There is a new regulatory framework governing hospital transfusion laboratory practice which was implemented after the publication of two European Union Directives: 2002/98/EC and 2004/33/EC.⁶ In the UK these are the Blood Safety and Quality Regulations 2005 (Statutory Instruments 2005/50, 2005/1098 and 2006/2013) and they set standards for quality and safety of human blood and blood components in hospital ‘blood banks’ as well as ‘blood establishments’ (the UK Blood Services).⁷ ‘Blood banks’ are regulated in their roles of storing, distributing and performing compatibility tests, on blood and blood components for use in hospitals. The implications for hospital transfusion practice are the requirement for ‘vein to vein’ traceability of blood components, the importance of maintaining the ‘cold chain’ for all therapeutic blood components, the need to store transfusion records for 30 years and the requirement for a quality management system.^6,⁷ UK laboratories have to assess themselves and submit an annual compliance report to the competent authority, which is the Medicines and Healthcare Products Regulatory Agency (MHRA).⁸ The MHRA carries out laboratory inspections to assess compliance with these regulations.

The UK government, through the National Blood Transfusion Committee, continues to promote the safe and effective transfusion of blood components and has published three Health Service Circulars.⁹ These documents aim to ensure that a team approach to blood transfusion safety is taken via the local clinical governance arrangements and to promote good transfusion practice via Hospital Transfusion Committees and Hospital Transfusion Teams supported by Regional Transfusion Committees.

Technology and automation in blood transfusion laboratories

Important changes have taken place in the blood transfusion laboratory in the last 10–15 years, as outlined in the previous edition of this book. As a result transfusion laboratory practice is safer, largely as a result of the implementation of new technologies for testing, automated systems to replace manual systems and the widespread use of information technology (IT) systems to support transfusion laboratory practice.

Barcoded labels on blood components, reagents, patient samples and equipment are now commonplace and these result in safer transfer of information, free from the transcription errors associated with manual methods.

Column agglutination (CAT) and solid-phase technology can both be used on automated machines and CAT can also be used by manual techniques. In UK hospital transfusion laboratories these technologies have replaced tube techniques and liquid-phase microplates for antibody screening and crossmatching (Figs 22.2, 22.3).¹⁰

Figure 22.2 Change in indirect antiglobulin test (IAT) technology for antibody screening. Data from UK NEQAS surveys 1998–2009.¹⁰ These surveys of UK practice show that technology used for antibody screening and serological crossmatching is now predominantly column agglutination (CAT) with very few laboratories using tube techniques. LPM, liquid-phase microplate.

Figure 22.3 Change in technology for serological compatibility testing. Data from UK NEQAS surveys 1998–2009.¹⁰ These surveys of UK practice show that technology used for antibody screening and serological crossmatching is now predominantly column agglutination with very few laboratories using tube techniques.

The currently available CAT systems include Ortho BioVue, which comprises a six-well cassette containing a glass microbead matrix, ID DiaMed cards utilizing Sephadex gel matrix, also with six wells, and an eight-well Sephadex gel matrix card from Grifols. Each offers a wide range of profiles and reagent systems.

The currently available solid-phase systems are Immucor Capture-R and Bio-Rad Solidscreen II. The Immucor system utilizes a range of red cell antigens adhered to the surface of a U-shaped microplate well. Sensitized red cells are then used as a marker. The Bio-Rad Solidscreen II is a solid-phase method for antibody screening and identification. The wells of the microplates are coated with protein A to allow the reaction to bind to the plate forming a solid-phase cell layer.

Individual laboratories need to make careful and informed decisions when selecting reagents for pre-transfusion testing. It is vital that any abbreviated testing in an automated or semiautomated system is carefully evaluated for the risks that could ensue if important controls were omitted when using this technology for blood grouping.¹¹

Laboratory information management systems (LIMS) store patient details and results of laboratory tests, allowing timely and accurate access to important information. In the transfusion department, IT systems have a much broader use and the updated BCSH guidelines for the specification and use of Information Technology (IT) systems in blood transfusion practice (2006) reflect this.¹² Where possible, using bidirectional or unidirectional interfaces to automated blood grouping analysers, IT systems are used to eliminate errors that can arise when a manual step is employed, including interpretation of test results.

Computer algorithms support the ‘electronic issue’ of blood to patients with a negative antibody screen without the need to perform an antiglobulin crossmatch and, in the UK in 2009, 46% of laboratories were using this system for some or all of their patients (Fig. 22.4). The use of automation for all aspects of compatibility testing is now recommended practice as it is recognized that it is safer.³ Automation brings several or all of the discrete activities of compatibility testing into a single platform process. It provides various levels of increased security over manual testing and may provide justification for abbreviated pre-transfusion testing (e.g. abandoning duplicate D, previously termed ‘RhD’, typing or reverse ABO grouping in the presence of a valid historical group). A risk assessment must be made and documented prior to any abbreviation of an established procedure, with consideration being given to the presence or absence of key functions in the automated equipment. The BCSH guidelines for compatibility procedures¹² and guidance from the MHRA¹³ give a list of factors to be taken into consideration and the reader is advised to consult these prior to implementing automated or semiautomated systems.

Figure 22.4 Change in proportion of UK laboratories issuing blood electronically 1998–2009. Data from UK NEQAS surveys indicating the increased use of electronic issue for compatibility testing in UK transfusion laboratories.

Pre-transfusion compatibility systems

The process of providing blood for transfusion involves many steps, all of which have to be reliably completed. These include the following:

• Blood samples have to be taken from the correct patient and labelled at the bedside in a single uninterrupted procedure. The sample must be identified by four core patient identifiers: the correctly spelled forename and surname, the exact date of birth and an accurate unique patient number (such as the NHS number or equivalent). The sample should be dated and signed by the person taking the sample.¹⁴ Some guidelines recommend that addressograph labels should not be accepted on blood samples for compatibility testing because it increases the risk of ‘wrong blood in tube’(WBIT).¹⁵ In an international study from the BEST collaborative, it was estimated that miscollected samples demonstrating WBIT occurred at a median rate of 0.5 per 1000, although there was great variation worldwide in the reported frequency of mislabelled samples, probably resulting from variation in policies for sample acceptance.¹⁶ Labels printed at the bedside using barcoded patient wristband and hand-held scanners are acceptable.¹⁴ The laboratory should have a policy for rejecting badly labelled samples, although in 2004 a survey of hospitals in England and North Wales showed considerable variation in the content and application of these policies.¹⁷

• A request for services should include the previously outlined four core patient identifiers as well as clear information about the source of the request and the location of the patient and clinical details including a detailed justification for the request. Previous transfusion history and any special considerations for the selection of blood (e.g. antigen negative if the patient has a clinically significant antibody, cytomegalovirus [CMV] tested or irradiated for certain groups of immunosuppressed patients) are also needed.

• An ABO and D group of the patient sample must be accurately performed.

• An antibody screen of the patient’s plasma (or mother’s plasma in the case of a neonate) should be able to detect any clinically significant red cell antibodies. In the event of a positive red cell antibody screen, antibody identification should be undertaken to assist the selection of compatible blood.

• There should be a check of existing transfusion records to compare current and historical results.

• The appropriate blood component should be selected and issued to a named patient using a serological crossmatch or electronic issue.

• Traceable documentation should exist to ensure that the results of laboratory compatibility procedures are available at the patient’s bedside to allow a check before transfusing the blood component. This should include a blood bag compatibility label (Fig. 22.5) and may include a compatibility form. The patient must be identified with a wristband and the blood component should be prescribed on a drug or fluid administration chart.¹⁴ In some countries, an additional bedside check of the patient’s blood group is undertaken prior to commencing the transfusion.

Figure 22.5 A unit of red cells showing the compatibility label.

Documentation of the Transfusion Process

All stages of the transfusion process must be clearly documented and these records must be kept. In addition to guidance from the UK Royal College of Pathologists on the retention of documents,¹⁸ the BSQR 2005 regulations stipulate that the records must be accessible for 30 years.⁷ This allows any blood component to be traced from the donor to the recipient should information come to light about any potential infective risks to the recipient.

Computer records are easier to search than paper records, but laboratory information systems are likely to become obsolete and be replaced several times within this mandatory 30-year period, so provision must be made to store historical data in an accessible format when procuring a replacement computer system.¹² Patient-held records are useful for patients who are treated in more than one institution, particularly if they have red cell antibodies and require phenotyped blood or if they have special requirements because of their underlying disease or its treatment. Credit card-sized records with corresponding patient information leaflets are issued by some transfusion centres to patients with red cell antibodies and similar cards exist for patients who require irradiated cellular blood components.¹⁹

Identification and Storage of Blood Samples

Depending on laboratory practice, blood transfusion tests use a clotted (serum) sample or EDTA-anticoagulated blood (plasma). Most laboratories using automated systems will use a plasma sample.

The reason for automated systems requiring plasma is because incompletely clotted blood samples may contain small fibrin clots that trap red cells into aggregates that could resemble agglutinates which could be falsely interpreted as a positive reaction. Clotted samples should be taken into a plain tube but not a tube with a separating gel.

The presence of complement in serum can cause lysis. If laboratory staff are used to recognizing agglutination as an indicator of a positive reaction they may fail to interpret the lysed red cells as an equally valid positive reaction. Therefore, when using serum for blood grouping and compatibility testing, any red cells in the test system should be washed and resuspended in saline which contains ethylenediamine tetra-acetic acid (EDTA) (see later). In addition, false-negative reactions may occur in immediate spin crossmatching with potent ABO antibodies, where rapid complement fixation causes a prozone effect (bound C1 inhibits agglutination). EDTA saline is not necessary when using plasma.

Throughout this chapter, for the sake of brevity, ‘plasma’ is used as the majority of UK laboratories are automated and therefore anticoagulated (plasma) samples are required. If serum samples are specifically required, this will be indicated in the text.

On being received in the laboratory, the details on the request form must be checked against the blood sample. Each blood sample must be labelled with a unique sample number. Barcode labels offer the advantage of positive sample identification and reduce the number of transcription errors. Samples inadequately or inaccurately labelled should NOT be used for pre-transfusion testing.¹¹

Great care must be taken to select and identify the sample prior to any testing. Transposition of samples in the laboratory can lead to an incorrect blood group being assigned to a patient, with serious consequences, including ABO incompatible transfusions. Where possible, the primary sample should be used for testing. If samples are separated, the plasma must be clearly and accurately identified and special precautions must be taken. If repeated testing on this sample is anticipated, storing separate small aliquots reduces the risk of sample deterioration, which occurs with repeated thawing/freezing of larger samples.

Whole blood samples should be tested as soon as possible because they will deteriorate over time. Problems associated with storage include lysis of the red cells, loss of complement in the serum and decrease in potency of antibodies. The BCSH guidelines ¹¹ indicate working limits as outlined in Table 22.1. If separated plasma or serum samples are stored for later serological crossmatch, care must be taken to ensure that the patient has not been transfused in the interim. It has been recommended that samples should be kept for a minimum of 7 days from group and screen, stored at 4°C. Samples should be retained post-transfusion for investigation of acute transfusion reactions and preferably stored for 7 days post-transfusion to enable investigation of delayed transfusion reaction.¹⁸

Table 22.1 Recommended limits for storage of samples used for compatibility testing ¹¹

ABO and D grouping

ABO and D grouping must be performed by a validated technique with appropriate controls. Before use, all new batches of grouping reagents should be checked for reliability by the techniques used in the laboratory. Grouping reagents should be stored according to manufacturer’s instructions.

ABO Grouping

ABO grouping is the single most important serological test performed in compatibility testing; consequently, it is imperative that the sensitivity and security of the test system are not compromised. The fact that anti-A and anti-B are naturally occurring antibodies allow the patient’s plasma to be tested against known A and B cells in a ‘reverse’ group.

This is an excellent built-in check for the ‘forward’ or cell group and has always been considered to be an integral part of ABO grouping, allowing the reading and recording of test results to be split into two discrete tasks. However, with secure, fully automated systems, linked to secure laboratory information management systems that, in combination, have the ability to prevent procedural ABO grouping errors, some laboratories now omit the reverse group when testing samples for which a historical group is available.¹¹ This should only be considered following a careful risk assessment and taking into account that the first sample taken may have been from the wrong patient. This may be in the order of 1:2000 samples.¹⁶ Any discrepancy between the forward and reverse groups should be investigated further and any repeat tests should be undertaken using cells taken from the original sample rather than from a prepared cell suspension.

Reagents for ABO Grouping

Monoclonal anti-A and anti-B reagents have replaced polyclonal reagents in routine grouping tests. A₁ and B cells are used for reverse grouping; group O cells or an auto control may be included to ensure that reactions with A and B cells are not a result of the presence of cold autoantibodies. A diluent control should be included where recommended by the manufacturer.

D Grouping

D grouping is usually undertaken at the same time as ABO grouping for convenience and to minimize clerical errors that may arise through repeated handling of patients’ samples. In the absence of secure automation, testing should be undertaken in duplicate because there is no counterpart of the ‘reverse’ grouping of ABO testing.

Reagents for D Grouping

Monoclonal reagents do not have the problem of possible contamination with antibodies of unwanted specificities, as was the case with polyclonal reagents. Therefore, the duplicate testing may be undertaken using the same anti-D reagent, although this should be dispensed as though it were two separate reagents.

DVI is the partial D with the fewest epitopes; therefore of all the D variants, DVI individuals are those most likely to form anti-D and a case of severe haemolytic disease of the fetus and newborn (HDFN) has been described.²⁰ For this reason, anti-D monoclonal reagents that do not detect DVI should be selected for testing patients’ samples.^21,²² The use of anti-CDE reagents has led to the misinterpretation of r′ and r″ cells in UK National External Quality Assessment Scheme (NEQAS) exercises and, because they are of no value in routine patient typing, their use is not recommended.^10,¹¹ Selection of high-avidity monoclonal anti-D reagents will allow detection of all but the weakest examples of weak D, negating the need to use more sensitive techniques to check the D status of apparent D negatives.

Some anti-D reagents have high levels of potentiator (e.g. polyethylene glycol) and should be used with caution; a diluent control is essential to demonstrate that the diluent does not promote agglutination of the test red cells as may happen if the patient’s cells are coated in vivo with immunoglobulin G (IgG); any positive reaction seen with the control, however weak, invalidates the test result. Anti-D reagents are provided in many different kits and those responsible for selection and purchase should make themselves aware of the content, specificity and potentiation of the chosen reagent.

Column agglutination techniques

CAT techniques (see Chapter 21, p. 502) are now the commonest method for grouping in the UK (80% of laboratories who responded to a UK NEQAS survey in 2009, see Fig. 22.2), especially where automated systems are in place; these should always be performed in accordance with the manufacturer’s instructions. There are several different profiles to choose from and some cards/cassettes include monoclonal antibodies to other blood group antigens (e.g. K) in addition (see Chapter 21, p. 502 and Fig. 22.6). Forward and reverse grouping may be undertaken in separate cards/cassettes.

Figure 22.6 Column agglutination technology: blood group O D positive. First box, α. Second box, β. Third box, D. Fourth box, Control. Fifth box, A₁. Sixth box, B.

Solid-phase techniques

ABO/D grouping using solid-phase techniques would usually be part of a fully automated system and testing should be accordance with the manufacturer’s instructions. Bio-Rad Erytype employs a standard agglutination technique for blood grouping performed in a microtitre plate. The reagents for the forward group are dried onto the wells. Uncoated empty wells are used with standard liquid red cells for a reverse group.

Controls

Positive and negative controls should be included with every test or batch of manual tests. In fully automated systems, the controls should be set up at least twice in a 24-h period. The timings should coincide with machine startup and changing of reagents, taking account of the length of time that reagents have been kept at room temperature on the machine. The control samples should be loaded in the same way as the test samples. The required controls are shown in Table 22.2. Where controls do not give the expected reactions, investigations should be undertaken to determine the validity of all tests undertaken subsequent to the most recent valid control results.

Table 22.2 Control cells for blood grouping

Reagent	Positive control	Negative control
Anti-A	A cells	B cells
Anti-B	B cells	A cells
Anti-D	D positive cells	D negative cells
A₁ cells	Anti-A	Anti-B
B cells	Anti-B	Anti-A

Causes of Discrepancies in ABO/D Grouping

False-Positive Reactions

Rouleaux

Rouleaux may occur in various clinical conditions, where the ratio of normal albumin to globulin is altered in plasma (e.g. multiple myeloma) and in the presence of plasma substitutes such as dextrans. The stacking of red cells on top of one another in columns may be misinterpreted as weak agglutination by inexperienced workers. Rouleaux will usually disperse on a slide if a drop of saline is added; alternatively, the reverse grouping can be repeated using plasma diluted 1 in 2 or 1 in 4 with saline.

Cold autoagglutination and cold reacting alloantibodies

Cold autoantibodies (usually anti-I) that are reactive at room temperature may lead to autoagglutination in the cell group (ABO and D) and panagglutination in the reverse group, causing a grouping anomaly (p. 277). The tests should be repeated using cells washed in warm saline and plasma pre-warmed to 37°C. An auto control should be included.

Cold reacting alloantibodies (e.g. anti-P₁) may cause agglutination of reverse grouping cells and, if this is suspected, reverse grouping should be repeated using pre-warmed plasma or grouping cells that lack the implicated antigen.

T-activation/polyagglutination

Polyagglutination ²⁵ describes agglutination of red cells by all or most normal adult sera but not by the patient’s own serum. This is as the result of IgM antibodies reacting with an antigen on the red cells which is usually hidden but can be exposed by enzyme activity. The most common form is T-activation, which occurs when the bacterial enzyme neuraminidase cleaves N-acetyl neuraminic acid from the red cell membrane, exposing the T antigen.

This used to be a problem when grouping with polyclonal reagents, which contain anti-T, but tests using monoclonal reagents are not affected by this phenomenon.

Acquired B

This arises in group A₁ patients where the expected reaction with anti-A is noted but an additional weak reaction with anti-B occurs. The blood group appears to be ‘AB’ but with anti-B in the reverse group. This discrepancy between the forward and reverse grouping may be overlooked if the patient’s own anti-B is weak or if the reverse group is omitted.

The acquired B antigen is usually caused by a bacterial deacetylase enzyme acting on A₁ red cells and producing a B-like substance. Some anti-B reagents react strongly with the acquired B antigen (e.g. those derived from the ES4 clone).²⁶ Such anti-B reagents are rare but should be avoided in routine blood grouping.

Potentiators

Red cells may be coated with IgG as a result of in vivo sensitization. The use of potentiated techniques, such as the antiglobulin test for D typing, or of potentiated reagents for ABO or D typing, may result in a false-positive reaction; the latter would also result in a positive reaction with the diluent control, but UK NEQAS data have shown that some laboratories fail to include an appropriate control or fail to understand the significance of a positive control.²⁷ For this reason, use of potentiated techniques or reagents for blood grouping is not advised.

In vitro bacterial contamination

In vitro bacterial contamination of reagents, patients’ red cells or reverse grouping cells may cause false-positive agglutination.

False-Negative Reactions

Failure to add reagents

The most likely cause of a false-negative grouping result is failure to add the reagent or test plasma. For this reason, in liquid-phase tube and plate tests, the serum or plasma should always be added first to the reaction chamber and a visual check should be made before red cells are added. The use of colour-coded reagents for ABO grouping is helpful in this respect. Automated systems have liquid level sensors resulting in an alert of the failure to add a reagent or test plasma.

Loss of potency

Inappropriate storage or freezing and thawing may cause a loss of potency of blood grouping reagents. The use of regular controls will alert the user to this problem.

Failure to identify lysis

In the presence of complement, anti-A and anti-B may cause in vitro lysis of reagent red cells. If lysis is not recognized as a positive reaction, falsely negative results may be recorded in reverse grouping tests. To avoid this, reverse grouping cells should be resuspended in EDTA saline, where serum rather than plasma is used.

Mixed-field appearance

This describes a dual population of agglutinated and non-agglutinated red cells which may be observed in both ABO and D grouping. It is important to recognize this as a mixed-field picture and not to confuse it with weak agglutination. The most likely cause of a mixed-field picture is the transfusion (either deliberate or accidental) of non-identical ABO or D red cells. Investigation will be required to determine the actual blood group of the patient, who may have been transfused in an emergency, at a different establishment or may have received an intrauterine transfusion. A mixed-field ABO group may be the first indication of a previous ABO-incompatible transfusion.

An ABO or D incompatible haemopoietic progenitor cell transplant will result in a mixed-field picture until total engraftment has occurred; the mixed-field picture may subsequently reappear when a graft is failing. Rarely, a dual population of cells is permanent and results from a weak subgroup of A (A₃) or a blood group chimerism.

Interpretation of a dual population of red cells will depend on the technique used. In a tube, microscopic reading will reveal strong agglutinates in a background sea of free cells. In column agglutination techniques there will be a line of agglutinated cells at the top of the column, with the non-agglutinated cells travelling through to the bottom of the column. In liquid-phase techniques if the reaction grade is not a strong positive or an obvious negative then the reactions requires further investigation, which may include microscopic examination. In a solid-phase technique a mixed field is seen as a dual population of cells, with the agglutination being surrounded by free cells. Automated systems should be set up to detect mixed-field pictures and this should be used in conjunction with local policies.

D variant phenotypes

Weak D phenotypes are where the entire D antigen is present but there are fewer D antigen sites per cell and most weak D types group as D positive with the currently available high-avidity commercial monoclonal anti-D reagents. Where differing reactions are obtained with two reagents, the patient may be a partial D (i.e. one or more of the epitopes of the D antigen is missing). It was generally thought that patients who are weak D are unable to make anti-D and may be treated as D positive whereas some patients with partial D may be capable of making immune anti-D following sensitization with the missing epitope. This concept has been challenged by reports of weak D patients with anti-D.²⁸

Detailed genotypic analysis combined with structural modelling of the D antigen in D variants have demonstrated change in the amino acid sequence in the extracellular domain in partial D, whereas weak D is associated with mutations resulting in change to the amino acid sequence in the intracellular or membrane-spanning domain.

A weak reaction with a single anti-D reagent should be investigated with a second anti-D reagent before assigning a result of D positive. It is essential to be able to distinguish between a weak reaction and a mixed-field reaction because the latter may be the result of a patient who is being transfused with blood of a different D type.

If in doubt, it is safer to call the patient D negative, at least until investigations have been undertaken by a reference centre. This will be of no clinical consequence because it is safe to transfuse D negative blood to a patient who is D positive, and a pregnant woman who is D positive and her unborn child would be unlikely to be harmed by the injection of prophylactic anti-D immunoglobulin.

Current advice about choice of D-typing reagents is that because DVI lacks the most epitopes, such individuals are likely to make anti-D when challenged by transfusion or pregnancy. For this reason, anti-D reagents for routine grouping of patients’ samples should not detect DVI.¹¹ There is little evidence to suggest that a DVI donor would elicit an immune response in a recipient who is D negative; however, weak D positive and partial D donors, including DVI donors, should be classified as D positive.²⁹ There are some important ethnic differences in the frequency of different Rh haplotypes, as shown in Table 22.3. Resolution of anomalous D grouping where a partial D is suspected now includes both serological testing and genotypic studies.³⁰

Table 22.3 Approximate frequencies of common haplotypes in selected populations ³⁰

Antibody screening

Antibody screening is usually undertaken at the same time as blood grouping and in advance of selecting blood for transfusion. Antibody screening may be more reliable and sensitive than crossmatching against donor cells because some antibodies react more strongly with red cells with homozygous expression (double dose) of the relevant antigen than with those with heterozygous expression (single dose) – most notably anti-Jk^a/Jk^b but also anti-Fy^a, -Fy^b, -S and -s. Screening cells can be selected to reflect this, whereas donor cells are usually of unknown zygosity. In addition, reagent red cells are easier to standardize than donor cells and there is potentially less opportunity for procedural error, particularly in automated systems.

Clinically significant antibodies are those that are capable of causing patient morbidity as a result of accelerated destruction of a significant proportion of transfused red cells. With few exceptions, clinically significant antibodies are those that are reactive in the indirect antiglobulin test at 37°C; however, it is not possible to predict serologically which of these antibodies will definitely be of clinical significance, so the term ‘of potential clinical significance’ is often used.

Red Cell Reagents

The patient’s plasma should be tested against at least two individual screening cells, used individually, not pooled. The screening cells should be group O and encompass the common antigens of the indigenous population.

In the UK, the following antigens should be expressed as a minimum: C, c, D, E, e, K, k, Fy^a, Fy^b, Jk^a, Jk^b, S, s, M, N and Le^a; one cell should be R₂R₂ and another R₁R₁ or R₁^wR₁. The following phenotypes should also be represented in the screening set: Jk(a+b−), Jk(a−b+), S+s−, S−s+, Fy(a+b−) and Fy(a−b+) (Table 22.4). These recommendations for homozygosity are based on UK data regarding the incidence of delayed haemolytic transfusion reactions, the need for high sensitivity in the detection of Kidd antibodies and the poorer performance of column agglutination techniques in the detection of some examples of Kidd antibodies using heterozygous cells.^11,²⁹ The requirement for the expression of C^w and Kp^a antigens on screening cells has been the cause of much debate but in the UK and the USA detection of anti-C^w or anti-Kp^a is not a requirement even in the absence of an antiglobulin crossmatch.^11,²⁹ This is because these are low-frequency antigens and the antibodies rarely cause delayed haemolytic transfusion reactions or severe haemolytic disease of the newborn.

Table 22.4 Expression of red cell antigens on screening cells ¹¹

Blood group system	Antigen	Homozygous cells recommended
Rh	C	Yes (R₁R₁ or R₁^wR₁)
	c	Yes (R₂R₂)
	D	Yes (R₁R₁ and R₂R₂)
	E	Yes (R₂R₂)
	e	Yes (R₁R₁ or R₁^wR₁)
Kell	K	No^a
	k	No
Duffy	Fy^a	Yes
	Fy^b	Yes
Kidd	Jk^a	Yes
	Jk^b	Yes
MNSs	M	No
	N	No
	S	Yes
	s	Yes
Lewis	Le^a	No

a Although desirable, KK cells are unlikely to be available.

Methods

Antibody screening should always be carried out by an indirect antiglobulin test as the primary method. Additional methods (e.g. two-stage enzyme or Polybrene) may also be used but are inferior for the detection of some clinically significant antibodies and should not be used alone. A large retrospective study showed that the vast majority of antibodies reactive only by enzyme technique are of no clinical significance.³¹ In Issitt’s study of 10 000 recently transfused patients, only one anti-c, initially unreactive by indirect antiglobulin test, caused a delayed haemolytic transfusion reaction.³² There has been one SHOT report (1998–1999) of an enzyme-only anti-E that became detectable by indirect antiglobulin test 7 days following the transfusion, causing a delayed haemolytic transfusion reaction and subsequent death of the patient due to renal failure.¹

For liquid-phase techniques, BCSH guidelines ¹¹ recommend the use of red cells suspended in LISS, rather than in standard normal ionic strength saline (NISS), because LISS increases the speed and sensitivity of detection of many potentially clinically significant antibodies. There are conflicting reports about whether sensitivity can also be improved by adding polyethylene glycol.³³

Indirect Antiglobulin Techniques

Column Agglutination

In many countries, including the UK, column agglutination is now more commonly used than traditional tube or liquid-phase microplate techniques (see Figs 22.2 and 22.3) because it has been shown to be at least as sensitive as a standard LISS spin-tube technique,^33,³⁴ it is simpler to perform because it requires no washing phase, it uses small volumes of plasma and reagents, it has a more objective reading phase and it is easy to automate. The antihuman globulin (AHG) incorporated in the matrix is available as either a polyspecific or anti-IgG reagent. Red cell concentrations and volumes can be critical and it is important to follow manufacturers’ instructions at all times.

Column agglutination crossmatching tests have been shown to be less sensitive than standard tube techniques in detection of weak ABO antibodies such as anti-A with A₂B cells and Kidd antibodies with heterozygous red cells.^27,³⁵^–³⁷ It has been suggested that these failures may be the result of shear forces occurring during centrifugation, which cause weak agglutinates to be disrupted, especially when the antigen site density is low.³⁸

Solid-Phase Systems

Solid-phase techniques (e.g. Immucor Capture-R and Bio-Rad Solidscreen II) are also becoming more popular because they have been shown to have a high level of sensitivity and they also lend themselves to full automation. In 2009 10% of UK laboratories were routinely using solid phase for antibody screening (see Fig. 22.3).

Liquid-Phase Techniques – Tubes and Microplates

Tube techniques are still used for antibody screening in some parts of the world. With LISS-suspension techniques it is important to keep a high serum:cell ratio, without affecting the ionic strength. Equal volumes of serum and 1.5–2% cells suspended in LISS will result in a serum:cell ratio of >60:1, ensuring optimal sensitivity. Reagents should be incubated for 15–20 min at 37°C and the cells then should be washed in PBS. After adding AHG, the red cells should be examined using a careful ‘tip and roll’ procedure to prevent disruption of weak agglutinates. Reading aids such a light box or concave mirror may also be used with this technique.

Liquid-phase microplate technology has never achieved a huge popularity for antibody screening because it is relatively difficult to introduce and standardize and it cannot be automated. Because it is becoming increasingly difficult to obtain AHG reagents standardized for use in microplates, no method is detailed here. However, further information is available in the BCSH guidelines, including a recommended method for screening in V-well plates, using a streaming technique.^11,²⁴

Controls

A weak anti-D should be used on a regular basis to ensure the efficacy of the whole procedure, although the exact frequency will depend on work patterns as described in the blood grouping section. Additional weak controls are also recommended to confirm the sensitivity of the procedure and the integrity of the red cell antigens throughout their shelf life; anti-Fy^a and/or anti-S are good examples because Fy^a and S antigens are protease labile and may deteriorate more quickly on reagent cells than the D antigen and their use will also ensure that enzyme-treated cells have not been used by mistake. Consideration should also be given to selecting controls which demonstrate that the correct screening cell has been added to the batch of tests. Anti-S can be used as a control following the use of bleach to clean automated blood grouping analysers to check no cleaning fluid remains; the S antigen is very sensitive to Clorox.

The use of red cells weakly sensitized with anti-D is essential to control the washing phase of every negative liquid-phase test because inadequate washing may result in complete or partial neutralization of AHG by unbound globulin. Any test that does not give a positive reaction at the expected strength, following addition of these cells (and subsequent centrifugation), indicates insufficient free anti-IgG and should be repeated. The washing phase of solid-phase systems is difficult to control and it may be necessary to add a weak control to every column to ensure that every probe has dispensed wash solution during each wash cycle.

Antibody identification

When an antibody is detected in the antibody screen, its specificity should be determined and its likely clinical significance should be assessed before blood is selected for transfusion or relevant advice is given during pregnancy. It is essential to use a systematic approach to antibody identification to ensure that all specificities of potential clinical significance are identified. It may be tempting to match a reaction pattern immediately and look no further, but this is likely to result in additional specificities being missed.

Principles

As a starting point, the test plasma should be tested against an identification panel of reagent red cells by the technique with which it was detected in the screen. The next section on reagents outlines the minimum requirements for the panel. Inclusion of an autoantibody test is helpful in distinguishing between an autoantibody, an antibody directed against a high-frequency antigen and a complex mixture of alloantibodies. The positive and negative reactions should be compared with the panel profile in conjunction with the screening results.

Each antibody specificity should be taken in turn and its presence should be systematically excluded, by identifying antigen-positive cells that have given negative reactions. Wherever possible a negative reaction should be obtained with a red cell with homozygous expression of the relevant antigen. For example, if a negative reaction has been recorded against a Jk(a+b−) cell, then the presence of anti-Jk^a can be excluded, but if the only negative reactions are against Jk(a+b+) cells, then anti-Jk^a cannot immediately be safely excluded. This will leave a list of potential specificities, which should be considered by matching the positive reactions to the antigen-positive cells to determine if any are definitely present. Once this process is complete with the initial screen and identification panel, further cells and techniques may need to be used to complete the exclusion process. For example, where anti-S has been identified as being present, an enzyme-treated panel may be required to exclude the presence of anti-E, where all of the E-positive cells were also S positive or a K+S− cell may need to be selected to exclude the presence of anti-K.³⁹ The more specificities present, the more complicated the process, and where resources are limited, samples may need to be sent to a reference centre to elucidate all specificities. If the reagents are available, phenotyping the patient’s red cells early on in the process will allow exclusion of specificities for which the patient is antigen positive.

The specificity of an antibody should only be assigned when it is reactive with at least two examples of reagent red cells carrying the antigen and non-reactive with at least two examples of reagent red cells lacking the antigen. This is because a single positive reaction could occur if the panel cell unexpectedly expressed a low-frequency antigen and a single negative reaction could occur if the panel cell lacked a high-frequency antigen.

Phenotyping

When an antibody has been identified, the patient’s own red cells should be phenotyped for the relevant antigen. If the patient’s red cells are negative for the antigen, this confirms that the patient is capable of making an antibody of that particular specificity. If the patient’s red cells are positive for the antigen this suggests one of the following:

1. The antibody is an autoantibody, in which case the direct antiglobulin test should be positive.

2. The patient has been transfused recently and the reagent is detecting transfused cells rather than the patient’s own cells. Care should be taken to look for a mixed-field reaction. Evidence of a delayed transfusion reaction should be sought.

3. The initial antibody identification result is incorrect.

4. The patient’s red cells are immunoglobulin-coated (positive DAT). Care should be taken, particularly when using reagents potentiated with PEG, to recognize this to prevent reporting a false-positive result.

Extended phenotyping can be helpful where a mixture of antibodies is present because it allows the exclusion of antibodies specific for antigens for which the patient is positive, reducing the number of additional reagent cells required.

Additional Panels/Techniques

The chances of identifying antibodies, where two specificities are present, are significantly improved by using a two-stage enzyme technique, where at least one of the relevant antigens is affected by enzymes.²⁷ For example, Rh antibodies are enhanced by proteolytic enzymes in routine use, whereas M, N, S, Fy^a and Fy^b antigens are destroyed. Similarly, two different panels of reagent cells provide an increased chance of excluding further specificities where a mixture of two antibodies has been identified.

Direct agglutination at room temperature or 4°C may be helpful to distinguish between an antibody of potential clinical significance and a cold reacting antibody; again this is particularly useful where a mixture of antibodies is present. Weak examples of Kidd antibodies are often enhanced by the use of an indirect antiglobulin test using enzyme-treated cells and this can be particularly helpful where the antibody is only reacting against homozygous cells by indirect antiglobulin test.

Reagents

An identification panel should consist of red cells from at least eight group O donors, although 10 is more common in commercial panels and allows easier elucidation of antibody mixtures. To be functional, the panel must permit confident identification of the most commonly encountered, clinically significant antibodies. The UK guidelines ¹¹ can be summarized as follows:

1. For each of the commonly encountered clinically significant red cells antibodies, there should be at least two examples of phenotypes lacking, and at least two examples of phenotypes carrying, the corresponding antigen.

2. There should be at least one example of each of the phenotypes R₁R₁ and R₁^wR₁. Between them, these two samples should express the antigens K, k, Fy^a, Fy^b, Jk^a, Jk^b, M, N, S and s.

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.

4. The following phenotypes should be expressed in those samples lacking both D and C antigens: K+, K−, Jk(a+b−), Jk(a−b+), S+s−, S−s+, Fy(a+b−) and Fy(a−b+). The panel should be able to resolve as many likely antibody mixtures as possible.

Antibody Cards

Delayed haemolytic transfusion reactions can occur when antibodies have not been detected in the current antibody screen or have been incorrectly identified, maybe by a different establishment.^1,² It has been suggested that antibody cards, produced either by the hospital or the reference centre, could be carried by the patient for presentation on admission to hospital. To be effective, such cards have to be accompanied by patient information leaflets, explaining the significance of the antibodies and preferably handed personally to the patient by someone with a clear understanding of blood transfusion practice. There are potential pitfalls with this suggestion, not least that the level of proficiency in identifying antibodies and appreciating clinical significance varies between establishments. A better long-term approach may be to have a national database of patients who have been identified to have clinically significant antibodies.

Selection and transfusion of red cells

Once the blood group of a patient has been established and any antibodies have been identified, a set of procedures is required to select units of red cells that are appropriate for transfusion. These include selecting ABO/D compatible units, which will also need to be negative for antigens to which the recipient has clinically significant red cell alloantibodies (Table 22.5); It is also important that consideration is given to certain clinical criteria dictating special requirements (e.g. the requirement for CMV-negative, irradiated or washed red cells). Selection according to these criteria depends on good clinical information, accurate ABO and D typing and a sensitive antibody screen.

Table 22.5 Recommendations for the selection of blood for a patient with red cell antibodies

System	Antibody	Recommendation
ABO	Anti-A₁	IAT crossmatch compatible at 37°C
Rh	Anti-D, -C, -c, -E, -e	Antigen negative*
Rh	Anti-C^w	IAT crossmatch compatible^†
Kell	Anti-K, -k	Antigen negative*
Kell	Anti-Kp^a	IAT crossmatch compatible^†
Kidd	Anti-Jk^a, -Jk^b	Antigen negative*
MNS	Anti-M (active at 37°C)	Antigen negative*
MNS	Anti-M (not active at 37°C)	IAT crossmatch compatible at 37°C
MNS	Anti-N	IAT crossmatch compatible at 37°C
MNS	Anti-S, -s, -U	Antigen negative*
Duffy	Anti-Fy^a, -Fy^b	Antigen negative*
P	Anti-P₁	IAT crossmatch compatible at 37°
Lewis	Anti-Le^a, -Le^b, Le^a+b	IAT crossmatch compatible at 37°C
Lutheran	Anti-Lu^a	IAT crossmatch compatible at 37°C
Diego	Anti-Wr^a (anti-Di3)	IAT crossmatch compatible^†
H	Anti-HI (in A₁ and A₁B patients)	IAT crossmatch compatible at 37°C
All	Other active by IAT at 37°C	Seek advice from blood centre

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.

Some patients are identified as definitely needing transfusion of blood, but others may be undergoing surgery where blood is crossmatched to ‘stand by’. Audits have shown that blood is often crossmatched but not transfused. The transfusion ratio, or crossmatch index, can be used to assess how well blood stocks are managed. One option is to perform a blood group and antibody screen on a patient and then save the sample, only crossmatching blood if certain pre-agreed transfusion triggers are met. Local policy should define a maximum surgical blood ordering schedule, indicating which surgical procedures can be ‘group and screen/group and save’ and how many units of blood need to be crossmatched for procedures with a high likelihood of intraoperative transfusion.⁴¹ The decision as to whether blood is crossmatched in advance may relate to local factors including the proximity of the blood transfusion laboratory to the operating theatre (or other location where blood is to be transfused) and the time it takes to provide compatible blood following a request.

Once the appropriate red cells have been selected, compatibility needs to be ensured. This is usually referred to as crossmatching, which may include an indirect antiglobulin test to check for incompatibility as a result of IgG antibodies and ABO antibodies or an immediate spin test to check for ABO incompatibility only. Instead of a serological test, an assessment of ABO compatibility may be based on a ‘computer check,’ usually referred to as ‘electronic issue’. Some transfusion laboratories, where surgery takes place in a different hospital, have extended electronic issue to the selection of compatible units, as dictated by the transfusion laboratory, from a remote issue refrigerator.⁴²

The process of crossmatching blood is to prevent the transfusion of incompatible red cells and a subsequent haemolytic transfusion reaction. The different types of crossmatch are outlined in the following paragraphs. Whichever crossmatch technique is chosen, it should be clear that all patients with known red cell antibodies of potential clinical significance, even if currently undetectable, should have an indirect antiglobulin crossmatch and are not suitable for electronic issue.^11,¹²

Crossmatching

Choice of Test

Indirect Antiglobulin Crossmatch

The arguments for retaining an indirect antiglobulin crossmatch are based on the failure to identify antibodies against low-frequency antigens in the antibody screen. However, there is evidence, summarized by Garratty,³¹ that the likelihood of missing a clinically significant antibody is approx. 1 in 10 615 crossmatches if the indirect antiglobulin test component is omitted and antibody detection relies on a sensitive antibody screen alone. Moreover, the usual outcome of transfusion if an antibody is present but undetected by the antibody screen is limited to shortened red cell survival.

An indirect antiglobulin crossmatch should always be performed if the patient has red cell antibodies of likely clinical significance, even if currently undetectable. The reasons for this are as follows:

1. It acts as a double check that the donation has been correctly phenotyped and labelled as negative for the corresponding antigen(s)

2. It ensures serological compatibility even if the identification of the antibody(ies) is incorrect or incomplete

3. It allows detection of antibodies to low-frequency antigens not present on the screening cells, which may be more likely to be present in a patient who is clearly a ‘responder’ and which may be masked by other alloantibodies.

Other circumstances in which an indirect antiglobulin crossmatch should be performed include the following:

1. 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 antibodies that may be produced by passenger lymphocytes in the transplanted organ.⁴³

2. The patient has an alloantibody of low clinical significance detectable in the routine antibody screen such as anti-C^w.

3. The patient has known cold antibodies. In this case blood may be issued if compatible in the antiglobulin crossmatch performed strictly at 37°C, without the need for antigen-negative blood.

Methods for indirect antiglobulin crossmatching are the same as those used for antibody screening. Crossmatching may be less effective than antibody screening at detecting incompatibility as a result of IgG antibodies. This is partly because the cells may only show heterozygous expression of an antigen to which the patient has an antibody, potentially leading to weaker or negative reactions, and also because the cell suspensions and other techniques (e.g. cutting pigtails from donations, labelling tubes, washing cells) are less likely to be standardized and present more opportunity for transposition errors than the screening processes.

Immediate Spin Crossmatch

The sole purpose of this technique is to detect ABO incompatibility. It can be used in order to issue blood for transfusion when the patient has a full blood group and negative antibody screen. It may be used to convert a group and save/screen when blood is required and is often used in addition to an antiglobulin crossmatch. The immediate spin crossmatch will ensure that the correct units have been selected and that the correct ABO group has been assigned to the unit of donor red cells. Clearly it is not a suitable test to use for detection of ABO incompatibility if the reverse blood group reveals a very weak anti-A or anti-B or if the patient falls into one of the categories described in the previous section.

There is evidence of poor standardization of this technique,⁴⁴ but its sensitivity can be optimized by selecting the appropriate cell suspension, incubation time and serum: cell ratio. The following tube method is recommended:

Mix 2 volumes of plasma with 1 volume of 2–3% cells suspended in PBS or LISS (or EDTA saline if serum is used).

Incubate at room temperature for 2–5 min to enhance the detection of weak ABO antibodies.

Centrifuge at 100 g for 1 min.

Read the reaction carefully using a ‘tip and roll’ technique.

False-negative results (in immediate spin crossmatch)

Incompatibilities between A₂B donor cells and group B patient sera are not consistently detected with this technique.⁴⁵ Of more concern is the potential failure of agglutination with potent ABO antibodies^46,⁴⁷ on account of rapid complement fixation with bound C1 interfering with agglutination if using serum. Red blood cells must therefore be suspended in saline containing EDTA.

False-positive results (in immediate spin crossmatch)

Cold reacting antibodies, other than anti-A and anti-B, may cause agglutination in an immediate spin crossmatch. This has the potential to cause delays to transfusion while further procedures are used to rule out ABO incompatibility.

Electronic Issue

In many countries electronic issue is a commonly used alternative to the immediate spin crossmatch for issuing blood for transfusion when the patient has had a full blood group, a current negative antibody screen and no history of clinically significant antibodies. ABO and D compatible units are selected and issued through a computer system, which contains logic rules to prevent the issue of ABO and D incompatible blood.

BCSH guidelines ¹² and AABB standards⁴⁸ require that there be concordant determinations of the patient’s ABO and D type either on two separate samples, at least one of which must be a current sample or on one current sample tested twice, depending on the security present in the system. In addition, there must be no clinically significant antibodies detected and no record of any having been detected previously. The AABB recommends that the group of donor units is rechecked, but the BCSH accepts written verification by the supplying transfusion service of the accuracy of the donor unit label. It is strongly recommended by the BCSH that ABO and D grouping procedures are automated with positive sample identification (e.g. bar-codes) and electronic transfer of results from the analyser to the transfusion laboratory computer, whereas the AABB does not have such a requirement. Ideally, computer algorithms should direct the procedure, only allowing issue if all the criteria are fulfilled. For example, issue of red cells for transfusion will be prevented if only one ABO and D group is on file or if the previous and current groups do not agree. Any manually controlled part of this process increases the risk of error. Electronic issue, based on fully validated systems, has been in place in several countries for some time and has proved to be clinically safe, providing the recommendations are rigidly adhered to.⁴⁹^–⁵¹

Emergency blood issue

In clinical emergencies in which immediate red cell support is required, there may not be time for full compatibility testing. Either abbreviated testing is employed and rapid techniques are used or group O red cells are issued. There must be a documented procedure for dealing with emergencies. Local policies on this should be formally assessed for risk and adequate training should be given to staff, particularly those providing the service out of routine laboratory hours. Out-of-hours staff should be included in internal proficiency testing as well as external quality assessment exercises designed to test emergency tests and techniques.^3,¹¹

The transfusion laboratory should be involved in the development of emergency procedures within the hospital, including massive haemorrhage protocols and the major accident plan. All staff should receive regular training and should take part in emergency practice drills. Clear and effective communication is key to the success of provision of blood components in an emergency and the transfusion laboratory needs to be fully informed about the current status of the patient or patients, so as to provide an efficient and timely service.

Rapid ABO and D Typing

In an emergency, a sample should be obtained prior to transfusion and the patient’s ABO and D type should be determined as rapidly as possible using the techniques already described. Tube and slide tests are most convenient.

Confirmation

A reverse group, a repeat cell group or an immediate spin crossmatch should be carried out on the sample before issuing blood of an appropriate ABO group. The ABO and D group must always be confirmed by a further test on a second aliquot from the sample. If an inadequately labelled sample is provided, group O units should be issued until a further sample can be tested.

Selection of Units

Some hospitals provide one or two units of O D negative red cells for use by clinicians pending the availability of ABO and D specific compatible red cells. Because of the relatively short supply of O D negative red cells (only 8% of Caucasian blood donors are O D negative in the UK) the National Blood Transfusion Committee has issued guidance to hospitals about the restrictions on the use of O D negative red cells in patients of another blood group.⁵²

Compatibility Testing

Group-specific red cells can be issued following an immediate spin crossmatch to check for ABO incompatibility or a LISS antiglobulin crossmatch and antibody screen can be done if more time is available. If no matching procedure is performed, the red cell units must be group-checked unless the supplier has indicated confidence in the validity of the donor unit labelling. Units issued following abbreviated testing must be labelled as such (e.g. ‘Selected for patient … but not crossmatched’). Cells from the donor units should be removed before issuing the units to enable retrospective crossmatch although a crossmatch is only necessary if the antibody screen is subsequently found to be positive.

Antibody Screening

Good practice requires a simultaneous crossmatch and antibody identification if the antibody screen is positive. If group-specific units have been issued without an indirect antiglobulin test crossmatch, an antibody screen must be performed as soon as possible. If the antibody screen is negative, it is not necessary to carry out a retrospective crossmatch. It is not acceptable to perform a crossmatch and not carry out an antibody screen. If the antibody screen is found to be positive, then contact the clinicians responsible for the patient to discuss how to proceed regarding the issue of potentially incompatible blood. Always follow agreed local procedure. Any untransfused incompatible units should be withdrawn from issue as soon as possible.

Massive Transfusion

Massive transfusion is defined as more than one blood volume within 24 h and is usually taken to mean 8 or 10 units for an adult.⁵³ After this volume of transfusion, it is no longer necessary to undertake an antiglobulin crossmatch, but immediate spin crossmatch or electronic issue is recommended to check for ABO compatibility. If ABO non-identical blood is given initially, blood of the same group as the patient should be used as soon as possible after the first transfusion. In some situations the recipient’s need for red cell transfusion may necessitate the use of incompatible units, but this is a clinical decision based on the need for blood balanced against the known clinical significance of the red cell antibody detected.

Selection of Platelets and Plasma

In patients with hypotensive shock and/or large volume red cell replacement, a coagulopathy develops that requires replacement of other blood components. The use of platelets, fresh frozen plasma and cryoprecipitate may initially be determined by clinical evaluation of the patient’s haemostatic state including observation of diffuse microvascular bleeding. Massive haemorrhage protocols should be developed to enable release of these coagulation factors at the request of the clinicians, in advance of the results of laboratory tests. Ongoing management of the patient can be guided by the results of these initial full blood count and coagulation screen tests, combined with frequent re-evaluation of both the laboratory tests and clinical evaluation of the haemostatic state.⁵³

Potential Errors

Errors leading to transfusion of incorrect blood components are more likely to occur out of routine laboratory hours; therefore it has been recommended that only genuine emergency transfusions should take place out of hours.^1,² There may, however, be circumstances where patients with haemoglobinopathies receiving regular transfusion support are given blood overnight to minimize disruption of their education or employment.

Antenatal serology and haemolytic disease of the newborn

Maternal ABO and D grouping and red cell antibody screening must be done early in pregnancy as a routine. This is the basis for the prevention, detection and, with antibody titration or quantification, the management of haemolytic disease of the fetus and newborn (HDFN).

Haemolytic Disease of the Fetus and Newborn

Haemolytic disease of the fetus and newborn is a haemolytic anaemia of the fetus and/or newborn infant that occurs when maternal alloantibody to fetal antigens crosses the placenta and causes haemolysis of fetal red cells or suppression of fetal red cell progenitors, the latter occurring with antibodies within the Kell system.^54,⁵⁵

As IgG is the only immunoglobulin that crosses the placenta, only red cell antibodies of this class are a potential cause of HDFN. Anti-D causes the most severe form of HDFN, but the success of prophylaxis with anti-D immunoglobulin for potentially sensitizing events in pregnancy and after delivery of a D positive baby has reduced the number of cases and routine antenatal anti-D prophylaxis (RAADP) has reduced it even further. The relative proportion of HDFN due to other IgG red cell antibodies has thus increased.⁵⁶ Although HDFN resulting from anti-D is the most severe form of the disease, anti-c can give rise to significant haemolysis in utero, sufficient in some cases to result in intrauterine death and therefore to warrant intervention in pregnancy. Anti-K has a different mode of action^54,⁵⁵ but can also result in a severely affected fetus. Other IgG antibodies (e.g. anti-E, anti-Ce, anti-Fy^a and anti -Jk^a) uncommonly give rise to fetal haemolysis of sufficient severity to merit antenatal intervention. Haemolytic disease of the newborn (HDN) as a result of ABO antibodies can also occur and is described later. For a detailed discussion of the investigation and management of HDFN, the reader is referred to the reviews by Kumar and O’Brien,⁵⁷ Bowman⁵⁸ and the textbook Mollison’s Blood Transfusion in Clinical Medicine.³⁰

Antenatal Serology

ABO and D Grouping and Antibody Screening

Maternal ABO and D grouping as well as antibody screening and identification are performed early in pregnancy (i.e. when first seen and ‘booked in’) and then at 28 weeks’ gestation. All pregnant women, whether D positive or D negative, should be screened for red cell antibodies.⁵⁹ Further testing depends on the specificity of any antibodies detected, whether they are capable of causing HDFN and the obstetric history.

Follow-Up Antibody Screening

Protocols for antenatal screening and follow-up vary from country to country. In the United Kingdom, the following is recommended by the BCSH:⁵⁹

1. All pregnant women have ABO, D and antibody screen in early pregnancy when ‘booked-in’ and at 28 weeks’ gestation. If the antibody screen at 28 weeks is negative, no further routine testing is required.

2. Pregnant women with anti-D, antibodies to Kell-related antigens and anti-c should be tested monthly to 28 weeks and then every 2 weeks to delivery. The tests should include antibody quantification or titration as well as testing for additional red cell antibodies.

3. Pregnant women with other red cell antibodies should have a titration done when booked in and again at 28 weeks.

4. All pregnant women with a previous history of HDFN or those who have a significant increase in anti-D, antibodies to Kell-related antigens or anti-c should be referred to a specialist fetal medicine unit for further assessment of the need for antenatal intervention.

5. Pregnant women who have red cell antibodies of other specificities, capable of giving rise to HDFN and which demonstrate a significant increase in titre over the course of the pregnancy, should have their condition discussed with their obstetrician. It is now appreciated that an increasing titre rather than an individual level is more predictive of an affected fetus.⁶⁰

Prediction of Fetal Blood Group

Partner Testing

The father’s blood group phenotype should be determined in all cases in which the mother has a clinically significant red cell alloantibody. If the father’s red cells lack the corresponding antigen, the baby is not at risk. However, caution is advised because the assumed father may not be the biological father of the fetus. It is useful to predict whether the partner of a woman who is D negative and who has immune anti-D is homozygous or heterozygous for the D antigen. This helps to forecast the chances of having children affected by anti-D HDFN.

The zygosity of the D antigen is usually predicted from the results of tests with anti-c, anti-C, anti-e and anti-E and from the likelihood of the homozygous or heterozygous association with these antigens (Table 22.6, see also Table 22.3). These data have been compiled for different racial groups. It is important, therefore, to tell the specialist laboratory the ethnic origin of the patient. Because the genetic basis for the common D types is now known, DNA typing provides a better alternative for predicting the potential for haemolytic disease of the newborn.⁶¹

Table 22.6 The possible genotypes and frequencies in an English population (accounting for >99% of all Rh genotypes in this population)⁶¹

Testing Fetal DNA in the Maternal Circulation

It is now possible to detect fetal DNA in the maternal circulation and, using DNA amplification techniques (see Chapter 8), to obtain D, c, E and K types on these cells. This has proved to be accurate at predicting the D type and, in the UK, it is now offered as a clinical service at the beginning of the 2nd trimester.⁶² This may replace more invasive tests and supplement partner typing. It can be especially helpful if the father is absent or unknown.

Fetal Blood Sampling

Using ultrasound guidance, it is possible to take a sample of fetal blood for blood grouping, but this carries some risks. Contamination by maternal blood can hinder analysis of the sample obtained, leading to false-negative results. In addition, the procedure itself can lead to fetomaternal haemorrhage (FMH) and hence further sensitization to fetal antigens. There is also a risk of miscarriage.⁵⁷

Antenatal Assessment of the Severity of Haemolytic Disease of the Fetus and Newborn

There has been considerable change in antenatal assessment of the severity of haemolytic disease of the newborn with non-invasive techniques being routinely used to assess the degree of fetal anaemia, with fetal blood sampling and, if necessary, intrauterine transfusion only being considered in cases suspected of having severe anaemia but before development of hydropic changes on the ultrasound scan.

Antibody Titrations during Pregnancy

Techniques for antibody titration are described in Chapter 21, but these have variable reproducibility and sensitivity. In many laboratories tube techniques have been replaced by column agglutination technology. Figure 22.7 shows the range of reaction grades used in titrations. The role of the serologist is to carry out serial antibody measurements to determine changes in the titre or concentration of the antibody. It is recommended that the technique chosen for titration should be validated against the National Institute for Biological Standards and Control anti-D standard.⁵⁹ Hence, laboratories should ensure that titres obtained with the anti-D standard are within one doubling dilution when it is used as an internal control. In addition, antibody titrations performed in pregnancy should be performed in parallel with the previous sample. Increases in titres of more than one doubling dilution should be monitored in conjunction with obstetricians.

Figure 22.7 Column agglutination technology showing reaction grades 4+ to 0. Box 1, 4+. Box 2, 3+. Box 3, 2+. Box 4, 1+. Box 5, 0. Box 6, blank.

Antibody Quantitation

Individual hospital transfusion laboratories should work closely with reference laboratories and obstetricians. Automated quantification is considered to be a more accurate predictor of when to proceed to more active investigation of the fetus but is usually only available for anti-D and anti-c. Results in iu (or μg per ml) are used as part of clinical algorithms to proceed to the next step of fetal investigation.⁵⁷

Assessment of Fetal Anaemia

In a mother with increasing antibody levels and a fetus suspected or known to carry the red cell antigen against which the antibody is directed, an assessment of the severity of haemolysis is required. Traditionally this was done using amniocentesis to measure the optical density of the amniotic fluid (Lilley’s lines) using spectrophotometry.

This, however, is an indirect measurement, whereas direct fetal blood sampling by ultrasound-guided cordocentesis provides not only direct diagnostic information but also a new approach to fetal therapy by direct fetal intravascular transfusion. However, both of these procedures carry the risk of miscarriage and further fetomaternal haemorrhage.⁵⁷ It is now common practice for fetal medicine units to offer non-invasive tests to determine fetal anaemia; middle cerebral artery Doppler studies have been very useful in this regard.⁶³ The incidence and severity of HDFN is declining and the increasingly specialized management of severely affected pregnancies has meant that these women are now being referred early in pregnancy to fetal medicine units who specialize in dealing with this condition, thus decreasing the involvement of the routine transfusion laboratory in any but the early stages.

Prevention of Haemolytic Disease of the Fetus and Newborn as a Result of Anti-D

Correct identification of women in early pregnancy who are D negative offers the chance to give intramuscular anti-D immunoglobulin to prevent sensitization to the D antigen at times during the pregnancy when significant fetomaternal haemorrhage is likely to occur, known as ‘potentially sensitizing events’ (PSE).^64,⁶⁵ Accuracy in D grouping is particularly important because women who are D negative, erroneously grouped as D positive, risk not receiving prophylactic anti-D immunoglobulin (or being transfused with D positive cells). Sensitization to the D antigen could then result in severe HDFN in subsequent pregnancies as a result of development of anti-D. Serious Hazards of Transfusion (SHOT) has a reporting category for anti-D errors which includes any adverse event relating to the prescription, administration or omission of anti-D immunoglobulin that has the potential to cause harm to the mother or fetus immediately or in the future (Fig. 22.8).²

Figure 22.8 SHOT adverse events to anti-D immunoglobulin 2009.²

Anti-D Prophylaxis

Anti-D immunoglobulin should be given routinely as soon as possible after delivery (but always within 72 h) to women who are D negative who deliver babies that are D positive. It should also be given at times during pregnancy when sensitization could occur, such as during medical or surgical therapeutic termination of pregnancy, chorionic villus sampling, amniocentesis and following any abdominal trauma. It should also be given for episodes of vaginal bleeding where the pregnancy remains viable.^64,⁶⁵ At delivery and for potentially sensitizing events after 20 weeks’ gestation, it is necessary to screen for fetomaternal haemorrhage (FMH) using an acid elution method and estimate the degree of FMH if fetal cells are seen. The BCSH guidelines recommend confirming any FMH >2 ml by flow cytometry so that additional anti-D immunoglobulin can be given if the standard dose in use does not cover the estimated bleed.⁶⁶

It takes 125 iu of anti-D immunoglobulin to cover a bleed of 1.0 ml fetal cells and preparations containing 250 iu, 500 iu, 1250 iu and 1500 iu are in routine prophylactic use.

Because of the risk of silent fetomaternal haemorrhage in pregnancy, routine antenatal anti-D prophylaxis (RAADP) is being offered to women in some countries. In the UK, this has been the subject of an appraisal by the National Institute for Health and Clinical Excellence ⁶⁷ which recommends that anti-D immunoglobulin should be given either as two doses at 28 and 34 weeks or a single larger dose at 28 weeks, in addition to the postnatal dose and doses to cover any potentially sensitizing events during pregnancy. Women can decline RAADP, e.g. if they know the father is D negative or if they do not want any further pregnancies. The typing of fetal DNA in the maternal circulation may be used in the future to select women with fetuses that are D positive who would benefit from this additional prophylaxis, but, at the moment, it is not universally offered.⁶⁸

The administration of prophylactic anti-D results in a positive maternal antibody screen due to passive anti-D. It may be difficult to distinguish between passive anti-D and low-level immune anti-D, particularly in the absence of a history of anti-D administration (this may have taken place at another institution). It is therefore important to take the 28-week sample for blood group and antibody screen before administration of anti-D. Although further antibody screens are not routinely required,⁵⁹ events in the 3rd trimester may result in a sample being sent to the transfusion laboratory. Laboratories need to have a strategy for dealing with these samples. Some implement screening against rr screening cells, whereas others proceed to full antibody identification on all samples.

Measurement of Fetomaternal Haemorrhage

The following tests are performed to estimate the quantity of fetal cells in the maternal circulation by the difference between fetal and maternal cells. Most commonly used is acid elution, also known as the Kleihauer test, which depends on the Hb F in fetal cells resisting the acid elution to a greater extent than the Hb A in maternal cells. The calculation of the volume of fetal cells is based on the work by Mollison,³⁰ which assumed that the maternal red cell volume is 1800 ml, fetal red cells are 22% larger than maternal cells and only 92% of fetal cells stain darkly (p. 338). Mollison’s formula for calculating volume of fetomaternal haemorrhage is as follows:

Corrected for staining efficiency (1.09):

Volume of fetomaternal haemorrhage (ml fetal cells) = J × 1.09.

Occasionally, the acid elution (or Kleihauer) test is used to investigate an intrauterine death or stillbirth where a large but silent fetomaternal haemorrhage is suspected as the cause of death. The D group of the mother is known but often not that of the fetus. In these circumstances, acid elution is the preferred test. Anti-D prophylaxis should be given to women who are D negative if the fetus is D positive or the D type of the fetus is unknown.

The flow cytometry method uses a fluorochrome-labelled anti-D antibody to measure a minority of D positive cells in the maternal D negative blood and is recommended for confirmation of a positive acid elution test where the estimated FMH exceeds 2 ml. However, flow cytometry may not always be available. Flow cytometric techniques using anti-Hb F have also been developed. The BCSH guidelines ⁶⁶ give full details on performance and use of all these tests.

Recommended Action at Delivery (or Potentially Sensitizing Event)

All women who are D negative should be given a standard intramuscular dose (minimum of 500 iu after 20 weeks and 250 iu before 20 weeks’ gestation) (into the deltoid muscle) of anti-D within 72 h of delivery (or potentially sensitizing event) unless the baby (or fetus) is known to be D negative.

On the basis of a confirmed FMH result, further anti-D immunoglobulin should be given if the FMH exceeds the volume covered by the standard anti-D immunoglobulin (Fig. 22.9). If the fetomaternal haemorrhage is more than 2 ml, the maternal sample, if possible, should be retested by a second technique such as flow cytometry; alternatively, the test should repeated on the same sample by a different operator.

Figure 22.9 Dosage of anti-D immunoglobulin to cover calculated fetomaternal haemorrhage.⁶⁵

A repeat sample for Kleihauer should be tested at 72 h (48 h if the anti-D immunoglobulin was given intravenously) to check for clearance of fetal cells. Further anti-D immunoglobulin may be required if fetal cells are present.

It is good practice to counsel women with a large fetomaternal haemorrhage about the risk of sensitization and an antibody screen 6-months postpartum can be arranged to see if sensitization to the D antigen has occurred although a negative antibody screen at this stage does not completely exclude sensitization.

ABO Haemolytic Disease of the Newborn

ABO incompatibility as a result of high-titre maternal IgG anti-A and anti-B antibodies can cause prolonged neonatal jaundice and anaemia associated with spherocytosis on the blood film in babies that are group A or B born to mothers that are group O. It should be distinguished from non-spherocytic haemolysis and from haemolytic disease of the newborn resulting from other red cell antibodies. Although confirmatory tests are best carried out on cord blood samples, it is often the case that babies have been discharged from hospital before the problem is detected. In Caucasian populations, about 15% of births are susceptible, but only about 1% are affected; even then the condition is mild and very rarely severe enough to need exchange transfusion. The condition is more common in other racial groups. A number of special factors combine to protect the fetus from the effects of ABO incompatibility. These include the relative weakness of A and B antigens on the fetal red cells and the widespread distribution of A and B glycoproteins in fetal fluids and tissues, which divert much of the maternal IgG antibody away from the fetal red cell ‘target’. ABO haemolytic disease of the newborn may occur in a first pregnancy. Antenatal prediction of ABO haemolytic disease of the newborn is not essential for medical management because there is time to observe the baby after birth and treat according to the severity of the condition.

Serological Investigation

ABO haemolytic disease is difficult to diagnose, especially in Caucasians, because the direct antiglobulin test may be negative or weak even in a case of severe haemolytic disease. Furthermore, anti-A or anti-B is present in the mother’s serum and special tests may be required to demonstrate high-titre IgG antibodies in the presence of IgM antibodies with the same specificity. The following are helpful pointers to diagnosis when ABO haemolytic disease of the newborn is suspected:

1. It is almost always confined to mothers who are group O because there are higher titres of IgG anti-A and anti-B in group O than in group A or B.

2. Because anti-A and anti-B are always present in mothers who are group O, evidence for ABO haemolytic disease of the newborn depends on demonstrating a high titre of IgG anti-A or anti-B by treating the mother’s plasma to remove the IgM antibodies (p. 289) and then testing by the antiglobulin technique against adult A₁, B and O cells.

3. The direct antiglobulin test on the cord blood or baby’s sample may be weak or negative; the latter at least reduces the likelihood of any other serological incompatibility.

4. The simplest evidence for the occurrence of ABO haemolytic disease of the newborn is obtained by testing plasma from the cord blood or baby’s sample for anti-A or anti-B by the antiglobulin technique against adult A₁, B and O cells. The sooner after birth these tests are done, the better. Delays will lead to absorption of the antibody and the destruction of the red cells.

5. The best diagnostic test of ABO haemolytic disease of the newborn is to prepare an eluate from the baby’s red cells and test it (together with the last wash supernatant as a control) by the antiglobulin test against adult A₁, B and O cells. In some cases reactions occur with both A₁ and B cells because of the presence of anti-A,B cross-reacting antibodies, although most severe cases of ABO haemolytic disease of the newborn contain separate specific anti-A and anti-B antibodies. The test with cells and the last wash control should be negative.

Compatibility testing in special transfusion situations

Neonates and Infants within First 4 Months of Life

Infants younger than 4 months of age do not generally make alloantibodies but may have passively acquired maternal antibodies (see sections on haemolytic disease of the newborn and ABO and other red cell antigens).

Investigations on the Maternal Sample

ABO and D group

Antibody screen.

Investigations on the Infant Sample

ABO and D group (cell group only), repeated on the same sample if no historical group

Antibody screen if mother’s sample not available

Direct antiglobulin test.

If the direct antiglobulin test is positive or any red cell antibodies are detected in the maternal or infant serum, the diagnosis of haemolytic disease of the newborn should be considered (see above).

Selection of Blood and Other Components

Where possible, the cellular component chosen should be ABO and D identical or an alternative compatible ABO group, taking into account the infant’s blood group and maternal blood group (i.e. presence of maternal anti-A and/or anti-B) (Table 22.7). Care should be taken when giving components containing group O plasma (containing anti-A and anti-B) to infants that are not group O. Despite high-titre anti-A and anti-B donors being excluded from donating components for this group, there have been reports of haemolysis of group A infants’ red cells when given group O platelets.^1,² Current production methods for fresh frozen plasma result in minimal red cell stroma which is not antigenic; therefore plasma components do not need to be D compatible. If the maternal and infant antibody screens are negative (including absent maternal IgG anti-A in the infant), ABO- and D-typed blood can be given with no serological crossmatch, even after repeated small volume transfusions, because formation of red cell antibodies is rare in infants younger than 4 months old. Repeat antibody screen is not necessary if repeat transfusions are required within the first 4 months of life.⁶⁹ In the case of haemolytic disease of the newborn (non-ABO or ABO) or maternal red cell antibodies, a crossmatch is required against maternal plasma using group O blood (which has been tested and shown to have low titres of anti-A and anti-B or ‘HT negative’) or other group compatible with red cell antibodies in the maternal serum. For neonates and infants, red cells for exchange transfusion should be <5 days old, negative for CMV antibodies, plasma reduced (or washed), Hb S negative, leucodepleted, K negative and irradiated.

Table 22.7 Choice of ABO group for blood components for administration to neonates and infants younger than age 4 months ⁶⁹

Intrauterine (Fetal) Transfusion

Intrauterine (fetal) transfusion is usually carried out in a fetal medicine unit where local protocols should be in place. It is important for maternity units and associated neonatal units to have information about previous intrauterine transfusion because it may influence the selection of appropriate blood components if transfusion is required after delivery (see above). This is particularly important when the clinical care takes place in different institutions which are serviced by different transfusion departments. Shared-care protocols are one way of ensuring effective communication. When providing red cells for intrauterine transfusion, an antiglobulin crossmatch should be performed using maternal plasma and this must be repeated with a fresh maternal sample with every transfusion. Intrauterine transfusion can result in fetomaternal haemorrhage and hence sensitization to new antigens, so antibody identification and quantification or titration must be performed on all maternal samples. Red cells selected should be group O D negative (except where mother has made anti-c, when it will be necessary to give D positive, c-negative blood) and K negative. Further selection of phenotyped blood will depend on the maternal red cell antibody profile. In addition to the stipulations listed earlier for selection of blood for infants and neonates, blood for intrauterine transfusions should also be leucodepleted, K negative and irradiated to prevent transfusion-associated graft-versus-host disease and should be transfused within 24 h of irradiation.⁶⁹

Patients Receiving Transfusions at Close Intervals

Patients who are acutely ill, particularly those on intensive care units and those undergoing intensive chemotherapy for haematological malignancy, may require frequent blood transfusions. This section does not include transfusions to neonates and infants within the first 4 months of life (see above) and other special considerations are necessary for recipients of a stem cell transplant (see below).

Alloantibodies can develop quickly following a transfusion. Therefore, a sample should be obtained for compatibility testing before each transfusion if they are separated by an interval of 3 days or more. There is no need for daily samples, but an antibody screen at least every 72 h is recommended as being practical and safe.

Chronic Transfusion Programmes

Examples of patients in whom a decision has been made that regular transfusions are required include those with β thalassaemia major, some patients with sickle cell anaemia and congenital or acquired bone marrow failure. It is important to establish a treatment plan for each patient with clear triggers for transfusion and regular checks for the adverse effects of transfusion, including iron overload. The risk of developing alloantibodies to red cell antigens on transfused blood influences the timing of blood samples (Table 22.1). If a less rigorous approach is taken for patients in whom repeated transfusions have not led to alloantibody formation, a mutual decision should be made by the clinician and the transfusion department after careful consideration of the risks.

A pre-transfusion Rh phenotype allows matching for D, C, c, E and e. Some ethnic groups commonly have the phenotype cDe (R_o) and D positive blood negative for C and E may be difficult to find, particularly if there are other red cell antibodies. In this situation D negative (cde/cde) blood is selected. Additionally, patients with haemoglobinopathies should have an extended red cell phenotype before they are first transfused to include the antigens K, Jk^a, Jk^b, Fy^a, Fy^b, M, N, S and s.^70,⁷¹ This may be used to select blood but is also helpful when investigating antibodies formed as a result of repeated transfusions. Although provision of red cells with a matched extended phenotype is undertaken by some units treating haemoglobinopathies,⁷² the degree of matching depends on local resources and should not impede the delivery of effective transfusion support. There is insufficient evidence to make this recommendation for other patients who have been chronically transfused.

Allogeneic Haemopoietic Stem Cell Transplantation

An allogeneic haemopoietic stem cell transplant may introduce a new blood group; an ABO antigen (major mismatch), ABO antibody (minor mismatch) or both. The recipient/donor pairs have different ABO and D groups in about 15–20% of sibling stem cell transplants and this is more common in unrelated donor stem cell transplants. If there is a major ABO mismatch and isoagglutinins persist in the recipient’s plasma, engraftment may result in a positive direct antiglobulin test when substantial numbers of donor red cells start to enter the circulation and some haemolysis may occur.

A minor ABO mismatch is when the donor has antibodies to the recipient’s red cells (e.g. donor O, recipient A). Transfuse red cells of the donor ABO group until the recipient’s own red cells are no longer detectable.

A major ABO mismatch is when the recipient has antibodies to the donor red cells (e.g. donor A, recipient O). Transfuse red cells of the patient’s own type until red cell antibodies are no longer detectable by the indirect antiglobulin test and the direct antiglobulin test is negative and then transfer to donor type.

A major plus minor mismatch is when there are antibodies to recipient and donor red cells (e.g. donor A, recipient B). Transfuse group O red cells until the recipient’s red cell antibodies are no longer detectable by the indirect antiglobulin test and the direct antiglobulin test is negative.

Prior to the transplant, recipient type red cells, platelets and fresh frozen plasma are given, but following engraftment, the choice of blood components depends on the ABO and D mismatch between the donor/recipient pairs.¹¹ For a recipient who is D positive with a donor who is D negative, D negative components should be used.

From the time of conditioning therapy, throughout the period of prophylaxis, all cellular blood components should be irradiated to prevent transfusion-associated graft versus host disease. The stem cell processing laboratory will ensure that products for infusion in the allogeneic setting do not contain significant numbers/amounts of red cells/plasma if there is the potential for a haemolytic transfusion reaction.

Investigation of a transfusion reaction

Adverse events related to transfusion can be acute (within 24 h) or delayed (Table 22.8). Transfusion laboratories should immediately be informed of a suspected transfusion reaction, being ideally placed to coordinate investigation, to communicate with clinicians and transfusion services and to advise about appropriate choice of blood components for subsequent transfusions. Serious adverse events should be reported confidentially to the national haemo-vigilance scheme, the local blood centre and the Hospital Transfusion Team. Acute transfusion reactions are easier to attribute to the transfusion than delayed reactions, although, in patients who are already very ill, they can go undiagnosed. The symptoms and signs of acute transfusion reactions are similar regardless of the cause so treatment and investigation of causes is, by necessity, simultaneous. It is easier to distinguish between the causes of delayed transfusion reactions, but it may be more difficult to recognize their relationship to the transfusion episode because of the delay in onset. The following scheme outlines the role of the laboratory in investigation and management of transfusion reactions and a very useful algorithm can be found in the Handbook of Transfusion Medicine.⁷³

Table 22.8 Types of transfusion reaction

Acute transfusion reactions	Delayed transfusion reactions
Acute haemolytic reaction	Delayed haemolytic reaction
Anaphylaxis	Transfusion transmitted infection
Bacterial contamination of blood component	Transfusion-associated graft-versus-host disease
Transfusion-associated acute lung injury	Post-transfusion purpura
Transfusion-associated circulatory overload	Iron overload
Allergic reaction	Immunosuppression
Febrile non-haemolytic transfusion reaction