ROUTINE TESTS – Lablink

RETICULOCYTE COUNT TEST METHOD

1. Filter 2–3 drops of the stain into a small tube or vial.

2. Add about 4 drops of EDTA anticoagulated blood or capillary blood and mix well.
The amount of blood used is not critical. Use at least twice the volume of blood to stain if the patient is severely anaemic.

3. Incubate at room temperature for 20 minutes or 10–15 minutes at 35–37 C.

4. Mix gently to resuspend the red cells and using a capillary or plastic bulb pipette, transfer a drop of the stained blood to each of two slides. Spread to make two evenly spread thin films. Wave the slides back and forth to air-dry the films. Protect
the films from dust and insects until the count can be performed.

5. Count the reticulocytes microscopically. Use the 10 objective (with reduced condenser iris diaphragm) to check the distribution of the red cells. Select an area where the red cells can be seen individually, add a drop of immersion oil,
and examine using the oil immersion objective (open more the condenser iris diaphragm).

6. Count systematically (i.e. consecutive fields), 500 red cells (1 000 if there are very few reticulocytes), noting the number that are reticulocytes.
Calculate the percentage of reticulocytes.

Appearance of reticulocytes

Reticulocytes appear as pale green-blue stained cells containing dark blue-violet inclusions in the form of small granules, distributed irregularly. Mature red cells stain pale green-blue.

Counting reticulocytes:

A convenient method of counting reticulocytes is to reduce the size of the microscope field by inserting in each eyepiece a circular piece of black (opaque) paper which has
a punched out hole of about 5 mm.
To calculate % of reticulocytes:
– Using a hand tally counter, count a total of 500 red cells, noting on paper the number of cells that are reticulocytes (alternatively use two hand tally counters or a white cell differential counter).
– Multiply the number of reticulocytes counted by 2.
– Divide the figure by 10 to obtain the percentage figure.

See also:

Reticulocytes count principle

SOURCES OF ERROR WHEN MEASURING ESR

● Using the wrong volume of blood to anticoagulant.

● Blood not sufficiently mixed with anticoagulant.

● Clots in the blood. Even the smallest fibrin clot in the sample will invalidate the test result.

● Air bubbles at the top of the column.

● Testing blood samples at the hottest time of the day, or leaving tests in direct sunlight. Temperatures over 25°C increase sedimentation.

● Using a pipette which is not clean or not dry.

● Pipette not positioned vertically. Even slight variations from the upright increase sedimentation.

● Not checking whether the ESR stand is level on the bench.

● Placing an ESR stand on the same bench as a centrifuge where vibration will interfere with sedimentation.

● Measuring the ESR when a patient is dehydrated.

SEROLOGY TESTS PART 2

Antistreptolysin O Test (ASOT)

This test is used to detect the presence of Antistreptolysin O (ASO) antibodies in the serum. It widely used to detect conditions resulting from streptococcal infection and conditions secondary to a streptococcal infection. It also assists in the diagnosis of rheumatic fever, glomerulonephritis, bacterial endocarditis and scarlet fever.

C-Reactive Protein

The protein rises during inflammation and tissue destruction. It thus tested to chart to he progress of Rheumatoid arthritis, acute rheumatic fever, widespread malignancy and bacterial infections.

Cold Agglutinins

This detects presence of antibodies called cold Agglutinins. It is performed by incubating the patient’s serum with erythrocytes as cold temperature. The agglutination confirms the presence of as antibodies. It used to diagnose Infectious mononucleosis, mycoplasma pneumonia, chronic parasitic infections and lymphoma.

ABO and Rh typing

It is done to prevent transfusion and transplant reactions and to identify problems such as hemolytic diseases of the Newborn.

Rh antibody Titre

Detects amounts of antibodies in the blood. The antibodies can occur in a pregnant woman who is Rh-negative and is carrying an Rh-positive foetus. Most frequently applied in detection of Rh incompatibility problem with a mother and her unborn child.

See also:

Serology tests part 1

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

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.

ABNORMAL SPECIMEN APPEARANCE

● A dark coloured urine may be positive for bilirubin or haemoglobin.

● A urine that contains whole blood may contain S. haematobium eggs.

● A black faecal specimen may contain occult blood due to gastrointestinal bleeding.

● A dark brown serum may indicate intravascular haemolysis due to sickle cell disease, severe malaria, or an incompatible blood transfusion.

● A lipaemic (fatty) serum is associated with raised triglycerides (above 3.4 mmol/l).

● A deep yellow (icteric) serum indicates that a patient is jaundiced.

● A serum sample that is abnormally viscous (thick) or turbid may contain paraproteins.

● A serum that becomes markedly turbid after being refrigerated may contain cryoglobulins or cold agglutinins.

● A blood sample that contains a high concentration of red cells from which little serum or plasma can be obtained indicates severe dehydration or a blood disorder.

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

SEROLOGY TESTS PART 1

Hepatitis Tests

It is performed to viral hepatitis and the type of hepatitis present e.g A, B, C & D

Syphilis Tests

The method used is Venereal Disease Research Laboratories (VDRL) and the rapid plasma reagin (RPR) test. The test results are reported as none-reactive, weakly reactive or reactive. Weakly reactive results are considered positive for to the presence of syphilis antibodies. This is a screening test thus a more specific testing for final diagnosis Incase of positive result.

Mono Test

It is used to detect the infectious mononucleosis via identification of heterophile antibody.

Rheumatoid Factor

Rheumatoid arthritis is a chronic inflammatory disease that affects the joints of the body. Rheumatoid factor is antibody produced in this condition hence is tested for diagnosis.

HOW TO PREPARE CULTURE MEDIA

● Prepare media made from dehydrated products in as damp-free an environment as possible. To prevent the risk of inhaling fine particles of dehydrated media, wear a dust mask while handling dehydrated media.

● Wash the hands immediately after preparing media.

● Once the ingredients are weighed, do not delay in making up the medium. Follow exactly the manufacturer’s instructions.

● Use completely clean glassware, plastic or stainless steel equipment that has been rinsed in pure water. The container in which the medium is prepared should have a capacity of at least twice the volume of the medium being prepared.

● Use distilled water from a glass still. Deionized water can also be used providing the exchange resins do not contain substances inhibitory to bacteria (preparation of deionized and distilled water in district laboratories is described in Water containing chlorine, lead, copper, or detergents must not be used. Besides containing substances harmful to bacteria, impure water can alter the pH of a medium or cause a precipitate to form.

● Add the powdered or granular ingredients to the water and stir to dissolve. Do not shake a medium but mix by stirring or by rotating the container.

● When heating is required to dissolve the
medium, stir while heating and control the heat to prevent boiling and foaming which can be dangerous and damage the medium, e.g. DCA or TCBS agar. Overheating a medium can alter its nutritional and gelling properties, and also its pH.

● Autoclave a medium only when the ingredients are completely dissolved. Always autoclave at the correct temperature and for the time specified

● Dispense medium in bottles or tubes in amounts convenient for use. Know the length of time prepared media can be stored without deteriorating (take into account storage temperature)

COLLECTION OF 24-HOUR URINE SPECIMEN

1. Sanitize your hands and assemble the equipments

2. Greet and identify the patient. Introduce yourself to patient and explain the procedure

Instruct the patient as follows:

3. When you get up the in the morning empty your bladder into the commode just as you normally do In other words, this urine is not to be saved. Make note of the time.

4. The next time you need to urinate, void the urine directly into the plastic container.

5. Tightly screw the lid onto the container, and put into refrigerator or into an ice chest.

6. Repeat steps 4 & 5 each time you urinate.

7. On the next morning, get up exactly the same time ( exactly 24hrs after beginning the test). Void into the container for the last time.

8. Put the lid on the container tightly. Return the urine collection container to the office the same morning you complete the urine collection.

Provide the patient with the collection container and written instructions.