Tissue Immunology Section

1. Human Leucocyte Antigen (HLA) Typing

(i) HLA Serology typing

The Tissue Immunology and Paternity Laboratory have a close partnership with the Sunflower Fund, and the South African Bone Marrow Registry (SABMR).

The Sunflower Fund is responsible for creating awareness and education in the community. Funds raised assist with the cost incurred for laboratory testing. Blood samples are collected from donors and subjected to HLA Class I typing (Locus A + B). The test results are then released to the SABMR who capture and include the typings on the register.

Should you require further information on how to become a bone marrow donor, please contact the SUNFLOWER FUND on 0800 121082

A link between some HLA types and certain disease has been researched and documented e.g. locus B27 is associated with Ankylosing Spondylitis. Class I typing by serology can then be performed to determine whether the patient posesses this antigen and therefore is at risk for the disease.

(ii) HLA Molecular Class I and II Typing

Molecular typing is used as a powerful tool to further tissue type patients requiring solid organ and bone marrow transplantation.

Potential donors who have HLA matched a patient at a serological level, are then further typed to confirm HLA compatibility.

Requests are received from the SABMR to contact bone marrow donors who have potentially matched a patient at a serological level. The laboratory then performs molecular typing to ascertain the HLA Class II type i.e. Locus DRB.

(iii) HLA antibody screening and identification

Renal patients are tested on a regular basis for the presence of HLA antibodies. These antibodies are produced as a result of sensitizing factors, such as transfusions and pregnancies. The antibodies are detected by an ELISA-based method and identified by a lymphocytotoxicity method.

There are two types of donors for these patients; the first is the related living donor (RLD). The second type of donor is the cadaver donor, who is a person who has been declared brain-dead and permission has been obtained from family for the use of his/her organs.

A crossmatch between the patient’s serum and donor’s T and B lymphocytes is performed by a complement-dependant cytotoxicity method. A positive T cell x-match is a contraindication for transplantation with the donor. A positive B cell x-match, when the patient has antibodies, also carries a high risk of antibody-mediated damage and possible organ rejection.

Patients awaiting solid organ transplantation regularly submit samples to the laboratory to monitor their antibody status and for inclusion onto the transplant list. All patients awaiting transplantation are crossmatched against cadaver lymphocytes, and those patients who are negative for T and B cell crossmatches and are then considered for transplantation. The decision on whom to transplant rests with the nephrologist and surgeon.

Contact details
Area 1 (Johannesburg) Tissue Immunology + Paternity Laboratory 011 761 9227
Area 2 (Durban) Tissue Immunology + Paternity Laboratory 031 719 6619

2. Platelet typing

Platelets play an integral part in a number of clinical conditions, the most commonly seen being Neonatal Alloimmune Thrombocytopenia (NAIT) and Platelet refractoriness (PR).

NAIT is the platelet equivalent of HDN, where maternal antibodies are responsible for the destruction of fetal cells. In cases of NAIT, the maternal platelet antibody crosses the placental barrier into the

fetal circulation and destroys the fetal platelets. The low platelet count is responsible for bleeding episodes in the fetus and can lead to intercranial hemorrhage (ICH) which can be life threatening and have serious neurological consequences for the infant.

Detection of antibodies to platelet antigens, HPA 1, 3, 4 and 5 is performed on mother’s serum in cases of suspected NAIT. If antibodies are detected, genotyping can be performed for platelet antigens HPA 1­5 and ­15 to confirm antibody status. Should the baby require a matched platelet transfusion, a donor can be obtained from the HPA –typed donor database available. The most commonly found antibodies causing NAIT in Caucasians are HPA 1a and HPA 5b.

In cases where patients are not responding to platelet transfusions, HLA or HPA antibodies, or both, may be suspected. The patient is tested for HPA and HLA antibodies, and then HPA and HLA typed. There are HPA and HLA ­ typed donor databases available.

In suspected cases of Auto­immune Thrombocytopenia, patients and also be tested for platelet auto antibodies. The diagnosis of this disease is one of exclusion, and the results obtained by this test cannot be used as the only supporting evidence for a positive diagnosis.

Contact details
Dr Zyta Foxcroft, Senior Biomedical Scientist Tissue Immunology, Area 1 011 761 9214
Area 1 (Johannesburg) Tissue Immunology + Paternity Laboratory 011 761 9227

3. Cyropreservation facility

This facility is responsible for the collection, processing and storage in liquid nitrogen of stem cells taken from patients suffering from haematological conditions such as Aplastic Anaemia, Fanconi’s Anaemia and a number of cancers such as Myelomas, Lymphomas and Leukaemias. These are mostly autologous and are harvested by SANBS staff at the apheresis clinics.

The facility collects processes and stores stem cells from allogeneic patients, e.g. bone marrow registry patients or HLA matched­related siblings.

The duties also include assisting in the re­infusion of frozen stem cells when requested by the clinician. The cells are transported by the staff member to the patient’s bedside where they are re­ infused under clinical supervision.

Contact details
Cryopreservation Laboratory 011 761 9215 or 011 761 9264

4. Cryogenic testing

Chromosomes carry the genetic information needed for the normal development from the egg to adulthood. One set of chromosomes is inherited from each parent during fertilization. If too few or too many chromosomes are inherited the foetus will be abnormal.

During chromosome testing cells are cultured then analysed to see whether the number and arrangement of chromosomes is correct. The cells can be derived from amniotic fluid, solid tissue or blood.

Cytogenetic testing is requested by the patient’s doctor or a clinic sister.

(i) Amniotic fluid

In the case of pregnant women at high risk of having a chromosomally abnormal child, an amniocentesis is performed between 14 and 18 weeks. A small volume of amniotic fluid is drawn off from around the foetus for culture and analysis. Fetal blood removed by cordocentesis, or chorionic villus samples are sometimes submitted as an alternative.

Amniocentesis results are usually available within 3 ­ 4 weeks.

The alphafetoprotein level is also routinely tested on amniotic fluid specimens to screen for neural tube defects. If this is significantly raised, then the acetylcholinesterase test is performed as a confirmatory test. This laboratory is the national reference lab for this test, which is positive in the presence of open neural tube defects.

(ii) Blood

In the case of adults and children who require chromosome testing, a small amount of blood is drawn, which is cultured and analysed. These results are usually available within a week

(iii) Tissue samples

In the case of an early miscarriage, products of conception are cultured, while tissue is removed from a fetus for culture in the case of a later miscarriage. Other tissue samples are also processed from time to time.

Results are given to the requesting doctor, and genetic counselling is offered to private patients at the SANBS headquarters in Pinetown

Hospital patients receive counselling at the genetic clinics that are held at some hospitals.

Contact details
Cytogenetics Laboratory 031 719 6607
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Immunohaematology Section

1. Antenatal testing

Antenatal tests are performed on all pregnant women in order to determine if obstetrically significant antibodies are present in their samples. The antenatal programme is therefore aimed at preventing or managing Haemolytic Disease of the Newborn (HDN).

Haemolytic disease of the newborn occurs when there is an incompatibility between the blood groups of a pregnant woman and the foetus that she is carrying. (The foetal red cell possesses a red cell antigen that the mother lacks). The mother’s immune system recognizes these antigens on the foetus’s red blood cells as foreign, and forms specific antibodies to them.

These antibodies may then cross the placenta and attach to the antigens on the foetal red cells (sensitizaion). These sensitized red cells are then removed from the foetal circulation and the foetus becomes increasingly anaemic. Intra­uterine death may occur in severe cases.

The antibody most commonly implicated in HDN is Anti­D. Rh negative individuals lack the D antigen on their red cells, and may develop Anti­D, if exposed to Rh D positive blood, either through transfusion or pregnancy. It is for this reason that an Rh phenotype should be performed on all pregnant women.

Rapid Rh D typing Kits are available in antenatal clinics, and are used to determine the Rh D type of the mother. Samples from all mothers who type initially as Rh D negative are referred to the Red Cell Serology Laboratories in Durban and Johannesburg. There the Rh D type is confirmed and antibody identification tests are performed to determine if Anti –D antibodies are present. A report is then issued to the clinic or doctor, accompanied by a letter which advocates the administration of Anti­D immunoglobulin in order to prevent HDN from occurring. Should antibodies be present in the maternal samples, further specimens are requested at regular intervals in order to monitor antibody activity, and arrangements are made to handle potentially affected infants.

Contact details
Red Cell Serology Laboratory 031 719 6685

2. Antibody Identification

In the event of an incompatibility detected in a Crossmatching Laboratory as a result of the presence of antibodies in a patient’s sample, compatible blood may not be immediately available for the patient. In such instances, the patient’s samples are referred to the Red Cell Serology Laboratories in Durban or Johannesburg, for antibody identification tests. Antibodies may also be detected in blood donations, and samples from these donations are also referred to the Red Cell Serology Laboratories.

Contact details
Area 1 (Johannesburg) Red Cell Serology Laboratory 011 761 9208
Area 2 (Durban) Red Cell Serology Laboratory 031 719 6685

3. Provision of blood for patients with rare blood types

The National Immunohaematology Reference Laboratory houses the South African Rare Donor File, which facilitates the provision of blood for patients with rare blood types, both nationally and internationally.

The South African Rare Donor File is linked to International Rare Donor Panel in the United Kingdom, which facilitates the international exchange of rare donations.

Fresh rare donations may be available in stock, or rare donors can be bled upon request. The central storage facility in Durban facilitates the storage of frozen rare donations. There is also an ongoing effort to identify rare donors; routine donations are screened with rare antisera, sera from patients with rare antibodies are used to screen and crossmatch blood, and family study investigations are performed on patients and donors found to have a rare blood type, in order to determine if any of their relatives have the same rare blood type. These patients and their relatives are enrolled as donors, where possible.

Contact details
Immunohaematology, Reference Laboratory 031 719 6544

4. Transfusion reaction investigations

An investigation is performed in every instance where a patient suffers an untoward reaction to the transfusion of blood and blood products. These investigation results are then referred to a central office and reported on as part of a National Haemovigilance Programme.
Contact details
Area 1 (Johannesburg) Red Cell Serology Laboratory 011 761 9208
Area 2 (Durban) Red Cell Serology Laboratory 031 719 6685

5. Blood typing reagents

The Reagents Laboratory in Durban provides various blood grouping antisera and reagent red cells to the various SANBS laboratories and to external customers for the testing of patient and donor samples. Included are the reagents required for automated blood grouping of all blood donations received by SANBS. The reagent red cells include panel and screening cells which are required for the identification of red cell antibodies in patient and donor samples. Rapid Rh Typing Kits are prepared for use in the Emergency Blood Banks located throughout the country.

Contact details
Reagents Laboratory 031 719 6689

6. Anti­-D programme

The programme is aimed at obtaining high titre Anti­D plasma from selected donors for the production of Anti­D Immunoglobulin. This Anti­D immunoglobulin is administered to Rh negative mothers who

give birth to Rh positive babies, and prevents them from forming Anti­D antibodies. (Refer to Antenatal Testing). The goal is to make South Africa self­sufficient in the production of this product and eliminate the need to import Anti­D immunoglobulin.

Donors are carefully selected, and divided into plasma and red cell donors. Plasma donors are immunised with red cells from matched red cell donors over a period of time, and stimulated to produce Anti­D. The plasma containing the anti­D antibodies is then harvested by plasmapheresis once suitable antibody levels are reached, and the plasma is supplied to the National Bioproducts Institute.

Contact details
Immunohaematology, Reference Laboratory

7. Evaluation of new reagents and techniques

An Evaluation and Projects Laboratory is available to perform evaluations of new reagents and techniques. These tests are generally not charged for.

The Reference Laboratory participates in international workshops to test new monoclonal and polyclonal blood grouping reagents.

Contact details
Evaluation and Projects Laboratory 031 719 6952


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Molecular research and development

1. Molecular research and development 

The Molecular Research and Development Section provides support to the Specialised Laboratory Services Department, by researching and developing current technologies that contribute to the molecular processes performed within the laboratories.

Keeping abreast with the new advances in technology allows us to achieve the level of excellence and service provided by blood transfusion services worldwide.

(i) Cytogenetics Laboratory

Introduction of the Aneuploidy PCR allows for the prenatal and postnatal detection of Trisomy.13 (Patau syndrome), Trisomy.18 (Edward syndrome), Trisomy.21 (Downs Syndrome), and the X and Y sex chromosome abnormalities such as Turner syndrome. Results are provided within 48 ­ 72hours.

Current research is underway for Bone Marrow PCR, that is a qualitative multiplex, nested RT­PCR test designed to detect 28 different translocations or chromosomal rearrangements found to be specific for various types of leukemia’s example, Acute Myelogenous Leukemia (AML), Chronic Myelomonocytic Leukemia (CML) are being researched.

(ii) Immunohaematology, Reference Laboratory

Initiating the BAGene Project where BAGene DNA SSP kits are used for the genetic determination of the ABO, Rh, Partial D, MNS, Kell, Kidd and Duffy blood group systems on a molecular genetic basis, to facilitate the clear typing of donors, recipients and pregnant women at the DNA level with a high resolution.

(iii) Tissue Immunology and Paternity Laboratory

Investigating the feasibility of Sequence Based Typing (SBT) which offers high resolution typing of HLA Class I Loci A, B, and C and Class II Loci DPB1 and DQB1 which is important in pre­organ transplant testing, more especially bone marrow transplantation,

New technologies are been evaluated and validated to improve the quality of results when screening and detecting Class I and Class II HLA antibodies which will aid in reducing the risk of organ rejection and Graft vs. Host Disease.

Contact details
Lavendri Govender, Senior Biomedical Scientist Molecular R + D 031 719 6953
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