- Label a small (e.g. 75×12 mm) clean glass tube with the number of the donor blood and write this number also on the patient’s blood transfusion request form.
- Pipette 3 volumes of patient’s serum the tube.
- Add 1 volume of donor’s washed 3% red cell suspension and mix.
- Centrifuge at slow speed e.g. at 150 g for 1 minute or 500 g for 10 seconds.
- Tilting the tube back and forth, examine for haemolysis or agglutination. Haemolysis or agglutination means that the donor blood is ABO incompatible. The blood MUST NOT BE GIVEN TO THE PATIENT. When there is haemolysis or agglutination, recheck the ABO group of the patient and donor blood and also check that the correct patient’s blood sample has been tested.
- When there is no agglutination, mix the contents of the tube and incubate at 37 C for 20–30 minutes.
- Centrifuge at slow speed. Tilting the tube back and forth, examine for haemolysis or agglutination. Haemolysis or agglutination indicate that the blood is incompatible and must not be given to the patient.
- When there is no haemolysis or agglutination, perform an indirect AHG test. Fill the tube with saline, centrifuge (high speed), and remove the supernatant fluid. Wash the cells a further 3 times. At the end of the final wash remove all the supernatant fluid. Careful washing of the cells is essential. Traces of globulin left in the tube will neutralize the AHG reagent.
- Resuspend the cells by tapping the bottom of the tube. Add 2 drops of AHG reagent and mix.
- Centrifuge at slow speed, e.g. at 150 g for 1 minute or at 500 g for 10–15 seconds.
- Tilting the tube back and forth, look for agglutination. When no agglutination is seen, transfer a few of the cells to a slide and check for agglutination microscopically using the 10 objective. When there is no agglutination, check that the AHG has not been neutralized by adding 1 drop of AHG control sensitized cells to the tube. Repeat steps 10–11. The control cells will show agglutination, providing the AHG is active and the test has been performed correctly. When there is agglutination after adding AHG reagent, this means that the patient’s serum contains an immune IgG antibody reactive against the donor’s cells which may cause a transfusion reaction.
- Enter the test results in the Blood Transfusion Records book.
Category Archives: LABORATORY PROCEDURES
CAUSES OF DISCREPANCIES IN ABO GROUPING
1. Deterioration of reagents
Occasionally difficulties in ABO grouping are caused by using expired or contaminated reagents or incorrectly prepared or heavily contaminated
physiological saline.
Correction: Appropriate controls should be used to check the reactions of antisera and test cells.
Prepare fresh saline if contamination is suspected (check a sample microscopically).
2. Rouleaux
Rouleaux causes red cells to stack together (like piles of coins), giving the appearance of agglutination when there is no true agglutination. It can occur when
a patient has a protein abnormality, e.g. myelomatosis or when dextran, PVP, or similar product has been given intravenously. In cord blood samples, rouleaux can be caused by contamination
of the sample with gel substances such as Wharton’s jelly when applied to the cord of a newborn (wash the infant’s red cells with saline).
Marked rouleaux can cause discrepancies in serum grouping (particularly when tile grouping) and occasionally in cell grouping when using whole blood, unwashed or insufficient washed red cells.
Rouleaux can usually be distinguished from true agglutination by examining the red cells microscopically.
Correction: Whenever suspected, add a drop of saline to the cells. Rouleaux usually disperses after 1–2 minutes following the addition of saline. Serum grouping should be repeated using serum diluted 1 in 2 in saline (mix 1 drop of saline with 1 drop of serum).
This will cause rouleaux to disperse
3. Autoagglutinins
Occasionally a patient’s serum may contain autoagglutinins which are antibodies that cause the agglutination of a person’s own and other red cells (autoagglutination). They are known to occur in lymphoma, leukaemia, virus pneumonia, systemic
lupus erythematosus and other autoimmune diseases, and occasionally in severe falciparum malaria. Autoagglutination may also occur following
treatment with some herbal preparations. Cold agglutinins active at room temperature are a frequent cause of autoagglutination.
When autoagglutinins are present, a patient’s red cells appear agglutinated before commencing grouping. This can be confirmed by setting up an autocontrol.
Correction: When autoagglutination due to cold agglutinins is suspected, the cell grouping should be repeated after washing the patient’s red cells in warm saline and the serum group and autocontrol
read after incubation at 37°C. The autocontrol should then be negative.
4. Faulty technique
When it is possible that a technical error in blood grouping has occurred, repeat the cell and serum grouping (using a spun tube technique).
ANT A & B SERA FOR ABO CELL & SERUM GROUPING
Whenever possible, use monoclonal blood grouping antisera. Most commercially available antisera are monoclonal. Anti-A antiserum is colour coded blue and anti-B antiserum is colour coded yellow.
Commercially produced antisera contain a preservative (e.g. 0.1% sodium azide) and most have a shelf-life of two years from the date of manufacture.
Antisera require storage at 2–8°C. Not all antisera can be frozen (this information can usually be found in the manufacturer’s literature).
When using a monoclonal anti-A antiserum it is not necessary to include an anti-AB antiserum in cell grouping.
Most monoclonal anti-A and anti-B sera are produced by tissue culture using hybridoma cell lines derived from fusing
(hybridising) mouse myeloma cells with specific antibody secreting lymphocytes (from immunized mice). The fused
cells are cloned and cultured.
Why use Monoclonal reagents?
- They are specific, stable and reproducible, giving consistent results.
- They agglutinate strongly rapidly (avidly) and are able to detect weak reacting antigen variants, e.g. A2 and other weak antigen A variants.
- Being of animal origin, they are free from infectious agents such as HBV and HIV.
- Polyagglutination due to the Thomsen phenomenon does not occur because anti-T antibody is not present in monoclonal antisera.
See also:
PART 2
9. Tie tightly the loose knot in the tubing or fold back the tubing and apply a sealing clip, about 20 cm from the needle.
10. Cut the tubing between the clamp and knot
11. Collect a blood sample from the donor into a plain tube for testing (grouping, HIV screening,
etc). Unclamp the tubing and allow 5–7 ml
blood to run out of the tubing into a tube or
vial. Reclamp the tubing.
12. Remove the pressure cuff. Take the needle out of the vein, applying pressure with cotton wool.
Ask the donor to continue applying pressure to the venepuncture site with his or her other hand. Dispose of the needle safely.
13. Mix the blood in the bag and push (‘strip’) the non-anticoagulated blood from the tubing into the blood bag. Mix and allow the tubing to refill with the anticoagulated blood.
14. Write clearly the identity number of the donor on the blood pack and sample tube(s). When a blood pack has already been given an identity number, e.g. from the blood transfusion service use this number on the sample tube(s). Do not
use the manufacturer’s lot or batch number
because this will be the same on several blood packs.
15. After making sure the bleeding has stopped, cover the venepuncture site with a pad of cotton wool and adhesive tape (advise the donor to remove the dressing the following day). Thank the person for donating.
16. Give the donor a drink (not alcohol) to make up his or her fluid loss.
Provide the donor with a dated certificate of blood donation and information concerning future donation.
17. Check that the blood pack and blood sample(s) have been labelled correctly and the same identity number is written on the blood donation certificate.
18. Refrigerate the blood after allowing time for it to cool and for natural bactericidal activity of white
cells (1–2 h). Do not leave a blood bag in direct sunlight. To avoid raising the temperature of the blood bank, first store the blood for 3–4 hours in another refrigerator (when the blood bank is
an absorption type refrigerator.
TECHNIQUE OF COLLECTING BLOOD FROM A DONOR
1. Apply a deflated pressure cuff to the upper arm about 6 cm above the elbow. Raise the pressure to between 60 and 80 mm Hg to enable the veins to be seen and felt. Select a large well situated vein for the venepuncture, usually near the bend of the elbow.
2. Clean very well the required part of the arm with cotton wool and 70% ethanol (alcohol). Wipe dry with a clean swab of cotton wool.
3. Take a blood collecting pack:
- Make a loose knot (without kinks) in the tubing. When sealing clips are available, there is no need to make a knot in the tubing.
- Suspend the bag on a stand (linked to a Balance) about 30 cm below the level of the donor’s arm.
- Clamp the tubing near to the needle guard. Whenever possible use plastic forceps to avoid damaging the tubing.
4. Make a venipuncture with the needle directed upwards in the line of the vein. Unclamp the tubing to allow the blood to flow. If necessary, secure the needle in place with a small strip of adhesive tape.
5. When the blood begins to flow, reduce the pressure of the cuff to 40–60 mm Hg, and ask the donor to squeeze slowly a small object.
6. When the blood enters the pack, gently mix it with the anticoagulant by lifting and tilting the bag. Do not squeeze the bag because this can damage the red cells. Mix the blood a further three times during the donation and when the donation is finished.
7. When the pack weighs 500–600g, the donation is complete, i.e. 450–495 ml blood has been collected.
8. Reduce the pressure in the pressure cuff to zero and remove the object from the donor’s hand. Clamp off the tubing 10–15 cm from the needle.
GENERAL GUIDELINES FOR BLOOD TRANSFUSION
Blood is used only when it is absolutely necessary after a careful clinical assessment and measurement of a patient’s haemoglobin (or PCV).
It is inappropriate to use blood to treat patients with:
- Stable anaemia without signs of heart failure or respiratory distress which can be treated by other means.
- Acute blood loss when it is possible to use replacement fluids (crystalloids or colloids) to maintain blood pressure and oxygenation.
Recipients may be placed at high risk when using blood that;
- Has been collected from a high risk donor.
- Has not been collected aseptically using a sterile technique.
- Has not been transported or stored correctly.
- Has not been screened for important pathogens using sensitive assays.
- Has not been typed (grouped) and compatibility tested correctly using standardized controlled procedures.
Measures taken to reduce the need for blood transfusion
- Reducing the prevalence and severity of anaemia, particularly among pregnant and young children.
- Avoiding obstetric related haemorrhages by improving antenatal care and attendance at clinics. Implementing effective malaria control.
- Using health education to improve nutrition and awareness of the causes of anaemia and its prevention.
- Reducing blood loss during surgery and ensuring alternatives to blood are available
BLOOD TRANSFUSION IN VARIOUS CASES
TREATMENT OF ANAEMIA
For adults, including pregnant women, blood transfusion is indicated when:
- A patient is in danger of dying of anaemic heart failure or hypoxia before specific medication can raise the haemoglobin.
- Obstetric delivery is imminent and the mother’s haemoglobin is below 70 g/l (7 g/dl).
- Emergency major surgery is essential and the haemoglobin is below 80 g/l (8 g/dl) with an anticipated blood loss of more than 500 ml.
In the above situations, the use of concentrated red cells (10 ml/kg body weight), is indicated to avoid cardiac overload. The transfusion should be administered slowly over 4–6 hours. When indicated, a rapidly acting diuretic should be administered. The pulse and respiratory rate should be monitored and the chest examined to detect volume overload.
.
For infants and young children, blood transfusion is indicated when:
- The haemoglobin is below 50 g/l (5.0 g/dl) and is associated with respiratory distress.
- The haemoglobin is below 40 g/l (4.0 g/dl) and is complicated by malaria or bacterial infection even without respiratory distress.
- The haemoglobin is below 30 g/l (3.0 g/dl) without apparent infection or respiratory distress.
In the above situations, transfusion with whole blood (not packed cells), 10 ml/kg body weight, without diruretics will be tolerated.
Children with respiratory distress but not profound anaemia should be treated with intravenous colloids, and be transfused only if the haemoglobin falls later to less than 50 g/l.
TREATMENT OF ACUTE HAEMORRHAGE
● Blood transfusion is indicated when there is acute haemorrhage with a loss of more than 30% of apatient’s total blood volume, and blood pressure and oxygenation cannot be maintained by crystalloid solutions (saline or Ringers’ lactate) or colloids (e.g. 5% dextran or 5% hydroxyethylstarch).
Acute blood loss should be managed by
replacement of volume. Only when shock
persists or worsens should whole blood be transfused.
Postpartum haemorrhage: Blood transfusion is indicated when hypotension and reduced cerebral function persist
after at least 50 ml/kg of volume replacement fluid has been given intravenously and all measures have been
taken to stop blood loss.
TREATMENT OF NEONATAL JAUNDICE
For newborn infants with a serum bilirubin above 300 µmol/l, an exchange blood transfusion is indicated.
HYPOGLYCAEMIA
A low blood glucose level is called hypoglycaemia. Persistent occurrences of hypoglycaemia with glucose levels less than 2.2 mmol/l accompanied by
symptoms such as fainting, fits, sweating, hunger, pallor, confusion, or violence, should be investigated. Causes of hypoglycaemia include severe malnutrition, kwashiorkor, severe liver disease, alcoholic excess, insulin secreting tumours, Addison’s disease, and certain drugs. Commonly, however, markedly
reduced blood glucose levels occur following the overtreatment of diabetes.
Neonatal hypoglycaemia
Newborn infants may suffer hypoglycaemia when blood glucose levels fall below 1.1 mmol/l. Infants particularly at risk are
underweight poorly nourished babies, twins, premature infants, and babies born of diabetic mothers. It is important to detect hypoglycaemia of the newborn because without treatment brain damage may
occur.
Malaria associated hypoglycaemia
In severe malaria, hypoglycaemia is a common finding and can increase mortality particularly in young children.
Hypoglycaemia can also occur in those being treated with quinine and quinidine.
False glucose values
A falsely high glucose level will result if a blood sample is collected from an arm receiving a glucose (dextrose) intravenous
(i.v.) infusion. A falsely low value may be obtained if the plasma is markedly icteric.
See also:
PREPARATION OF GLUCOSE CALIBRATION GRAPH
1. Take six tubes and label them ‘B’ (reagent blank) and 1 to 5. Pipette 1.5 ml of protein precipitant reagent into each tube. Note: The use of the protein precipitant reagent is required because it forms part of the buffer system and contains phenol which is needed for the colour reaction.
Add to each tube as follows:
Tube
B …………………….. 0.05 ml distilled water
1 ……………………. 0.05ml standard, 2.5 mmol/l
2 ……………………..0.05ml standard, 5 mmol/l
3 ……………………..0.05ml standard, 10 mmol/l
4 ………………………0.05ml standard, 20 mmol/l
5 ……………………..0.05ml standard, 25 mmol/l
3. Continue as described in steps 5 to 7 of the glucose method.
4. Take a sheet of graph paper and plot the absorbance of each standard (vertical axis) against its concentration in mmol/l (horizontal axis). A linear calibration should be obtained.
The useful working limit (linearity) of the glucose oxidase method is about 25 mmol/l.
Check the calibration graph by measuring a control serum. A new calibration graph should be prepared and checked against controls whenever stock reagents are renewed.
THROMBIN TIME (TT) TEST PROCEDURE
(Courtesy of Diagen Thrombin Test Time Kit. Test )
Test the patient’s plasma and
control plasma in duplicate.
- Pipette 200 l (0.2 ml) of plasma into a small glass tube. Incubate at 37°C for 1–2 minutes.
- Add (0.1 ml) of thrombin, 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 stopwatch and record the time in seconds.
- Report the patient’s TT (average of the duplicate tests) providing the TT of the control plasma is satisfactory.
What are the normal ranges for TT?
12–15 seconds
What are causes of prolonged TT?
- DIC and other conditions which produce a low fibrinogen level
- Abnormal fibrinogen
- Treatment with heparin
- Liver failure
- Presence of inhibitors of thrombin such as FDPs
See also:
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