Guidelines for compatibility procedures in blood transfusion laboratories

  • Writing party members: J. F. Chapman, C. Elliott, S. M. Knowles, C. E. Milkins, G. D. Poole.

    Transfusion Task Force members: J. Duguid, F. Boulton, B. McClelland, N. Smith, H. Cohen, M. Rowley, J. Taylor.

Section 1 Introduction

  • 1.1 Purpose of the guidelines
  •  The effective development and maintenance of satisfactory standards in pretransfusion testing requires a structured approach in the adoption of a quality system. Technical errors, clerical errors, the use of nonvalidated techniques or equipment and noncompliance with established procedures may result in missed incompatibilities and immediate or delayed haemolytic transfusion reactions (Love et al., 2002). The purpose of these guidelines, which replace those previously published in 1996 (BCSH, 1996b), is to define the laboratory processes and procedures that should be adopted to undertake pretransfusion testing.

  •  These guidelines are formulated from expert opinion and based on the recommendations of Clinical Pathology Accreditation (CPA, UK), Guidelines for Blood Transfusion Services in the UK and data from UKNEQAS (BTLP) (Knowles et al., 2002) and the Serious Hazards of Transfusion (SHOT) haemovigilance scheme. Where evidence exists to support new and potentially contentious recommendations, this is referenced in the text.

  • 1.2 Elements in pretransfusion testing
  • 1.2.1 ABO and D grouping of the recipient.
  • 1.2.2 Antibody screen of the recipient, or mother in the case of neonatal transfusion, which, in the event of a positive screen, should be followed by antibody identification.
  • 1.2.3 A computer or manual comparison of current results with any historical record.
  •  These three elements constitute a group and screen.

  • 1.2.4 Donor red cell selection and crossmatching, which may involve serological testing between patient serum/plasma and donor red cells or electronic selection and issue.
  • 1.2.5 There should be a documented procedure for dealing with emergencies. In clinical emergencies, the recipient's need for immediate red cell support may dictate that pretransfusion testing is abbreviated or even that group O blood be issued.
  • 1.3 Clinical significance of red cell antibodies
  • 1.3.1 Clinically significant antibodies are those that are capable of causing patient morbidity due to accelerated destruction of a significant proportion of transfused red cells.
  • 1.3.2 Anti-A, anti-B and anti-A,B must always be regarded to be of clinical significance.
  • 1.3.3 With few exceptions, red cell antibodies that are potentially clinically significant are only those which are reactive in the indirect antiglobulin test (IAT), performed strictly at 37 °C.
  • 1.3.4 In certain clinical circumstances, e.g. massive blood loss, the recipient's need for red cell transfusion may necessitate the use of incompatible units.
  • 1.3.5 Recommendations for the selection of red cells for transfusion to patients with alloantibodies are given in Table 1.
  • 1.3.6 The use of antibody cards, produced by the laboratory or by the blood services and held by the patient, should be considered. They may alert the clinician or laboratory to the presence of clinically significant antibodies. However, there are potential pitfalls in the use of written records such as these, and the laboratory should carefully consider procedures for their production and issue including validation of the information. A patient information leaflet detailing information such as the significance of the presence of antibody and why its presence is important should accompany antibody cards. Links between National Health Service (NHS) computer systems, or the growth of national databases such as those operated by the UK Blood Services accessed via a web browser, provide important opportunities for hospital transfusion laboratories to check on historical information such as red cell antibody specificity.
Table 1.  Recommendations for the selection of blood for patients with red cell alloantibodies
  • The above guidance is also suitable for patients undergoing hypothermia during surgery (Mollison et al., 1993).

  • IAT, indirect antiglobulin test.

  • *

    Antigen negative and crossmatch compatible.

  • These recommendations apply when the antibody is present as a sole specificity. If present in combination, antigen-negative blood may be provided by the blood centre, to prevent wastage of phenotyped units.

ABOAnti-A1IAT crossmatch compatible at 37 °C
RhAnti-D, -C, -c, -E, -eAntigen negative*
RhAnti-CwIAT crossmatch compatible
KellAnti-K, -kAntigen negative*
KellAnti-KpaIAT crossmatch compatible
KiddAnti-Jka, -JkbAntigen negative*
MNSAnti-M (active 37 °C)Antigen negative*
MNSAnti-M (not active 37 °C)IAT crossmatch compatible at 37 °C
MNSAnti-NIAT crossmatch compatible at 37 °C
MNSAnti-S, -s, -UAntigen negative*
DuffyAnti-Fya, -FybAntigen negative*
PAnti-P1IAT crossmatch compatible at 37 °C
LewisAnti-Lea, -Leb, -Lea + bIAT crossmatch compatible at 37 °C
LutheranAnti-LuaIAT crossmatch compatible at 37 °C
DiegoAnti-Wra (anti-Di3)IAT crossmatch compatible
HAnti-HI (in A1 and A1B patients)IAT crossmatch compatible at 37 °C
AllOthers active by IAT at 37 °CSeek advice from Blood Centre

Section 2 Quality Assurance in pretransfusion Testing

  • 2.1 Laboratory aspects of quality assurance
  • 2.1.1 In keeping with all other clinical laboratories, the transfusion laboratory should document its quality management system, clearly defining the organizational structure, procedures, processes and resources necessary to meet the requirements of its users, to accepted standards such as those specified by CPA (UK) Ltd.
  • 2.1.2 Transfusion laboratories should use equipment, information systems and test systems that have been validated against the documented requirements of the laboratory.
  • 2.1.3 The systems should enable a full audit trail of laboratory steps, including original results, cross-referenced to associated internal controls, interpretations, amendments, authorizations and all staff responsible for conducting each critical step.
  • 2.1.4 The laboratory must identify all critical points in pretransfusion testing (e.g. bar-code labelling and editing of automated groups) and build in security at these points.
  • 2.1.5 Where abbreviated testing, e.g. electronic issue, is carried out in patients with historical data, the algorithms should be computer controlled (level 3 evidence, grade B recommendation).
  • 2.1.6 A programme of regular independent internal audits should be instituted to assess compliance with laboratory processes.
  • 2.1.7 The laboratory management should conduct regular reviews of untoward laboratory incidents (including those reported to SHOT), complaints, external quality assessment reports and internal audits of the laboratory procedures.
  • 2.1.8 The laboratory should participate in relevant accredited external quality assessment schemes.
  • 2.1.9 Staff training and proficiency:
  • (i) There must be a documented programme for training laboratory staff, including on-call staff not routinely working in the laboratory, which covers all tasks and testing performed appropriate to the grade of staff and which fulfils the documented requirements of the laboratory. It should also include handling major incidents and emergency situations.
  • (ii) Laboratory tasks should only be undertaken by appropriately trained staff.
  • (iii) There should be a documented programme for assessing staff proficiency in all laboratory tasks, which should include details of the action limits for retraining.
  • (iv) Software upgrades to equipment should be followed by a training programme agreed with the manufacturer.
  • 2.1.10 Reagents/proprietary test systems
  • (i) All reagents or test systems should be stored and used in accordance with the manufacturer's instructions.
  • (ii) Where reagents cannot be used in accordance with the manufacturer's instructions and there is no appropriate alternative, their performance should be validated in accordance with the British Committee for Standards in Haematology Guidelines for evaluation, validation and implementation of new techniques for blood grouping, antibody screening and crossmatching (BCSH, 1995).
  • (iii) Laboratories should ensure that they are informed by the manufacturer of any change in the source of raw material used for reagents or test systems, and if this occurs, the performance of the system should be revalidated.
  • (iv) There should be a record of all batch numbers and expiry dates of all reagents used in the laboratory.
  • (v) Where testing is not conducted using proprietary test systems (test kits), all techniques used should be in accordance with recommended practice.
  • (vi) All changes in the testing process, e.g. change in reagent manufacturer, must be thoroughly validated in accordance with the BCSH Guidelines for evaluation, validation and implementation of new techniques for blood grouping, antibody screening and crossmatching before being introduced into routine use (BCSH, 1995).
  • 2.1.11 All laboratory equipments should be regularly maintained in accordance with the manufacturer's instructions. There should be a record of instrument failure, subsequent corrective action and downtime.
  • 2.1.12 There should be a documented programme to ensure the efficacy of the IAT, including cell washers (Voak et al., 1988).
  • 2.1.13 Information systems
  • (i) The hospital blood bank computing system should comply with the requirements described in the BCSH Guidelines for blood bank computing (BCSH, 2000).
  • (ii) The system must be validated prior to the introduction of electronic issue by challenging it with routine clinical scenarios.
  • (iii) The entire system should be revalidated following the installation of any upgrades or changes in interface.
  • 2.1.14 Automated equipment
  • (i) Prior to use, the system should be validated by challenging it with known clinical samples.
  • (ii) The system should be revalidated following servicing and any software upgrades.
  • (iii) The laboratory must have a policy with respect to the seniority of staff permitted to manually edit and authorize test results. Algorithms for editing and retesting should be documented, and designated staff should be trained in these procedures.
  • (iv) There should be a validated manual backup system and a documented procedure to define the circumstances in which it should be used.
  • 2.2 Role of the hospital transfusion committee
  • 2.2.1 This document is primarily concerned with the laboratory aspect of pretransfusion testing. However, every hospital or Trust should have a multidisciplinary hospital transfusion committee to oversee the provision of safe and appropriate transfusion support (Department of Health, 1998,2002). The remit of the hospital transfusion committee should comply with the requirements of these documents and the standards provided by the Research Unit of the Royal College of Physicians (1995).
  • 2.2.2 The hospital transfusion committee should specifically support laboratory pretransfusion testing by:
  • • endorsing local policies for the administration of blood including requesting, patient sampling, collecting and handling of blood components;
  • • agreeing specific procedures for the management of massive transfusion;
  • • ensuring that all relevant groups of staff are trained in accordance with local policies;
  • • formulating and periodically reviewing maximum surgical blood order schedules (MSBOS);
  • • commissioning audits of compliance with these policies and procedures;
  • • ensuring that investigations of adverse events and errors are conducted, which, in turn, may focus on the need for further education or amendments to existing procedures;
  • • ensuring that transfusion incidents are reported to SHOT;
  • • regularly reviewing hospital performance, which impacts upon the laboratory, e.g. wastage rates;
  • • reviewing the operational effectiveness of the laboratory service, e.g. response times for emergency requests.

Section 3 Automation

  • 3.1 Introduction
  •  The application and use of automation for compatibility testing continues to increase. Manual techniques allowed separation of processes into discrete activities as represented within the guideline. Automation throughout the compatibility procedure brings several or all activities into a single-platform process.

  • 3.2 Abbreviated testing
  • 3.2.1 Automation provides various levels of increased security over manual testing and may provide justification for abbreviated pretransfusion testing, e.g. abandoning duplicate D grouping or reverse ABO grouping. A risk assessment should be made and documented prior to any abbreviation of an established crossmatch procedure, with consideration given to the presence or absence of key functions in automated equipment. The following list is not exhaustive, as additional safety factors may be incorporated over time by equipment manufacturers:
  • • positive patient identification, including in situ validation of sample and/or reagent position and revalidation following a break in processing;
  • • notification of failure to dispense and/or aspirate samples or reagents or wash solutions;
  • • presence of a level check on final test mixture;
  • • password controls for different access levels;
  • • identification of noncompliant quality control results;
  • • unidirectional and bidirectional interfaces;
  • • the ability of on-board software to detect, monitor, control and record nonstandard events;
  • • availability of check digits on sample number bar codes;
  • • the use of bar-coded reagent containers and test chambers (e.g. microplates and cassettes).
  • 3.2.2 The risk assessment should take into account the potential for a sample being labelled from a patient other than the one intended (Love et al., 2002) and the potential for a subsequent grouping anomaly (see§5.8). This may be particularly pertinent in the event of a mixed-field ABO group resulting from an ABO-incompatible transfusion.
  • 3.2.3 Even in the presence of clot and air bubble sensors, it is advisable to centrifuge all patient samples prior to testing in automated systems to reduce the likelihood of inadequate sampling due to air bubbles, fibrin or other deposits. This is of particular importance in the absence of an IAT crossmatch.

Section 4 Samples/Documentation

  • 4.1 Introduction
  • Errors in patient identification and sample labelling may lead to ABO-incompatible transfusions. Evidence for this is well documented in the annual reports of the SHOT steering group Williamson et al. (1998,1999), Love et al. (2001,2002) and Sazama (1976).

  • 4.2 Written/electronic requests
  • 4.2.1 There must be written policies for generating blood transfusion requests and for the collection of blood samples for pretransfusion compatibility testing. This should specify grades of staff authorized to request blood and to take samples for pretransfusion testing.
  • 4.2.2 It is essential that the request form and sample contain the following minimum patient identification (PIN) as described in BCSH Guidelines for the administration of blood and blood components and management of transfused patients (BCSH, 1999).
  • (i) surname/family name (correctly spelt);
  • (ii) first name(s) in full;
  • (iii) date of birth (not age or year of birth);
  • (iv) hospital number/accident and emergency number/NHS number/major incident number.
  • The sample should be dated, labelled and signed by the person taking it. The request form should also include the patient's location and the location where blood units should be sent and the signature/name of the person making the request.

  • A local standard operating procedure should be in place for the procedure for dealing with inadequately labelled samples.

  • 4.2.3 Information concerning the sex of the patient and obstetric and recent transfusion history should be obtained wherever possible and is essential when there are anomalous pretransfusion testing results.
  • 4.2.4 Requests should also include the date and time that the blood is required, the number or volume and type of component required, the full reason for request (‘Pre-op’ is insufficient) and any other specific requirements relating to the patient or request such as cytomegalovirus (CMV) negative or irradiated products.
  • 4.2.5 Provided that sample labels are printed and attached to the bottle next to the patient at the time of phlebotomy, hand-held bedside/chair-side scanners and printers utilizing bar-coded wristbands may increase security during phlebotomy. Labels produced in this way are not the same as addressograph labels, which are more likely to result in inadequate checking of PIN at the bedside (Sharp & Cummins, 2001). It is therefore recommended that any labels preprinted away from the bedside should not be accepted for either grouping or pretransfusion testing samples.
  • 4.2.6 Electronic ward requesting should comply with all the same minimum standards as in §4.2.1–§4.2.5, although in the absence of a facility for signing the request form, the identity of the requester must be clear.
  • 4.2.7 Samples received from trauma or unconscious accident and emergency patients are unlikely to contain the full PIN. There, however, must be at least one unique identifier, usually an accident and emergency or trauma number, and the sex of the patient identified on the sample label. The sample should be taken and labelled and the form and sample should be signed by the prescribing medical officer as one continuous procedure.
  •  In the event of there not being at least one unique identifier on the sample in a life-threatening situation, group O blood only must be issued until a suitably labelled sample is available. If the patient is a female under 60 years old, group O D-negative blood should be given.

  • 4.3 Telephone requests
  • 4.3.1 There should be a policy for documenting telephone requests (BCSH, 1999).
  • 4.3.2 Requests must be made by a medical officer or delegated individual. The identity of the person or people initiating and making the request should be documented. The identity of the individual receiving the request should also be documented.
  • 4.3.3 The following minimum information must be given and confirmed (e.g. by reading back to the person giving the information):
  • (i) surname/family name
  • (ii) forename
  • (iii) hospital/accident and emergency number/trauma number
  • (iv) location
  • (v) number/volume and type of product
  • (vi) reason for request
  • (vii) date and time required.
  • 4.4 Duplicate records
  •  Duplicate patient records must be avoided; otherwise, essential transfusion or antibody history may be overlooked. It is therefore necessary, at the time of the request, to identify and link separate records that exist for each patient.

  •  If a computer system is in use, the user must be alerted at the time of a request entry that there are existing records for patients with the same name and date of birth. If a computer system is not in use, manual records need to be checked by name and date of birth for previous encounters (BCSH, 2000).

  • 4.5 Sample requirements
  • 4.5.1 Clotted or ethylenediamine tetra-acetic acid (EDTA) samples may be used for pretransfusion testing. If a change in protocol is made from the use of serum to plasma, appropriate validation using weak examples of antibodies must be performed to ensure that the detection of clinically significant antibodies is not compromised. Guidance can be found in the BCSH Guidelines for evaluation, validation and implementation of new techniques for blood grouping, antibody screening and crossmatching (BCSH, 1995).
  • 4.6 Timing of sample collection in relation to previous transfusions
  • 4.6.1 Transfusion or pregnancy may stimulate the production of unexpected antibodies through either a primary or secondary immune response. The timing of samples selected for crossmatching or antibody screening must take account of this, as it is not possible to predict when or whether such antibodies will appear. It is also important to note that any component containing residual red cells can elicit an immune response.
Table 2.  Evidence from SHOT (Love et al., 2002) with respect to delayed transfusion reactions has been influential in providing evidence for timing of new samples when a transfusion has been given more than 72 h earlier:
Patient transfused withinSample to be taken not more than
3–14 days24 h before transfusion
15–28 days72 h before transfusion
29 days to 3 months1 week before transfusion
  •  In situations where patients are being repeatedly transfused, a daily sample is not a requirement. These patients should be screened for the development of irregular antibodies at least every 72 h. This interval has been selected to be both practical and safe (see§8.2.6).

  •  It is recognized that for some individuals, e.g. thalassaemic patients, who have been repeatedly transfused for several years and who have not had an antibody response, a more tolerant approach may be taken.

  • 4.6.2 It is recognized that there may be a problem obtaining samples from pregnant women, who, for example, are booked for elective caesarean section but may not arrive in the hospital until shortly before surgery. As immunization is more likely to occur during the last trimester of pregnancy, samples used for pretransfusion testing in these patients should never be more than 7 days old and must meet labelling requirements. Where possible, it is advisable that a sample taken immediately before transfusion be also made available for retrospective testing in the event of a transfusion reaction occurring.
  • 4.7.1 Whole-blood samples will deteriorate over a period of time. Problems associated with storage include red cell lysis, bacterial contamination, loss of complement in serum and decrease in potency of red cell antibodies, particularly immunoglobulin M (IgM) antibodies.

The following are suggested as working limits:

 18–25 °C4 °C−30 °C
EDTA whole bloodUp to 48 hUp to 7 daysNA
Separated plasma/serumNAUp to 7 days6 months
  • 4.7.2 Although antibodies are probably stable for up to 6 months in frozen-stored samples, the risk of intervening transfusion or pregnancy and the risk associated with the sample identification of separated plasma/serum samples should be assessed before considering the use of stored samples for crossmatching.

Section 5 Abo and D Grouping

  • 5.1 Introduction
  •  ABO grouping is the single most important serological test performed on pretransfusion samples, and the sensitivity and security of testing systems must not be compromised.

  • 5.2 ABO grouping
  • 5.2.1 Patients' red cells should be tested against monoclonal anti-A and anti-B blood-grouping reagents. The anti-B reagent should not react with acquired-B.
  • 5.2.2 A reverse ABO group using A1 and B reagent red cells must always be performed except:
  • (i) when secure automation is used and a risk assessment has been undertaken (see§3.2.2). In this case, there must be at least one historical record where testing included a reverse group, and the current cell group must be identical with all historical records. It should be noted that there are risks involved with dropping the reverse group with only one historical record, as the first sample may have been taken from the wrong patient and a mixed-field reaction on a second sample may be the only indication of an ABO-incompatible transfusion.
  • (ii) on samples from neonates.
  • 5.2.3 In the absence of a reverse group, a negative diluent control reagent should always be tested against the patient's red cells as part of the ABO grouping procedure.
  • 5.2.4 In order to prevent misinterpretation of results due to haemolysis where serum is used, it is recommended that the diluent used for resuspension of reverse grouping cells contain EDTA.
  • 5.3 D grouping
  • 5.3.1 In the absence of secure automation, each sample should be tested in duplicate with IgM monoclonal anti-D blood-grouping reagents, which should not detect DVI (see§5.10.1).
  • 5.3.2 The antiglobulin test should not be used for D-grouping patient samples for the purposes of transfusion.
  • 5.3.3 Anti-CDE reagents are of no value for routine typing of patients' red cells and have led to misinterpretation of r′ and r′′ red cells as D positive. It is therefore recommended that anti-CDE reagents should not be used for routine typing of patients (level 3 evidence, grade C recommendation).
  • 5.3.4 Those responsible for choosing blood-grouping reagents should be aware of the content and specificity of reagents used, including any potentiators. Ideally, blood-grouping reagents should not contain any additives that could result in false-positive results due to in vivo IgG coating of the patient's cells.
  • 5.4 D grouping of neonates for maternal postnatal  anti-D prophylaxis
  • 5.4.1 There is minimal evidence that DVI or weak D on fetal red cells can cause maternal sensitization. Testing cord samples for DVI requires the use of different reagents or techniques and, because there may be an added risk of these being used inappropriately on adult samples, is not currently recommended. Most examples of weak D, however, can be easily detected by selecting high-affinity monoclonal reagents for routine D typing. It should be noted that weak D fetal cells may be cleared from the maternal circulation poorly by standard anti-D Ig preparations administered to the mother (Mollison et al., 1993).
  • 5.5 Controls
  • 5.5.1 Positive and negative controls should be used on a regular basis; the exact frequency will depend on work patterns.
  • 5.5.2 When using automated systems, control samples should be loaded onto the machine in the same way as patient samples. Controls should be included at least twice daily. Timings should take into account the length of time that reagents have been on the analysers.
  • 5.5.3 When working manually in batches (one or more samples at a time), controls should be included in every batch.
  • 5.5.4 Controls should always be included when changing reagent batches and when starting up a machine.
  • 5.5.5 Controls for manual and automated procedures should be as follows:
ReagentPositive control cellsNegative control cells
Anti-DD positiveD negative
  •  Where these do not give the expected reactions, investigations should be undertaken to determine the validity of all test results subsequent to the most recent valid control results.

  • 5.5.6 Where recommended by the manufacturer, a diluent control reagent should always be tested against the patient's red cells, as part of the ABO and/or D grouping procedure. If positive (even weakly), the test result is invalidated.
  • 5.5.7 A diluent control(s) must always be used as part of the ABO/D grouping procedure in cases where a strong cold autoantibody may be present or when auto-agglutination in the patient's sample has been detected. In such cases, washing the cells with warm saline prior to testing may also be helpful (see§7.9.4)
  • 5.6 Interpretation of results
  • 5.6.1 Manual reading. Documentation errors may occur during manual reading and interpretation of ABO and D groups. The risk of error can be minimized by separating the procedure into distinct tasks and, wherever possible, using different members of staff to perform each task.
  •  Suggested options for achieving this are:

  • (i) separating the documentation of reaction patterns from the final interpretation;
  • (ii) separating the interpretation and documentation of the cell and reverse groups.
  • 5.6.2 Automated reading. Automated readers are frequently used to interpret individual reactions and reaction patterns, when using microplate or microcolumn techniques. The system must be validated against manual systems prior to routine use.
  • In the absence of a fully automated system (e.g. where there is no integrated bar-code reader), procedures including double-checking of plates or cards should be in place to prevent misidentification.

  • A visual inspection of results is still necessary when using automated readers that are unable to interpret mixed-field reactions.

  • 5.7 Verification of results
  • 5.7.1 Prior to validation of results, the PIN on the sample must be checked against the request form/computer-generated worksheet to ensure that they match and no errors have been made at the booking in stage.
  • 5.7.2 The ABO and D group must, wherever possible, be verified against previous results for the patient.
  • 5.7.3 Any discrepancies must be resolved prior to transfusion of red cells or red-cell-contaminated components.
  • 5.7.4 Any manual editing of results should be performed in accordance with §2.1.14.
  • 5.8 Grouping anomalies
  •  The following are some examples of blood-group anomalies.

  • 5.8.1 Acquired B. Some anti-B reagents may react strongly with the acquired-B antigen. This usually leads to a discrepancy between cell and reverse groups, and such anti-B reagents should not be used. If the patient's own anti-B is only weakly detectable, an incorrect interpretation may result. Particular care must be taken if an antiglobulin test is not performed as part of the crossmatch as ABO incompatibility may be missed.
  • 5.8.2 Unexpected mixed-field reactions. Any tests showing mixed-field reactions must be repeated and/or investigated prior to group authorization or issue of red cells. These reactions may represent an ABO/D-incompatible transfusion or bone marrow/stem cell transplant.
  • 5.8.3 Partial or weak D (see§5.10).
  • 5.8.4 Intrauterine transfusions. For a period of several months after delivery, neonates who have received intrauterine transfusions may appear to have the same ABO and D group as that of the transfused red cells, due to bone marrow suppression.
  • 5.8.5 Presence of cold-active alloantibodies. A reaction with the reverse grouping cells may be obtained if these cells contain an antigen for which a cold-active alloantibody is present in the patient's plasma other than anti-A or anti-B. Where possible, the reverse group should be repeated at a higher temperature or using reverse grouping cells that lack the implicated antigen.
  • 5.9 Resolution of grouping anomalies
  • 5.9.1 ABO and D groups must be repeated when a discrepancy (anomaly) is found.
  • 5.9.2 Repeats should be performed using washed cells. To prevent the perpetuation of mistakes, the cells used should be taken from the original sample, rather than from a suspension made previously. An autocontrol should be included.
  • 5.9.3 Repeatable anomalous results should be referred to a senior person in the laboratory. It may also be necessary to obtain a fresh sample and refer to a reference laboratory.
  • 5.9.4 If it is not possible to obtain a reliable reverse grouping result due to the age of the patient or to insufficient sample, and there is no historical group against which to validate, the cell group must be repeated (see§5.2.3 and §5.7).
  • 5.9.5 Where verification checks against historical results reveal a discrepancy, a further sample must be obtained and tested immediately.
  • 5.10 Partial and weak D
  • 5.10.1 It is important to note that monoclonal anti-D reagents vary widely in their ability to detect both partial and weak D. If using two different reagents, it may be helpful to use those of similar affinity to reduce the number of discrepancies due to the detection of weak D. Where there is a discrepancy in grouping, the patient should be treated as D negative until the D status is resolved. The sample may need to be sent to a reference laboratory for investigation.
  • 5.10.2 Patients with a known partial D status should be regarded as D negative, but the findings should be explained clearly to the patient in order to prevent misunderstandings.
  • 5.10.3 Patients should not be classified as D positive on the basis of a weak-positive result using a single anti-D reagent (or a pool of more than one reagent). If clear-cut positive results are not obtained with modern monoclonal anti-D reagents, it is safer to classify the patient as D negative until a reference laboratory has undertaken confirmatory D grouping.
  • 5.10.4 Patients of category DVI status are those most likely to make anti-D. Reagents used for D grouping of patients must not detect category DVI.
  • 5.11 Infants in the first 4 months of life
  • 5.11.1 It is important to distinguish cord samples from maternal samples to prevent misgrouping (e.g. by using the alkali denaturation test), particularly if maternal and cord sample are the same ABO and D group. A direct antiglobulin test (DAT) should be performed at the same time to detect any IgG antibody coating of cord cells in vivo (see§8.2.7).
  • 5.12 ABO/D grouping in urgent situations
  • 5.12.1 When blood or blood products are required urgently, there may be insufficient time for routine ABO/D grouping prior to selection of blood products.
  • 5.12.2 Emergency groups performed in these circumstances must include a test against anti-A, anti-B and anti-D, with appropriate controls or a reverse group. Care should be taken when results indicate that the patient is group AB D positive, as anomalies such as cold agglutinins may not be detected without adequate controls.
  • 5.12.3 The result must be documented and confirmed as soon as possible by routine methods if these differ from emergency procedures.
  • 5.12.4 Compatibility testing must be completed as defined in §8.6.

Section 6 Antibody Screening

  • 6.1 Introduction
  • 6.1.1 Antibody screening undertaken in advance of the requirement to provide blood for transfusion alerts the clinician to possible delay in the supply of compatible blood if the antibody screen is positive. It also provides the laboratory with time to identify irregular antibodies and select suitable units.
  • 6.1.2 Antibody screening may be more reliable and sensitive than crossmatching against donor red cells, and it is therefore recommended that antibody screening should be performed in all pretransfusion testing (see§6.3.1).
  • 6.2 Choice of techniques
  • 6.2.1 Indirect antiglobulin test. The IAT using red cells suspended in low-ionic-strength solution (LISS) is considered to be the most suitable for the detection of clinically significant antibodies, because of its speed, sensitivity and specificity. Column agglutination methods have been shown to be simple, robust and reliable. Liquid-phase tube methods have an equal sensitivity but require a careful washing and reading procedure (BCSH, 1991a). Solid-phase methods have a higher sensitivity for some red cell antibodies but require careful validation and thorough quality assurance (Poole et al., 1996).
  • 6.2.2 Additional techniques. Additional techniques, such as two-stage enzyme and Polybrene methods, may be used. However, these methods are inferior to the IAT for some clinically significant antibodies. Proficiency in the performance in the IAT is therefore of overriding importance.
  •  References for these methods will be found in Mollison et al. (1993) and Scott et al. (1994).

  • 6.3 Reagent red cells for use in antibody screening
  • 6.3.1 Antibody screening provides the laboratory with the most reliable and sensitive method of detecting a red cell antibody. Crossmatch methods using red cells from donor units are often less reliable because the expression of blood-group antigens varies according to genotype: for example, the homozygous genotype JkaJka often results in a higher expression of the Jka antigen than the heterozygous genotype JkaJkb. In addition, it is easy to transpose donor cell suspensions and there is less standardization in the procedure. Red cells for antibody screening should be preserved in a temperature-controlled environment in a medium shown to minimize loss of blood-group antigens during the recommended storage period. For these reasons, an antiglobulin crossmatch using donor cells is not the most effective way of detecting a serological incompatibility between a patient and a donor.
  • 6.3.2 As a minimum, the following antigens must be expressed within the screening cell set: C, c, D, E, e, K, k, Fya, Fyb, Jka, Jkb, S, s, M, N and Lea. Reagent red cells from different donors should not be pooled in order to achieve the required representation of antigens.
  • 6.3.3 It is recommended that one of the cell samples used in the screening cell set should have the phenotype R1R1 or R1wR1 and another should be R2R2. It is also recommended that the following phenotypes should be represented in the screening cell set: Jk(a+b–), Jk(a–b+), S+s–, S–s+, Fy(a+b–) and Fy(a–b+). These recommendations are, in part, based on UK data regarding the incidence of delayed transfusion reactions and the need for a high sensitivity in the detection of Kidd antibodies (Love et al., 2002).
  • 6.3.4 Anti-Kpa and anti-Cw are rarely of clinical significance, and therefore, the inclusion of screening cells that are Kp(a+) and Cw+ is not considered essential (Garratty, 2003).
  • 6.4 Controls (see also§5.5)
  • 6.4.1 An autologous control or DAT need not form a part of antibody screening.
  • 6.4.2 A weak anti-D control serum or reagent (containing anti-D at a level of less than 0.1 IU mL−1) should be used on a regular basis to assure the efficacy of the whole test procedure, including antigen expression on screening cells (see§5.5.1–5.5.4).
  • 6.4.3 The use of further control sera or reagents, containing weak examples of specificities known to be clinically significant when present in patients' plasma (e.g. anti-Fya), is also recommended to assure the sensitivity of the test procedure and the integrity of antigen expression of reagent red cells during storage.

Section 7 Antibody Identification

  • 7.1 When an alloantibody is detected in the screening procedure, its specificity should be determined and its likely clinical significance should be assessed.
  • 7.2 If the patient is known to have a red cell alloantibody, the serum/plasma should be checked on each occasion of testing to exclude the development of further alloantibodies. The only exception is where the patient has received a massive blood transfusion (see §8.2.6).
  • 7.3 A blood sample should be referred to a red cell reference laboratory if there is any doubt concerning the identities of any antibodies present or lack of exclusion of clinically significant antibodies.
  • 7.4 Laboratories that are not registered for antibody identification in an accredited External Quality Assessment scheme should refer all sera that have given positive results in the antibody screen to a laboratory that is registered for antibody identification.
  • 7.5 It is important to recognize the limitations of the panel in use. A single panel may be unable to identify some common combinations of antibodies. A selection of two different panels increases the probability of being able to confidently identify a mixture of antibodies and is strongly recommended for laboratories that do not regularly refer samples to a reference laboratory, so that additional antibodies of clinical significance can be excluded.
  • 7.6 There are now software packages available for interpretation of reaction patterns in antibody identification. These should be validated and require experienced staff to interpret the result options offered by the software.
  • 7.7 Principles of antibody identification
  • 7.7.1 The patient's serum or plasma should be tested by an appropriate technique against an identification panel of reagent red cells. As a starting point, the technique by which the antibody was detected during screening should be used. Inclusion of the patient's own red cells may be helpful, for example, in the recognition of an antibody directed against a high-frequency antigen.
  • 7.7.2 The specificity of the antibody should only be assigned when it is reactive with at least two examples of reagent red cells carrying the antigen and nonreactive with at least two examples of reagent red cells lacking the antigen. Note that, wherever possible, the presence of anti-Jka, anti-Jkb, anti-S, anti-s, anti-Fya and anti-Fyb should be excluded using red cells having homozygous expressions of the relevant antigen (see§6.3.1 and §6.3.3).
  • 7.7.3 Antibody screening results can contribute to assigning the antibody specificity. A check should be made to ensure that the panel results do not conflict with the antibody screening results. Negative results with all panel cells may be the result of using an incorrect sample or the correct sample containing an antibody directed against a low-frequency antigen. A positive control (see§6.4.3) set up with the panel will identify a failure in the antiglobulin procedure.
  • 7.7.4 When one antibody specificity has been identified, it is essential that the presence of additional clinically significant antibodies is not missed. Multiple antibodies can only be confirmed by choosing cells antigen negative for the recognized specificity but positive for other antigens to which clinically significant antibodies may arise. Failure to recognize all of the antibody specificities within a sample may lead to a haemolytic transfusion reaction (Love et al., 2002). Note that the requirements in §7.7.2 need to be met for each antibody detected.
  • 7.7.5 The use of a panel of enzyme-treated cells is strongly recommended for antibody identification, particularly when an antibody weakly reactive by the antiglobulin technique, or a mixture of antibodies, is present. The use of monospecific antiglobulin reagents in place of polyspecific reagent is beneficial when determining the presence of IgG antibodies in serum samples containing complement-binding antibodies. The use of low-temperature saline techniques may also be helpful.
  • 7.7.6 The majority of antibodies detectable only by an enzyme technique are unlikely to be of clinical significance (Issitt et al., 1993). Specific antibodies detectable only by the enzyme technique should not be ignored unless procedural errors in the antiglobulin test have been ruled out. This is best achieved by retesting the serum/plasma against red cells possessing a homozygous expression of the relevant antigen using an antiglobulin technique.
  • 7.7.7 The patient's red cells should be phenotyped using a reagent of the same specificity as the assigned specificity. If this test is positive: (a) the antibody is an autoantibody (in which case the patient's cells will normally be DAT positive), and/or (b) if an antiglobulin or potentiated test method has been used, the patient's cells may be coated with globulin components (in which case the cells will be DAT positive); or (c) the assignation of antibody specificity may be incorrect. The incorporation of a reagent control or AB serum control used by the same technique as the phenotyping reagent is recommended, unless the reagent is known to contain unpotentiated IgM antibody. A positive result in this control test will invalidate the phenotyping test results.
  • 7.7.8 When red cells taken from a blood donation unit are found to be positive in an antiglobulin crossmatch against patient's serum/plasma, but no activity is detected in the serum/plasma against red cells in an identification panel, it is likely that: either (a) the serum/plasma contains an antibody to a low frequency antigen; or (b) the red cells in the donation are DAT positive; or (c) blood of the wrong ABO group has been selected for crossmatching.
  • 7.7.9 Further details of antibody identification can be found elsewhere (AABB, 2002).
  • 7.8 Reagent red cells for use in antibody identification
  • 7.8.1 An identification panel should consist of red cells from eight or more group O donors. For each of the more commonly encountered clinically significant red cell antibodies, there should be at least two examples of phenotypes lacking, and at least two examples of phenotypes carrying, the expression of the corresponding antigen. In addition, the panel should be able to resolve as many likely antibody mixtures as possible.
  • 7.8.2 There should be at least one example of each of the phenotypes R1R1 and R1wR1. Between them, these two samples should express the antigens K, k, Fya, Fyb, Jka, Jkb, M, S and s.
  • 7.8.3 There should be at least one example of each of the phenotypes R2R2, r′r and r′′r and at least two examples of the phenotype rr. The following phenotypes should be represented in those samples lacking both C and D antigens: K+, K–, Jk(a+b–), Jk(a–b+), S+s–, S–s+, Fy(a+b–) and Fy(a–b+).
  • 7.9 Autoantibodies
  • 7.9.1 Many autoantibodies cause no clinical problems. In patients with autoimmune haemolytic anaemia (AIHA), autoantibodies directed against red cell antigens are responsible for shortening red cell survival that may lead to severe anaemia.
  • 7.9.2 Serological investigations in AIHA should focus on the determination of the correct ABO and D group of the patient and determination of the possible presence of an alloantibody. An autoantibody may mask the presence of an underlying alloantibody. It may be necessary to refer cases of AIHA to a red cell reference laboratory in view of the complexity of the investigations required.
  • 7.9.3 Selection of blood for transfusion may be influenced by the presence of an autoantibody of simple specificity, but extensive investigations to determine complex specificities of autoantibodies are rarely of value.
  • 7.9.4 Cold-type AIHA or cold haemagglutinin disease. The red cells from the patient will usually have a strongly positive DAT due to coating with complement (C3d) components (the red cells should be washed at 37 °C to remove IgM autoantibody). It is important to exclude the presence of alloantibodies using cells and serum separately prewarmed to 37 °C. The use of anti-IgG in place of polyspecific antiglobulin reagent may also be helpful when serum, rather than plasma, is used.
  • 7.9.5 Warm-type AIHA. Red cells from the patient will usually have a positive DAT, because of coating with IgG and, sometimes, complement components. Autoabsorption using the patient's red cells may be necessary to permit the recognition of underlying alloantibody. Removal of autoantibody from the patient's red cells and enzyme treatment of the cells improve the efficiency of autoabsorption and may be performed in a single stage using the ‘ZZAP’ method (Branch & Petz, 1982). In some circumstances, autoabsorption may be difficult or undesirable (e.g. when there is a limited amount of patient's red cells or within 3 months of a transfusion). Such cases should always be referred to a red cell reference laboratory to allow allogeneic absorptions using selected red cells to be performed, in order to facilitate the exclusion of alloantibodies.
  • 7.10 Antibodies showing no obvious specificity at 37 °C
  • 7.10.1 Laboratories using solid-phase methods may find a higher proportion of serum or plasma samples that give a positive screening result but for which no specificity can be determined, when compared with laboratories using column agglutination or tube methods (Poole et al., 1996). There is no evidence to suggest that such antibodies that are detected by solid-phase, but are not detected by IAT using column agglutination or tube methods, cause accelerated destruction of red cells. It is therefore acceptable, in these circumstances, to provide blood without an IAT crossmatch, provided the requirements in §7 are met.
  • 7.10.2 Antibodies reacting preferentially at temperatures below 37 °C may sometimes be detected in antibody screening. In many cases, specificity is determined using standard IAT methods, but sometimes it is not possible to conclude an unequivocal specificity. If it is suspected that a cold-active antibody is present in a patient's sample, it is unnecessary to identify the specificity if the antibody screen, using cells and serum prewarmed to 37 °C, is negative. Further guidance on the selection of blood for patients with some cold-active antibodies is given in §1.

Section 8 Crossmatching

  • 8.1 Introduction
  •  The crossmatch is defined in this document as a procedure to exclude incompatibility between donor and recipient. Until recently, the inclusion of an IAT was considered to be the most important feature of pretransfusion testing, particularly in the crossmatch. As the sensitivity of the antibody screen has improved, abbreviated serological techniques such as immediate-spin and non-serological methods, e.g. electronic issue, have replaced the serological IAT crossmatch. In view of the lack of standardization of the immediate-spin technique (O'Hagan et al., 1999), it is recommended that computer issue without a serological crossmatch is the method of choice when a laboratory wishes to replace the IAT crossmatch with an alternative method. The requirements identified in the Guidelines for blood bank computing should be met (BCSH, 2000). Whatever technique or combination of techniques are used, they must be capable of detecting an ABO incompatibility.

  • 8.2 Selection of blood
  • 8.2.1 There should be clear written policies and continuing education on the selection of blood (Love et al., 2002).
  • 8.2.2 Red cell components of the same ABO and D group as the patient must be selected whenever possible. If ABO-identical blood is not available, group O blood may be used, provided it is plasma depleted or does not contain high-titre haemagglutinins. Group AB should be used for AB patients, but if unavailable, group A or B, rather than group O red cells, should be used for blood conservation reasons.
  •  When supplies of D-negative blood are limited, D-positive blood may be selected for D-negative recipients. It is important that D-positive cellular components are not issued to D-negative females under 60 years old.

  • 8.2.3 Patients with clinically significant red cell alloantibodies. Blood should be selected which has been tested and found negative for the relevant antigen. Antigen-negative blood should also be selected when a clinically significant antibody has been previously identified but cannot be detected or identified in the current sample. Patients with anti-D should receive rr blood because anti-C may also be present (see§1.3).
  • 8.2.4 Patients with alloantibodies considered not clinically significant or with antibodies against low-frequency antigens. IAT crossmatch compatible blood may be issued. Pre-warming to 37 °C is advised in some instances (see§1.3).
  • 8.2.5 Patients with autoimmune haemolytic anaemia. Patients should be investigated for the presence of alloantibodies (see§7.9).
  •  Unless clinical need indicates that the patient requires blood urgently, it is unacceptable simply to crossmatch and issue blood as compatible as the patient's own cells and serum. In regularly transfused patients, frequent repeat testing is not necessary after initial testing has taken place; however, it is advisable to provide Rh-matched and K-matched units (Sanguin et al., 2001).

  • 8.2.6 Massive blood transfusion. Where the volume of blood transfused in any 24 h period is equivalent to the patient's own blood volume, ABO group identical blood can be issued without the need for a serological crossmatch and ABO incompatibility must be excluded using serological testing or electronic issue. If ABO non-identical blood has to be transfused, blood of the same group as the patient should be used as soon as possible after the first transfusion. There is no need to persist with the ABO group originally transfused.
  • 8.2.7 Fetal/neonatal transfusions.
  • 8.2.8 Chronically transfused patients
  • (i) Sickle cell anaemia patients. The incidence of alloimmunization in multiply transfused sickle cell anaemia patients varies (Vichinsky et al., 2001). It is desirable to phenotype the patient's red cells as fully as possible prior to transfusion and to match for D, K, C, c and E and e antigens, provided this does not impede the delivery of effective transfusion support. Many patients are phenotypically Dce, and D-positive blood negative for C and E may be difficult to find, particularly if there are other antibodies. D-negative blood should be supplied in these circumstances. HbS-negative blood should be selected.
  • (ii) Chronically transfused haematology patients. It is prudent to phenotype as fully as possible prior to transfusion. It may also be reasonable to provide units matched for D, K, C, c, E and e antigens where these are readily available, although there is insufficient evidence to make this a recommendation.
  • 8.2.9 Recipients of allogeneic haemopoietic stem cell grafts
  •  Recipients of allogeneic transplants present unusual grouping and crossmatching problems. The transplant may introduce a new ABO antigen (major mismatch) or a new ABO antibody (minor mismatch) or both. All cellular products should be irradiated to prevent graft-versus-host disease. See Guidelines on gamma irradiation of blood components for the prevention of transfusion-associated graft-versus-host disease (BCSH, 1996a).

  • (i) Major ABO mismatch. Red cells and products contaminated with red cells should be of the patients' own ABO group until their antibodies are no longer detectable by the IAT, the DAT is negative and donor ABO group is established.
  • (ii) Minor ABO mismatch. Red cells and products contaminated with red cells should be of donor ABO group and plasma depleted until the recipient's original red cells are no longer detectable.
  • (iii) Combined ABO mismatch. Before graft, red cells of the recipient's own group should be selected. After graft, group O blood should be used until ABO antibodies to the donor type are no longer detectable by the IAT and the DAT is negative.
  • (iv) D-positive recipient with D-negative donation or graft. D-negative components should be transfused.
  • 8.3 Procedure
  • 8.3.1 One person should carry out the crossmatching procedure from beginning to end. Where this is not possible, there should be an audit trail of all individuals involved in each stage of the procedure.
  • 8.4 IAT crossmatch
  • 8.4.1 A crossmatch, which includes an IAT, must be used if:
  • (i) the patient's plasma/serum contains, or has been known to contain, clinically significant red cell alloantibodies. This recommendation is based on the need to (a) assure the correct phenotype of the donor red cells, (b) potentially detect additional specificities, which may have been masked in antibody identification and (c) assure that the assigned specificity is correct;
  • (ii) the antibody screen does not conform to the recommendations in §6;
  • (iii) the patient has had an ABO-incompatible solid-organ transplant and is being transfused within 3 months of the transplant. This is necessary to detect IgG anti-A or anti-B produced by passenger lymphocytes in the transplanted organ (Mollison et al., 1993);
  • (iv) fetuses or neonates are being transfused for alloimmune disease, in which case maternal serum plasma should be used.
  • 8.5 Computer issue without a serological crossmatch
  •  The correct determination of the ABO and D group and robust antibody screening procedures that comply with §6 are vital when computer issue without a serological crossmatch is used. It is equally essential that the computer system in use be locally validated to meet the guidelines. Recommendations for the process of electronic selection and issue of units without serological testing between patient and donor red cells are given in Guidelines for blood bank computing (BCSH, 2000).

  • 8.6 Immediate-spin crossmatch
  • 8.6.1 The immediate-spin crossmatch may be used on all patients, except those described in §8.4.1 and except in patients where ABO grouping reveals very weak anti-A or anti-B (except in group AB patients).
  • 8.6.2 There is a lack of standardization in respect of immediate-spin methodology (O'Hagan et al., 1999). It is important for sensitivity that the optimum ratio of serum to cells and appropriate cell suspension and centrifugation speed and time are used. There are no recommended procedures for performing immediate-spin techniques other than by tube.
  • 8.6.3 A 2 : 1 ratio of serum/plasma to cells and a 2–3% red cell suspension are recommended.
  • 8.6.4 A short incubation time of 2–5 min before centrifugation is recommended to enhance the detection of weak ABO antibodies.
  • 8.6.5 EDTA saline should be used as group O sera can show prozone owing to steric hindrance of agglutination by C1 (Judd et al., 1988).
  • 8.6.6 The appropriate centrifugation speed and time should be determined.
  • 8.6.7 Readings should be taken carefully using a tip-and-roll technique.
  • 8.7 Emergency situations
  • 8.7.1 The sample should be obtained and labelled as in §4.2.7.
  • 8.7.2 The sample should be ABO and D grouped by rapid (quick spin) techniques. The patient's cell group must include testing with anti-A, anti-B and anti-D. Appropriate controls should be included.
  • 8.7.3 A reverse group or a repeat cell group, in either case using resampling, or an immediate-spin crossmatch must also be carried out before ABO-matched blood is issued.
  • 8.7.4 An antibody screen should be performed. If this is negative, it is not necessary to carry out a retrospective IAT crossmatch. It is not acceptable to perform a crossmatch and not carry out an antibody screen (see§6.1.2).
  • 8.8 The compatibility report
  • 8.8.1 A compatibility report must be issued before or with the first unit of blood issued for transfusion. Information should include the patient's surname, first name, hospital number, date of birth, ward and blood group and component details to include the product type, donation number and blood group.
  • 8.8.2 If the blood group of the unit and the patient are not identical, a comment should be made in the report indicating that, although not of an identical group, the blood is suitable for transfusion.
  • 8.8.3 If routine pretransfusion testing has not been carried out, this should be stated in the report. The report may be used as a record in the patient's notes that pretransfusion testing has not been carried out in full.
  • 8.9 The compatibility label
  • 8.9.1 There must be a compatibility label, which should be securely attached to the blood bag. Information should include the patient's surname, first name, hospital number, date of birth, patient's blood group, donation number and blood group and the date that the blood is required/crossmatched (BCSH, 1999). Previous compatibility labels should be removed or obliterated so that the identity of previous potential recipients is no longer visible.
  • 8.10 Visual inspection of the red cell unit
  • 8.10.1 Before the unit is placed in the blood issue refrigerator, it should be inspected for:
  • (i) integrity of the pack by checking for leaks at the ports and seams;
  • (ii) evidence of haemolysis in the plasma or at the interface between the plasma and red cells;
  • (iii) evidence of discoloration of the red cells;
  • (iv) presence of large clots in the red cells.
  •  If there is any evidence of the above, the unit should not be used and the local blood centre should be informed.

  • 8.11 Removal of blood from the issue refrigerator should be in accordance with a written procedure (BCSH, 1999).


Whilst the advice and information contained in these guidelines is believed to be true and accurate at the time of going to press, neither the authors nor the publisher can accept any legal responsibility for any errors or omissions that may be made.