Category Archives: HEMATOLOGY

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

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.

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.

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.

View Previous Steps

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.

Continue to next steps……

TYPES OF BLOOD COLLECTION PACK

● Single bag collection pack

For collecting 450 ml blood. When concentrated red cells are required, the plasma can be removed and discarded following sedimentation of the red cells or the transfusion can be stopped when the plasma level is reached.

● Double bag collection pack

Collects 450 ml blood which enables plasma to be saved following its transfer (in a closed system) to the attached sterile bag. A double bag pack costs twice the price of a single bag pack.

● Paediatric quadruple bag collection pack


Consists of a blood collecting bag (usually for 250 ml blood) with three small sterile bags attached.
Following blood collection, the well mixed blood is subdivided in a closed sterile system between the three attached sterile bags, providing individual small volume packs. When concentrated red cells only are required, the transfusion can be stopped when the plasma level is reached (packs need to be stored inverted). A paediatric bag pack costs about three times the price of a single bag collection pack.

SOP REQUIREMENTS IN BLOOD TRANSFUSION (PART 2)

Compatibility testing (cross-matching) of blood SOPs should include:

  • Details of the request form and patient’s
  • blood sample.
  • Procedure for compatibility testing including use of controls, interpretation and recording of test results.
  • Procedure for emergency compatibility testing.
  • Labelling compatible blood.
  • Preparation of concentrated red cells.
  • Procedure for investigating a transfusion reaction.

Safety issues SOPs should include:

  • Safe handling of blood and blood products.
  • Decontamination of work surfaces and laboratory-ware and preparation of sodium hypochlorite solutions
  • Disposal of ‘sharps’
  • Disposal of contaminated and expired blood.

Procurement of supplies SOPs should include:

  • Procedures for ordering essential reagents, HIV and other test kits.
  • Recording expenditures and keeping financial accounts.
  • Reliable systems for transporting essential supplies.
  • Checking expiry date and specifications, and recording supplies upon their receipt.
  • Storage requirements of antisera reagents.

See also:

SOP REQUIREMENTS PART 1