HDN DUE TO RHESUS INCOMPATIBILITY


Rhesus HDN is usually caused by immune anti-D and less commonly by other Rhesus antibodies. It occurs when a Rh negative mother with circulating IgG anti-D antibody (formed from a previous Rhesus
incompatible pregnancy) becomes pregnant with a Rh positive infant and IgG anti-D passes into the fetal circulation, destroying fetal cells. The infant can
be born severely anaemic and jaundiced.

The severity of disease increases with each Rh positive pregnancy. Infants with Rhesus HDN are usually more severely affected than infants with ABO HDN.
Note: In most cases of Rhesus HDN, maternal IgG anti-D will have been detected by the laboratory during routine antenatal visits, and the strength of the antibody (titre), monitored.
When Rhesus testing is not performed routinely (due to low frequency of Rh negative persons in the population) and an infant is born severely anaemic
and jaundiced, the laboratory may be asked to investigate the possibility of Rhesus HDN.

See also:laboratory investigation of HDN

Requirements for Single tube compatibility technique using AHG reagent

The following are required:
1. Patient’s serum

2. Donor’s washed 3% red cell suspension
prepared as follows:

  • Transfer 0.2–0.5 ml of red cells from the pilot tube of the donor blood into about 5 ml of physiological saline  and mix. 
  • Centrifuge at high speed (e.g. 1 000 g) for about 2 minutes.
  • Discard the supernatant fluid and resuspend the cells in a further 5 ml of saline. Mix, centrifuge, and discard the supernatant fluid.                                        
  • Prepare a 3% red cell suspension by adding 1 volume of packed cells to 30 volumes of saline.

3. Antiglobulin polyspecific (Broad-spectrum) reagent, usually colour coded green. AHG reagent will agglutinate red cells sensitized with antibodies and/or coated with detectable levels of complement components.

4. Anti-D serum to make AHG control cells.

5. AHG control IgG sensitized red cells, prepared as follows:

  • Wash group O Rh positive red cells (obtained from a group O Rh positive person) three times in saline. Discard the final saline supernatant fluid.
  • Add an equal volume of IgG anti-D to the packed red cells and mix.
  • Incubate at 37 C for 30 minutes. Wash the cells four times in saline. Remove the final supernatant fluid.
  • Suspend the packed cells in saline to make a 5% red cell suspension. When added to AHG reagent, the sensitized cells should show visible agglutination
  • Store the sensitized cells at 2–8 C. They can be kept for 2–3 days.

INVESTIGATION OF HEMOLYTIC DISEASE OF NEWBORN (HDN)

1. Carry out ABO and Rh grouping of the mother and infant.
There can be no Rhesus incompatibility caused by anti-D antibody unless the mother is Rh negative and the infant is Rh positive.
occasionally, when grouping the infant’s
cells they may not appear Rh positive when antigen D receptors on the baby’s cells have been coated with maternal anti-D.


3.  Measure the infant’s haemoglobin and serum bilirubin


2. Carry out a DAT test on the infant’s cord cells. The DAT will be positive in Rhesus HDN.


4. Examine a Romanowsky stained blood film for the features of HDN, including spherocytosis which is usually less marked than in ABO HDN, polychromasia (reticulocytosis) and many nucleated red cells.

With Rhesus HDN, the infant’s haemoglo-
bin is usually below 140 g/l (14 g/dl) and the serum unconjugated bilirubin may rise to over 340 µmol/l (20 mg%). Such high levels of unconjugated bilirubin can cause irreversible brain damage (kernicterus).


5. Test also the mother’s serum for anti-D antibody when this has not been tested previously.

ABO GROUPING


ABO grouping consists of:

● Cell grouping in which the red cells are tested for antigens A and B using anti-A and anti-B sera.
● Serum grouping (reverse grouping) in which the serum is tested for anti-A and anti-B antibodies using known A and B red cells.

Why perform both cell and serum grouping ?

  • It greatly reduces the risk of errors in ABO grouping (serves as a double check).
  • There is less risk of misgrouping a group A person with weak antigen A as group O (or group AB as group B) because the error will be detected when serum grouping.
  • Errors due to autoagglutination will also be detected more easily.
  • Serum grouping using a tube will also detect the presence of anti-A and anti-B haemolysins in group O donor blood.

Grouping infants and elderly patients

Serum grouping is not performed when grouping infants below 4 months of age because naturally occurring anti-A and anti-B antibodies are only formed 3–4 months after birth.

When ABO grouping elderly people or persons with a gamma globulin deficiency, anti-A and anti-B may react weakly in the serum group and therefore cell grouping will be more reliable.

Method of performing Single tube compatibility test

  1. 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.
  2. Pipette 3 volumes of patient’s serum  the tube.
  3. Add 1 volume of donor’s washed 3% red cell suspension and mix.
  4. Centrifuge at slow speed e.g. at 150 g for 1 minute or 500 g for 10 seconds.
  5. 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.
  6. When there is no agglutination, mix the contents of the tube and incubate at 37 C for 20–30 minutes.
  7. 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.
  8. 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.
  9. Resuspend the cells by tapping the bottom of the tube. Add 2 drops of AHG reagent and mix.
  10. Centrifuge at slow speed, e.g. at 150 g for 1 minute or at 500 g for 10–15 seconds.
  11. 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.
  12. Enter the test results in the Blood Transfusion Records book.

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:

Blood sample for ABO grouping

FORMATION OF RED CELL ANTIGENS A AND B FROM H SUBSTANCE

Dominant H gene (located on chromosome 19) encodes for an enzyme which converts a carbohydrate precursor substance in red cells into H substance (H antigen). A and B genes encode for specific transferase enzymes which convert H substance into A and B red cell antigens. O gene encodes for an inactive transferase enzyme which results in no conversion of the H substance in group O red cells.

Persons who do not inherit H gene (very rare hh genotype) are unable to produce H substance and therefore even when A and B genes are inherited, A and B antigen
cannot be formed. This rare group is referred to as Oh, or Bombay group (originally recognized in Marathi-speaking
people of India). Antibodies anti-A and anti-B are present in the blood of Oh Bombay persons.

Secretors: Up to 80% or more of people inherit the secretor gene Se and secrete water soluble H, A, and B antigens in
their saliva, plasma, and other body fluids in addition to expressing the antigens on their red cells. Knowing whether a
person is a secretor is not important in routine blood transfusion practice.

GUIDELINES WHEN USING ABSORPTION GAS REFRIGERATOR IN BLOOD BANK

It is important to ensure that the refrigerator:

Is fitted with a gas thermostat and whenever possible is tropicalized, i.e. very well insulated.

Is sited in a cool, airy, secure place away from direct sunlight. It must be level (checked by using a spirit level) and correctly installed.

Maintains the correct temperature for storing blood, ideally 4–6 C and not outside the range 2–8 C. A thermometer (preferably maximum/ minimum type) must be kept inside the refrigerator in a bottle of water and each morning the temperature should be checked and recorded on a chart.

Is correctly and safely connected to a gas supply.

The gas must not be allowed to run out.

Whenever possible two cylinders with an automatic change-over valve should be used. When this is not possible, the amount of gas in the cylinder must be monitored. In practice, the amount of gas used by the refrigerator over a period of time becomes known so that a judgement can be made when to change a cylinder in advance of the gas running out. Any gas remaining in the former cylinder can be used elsewhere in the hospital.

Is maintained as recommended by the manufacturer.

SCREENING DONOR BLOOD FOR INFECTIOUS DISEASES


● Human immunodeficiency virus (HIV) 1 and 2:

The risk of developing HIV disease/AIDS after being transfused with HIV infected blood is high (greater than 95%). All donor blood must be screened for antibody to HIV-1 and HIV-2 using a sensitive test. Transmission of HIV in donor blood can be minimized by using low risk voluntary unpaid donors and giving donors the opportunity to self-exclude when they suspect infection with HIV.

Even when an HIV antibody screening test is negative, blood may still contain HIV. This can happen when blood is collected during the ‘window period’, i.e. soon after a donor becomes infected with HIV when antibody to the virus is
not yet detectable in the serum.

● Hepatitis B virus (HBV):

Those at greatest risk of developing viral hepatitis from HBV infected blood
and blood products are young children and
those without effective immunity. Tests to screen donor blood for HBV are based on the detection of hepatitis B surface antigen (HBsAg).

● Hepatitis C virus (HCV).

This can cause viral hepatitis in recipients but it is not as infectious as HBV. Following acute infection, 70–80% of individuals become chronic HCV carriers with the
risk of developing liver cirrhosis and liver cancer later in life. Information on the epidemiology of HCV in tropical countries is incomplete. Where the prevalence of HCV is known to be high, donor blood should be screened for antibody to HCV when this can be afforded.

● Treponema pallidum

Transfusing blood containing T. pallidum can cause syphilis in recipients but
the risk of transmitting the disease is low, particularly when donated blood is stored at 2–8 C for 48–72 h which inactivates T. pallidum.

● Plasmodium species

Transfusing blood containing malaria parasites can cause malaria in recipients without effective immunity, e.g. young
children and pregnant women. Blood should not be collected from donors with suspected malaria.

In malaria endemic areas it is not feasible to screen all donor blood for malaria parasites or reject donors who have had malaria previously. In some malaria endemic areas, it is the policy to give curative antimalarial drugs followed by prophylactic drugs for 3 weeks to all recipients of blood. Tests to detect malaria parasites in blood and rapid antigen tests to diagnose malaria are available.

● Trypanosoma cruzi:

Transfusing blood containing
T. cruzi can cause Chagas’ disease
T. cruzi is endemic in South and Central American countries from Mexico to Argentina. Donors infected with
T. cruzi are often asymptomatic and therefore in endemic areas, donor blood must be screened for T. cruzi. Guidelines regarding the most appropriate test to use in a particular area should be obtained from the nearest Chagas’ Disease
Reference Laboratory or regional blood transfusion.

● Human T cell lymphotropic virus (HTLV)1

This virus can cause HTLV disease, adult T-cell leukaemia/lymphoma (ATLL), or tropical spastic paraparesis (TSP). It has a high prevalence in parts of Central and South America, the Caribbean, and parts of sub-Saharan Africa. It is estimated that about 60% of recipients receiving HTLV infected blood actually seroconvert. The risk of developing disease later in life is thought to be low. HTLV antibody screening tests are expensive.