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Introduction

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

Pre-transfusion compatibility testing is a series of procedures and processes to select, for each recipient, donor red cells that, when transfused will have an acceptable survival and not lead to clinically significant destruction of those transfused red cells. If performed correctly this should detect ABO incompatibilities between donor and recipient and clinically significant red cell antibodies in patient’s serum/plasma.

There are many different techniques for serological compatibility testing incorporating the use of tiles, tubes, solid phase and more recently column agglutination. Over the years pre-transfusion tests have undergone considerable review and modifications. In the past, it was common to use, and no matter at what cost, all available techniques to detect as many red cell antibodies and serological incompatibilities as possible irrespective of their clinical significance. Since the 1980s pre-transfusion serological testing protocols have been simplified because we now have a better understanding of what constitutes a clinically significant antibody which has lead to further rationalization of pre-transfusion testing.

In general an antibody may be considered potentially clinical significant if it is reactive at 37°C and/or by indirect antiglobulin test or has caused haemolytic transfusion reactions or lead to decreased red cell survival or known to have caused Haemolytic Disease of the Fetus and Newborn (HDFN).

Many of the early methods for antibody screening and crossmatching included testing at room temperature (RT) and 37°C, using enzyme treated red cells, including auto controls and the indirect antiglobulin test (IAT). The antibody screen has now evolved to be more effective than the crossmatch for detecting clinically significant antibodies and has replaced the crossmatch as the key step in pre-transfusion compatibility testing. No longer is there need to undertake antibody screening by enzyme or at RT or include auto controls. More recently it has been recommended that the indirect antiglobulin crossmatch could be replaced by an abbreviated crossmatch in patients with a negative antibody screen and no history of clinically significant antibodies.

In the early 1960s, many blood banks carried out minor crossmatching (testing donor’s serum against patient’s red cells) in addition to major crossmatching (testing patient’s serum against donor red cells). It was only in the mid-1970s that the minor crossmatch was finally abandoned as antibody screening of donor blood became routine.

The last three decades have seen technical advances such as the use of low ionic strength solutions (LISS). This has allowed IAT incubation times to be decreased from 60–90 min to 10–20 min, while still retaining the sensitivity and specificity required to detect clinically significant antigen/antibody reactions.

Traditionally, a patient’s serum was screened by tube (or tile) techniques against pooled screen cells whereas now EDTA plasma or serum is tested against 2–3 extensively phenotyped individual group O reagent red blood cells possessing specific blood group antigens by manual methods using column agglutination technology, microplates, or solid-phase. In more recent times fully automated bi-directionally interfaced systems have been developed that perform all the steps of these different techniques using column agglutination, solid phase or micro-titre plates requiring minimal technical intervention.

Today, most large and medium-sized blood banks and transfusion services screen and identify patients’ alloantibodies using plasma in automated analysers, often with neural networks capable of interpreting agglutination patterns, identifying antibodies according to internal programs, and delivering the results to the laboratory’s information system via an electronic interface. Currently, some advanced transfusion services augment serological screens for blood group antibodies using molecular analysis to determine the patient’s potential for forming antibodies by genotyping.

Procedures have now been developed and streamlined, both in the interests of cost effectiveness and patient safety based on a balance between:

  •  Patient safety.
  •  The number of unwanted reactions eliminated.
  •  The speed at which tests can be performed.
  •  The technology used.
  •  Patient identification.
  •  Specimen collection.
  •  Review of patient history.

Pre-transfusion testing

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

Pre-transfusion compatibility testing is extremely important and entails several key elements including:

  •  Positive identification of the patient and patient’s blood sample.
  •  Review of historical transfusion records.
  •  ABO and RhD grouping of the patient’s blood sample.
  •  ABO and RhD grouping of the donor red cells.
  •  Antibody screening for unexpected alloantibodies.
  •  Antibody identification if the antibody screen is positive.
  •  Selection of blood.
  •  Serological crossmatching the patient’s plasma/serum against ABO compatible donor red cells or
  •  Electronic release of ABO RhD compatible red cells.
  •  Labelling the red cells and completing all records.

The need to undertake a serological crossmatch in all situations has now been modified. Electronic issue of ABO RhD compatible red cells may be used instead of an indirect antiglobulin crossmatch when specific rules or conditions have been met. If these conditions are not met then antigen negative red cells must be crossmatched by IAT.

With technical advances such as the use of LISS and column agglutination technology it then follows that blood should be available for transfusion within 40 min of a specimen reaching the laboratory when using these techniques. If a patient has had a routine group and screen, with no antibodies found then rapid-spin crossmatch methods such as immediate spin crossmatch (IS-XM) have the capacity to reduce the overall time to approximately 20 min. The use of electronic release of red cells can further minimise the time required to provide blood following a negative antibody screen.

Blood sampling and clerical checking

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

In pre-transfusion testing, the importance of careful clerical checking cannot be overemphasized. From the outset, collecting and properly labelling blood samples from the correct patient is critical to accurate serological testing and safe blood transfusion. Today the risk of haemolytic transfusion reactions occurring are more likely to be the result of mis-identification of the patient rather than failing to detect clinically significant antibodies.

When a sample is received in the blood bank all details on the sample and transfusion request form must be checked to confirm the details are identical. Any discrepancies must be resolved, usually by requesting a new specimen and request form, before pre-transfusion testing can proceed. The patient’s transfusion history must also be checked to determine the validity of the sample.

Sample validity

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

The sample validity period depends on the patient’s transfusion and obstetric history. Red cell antibodies can rapidly appear in response to stimulation by transfused red cells or as a result of pregnancy. Consequently, if the patient has been transfused with red cells, or is currently (or has been) pregnant in the 3 months preceding pre-transfusion sample collection, the sample will have a validity of 72 h. A new sample will need to be collected once 72 h elapses if further transfusion is required. Longer expiry times are often applied if the patient does not have a history of transfusion or pregnancy.

ABO and RhD grouping

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

Determination of the patient’s correct ABO group is the most critical pre-transfusion serological test. It is essential to perform the patient’s ABO and RhD blood group on two separate occasions either using another sample collected separately, retesting the original sample or comparing the current ABO RhD result with historical records.

ABO grouping must include both testing of the patient’s red cells (cell group) with polyclonal or monoclonal anti-A and anti-B and testing of the patient’s plasma or serum (reverse group) with A1 and B cells. For infants less than four months of age a reverse group is not required because ABO antibodies have not yet fully developed.

If the cell and serum grouping results do not agree, additional testing must be conducted to resolve the discrepancy. If done incorrectly or on an improperly identified specimen, the result could be the transfusion of incompatible red cells and consequent patient fatality. If the patient’s ABO group cannot be satisfactorily determined and immediate transfusion is essential, group O red blood cells should be used.

RhD grouping is performed by testing the patient’s red cells with anti-D (polyclonal or monoclonal). Unless otherwise indicated by the manufacturer of the anti-D, a control system appropriate to the anti-D reagent must be used to avoid false positive results. Individuals typing as RhD negative in direct testing should receive RhD negative red cells and those typing as RhD positive in direct testing should receive RhD positive red cells.

Testing for weak D antigen expression is not required for pre-transfusion purposes (although some transfusion services may choose to perform weak D testing). As weak D is considered RhD positive, these patients may receive RhD positive blood during transfusion.

If the RhD group of the recipient cannot be determined and transfusion is essential, RhD negative blood should be given.

Antibody screening

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

The goals of antibody screening are:

  •  To detect as many clinically significant antibodies as possible.
  •  To detect as few clinically insignificant antibodies as possible.

There are several techniques used to detect antigen–antibody reactions. These have been based on various factors that are known to affect the antigen–antibody reaction. The techniques include IAT, enzyme pre-treatment of red cells, or use of low ionic strength solutions, polyethylene glycol or polybrene. Regardless of the method or enhancement media used, the method must be capable of detecting clinically significant antibodies. The antibody screen method must include a 37°C incubation phase with reagent red cells that have not been pooled followed by an IAT using either polyspecific antiglobulin reagent (anti-IgG and anti-C3) or monospecific reagent (anti-IgG), or an alternate method that has documented capability to provide comparable sensitivity.

Current antibody screening tests cannot detect all clinically significant antibodies. For instance, antibodies against low-incidence antigens are likely to be missed. Other antibodies may manifest a dosage effect dependent on the homozygosity of genes controlling expression of antigens on the reagent screening cells. Examples of such dosage-effect antibodies include anti-Jka, anti-Jkb, anti-Fya, anti-Fyb, anti-C, anti-E, and anti-c. Using two, preferably three group O screen cells improves the chances of having all major red cell antigens (D, C, E, c, e, M, N, S, s, K, k, Fya, Fyb Jka, Jkb) present in their homozygous forms during testing. Selecting such reagent cells ensures they carry the blood group antigens necessary for detecting the most important clinically significant red cell alloantibodies, namely, those antibodies reactive at 37°C and in the antiglobulin test as opposed to those reactive at room temperature.

Antibody investigation

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

In cases of positive antibody screening, further serological testing with an expanded panel of reagent red cells for the identification of clinically significant antibodies is required. Transfused patients may experience potentially life-threatening haemolytic transfusion reactions if clinically significant red cell antibodies are misidentified or unidentified.

Therefore, once the specificity of the antibody is known, donor units must be screened for the corresponding antigen to select those units that lack the corresponding antigen.

Before beginning to identify antibodies, available patient information must be reviewed. Many factors can provide valuable insights to help resolve the problem:

  •  Patient history (transfusion history; obstetrical history; diagnosis).
  •  Patient demographics.
  •  Sample characteristics.
  •  Initial serological results and characteristics.

Usually, the same method used for antibody screening and compatibility testing is used for identification. The result is often identification of a probable antibody with several antibodies requiring further exclusion and therefore requiring additional tests to eliminate.

Group and screen

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

Group and screen (G&S) is the first step in the ordering process for red cells. Many blood banks perform the G&S and, if the antibody screen is negative, do not provide compatible blood until a specific request for red cells is received. This approach helps to maintain the blood supply and minimises unnecessary holding of crossmatched blood. A G&S involves:

  •  Determining the recipient’s ABO and RhD group.
  •  Performing a red cell antibody screen.

Patients who have a history of clinically significant antibodies or a positive antibody screen require antibody identification and antigen negative red cells. A more comprehensive crossmatch using an IAT must be performed in this situation with antigen negative blood.

Crossmatch

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

Compatibility testing normally comprises ABO and RhD group, antibody screen and some form of crossmatch, whether serological or electronic. The serological crossmatch involves testing the donor’s red cells against the patient’s plasma or serum. Traditionally this was completed at different incubation temperatures such as room temperature, 37°C and following addition of polyspecific antiglobulin reagent. Now, in the IAT crossmatch (IAT-XM) monospecific antiglobulin reagent may be used without loss of sensitivity. The room temperature crossmatch was eliminated in the 1980s and has been replaced by an immediate spin crossmatch.

Crossmatching blood in advance for patients undergoing surgical procedures that usually do not require blood transfusion is unnecessary. In these circumstances the patient’s blood is group and screened for the presence of red cell alloantibodies and, if there is no alloantibody or history of antibodies, red cells are not routinely crossmatched. When blood is required it may be crossmatched by IS-XM or electronically released within 5–10 min. If the antibody screen is positive or there is a past history of clinically significant antibodies then antigen negative blood should be crossmatched by IAT in advance of surgery.

Some patients form multiple antibodies or antibodies to high frequency antigens making compatible (antigen-negative) red cells difficult to find. In such cases, rare donor files may be able to help find antigen-negative donors through their database or by mass phenotyping of donors.

Sometimes the identity of an antibody cannot be determined or, when determined, the antibody is found to be clinically insignificant. In these cases, crossmatch-incompatible red cells may be issued according to laboratory policy if transfusion cannot be avoided.

Serological crossmatch

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

These techniques have been simplified in recent years, and only the 37°C IAT-XM and IS-XM remain in common use. The IAT-XM is performed when the patient’s serum/plasma contains clinically significant red blood cell antibodies whereas the IS-XM has been retained primarily to detect ABO incompatibility in the setting of a negative antibody screen and no history of clinically significant antibodies. If the patient’s expected ABO antibodies are not reactive or weak in the reverse group then donor units should be crossmatched by IAT. Occasionally the IS-XM detects cold reactive (clinically insignificant) antibodies that react at room temperature which is inconvenient.

Electronic release

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

Using a validated computer system, patients with no history of clinically significant antibodies and a negative antibody screen on a current specimen may be issued ABO RhD compatible red cells following electronic checking of both the donor and patient’s blood group records for compatibility. Electronic release can only be permitted if the following conditions have been met:

  •  The patient’s ABO RhD group has been determined twice (once on a current sample; a second time on the same sample, on a second current sample, or by comparison with previous records).
  •  The computer system’s database contains the donor unit number, the component name, the ABO RhD group of the component, blood-group confirmatory-test interpretation and identification, expiry date and the ABO RhD group of the patient.
  •  A method is in place to ensure correct data entry.
  •  The system must prevent the electronic release of ABO incompatible blood to the patient, alert the operator to any errors or discrepancies between donor unit labelling and blood-group confirmatory-test interpretation and
  •  Prevent electronic issue of red cells to patients with clinically significant antibodies.

Advantages

  •  Donor units may be prepared quickly.
  •  Only a group and screen is required for most patients.

Disadvantages

  •  Will NOT detect antibodies to low incidence antigens present on donor cells that are not present on screening cells.
  •  Will NOT detect donor cells with a positive direct antiglobulin test.
  •  Donor unit must be ABO RhD confirmed prior to placing in available inventory.

Automated testing

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

Fully automated bi-directionally interfaced systems under computer control using bar-code identification of reagents and samples have been developed that perform all the steps of different pre-transfusion techniques using column agglutination, solid phase, micro-titre plates or other technologies that require minimal technical intervention. Today, many large and medium sized transfusion services perform group and screen using plasma in automated analysers, often with the capability of interpreting agglutination patterns, identifying antibodies according to internal logic, and transferring the results to the laboratory’s information system via a bi-directional electronic interface.

Emergency transfusions

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

During an emergency, depending on its degree it is best to do as much as the time allows. Blood may be issued as uncrossmatched O RhD negative, ABO RhD specific or crossmatched. Compatibility testing must be completed as soon as possible after the blood has been issued. Minimum immediate testing should include rapid ABO RhD grouping of patient and donor units and an immediate spin crossmatch. Antibody screening on pre-transfusion samples must be completed. If the antibody screen is negative, additional units can be issued electronically or after IS-XM. If antibody screening detects the presence of an antibody, additional compatibility testing must use an IAT-XM. If incompatibility is detected at any stage of testing, the treating clinician must be immediately notified.

Conclusion

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures

Pre-transfusion testing is intended to guarantee the normal survival of transfused red cells at minimum cost, yet how best to do this remains problematic. While the importance of careful clerical checks is not questioned as well as accurate ABO and RhD grouping, and antibody screening, there has been an increasing trend toward abbreviating the crossmatching procedure without compromising patient safety. As we become more aware of the economy of health care delivery, it is important that we continue to re-examine the rationale for performing some steps in pre-transfusion testing.

Suggested reading

  1. Top of page
  2. Introduction
  3. Pre-transfusion testing
  4. Blood sampling and clerical checking
  5. Sample validity
  6. ABO and RhD grouping
  7. Antibody screening
  8. Antibody investigation
  9. Group and screen
  10. Crossmatch
  11. Serological crossmatch
  12. Electronic release
  13. Automated testing
  14. Emergency transfusions
  15. Conclusion
  16. Suggested reading
  17. Disclosures
  •  BCSH. Guidelines for compatibility procedures in blood transfusion laboratories. Transf Med 2004; 14:59–73.
  •  Poole J, Daniels G: Blood group antibodies and their clinical significance in transfusion medicine. Trans Med Rev 2007; 21:58–71.
  •  Contreras M: ABC of Transfusion, 4th edn. UK, Wiley-Blackwell, 2009.
  •  McCullough J: Transfusion Medicine, 2nd edn. UK, Elsevier/Churchill Livingstone, 2005.
  •  Armstrong B, Wilkinson R, Smart E: Compatibility testing. ISBT Sci Ser 2008; 3:197–215.
  •  Roback J, Combs M, Grossman B, Hillyer C (eds): Technical Manual, 16th edn. Bethesda, USA, AABB, 2008.
  •  McClelland D: Handbook of Transfusion Medicine, 4th edn. London, UK, TSO, 2007.
  •  Clinical significant antibodies.
  •  BCSH. The Specification and Use of Information Technology (IT) Systems in Blood Transfusion Practice. London, UK, 2006.
  •  Sandler S, Abedalthagafi M: Historic milestones in the evolution of the crossmatch. Immunohaematology 2009; 25:147–151.
  • http://www.shotuk.org– SHOT Annual Reports.