Laboratory Aspects of Blood Transfusion

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, the introduction of 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. It is estimated that in the UK, 97% of routine group and screens are undertaken using full automation.

Column agglutination (CAT) and solid-phase technology can 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.

The currently available CAT systems include Ortho BioVue ( www.orthoclinical.com ), which comprises a six-well cassette containing a glass microbead matrix, ID Bio-Rad cards utilising Sephadex gel matrix, also with six wells, and an eight-well Sephadex gel matrix card from Grifols ( www.grifols.com ). Each offers a wide range of profiles and reagent systems. Other similar formulations produced by different commercial companies are available outside of the UK.

The currently available solid-phase systems are Immucor Capture-R ( www.immucor.com ) and Bio-Rad Solidscreen II ( www.bio-rad.com ). The Immucor system utilises a range of red cell antigens that have adhered to the surface of a U-shaped microplate well. Sensitised 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 so that the reaction results in a solid-phase cell layer bound to the plate.

Individual laboratories need to make careful and informed decisions when selecting reagents for pretransfusion 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.

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 IT systems in blood transfusion practice (2015) reflect this. 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 in 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 2014, 53% of laboratories were using this system for some or all of their patients ( Fig. 22-2 ). The use of automation for all aspects of compatibility testing is now recommended practice and recognised as safer than manual techniques. 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 pretransfusion testing (e.g. stopping duplicate D 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 pretransfusion compatibility procedures and guidance from the MHRA give a list of factors to be taken into consideration. The reader is advised to consult these prior to implementing automated or semiautomated systems.

Pretransfusion 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 handwritten on the sample label: the correctly spelled first name and last name, the exact date of birth and an accurate unique patient number (ideally the NHS number or equivalent, but a hospital medical records number is acceptable). The sample should be dated and signed by the person taking the sample. The laboratory should have a policy for rejecting badly labelled samples. In a UK national comparative audit of collection and labelling of samples sent for pretransfusion testing, 2.99% were rejected. The hospitals participating in this audit showed considerable variation in the content and application of sample acceptance policies. UK guidelines recommend that preprinted ‘addressograph’ labels are not accepted on blood samples for pretransfusion compatibility testing, because acceptance increases the risk of ‘wrong blood in tube’ (WBIT). In an international study from the Biomedical Excellence for Safer Transfusion (BEST) collaborative, it was estimated that these miscollected samples occurred at a median rate of 0.5 per 1000, with a great variation worldwide in the reported frequency of mislabelled samples, probably resulting from variation in sample acceptance policies. Labels printed at the bedside using barcoded patient wristband and hand-held scanners are acceptable. Unless secure electronic patient identification systems are in place, it is recommended that a second sample collected at a different time is taken for confirmation of the ABO group of a first-time patient prior to transfusion, as long as this does not impede the delivery of urgent red cells or other components.

• A request for testing or issue of blood components can be made electronically, in writing or exceptionally in an urgent situation, by phone. In addition to the previously outlined four core patient identifiers, the request should include clear information about the source of the request and the location of the patient. Clinical details should include the justification for the request, such as the indication for transfusion. Previous transfusion history and any special considerations for the selection of blood components are also needed. Specific requirements could include antigen-negative red cells if the patient has a clinically significant antibody, extended matched red cells in a patient with a haemoglobinopathy or irradiated cellular blood components for certain groups of immunosuppressed patients. The use of ‘order-comms’ or computerised provider order entry (CPOE) systems can improve the accuracy and completeness of transfusion requests as well as being of use to challenge requests that do not comply with the hospital’s transfusion policy.

• 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 red cell antibodies of potential clinical significance. 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. When LIMS are upgraded or replaced, results of previous ABO and D groups, antibody identification and specific requirements on the legacy system should preferably be migrated to the new LIMS, but alternatively should be accessible in real-time to support the correct selection of blood components for issue to named patients.

• 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. 22-3 ) and may include a compatibility form. The patient must be identified with an identification wristband containing the four core identifiers, and the blood component should be prescribed on a drug or fluid administration chart. In some countries, an additional bedside check of the patient’s blood group is undertaken prior to commencing the transfusion.

  

quality assurance in the transfusion laboratory

Test systems used for pretransfusion testing are important because errors can and do lead to patient morbidity and mortality. Chapter 25 covers the general aspects of a laboratory quality assurance system, and specific guidance for transfusion laboratories can be found in the BCSH guidelines for validation and qualification as well as the guidelines for pretransfusion compatibility procedures in blood transfusion laboratories.

The quality management system for a hospital transfusion laboratory should include the following:

• Quality control: The appropriate use of internal quality controls and participation in external quality assessment exercises where available.

• Validation: Automated equipment and computer systems should be validated to ensure that they function as specified and back up procedures should be in place to cover the failure of automated equipment and computers.

• Policies and procedures: There should be standard operating procedures (SOPs) available which cover all aspects of the laboratory work, and these must be reviewed and updated regularly.

• Reagents, calibrators and control materials: They should correspond to the specification given in the Guidelines for the Blood Transfusion Services and should be used in accordance with the manufacturer’s instructions. As they are included under the In-vitro Diagnostics (IVD) Medical Devices Directive they must be CE (Conformité Européenne) marked ( www.gov.uk/ce-marking ).

• Preventative maintenance: The regular checking and maintenance of all laboratory equipment must be documented.

• Training and competency testing: Laboratory tasks should only be undertaken by appropriately trained staff, and there should be a documented programme for training laboratory staff, which covers all SOPs in use and which fulfils the documented requirements of the laboratory. There must be a documented programme for assessing staff proficiency, which should include details of the action limits for retraining.

• Quality incidents and exceptions: There should be a system in place for documenting and reviewing all incidents of noncompliance with procedures. A programme of independent audits should be conducted to assess compliance with documented ‘in-house’ procedures.

ABO and RhD grouping

Fully automated systems should be used where possible to reduce the risks of interpretation and transcription errors. ABO and RhD (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 the manufacturer’s instructions.

D grouping

D grouping is usually undertaken at the same time as ABO grouping for convenience and to minimise clerical errors that may arise through repeated handling of patients’ samples. In the absence of secure automation, testing should be undertaken in duplicate, as 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. With secure automation, a single IgM monoclonal anti-D reagent that does not detect DVI (see below) may be used. If secure automation is not in use, each sample should be tested in duplicate with either the same anti-D reagent or with two different IgM monoclonal anti-D 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. The use of anti-CDE reagents has led to the misinterpretation of r′ and r″ cells as D positive in UK National External Quality Assessment Service (NEQAS) exercises, and because they are of no value in routine patient typing, their use is not recommended. ^, 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. However, monoclonal anti-D reagents vary widely in their ability to detect both partial and weak D, so it is helpful to use two of a similar affinity to reduce the number of discrepancies due to the detection of weak D.

Some anti-D reagents have high levels of potentiator (e.g. polyethylene glycol, PEG) 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 IgG; any positive reaction seen with the control, however weak, invalidates the test result. Anti-D reagents are provided by many manufacturers, and those responsible for selection and purchase should make themselves aware of the content, specificity and potentiation of the chosen reagent.

Methods

There are several techniques available for routine ABO and D grouping, including both manual tube and slide tests and liquid-phase and solid-phase microplates and columns that can be used in both manual and automated settings. Care should be taken to use the appropriate reagent, because not all reagents have been validated by the manufacturer for all techniques.

The techniques are described above (in the section on Technology and Automation in Blood Transfusion Laboratories, p. 472 ) and also in more detail in Chapter 21 . Molecular techniques for ABO blood grouping are not in routine use because serology is superior, being quicker, cheaper and accurate. ABO genotyping can be helpful to investigate anomalous ABO groups and is in use in organ transplantation where red cells from the donor are not available as well as in forensic practice and paternity testing. ^,

Tube and slide tests

Spin-tube tests may be used for urgent testing, where small numbers of tests are performed at once. Slide or tile techniques are widely used in under-resourced countries for ABO and D grouping. Immediate spin tests may be used in an emergency, whereas routine tests are usually left for 15 min at room temperature (about 20 °C) before centrifugation for 1 min at 150 g. Equal volumes (1 or 2 drops from either a commercial reagent dropper or a Pasteur pipette) of liquid reagent or plasma and 2% cell suspensions are used. The patient’s red cells (diluted in phosphate buffered saline, PBS) should be tested against monoclonal anti-A and anti-B grouping reagents. The patient’s plasma should be tested against A ₁ and B reagent red cells (reverse grouping). In addition, the plasma should be tested against either the patient’s own cells or group O cells (i.e. a negative control) to exclude reactions with A and B cells as a result of cold agglutinins other than anti-A or anti-B in the patient’s sample. Mix the suspensions by tapping the tubes and leave them undisturbed for 15 min. Agglutination should be read as described on p. 451.

Slide method

In an emergency, rapid ABO grouping may be carried out on slides or tiles. The method is satisfactory if potent grouping reagents are used. An immediate spin-tube test is preferable.

Liquid-phase microplate methods

Liquid-phase microplate technology provides a relatively inexpensive and secure method for batch testing when semiautomation is utilised for dispensing and reading, but it is no longer the grouping technique of choice in the UK. In 2015 a UK NEQAS survey showed that < 1% of responding laboratories were using liquid phase microplates for manual grouping, down from 41% in a similar survey in 2002.

Column agglutination techniques

Column agglutination techniques are now the commonest method for grouping in the UK (85% of laboratories who responded to a UK NEQAS survey in 2014), especially where automated systems are in place. There are several different profiles to choose from, and some cards/cassettes include monoclonal antibodies to other blood group antigens (e.g. K). The manufacturer’s instructions should always be followed. An example is shown in Figure 22-4 , and more details can be found in Chapter 21 .

Solid-phase techniques

ABO/D grouping using solid-phase techniques would usually be part of a fully automated system, and testing should be in accordance with the manufacturer’s instructions. Bio-Rad Erytype ( www.bio-rad.com ) 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 reagent red cells for a reverse group.

Controls for ABO and D grouping

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 take account of the length of time that reagents have been kept on the analyser. Controls should be run at start-up and when changing reagent lot numbers. Control samples should be loaded in the same way as the test samples. Where controls do not give the expected reactions, investigations should be undertaken to determine the cause of the problem and also to confirm the validity of all tests undertaken subsequent to the most recent valid control results.

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. in 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. 262). The tests should be repeated using cells washed in warm saline and plasma prewarmed to 37 °C. An auto control should be included.

Cold reacting alloantibodies (e.g. anti-P ₁ ) may cause agglutination of reverse grouping cells, and if this is suspected, reverse grouping should be repeated using prewarmed plasma or grouping cells that lack the implicated antigen.

T-activation/polyagglutination

Polyagglutination describes agglutination of red cells by all or most normal adult sera but not by the patient’s own serum. This is 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 A ₁ 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 A ₁ 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). 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 sensitisation. 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. 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 indicating failure to add reagent or test plasma.

Loss of potency

Inappropriate storage or freezing and thawing may cause a loss of potency of blood grouping reagents. The regular use of 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 recognised 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 nonagglutinated red cells, which may be observed in both ABO and D grouping. It is important to recognise 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 nonidentical 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 or at a different establishment or who 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 stem cell transplant will result in a mixed-field picture until total engraftment has occurred; the mixed-field picture may subsequently reappear if the graft is failing. Rarely, a dual population of cells is permanent and results from a weak subgroup of A (A ₃ ) or a blood group chimaerism.

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 CAT cards/cassettes there will be a line of agglutinated cells at the top of the column, with the nonagglutinated cells travelling through to the bottom of the column ( Fig. 22-5 ). In liquid-phase techniques, if the reaction grade is not a strong positive or an obvious negative then the reaction 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

Most people are either D positive or D negative, but a minority have a variant D type, historically categorised as weak or partial D. People with a partial D antigen can make anti-D to the epitope(s) of the D antigen which they lack, when sensitised to D-positive red cells by transfusion or pregnancy. Historically, those classified as weak D, with fewer D antigen sites per red cell, but no missing epitopes, were considered unable to make anti-D, and could therefore be treated as D positive. There are two problems with using this concept to decide on whether to treat a patient as D positive or D negative: firstly, it is not always possible to distinguish serologically between weak and partial D, as many partial D antigens are also phenotypically weak; secondly, there have been many reports of patients with a variant defined as weak D having made anti-D. Daniels has suggested replacing the terms weak and partial D with a single term of D variant.

In the previous edition it was recommended that 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. However, in the UK, a new patient-based algorithm has been recommended ( Fig. 22-6 ), whereby following a weak reaction with anti-D, samples from children, from women of child-bearing potential and from any patient likely to be transfusion dependent, should be investigated to determine the specificity of the D variant, with the patient being treated as D negative in the meantime. All other patients can be reported as D positive, without further investigation. Weak D types 1, 2 and 3 are the most common weak D types in a Caucasian population and rarely, if ever, make anti-D. Types 1 and 2 can be distinguished from other D variants using a panel of monoclonal antibodies, and such patients can be regarded as D positive. Identification of type 3 requires molecular testing, so if this is unavailable or not cost effective, a practical guide is to regard vulnerable patients (see above) other than those confirmed as types 1 and 2 as D negative. If in doubt, it is safer to call the patient D negative. 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 Ig.

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 having been transfused with blood of a different D type.

Because DVI lacks the most epitopes, such individuals are likely to make anti-D when challenged by transfusion or pregnancy. For this reason, current advice about choice of D-typing reagents is that anti-D reagents for routine grouping of patients’ samples should not detect DVI. 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. There are some important ethnic differences in the frequency of different Rh haplotypes, as shown in Table 22-2 .

Table 22-2

Approximate frequencies of common Rh haplotypes in selected populations

Haplotype	Approximate Frequencies
	English	Nigerian	Chinese
DCe R	0.421	0.060	0.730
dce r	0.389	0.203	0.023
DcE R	0.141	0.115	0.187
Dce R 0	0.026	0.591	0.033
dcE r″	0.012	0	0
dCe r′	0.010	0.031	0.019

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 the zygosity of donors is usually unknown and some will be heterozygous. Red cells for antibody screening are preserved in diluents shown to minimise loss of blood group antigens during storage. In addition, reagent red cells are easier to standardise 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 haemolytic transfusion reactions or haemolytic disease of the fetus and newborn (HDFN). With few exceptions, clinically significant antibodies are those that are reactive in the IAT 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.