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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.

TYPES OF FUNGAL MEDIA

1. Czapek’s agar:

It is used for the subculture of Aspergillus species for their differential diagnosis. It contains sucrose as C-source and nitrate as the sole source of nitrogen, useful for the general cultivation of fungi, yeasts and soil bacteria.

2. Potato Dextrose Agar (PDA):

It is a relatively rich medium for growing a wide range of fungi. Many standard procedures use a specified amount of sterile tartaric acid (10%) to lower the pH of this medium to 3.5 +/- 0.1, inhibiting bacterial growth. Chloramphenicol acts as a selective agent to inhibit bacterial overgrowth of competing microorganisms from mixed specimens, while permitting the selective isolation of fungi.

3. Sabouraud’s dextrose agar (SDA): Sabouraud Dextrose Agar (SDA) is a selective medium primarily used for the isolation of dermatophytes, other fungi and yeasts but can also grow filamentous bacteria such as Nocardia. Sabouraud agar is used to culture fungi and has a low pH that inhibits the growth of most bacteria; it also contains the antibiotic gentamicin to specifically inhibit the growth of Gram-negative bacteria. Hay infusion agar is specific for the culturing of slime moulds. The acidic pH of this medium (pH about 5.0) inhibits the growth of bacteria but permits the growth of yeasts and most filamentous fungi. Antibacterial agents (with antibiotics) can also be added to augment the antibacterial effect.

4. Brain-heart infusion agar (BHIA). Malt extract and less commonly brain heart infusion medium. To prevent contamination of the medium by bacteria, chloramphenicol is used, but prevents the growth of Actinomyces, which others grows well on Sabouraud dextrose agar.

HOW TO TEST A SELECTIVE MEDIUM

1. Prepare a 5 h broth culture of the organism to be selected and a 5 h mixed broth culture of the organism(s) to be
inhibited.

2. Take three sterile tubes and label 1 to 3. Using a sterile Pasteur pipette, place in each tube, 5 drops of the broth
culture containing the organism to be selected.

3. Using a second sterile Pasteur pipette, add to each tube the following drops of the mixed broth culture containing
the organisms to be inhibited:
Tube 1: 5 drops
Tube 2: 10 drops
Tube 3: 15 drops

4. Divide a plate of the medium to be tested into three segments and label 1, 2, and 3. Using a small sterile loop, inoculate the appropriate segments of the plate with a loopful of the organism suspension from the tubes (i.e. 1 loopful from tube 1 in segment 1, etc).

5. Inoculate a second plate of the medium with a loopful of the pure 5 h broth culture of the organism to be selected.

6. After overnight incubation at 35–37 C, record the degree of selectivity of the medium (from the segmented plate)
and the size and appearance of the colonies of the selected organism (from the pure culture plate). Compare with the results of previous performance tests.

VENIPUNCTURE TECHNIQUE

1. Select a sterile, dry, preferably plastic syringe of the capacity required, e.g. 2.5 ml, 5 ml, or 10 ml.
Attach to it a 19 or 20 SWG needle (preferably a disposable one). If the patient is a child or adult with small veins, use a 23 SWG needle.

Note: When not using a disposable syringe or needle, check the syringe for good suction and the needle for any blockage, directing the syringe and needle safely away from the patient.
Ensure all air is expelled from the syringe.
Whenever possible use a disposable needle and syringe.

2. Apply a soft tubing tourniquet or velcro fastening arm band to the upper arm of the patient to enable the veins to be seen and felt.
Do not apply the tourniquet too tightly for longer than 2 minutes. Ask the patient to make a tight fist which will make the veins more prominent.

3. Using the index finger, feel for a suitable vein, selecting a sufficiently large straight vein that does not roll and with a direction that can be felt.
If a vein cannot be felt, apply a pressure cuff above the elbow and raise the pressure to 80 mm (deflate the cuff once the needle is in the vein).

4. Cleanse the puncture site with 70% ethanol and allow to dry. Do not re-touch the cleansed area.

5. With the thumb of the left hand holding down the skin below the puncture site, make the venepuncture with the bevel of the needle directed upwards in the line of the vein. Steadily withdraw the plunger of the syringe at the speed it is taking the vein to fill. Avoid moving the needle in vein

If the plunger is withdrawn too quickly this can cause haemolysis of the blood and the collapse of a small vein.

6. When sufficient blood has been collected, the tourniquet and instruct the patient to open his or her fist. Remove the needle and immediately press on the puncture site with a piece of dry cotton wool. Remove the tourniquet completely. Instruct the patient to continue pressing on the puncture site until the bleeding has stopped.

7. Remove the needle from the syringe and carefully fill the container(s) with the required volume of blood. Discard the needle safely.

Do not attempt to re-sheath it because this can result in needle-stick injury.
Important: Do not fill a container with the needle attached to the syringe. Forcing the blood through the needle can cause haemolysis.

8. Mix immediately the blood in an EDTA or citrate anticoagulated container. When required, make a thick blood film from the blood remaining in the syringe. Immediately label carefully all the
blood samples.

9. Check that bleeding from the venipuncture site has stopped. Cover the area with a small dressing

STERILE TECHNIQUES FOR TRANSFERRING FUNGAL CULTURE

1.Take an inoculating needle, usually a thin needle or wire at the end of a long pencil-like handle, and heat it in an alcohol or gas flame until it glows bright red

2. Allow the needle to cool for about 15 seconds. (A hot needle will kill the mould that is to be transferred).

3. Open the Petri dish containing the culture just wide enough to allow entry of the needle.

4. With the heat-sterilized needle, cut out a small portion of the colony margin. Hyphal tip transfers work best as they are usually the most active parts of the culture; in addition, transfers from the heavily sporulating central portions will result in spores being spread into the air. Especially in medical work, hyphal tip transfers are essential. The excised colony margin should be only about 1 mm square.

5. Transfer the square of colony margin to the sterile plate, making sure that the lid is opened only wide enough to admit the needle and make the transfer. Place the block at the centre, withdraw the needle and flame it until it is red hot, to kill all adhering spores and hyphae.

6. Close the lid; label the plate with a marking pen, including name of culture and date. We usually wrap a thin strip of paraffin film around the sides of the plate to cover the opening, but this is not absolutely necessary; just a couple of pieces of masking tape to hold the lid down will do.

7. Leave the culture to grow in a protected place that has as little air movement as possible.

HOW TO COLLECT CAPILLARY BLOOD

Capillary blood can be collected from:

The ‘ring’ finger of a child or adult
Do not stick the thumb or index finger
as these are the most sensitive.

The heel of an infant up to one year old Care must be taken not to damage the heel by sticking it too near the edge or by holding it too forcibly

Make sure the puncture area is warm to allow the blood to flow freely. On cold days soak the hand or foot of an infant in warm water prior to collecting a sample.

1 Cleanse the puncture area with 70% ethanol. Allow the area to dry.

2 Using a sterile pricker or lancet, make a rapid puncture, sufficiently deep to allow the free flow of blood

3 Wipe away the first drop of blood with a dry piece of cotton wool and use the next few drops for the test. Do not squeeze too hard because this will result in an unreliable test result.

4 When sufficient blood has been collected, press a piece of dry cotton wool over the puncture area until bleeding stops.

CHRONIC LYMPHOID LEUKEMIA

Marked lymphocytosis
Smugde cells are present
CD 19+ and weak IgM & IgD expression
Normochromic normocytic anaemia Reduced concentration of serum immunoglobulins
Neutropenia and red cell aplasia
Nodullar diffuse bone marrow, high concentration of lymphocytes
White pulp in the skin

Clinical Features

Lymphadenopathy
Splenomegaly (less common)
Hepatomegaly( late stages)
Bruising
Immunosupression

Staging

Lymphocytosis >5×10^9/L
1 + enlarged lymph
2 + enlarged liver and spleen
3 +anemia and splenomegaly
4 + thrombocytopenia

CHRONIC MYELOID LEUKEMIA

Arises due to Philadelphia chromosome t(22;9) The BCL- ADR oncogene

Marked leukocytosis with left shift (<200×10^9)
Normocytic normochromic anaemia
Increase in basophils and platelets
Hypercellular bone marrow (granulopoietic predominant)
High serum uric acid

Clinical Features

Hypermetabolism
Splenomegaly
Features of anaemia
Abnormal platelet count
Renal diseases
Rare visual disturbance, priapism

Staging

Chronic phase: more than 10% blasts
Accelerated phase: 15-30% blasts, 20% basophils and thrombocytopenia
Blasts phase: more than 20% blasts, it is acute and respond poorly to treatment

SERUM CREATININE TEST

Serum creatinine is a better indicator of overall renal function and progression to renal failure.

Serum creatinine levels are less affected than urea levels by age, dehydration, and catabolic states, e.g. fever, sepsis, and internal bleeding as compared to UREA

Creatinine levels are also less influenced by changes in diet such as low intake of protein (providing this is not prolonged).

Increasingly the measurement of serum creatinine is being used to investigate HIV associated renal disease and to monitor patients being treated
with nephrotoxic antiretroviral drugs, e.g. tenofovir.

What are normal Ranges?

Males: 60–130 mol/l 0.7 to 1.4 mg/100 ml

Females: 40–110 mol/l 0.4 to 1.2 mg/100 ml

Lower in children depending on
muscle mass.

What does results indicate?

Increase in serum creatinine levels is associated with diseases that cause renal failure:

Diseases that can cause renal failure with a reduced GFR include glomerulonephritis (inflammation of the kidney glomeruli), pyelonephritis (inflammation of the pelvis of the kidney), and renal tuberculosis.

SERUM UREA TEST

Is an indicator of renal function but is more affected by state of hydration and dietary intake. CREATININE is therefore more preferred.

What are normal ranges for UREA TEST?

Adults: 3.3–7.7 mmol/l OR 20–46 mg/100 ml

Infants: 1.3–5.8 mmol/l OR 8–35 mg/100 ml

Values are higher in the elderly and slightly lower in females.

What causes rise in serum Urea levels?

Renal causes of increase are same as those of CREATININE
Non-renal causes: Slight increases in urea (not more than three times the upper limit of the reference range) may occur when there is:
– Dehydration
– Diuretic therapy
– Gastrointestinal blood loss
– Any condition associated with increased protein breakdown such as pneumonia, malaria, meningitis, typhoid, major trauma, and surgical operations.

What causes low serum levels?

Low urea levels may be found in:
– Pregnancy
– Malnutrition and AIDS
– Severe liver disease
– Water overload