically. Reticulocytes are recognized by the violet-blue stained granules of ribosomal RNA (reticulin) they contain.

The reticulocyte count is expressed as a percentage, or preferably in absolute numbers when an electronic analyzer RBC count is available.

See also:

Procedure for Reticulocytes count

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

THROMBOCYTOSIS

Occurs when platelet count rises above the normal range of 150–400 10^9 per litre of blood

What causes Thrombocytosis

● Chronic myeloproliferative diseases, e.g. essential thrombocythemia, polycythaemia vera, chronic myeloid leukaemia, myelofibrosis.
● Carcinoma (disseminated)
● Chronic inflammatory disease, e.g. tuberculosis
● Haemorrhage
● Sickle cell disease associated with a nonfunctioning spleen or after splenectomy.
● Iron deficiency anaemia, associated with active bleeding.

SOURCES OF ERROR WHEN MEASURING HAEMOGLOBIN PHOTOMETRICALLY

● Not measuring the correct volume of blood due to poor technique or using a wet or chipped pipette.

● When using anticoagulated venous blood, not mixing the sample sufficiently.

● Not ensuring that the optical surfaces of a
cuvette are clean and dry and there are no air bubbles in the solution.

● Not protecting a colorimeter or haemoglobin meter from direct sunlight and not checking the performance of an instrument or maintaining it
as instructed by the manufacturer. A common error when using a filter colorimeter is using a glass filter which is not clean.

● Not checking a diluting fluid such as Drabkin’s for signs of deterioration as explained in the HiCN techniques.

Technique to prevent cuvette-related errors

1. Hold a clean cuvette only by its frosted (matt) or ridged sides. When transferring a solution to a cuvette, allow the fluid to run down the inside wall of the cuvette. This will help to avoid air bubbles in the solution. Do not fill a cuvette more than three quarters full.

2. Using a tissue or soft clean cloth, wipe clean the clear optical surfaces of the cuvette.
Carefully insert the cuvette in the colorimeter or haemoglobin meter (optical surfaces facing the light source).
Note: Ensure a solution is at room temperature before reading its absorbance otherwise condensation will form on the outside of the cuvette which will give an incorrect reading.

HAEMOGLOBIN MEASUREMENT USING HEMOCUE METER

1. Pull out the microcuvette holder to its loading position.

2. Press and hold down the left button of the meter until the display is activated. the meter automatically carries out a performance check. after 10 seconds the meter will show three flashing dashes, indicating it is ready for use.

3. Fill a microcuvette in one continuous process with capillary blood or well mixed venous blood.

4. Wipe off excess blood from the outside of the microcuvette tip, making sure no blood is withdrawn.

5. Within 10 minutes of filling the microcuvette, measure the haemoglobin. Place the microcuvette in the microcuvette holder and push in the holder to its measuring position.
After 15–60 seconds, the haemoglobin value
will be displayed (it will remain displayed for as long as the holder is left in its measuring position.)

THE EDTA ANTICOAGULATED BLOOD

When EDTA anticoagulated blood cannot be tested within 1–2 hours, it must be refrigerated at 4–8 C to prevent cellular changes affecting test results.
Manual or automated blood cell counts, reticulocyte counts, and PCV change little in EDTA blood at
4–8 C when stored for up to 24 hours. Haemoglobin concentration is stable for 2–3 days at 4–8 C providing there is no haemolysis

Blood films:

In EDTA anticoagulated blood, morphological blood cell changes occur soon after blood is collected when it is stored at room temperature (18–25 C) and within 3 hours when stored at
4–8 C.

It is therefore recommended that blood films be made and methanol-fixed as soon as possible after blood is collected and never made after overnight storage. Some of the blood cell changes which occur in EDTA blood include:

● Neutrophil degeneration with neutrophils
becoming more irregular in shape, nuclear lobes separating, and vacuoles appearing in the cytoplasm. There is also loss of granules.

● Segmentation (budding) of the nucleus of lymphocytes and monocytes and vacuoles appearing in the cytoplasm.

● Erythrocytes becoming crenated and spherocytic.

● Platelets disintegrating.

HOW CAN YOU AVOID HAEMOLYSIS OF BLOOD SAMPLES?

Haemolysis can be avoided by:

● Checking that the syringe and needle are dry and that the barrel and plunger of the syringe fit well.

● Not using a needle with too fine a bore.

● Not withdrawing the blood too rapidly or
moving the needle once it is in the vein. Frothing of the blood must be avoided.

● Removing the needle from the syringe before dispensing the blood into the specimen container and allowing the blood to run gently down the inside wall of the container.

● Adding the correct amount of blood to anticoagulant. Do not shake the blood but gently mix it with the anticoagulant.

● Using clean dry glass tubes or bottles for blood from which serum is required. Allow sufficient time for the blood to clot and clot retraction to take place. Red cells are very easily haemolyzed by the rough use of an applicator stick to dislodge a clot.

● Centrifuging blood samples for a minimum period of time. Centrifuging for 5 minutes at about 1000 g is adequate to obtain serum or plasma.

● Not storing whole blood samples in, or next to the freezing compartment of a refrigerator.