Introduction
A careful evaluation of the presence of irregular red cell antibodies must be carried out in patients undergoing transfusion therapy1–3. For immunised patients, the availability of donors of rare blood groups and access to banks and international registries enables compatible blood components to be found and assigned correctly even in the most difficult cases. The accreditation of laboratories that provide these services indicates not only fulfilment of the accreditation body's requisites regarding the quality system, structure and organisation, but also those related to the technical and professional capacities of the laboratory and its staff.
The requisites on which accreditation is based are technical (warranting satisfactory levels of performance), economic (controlling the appropriateness of the services) and educational (promoting continuous training and exchange of knowledge and experience). Accreditation also facilitates the evaluation and comparison of structures, since the services are delivered and controlled according to internationally recognised models.
Our experience
Established at the time of the creation of the Transfusion and Transplant Immunology Centre in 1974 for the identification of red blood cell antibodies and the laboratory diagnosis of cases of paroxysmal nocturnal haemoglobinuria, in 1992 our immunohaematology laboratory acquired its current status: it is a highly specialised area that supplies an essential diagnostic service for patients with antibodies to red blood cells and platelets and finds, when necessary, units of compatible blood components. The immunohaematology laboratory has the taskof refining immunohaematological methods and supplying specialist diagnostic and advisory services in the field of immunohaematology for subjects referred by their General Practitioners for, those using the facilities of the Foundation itself and other regional health care services as well as for patients from national health care structures that lack an immunohaematology reference laboratory.
This requires the combined use of methods based on different technologies, the use of rare reagents, sera and cells, the maintenance of a bank of donors with rare or uncommon red cell and platelet antigen phenotypes, collaboration with international centres, and the development and evaluation of new techniques.
The laboratory staff currently comprises four biologists (2 permanent and 2 temporary), five technicians (3 permanent and 2 temporary) and one administrator.
With this staff, the immunohaematology laboratory ensures continuous coverage of the following services: investigations in the case of complex red cell immunisation for admissions, for institutes with stipulated arrangements and for the Departments of Transfusion Medicine and Haematology (DMTE) of the Region of Lombardy; consultation on problems regarding red cell and/or platelet immunohaematology from the admissions sector during emergency shifts; investigations in urgent cases of suspected neonatal alloimmune thrombocytopenia and post-transfusional thrombocytopenic purpura requiring transfusion of platelet concentrates; consultation advise for the admissions sector for the allocation/delivery of units of rare blood; finding units of rare blood for the admissions sector and for institutes with stipulated arrangements when such units are not available in liquid or frozen form in the inventory and transfusion cannot be delayed; finding units of rare blood for the DMTE of the Region of Lombardy and processing of requests of units of rare blood from the American Rare Donor Program.
The on-call services are provided by the staff of the immunohaematology laboratory from 8 p.m. to 8 a.m. from Monday to Friday (1 biologist) and from 8 a.m. to 8 p.m. on Saturday, Sunday and Bank Holidays (1 biologist and 1 laboratory technician). The on-call staff can be contacted by the graduate on duty in the admissions sector or by a colleague from the DMTE of the Region of Lombardy.
On the 11 July, 2003 the immunohaematology laboratory achieved an important goal: accreditation as an immunohaematology reference laboratory (IRL) by the American Association of Blood Banks (AABB).
This was the culmination of a long and demanding process, started in 2000, aimed at producing a high quality system for diagnostics in red cell immunohaematology.
The choice of organisation for the accreditation was not casual. The AABB, with its headquarters in Bethesda (USA) is, in fact, one of the most important international organisations bringing together more than 2,200 institutes and 8,500 graduates, technicians and administrators working in the fields of transfusions, transplantation and donor recruitment. Members live and work in 50 states in the USA and in 80 countries throughout the world and American AABB centres separate into components and transfuse about 80% of the 14 million units of whole blood collected each year in the USA.
The AABB establishes and adopts high quality standards and, since 1957, has been the leader in proposing accreditation programmes, designed by experts, aimed at improving quality and safety and reaching levels of excellence in the fields of transfusion and transplantation.
Among the various accreditation programmes of the AABB, the one related to IRL is very important for those structures that have a red cell immunohaematology laboratory that acts as a reference centre for other transfusion facilities.
At present 50 laboratories have been accredited as IRL in the USA and our laboratory is the first Italian laboratory to have received this accreditation.
The stages of the accreditation
Although IRL are usually part of a Transfusion Centre, the AABB has recognised their operative autonomy and the importance that they comply with precise standards that define procedures specific to this type of structure. These standards, decided by a committee of the AABB, indicate the indispensible operative procedures for accreditation and are defined considering not only technical and qualitative aspects but also medical and scientific issues, as well as the legislation and regulations related to this field of activity4–6.
In the long process that led to the accreditation, the starting point was a detailed analysis of these standards and whether the technical and organisational set-up of the laboratory was compatible with the accreditation requisites. Only after this verification had been carried out the request for accreditation was made, in the stages that we describe below.
Self-assessment
A huge questionnaire was completed on the laboratory's organisation, resources in terms of reagents and staff, experience and techniques used when dealing with complex cases, and quality systems in place. The self-assessment was considered satisfactory by the AABB, which, therefore, allowed the start of the subsequent stages.
External evaluation of competence
The AABB periodically sends samples presenting particular immunohaematological problems that the laboratory must identify and resolve. The accreditation body uses these tests to evaluate the global competence of the laboratory, the experience of staff and supervisor, the techniques employed, the capacity to identify compatible donors for a transfusion and adherence to the IRL standards. The cases to resolve are usually complex and the review of the results by the AABB is very critical and severe. In order to understand the importance of this stage it is sufficient to appreciate that already accredited laboratories are permitted only one error every six samples: two or more errors lead to a new inspection and the possible suspension of the accreditation.
Inspective audit
The inspective audits are usually carried out over two to three days by experts sent by the AABB. The applications of the quality system used both in the laboratory and the Transfusion Centre and the specific activity of the immunohaematology laboratory are considered. The inspectors verify the correct implementation of the IRL standards in the general management of the laboratory, of the inventory and reagents and materials and of the equipment. Particular attention is given to the management of cases of complex immunisation and to donors of rare blood groups, to staff training and to programmes of continuing education. The laboratory must document a programme of external quality audits which each member of staff qualified to perform the tests must carry out in turn. Among the various external quality audits, the laboratory must undergo at least three each year approved by the IRL of the AABB Accreditation Programme. Appropriate controls are made of the procedures, working instructions and related forms to ensure the best quality of the tests and the final reports. The laboratory must record all nonconformities with the standards in order to be able to take any necessary corrective actions subsequently. These inspective audits require the active involvement of all the laboratory staff.
The inspectors reported on both skills and availability: at the end of the audit they expressed their appreciation of the professionality of the staff and the organisational and technical choices applied in the laboratory and in the whole transfusion Centre. Slight non-conformities were found for which the description of the corrective interventions was sent. The approval of these corrective actions made it possible for the AABB to formalise the accreditation. It should, however, be remembered that the inspective audits are repeated periodically every 2 years: if these audits do not give positive results, the accreditation can be suspended. Following the initial accreditation of the laboratory in 2003, we have passed two other inspective audits in 2005 and 2007; the next one will take place in 2009.
Safe transfusion: an international network for the most complex cases
The AABB, like the main international transfusion organisations, identifies the IRL as a highly specialised area that carries out investigations aimed at identifying antibodies and finding units of compatible blood components for patients with complex profiles of immunisation to red cell antigens.
The IRL are capable of using multiple methods of analysis, from the simplest to the most sophisticated, and have a broad inventory of reagents and cells not easily found, which can only be obtained through adherence to collaborative programmes with other accredited Centres.
Furthermore, given that the transfusion of subjects with complex immunisation profiles is possible only through the use of units with rare antigen profiles, in addition to the serological techniques, the reference laboratories regularly carry out programmes of typing red cell antigens in large number of donors.
In order to achieve a service at a regional level, the Region of Lombardy established the ‘Bank of Rare Group Blood Components' at the Transfusion and Immunohaematology Centre of the IRCCS Policlinico Maggiore Hospital, Mangiagalli and Regina Elena Foundation in Milan as the reference Centre for the Region.
Report on the activity of the Bank of Rare Group Blood Components
The results obtained in the last 3 years in the Bank of Rare Blood Group Blood Components are summarised in table I.
Table I.
Summary of activity (January 2005 – June 2008)
| Number | |
|---|---|
| - Donors typed | 41,437 |
| - Red cell typing | 563,354 |
| - Rare donors identified | 6,033 |
| - Rare donors for combinations of antigens | 5,713 |
| - Rare donors for high-incidence antigens | 295 |
| - Donors of rare Rh phenotypes | 25 |
| - Requests for studies of complex immunisation | 258 |
| of which: in the setting of the Region of Lombardy | 191 |
In detail, from January 2005 to June 2008, 563,354 red cell typings were carried out on 41,437 blood donors and 6,032 rare donors (14.6%) were identified. Of these, 5,713 were negative for combinations of antigens: 4,403 were S-s+, while 1,310 were S+s-. Furthermore, another 295 donors negative for high-incidence antigens and 25 donors of rare Rh phenotypes were identified. As regards the high-incidence antigens, the following donors were identified: 26 Lu(a+b-), 21 Lu(a-b-), 81 k-, 1 Kp(b-), 41 Fy(a-b-), 1 Fy(a-b-) and U(-), 2 Fy(a-b-) and Js(b), 74 Yt(a-), 2 Co(a-), 21 Co(a-b+), 1 Co(a-b-), 3 Ge2(-) and 21 Vel(-). As regards the Rh system, 14 CCdee, 4 ccdEE, 1 CCdee, 4 CCDEE and 2 –D–donors were found.
Besides carrying out red cell typing of donors on a large scale in the Region of Lombardy, the Bank also has the duty of identifying antibodies present in patients with complex immunisation referred to hospitals in the Region and of supplying these patients with compatible blood components.
In the same period, the IRL received 258 requests for investigations of complex profiles of red cell immunisation, of which 191 were from within the Region of Lombardy. With regards to these 258 investigations, the selection of compatible blood was required in 99 cases. The characteristics of the complex cases are summarised in table II.
Table II.
Main forms of complex cases of red cell immunisation
|
Of the 258 requests investigated, 49 concerned mixtures of antibodies against common antigens, 37 antibodies against high-incidence antigens, 28 antibodies against low-incidence antigens, 37 autoantibodies and 30 mixtures of allo-autoantibodies. The other 77 requests concerned patients with ABO and Rh variants of with simple antibody specificities.
During the same period, the Bank selected 4,465 units of rare blood group: 90 of these were extremely rare frozen units, deglycerolized prior to release. In detail, these included units with a rare Rh phenotype and/or particular combinations of antigens, and PP1P k (-), K0, Co(a-), Kp(b-), Ge:-2, Yt(a-), Lu(b-), Sc:-1, Lu(a-b-) and Fy(a-b-) units.
At present the inventory contains 351 units of frozen rare blood: 286 units for homologous use and 65 units for autologous use. Thanks to these stores, the laboratory has had to resort to international collaboration in only two particular cases: the first was a request made for a young woman with thalassaemia with anti-Sc1 red cell alloantibodies; the second was made with the prospect of having to transfuse a neonate whose mother, of African origin, was immunised against an anti-P haemolytic high-incidence red blood cell antibody. Our bank had blood for the group A positive mother, but not for the group O positive baby.
Evaluation of methods and reagents
The laboratory follows evaluation protocols adopted by the most important and accredited international laboratories (protocols proposed by the International Society of Blood Transfusion and by the International Committee for Standardisation in Haematology), which examine aspects related to the quality of tests (specificity and sensitivity), technical work, costs and routine applicability, comparing the results of the method under evaluation with those of standard methods used for a given diagnostic service7,8.
Molecular biology techniques
Recent literature and the experience of some of the most important international transfusion facilities show that molecular biology is destined to replace traditional serological tests in immunohaematology in many cases9–11. This is also supported by the fact that, in some situations, agglutination studies have some limitations. For example, in haemolytic disease of the foetus, agglutination methods can only give an indirect measure of the potential complications in a pregnancy at risk and cannot indicate exactly the RhD zygosity in D+ subjects, they cannot be used to type recently transfused patients or patients with a positive direct antiglobulin test, they sometimes require antisera that are not commercially available and sometimes are not able to resolve serologically complex cases.
In recent years the development of high throughput molecular biology methods and instruments, able to type samples and donors for a large number of clinically relevant antigens, has made the application of genomic typing feasible also for banks and registries of donors of rare blood groups.
The antigenic profile of donors of rare blood groups for high-incidence antigens is currently determined using commercial kits based on PCR-SSP methodology (Seltene Blutgruppen-SSP, Inno-Train Diagnostik GmbH, Germany), while that of rare donors for combinations of antigens is determined using technology based on Luminex microarrays (Luminex xMAP, Luminex Corp., USA), produced by our Centre, involving microspheres conjugated to various fluorochromes12,13.
The biobank and collaborative groups
Since the key element for diagnostics is the availability of an adequate inventory of reagents, the laboratory has created a biobank of materials to use for subjects under investigation and to validate new methods. The current inventory of rare materials available in the biobank is superior to that required by the minimum standards for accreditation.
The establishment of collaborations with other international organisations and suppliers with large inventories of uncommon reagents has been of fundamental importance. In this regard, since 1980 the laboratory has been a member of the international SCARF (Serum, Cells and Rare Fluids) Exchange, whose purpose is to foster exchanges of red blood cells and DNA from subjects with rare phenotypes and sera containing rare antibodies between reference laboratories. It is the most important international organisation, formed of 150 laboratories through the world, which has facilitated the distribution of rare samples since 1972. Participation in the programme has gradually enabled us to create a cell and serum biobank containing numerous samples, of great help in the resolution of particularly complex cases. One of the main requirements of the AABB for accreditation of an immunohaematology laboratory is to have a minimum inventory of rare cells and sera listed in the standards.
Particular attention must be given to the choice of samples to request since biological samples can be useful for different tests. For example, whole blood is used in platelet and red blood cell serology or as a source of DNA; serum, plasma, eluate, human biological fluids or particular chemical substances are used for antibody identification (antibody neutralisation and inhibition and antigen inactivation), while rare antisera are used for typing platelets and red blood cells. The archive of samples is achieved by freezing or storage at +4°C and the inventory is managed using standard electronic spreadsheets.
The laboratories have access to international banks and registries of donors of rare blood groups, which they can ask for units not locally obtainable, in that they are able to guarantee both the correct performance of the investigations and the appropriateness of the request. In this perspective, all the IRL of the AABB are members of the American Rare Donor Program of Philadelphia, a collaborative registry established by the AABB and the American Red Cross, which currently includes about 30,000 donors of rare blood groups and which coordinates the inventory of rare blood in the USA. Other international banks of rare blood have developed in Europe14: the most important of these are in France (www.ints.fr), Germany (www.uni-ulm.de/~wflegel/RARE), the Netherlands (www.sanquin.nl) and England (www.ibgrl.blood.co.uk). Furthermore, Japan, South Africa, Korea and Australia have contributed to the international programme.
Participation in international programmes fosters the activation of a network of high-quality collaboration able to tackle the transfusion problems raised by the most complex cases of immunisation.
Future prospects
For the next 3 years our plan is to maintain the current organisation of our Reference Centre, and to develop the following new areas of activity: immunohaematological diagnostics for cases of complex platelet immunisation and identification of donors of rare platelet antigen groups, identification of IgA-deficient donors, identification of donors with low titres of anti-T antibodies and immunohaematological diagnostics for patients with T-activated red blood cells, management of the regional register of donors of rare platelet antigen groups, IgA-deficient donors and donors with low titres of anti-T antibodies. In particular, we shall be focusing on the use of a new molecular biology technique based on the use of microarrays able to carry out large-scale red cell and platelet typing of blood donors in our Region15,16.
Conclusions
The accreditation programme has been a fundamental step for our immunohaematology laboratory and a stimulating educational and professional challenge, with the aim of improving the quality of the services provided.
The accreditation represents the evolution of a process started in 1997 when the Transfusion Centre received its first ISO 9000 certification17. Despite its importance, ISO 9000 certification has the limitation of not considering the quality of the results obtained. Only a critical evaluation carried out by a organisation with specific understanding of the transfusion world can provide a valid attestation of the professional and scientific quality of an immunohaematology laboratory and that its activities meet internationally recognised standards.
In conclusion, the process for accreditation as an IRL by the AABB led to a thorough review of activities and performance improvements. The laboratory has been able to show that it on the level with the best facilities in the world, offering safe transfusions and high-quality services not only to patients at the hospital in Milan, but also to those in other regional or national structures.
Finally, it should be remembered that accreditation is only a starting point and the periodic review of the standards and maintenance of the quality of services must continue.
Acknowledgments
This work is dedicated to the memory of Dr. Fernanda Morelati, founder and creator of the Immunohaematology Laboratory.
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