New technologies in immunohaematology

Introduction

Since the discovery of the ABO system, numerous important innovations have contributed to a continuous, rapid evolution in the diagnostic methods for in vitro measurements of the antigen-antibody reaction, allowing a significant improvement in the compatibility between blood from donors and the recipients. Apart from the introduction of ABO typing, these methods include the determination of Rh type and phenotype, the direct and indirect antiglobulin tests, cross-matching and consequent identification of antigens and antibodies of clinical relevance, the use of low ionic strength additives and enzyme treatments, the development of monoclonal reagents and solid-phase and microcolumn platforms for performing the pre-transfusion tests.

Since transfusion safety depends on a series of strictly inter-related processes¹, among which pre-transfusion tests have a predominant role, in recent years some of the new technologies that integrate the classical techniques in immunohaematology have become valid instruments for improving the safety of transfusions. The aim of this review is to illustrate the principles and practical applications of these emerging techniques used in our laboratory to identify antigens and antibodies, in cases of red cell or platelet immunisation.

Automation for complex cases

The most recent data in the literature² indicate that, still nowadays, incorrect identification of samples and errors in performing tests are the most frequent causes of transfusion reactions and complications, with sometimes dramatic consequences³ for health.

The use of completely automated systems, indivisible from the use of information technology, is the most efficient strategy for achieving two main goals in the field of immunohaematology:

• - reducing transfusion risks related to human errors, by automating the stages related to identifying samples, selecting reagents, performing and interpreting results and transferring data to the laboratory's information management system;

• - guaranteeing the traceability of all the elements involved in the analytic process, which can be archived and remain accessible after the test has been performed.

Following the 1990s the use of automated systems increased in all industrialised countries in parallel with the development and marketing of new technologies; these systems have increased the objectivity and stability of the results and the standardisation of the process with respect to the traditional liquid phase methods.

The most widely used systems are based on the use of:

• - microcolumns, with different types of commercial products, which enable the results to be seen after the passage of red blood cells through a matrix containing the reagents; the main advantage of this technology, which has led to its widespread use, is mainly related to the fact that the antiglobulin test can be carried out without washing steps;

• - polystyrene microplates with wells pre-coated with lyophilised red bloods or platelets, or anti-erythrocyte or anti-platelet antibodies: the antibodies present are revealed by immuno-adherence after addition of red blood cells coated with an anti-IgG human antiglobulin; a more recent system, based on the use of microplates sensitised by an anti-IgG human antiglobulin, enables the reaction to be visualised through magnetised red cells and for the antiglobulin test to be carried out without washing steps.

The combined use of these techniques and latest generation, completely automated instruments has enabled automation of even more sophisticated immunohaematology tests. These tests can be used in particular conditions to resolve the most complex cases. In our Centre, full automation has been efficiently applied in the following conditions.

1) Large-scale cell phenotyping

A fully automated high output system based on solid -phase technology ^4–7 is currently used for the red cell extended phenotype.

The system enables typing of 14 red blood cell antigens of the greatest transfusional relevance, using samples of blood in anticoagulated (EDTA) blood, processed within 3–6 days of collection, and a combination of:

1. polyclonal antisera (anti-Fy^a, anti-Fy^b, anti-Jk^a, anti-Jk^b, anti-S, anti-s, anti-Co^a, anti-Js^b, anti-Lu^b, anti-Kp^b Immucor, Norcross, GA, USA) prepared for use with an automated instrument and the solid phase method; the results are confirmed using the same working conditions and polyclonal antisera of the same specificities prepared for the test-tube method;

2. plasma from immunised donors (anti-Ge2, anti-PP₁P^k, anti-U, anti-Vel), diluted 1:5 in saline and stored at +4 °C until use.

The instrument processes samples in batches of 50–100, dispensing 12 samples, 7 typing reagents and 1 negative control/sample for each plate.

Over a period of 12 months, this procedure was used to carry out 134,129 typings on 12,644 blood donors attending the 'Rare Blood Group Bank – Reference Centre, Region of Lombardy'. In 1% of the cases (1,339 typings) the result was not conclusive (indeterminate/doubtful/invalid) at the first test.

The commercial antisera were the cause of inconclusive results in 156 (0.12%) typings and human plasma in 1,183 (0.9%) typings.

No inconclusive results were observed with anti-Fy^b, anti-K, and anti-k specificities. A high percentage of repetitions were required after the first test with the anti-^k plasma samples (803 tests for anti-Vel, Vel and anti-PP₁P^k), related to the peculiarity of the reactions 161 for anti-PP₁P of the antibodies themselves. In 233 (0.17%) of these cases a manual method had to be used in order to identify the antigens.

2) Identification of red blood cell antibodies

The possibility of automating this complex process was evaluated in a pilot study^8,9 carried out in 2004 using a completely automated instrument based on standard commercial panels and microcolumn technology.

One of the most important difficulties in the identification of the red cell antibodies was related to the antigen profile of the commercial panels, which was scarcely useful when mixtures of antibodies were present.

In theses cases, further extensively typed red blood cells are necessary to achieve complete identification of the specificities involved.

Two new solid-phase panels^{4–6, 10}, selected for Rh phenotype and also prepared for use in a completely automated instrument, recently became available. These were evaluated for their performance in an automated process when mixtures of red cell antibodies are present, that include also Rh specificities.

Two 14-cell panels were used for this evaluation: the first panel consisted of homozygous cells for the C and E antigens and the second comprised 13 cells negative for the Rh(D) antigen and a control, Rh(D) positive cell.

The panels were used to test samples from 61 non-immunised subjects and 104 immunised subjects, who had undergone complete immunohaematological investigations, prior to the evaluation.

Among the subjects investigated, 75 had single antibody (28 anti-D, 7 anti-CD, 2 anti-CDE, 26 anti-E, 1 antie, 1 anti-C, 6 anti-c, 1 anti-K, 2 anti-Jk^a, and 1 anti-M) while the other 29 patients had mixtures of antibodies (4 anti-D, 20 anti-E, 3 anti-C, 8 anti-c, 5 anti-C^w, 11 anti-K, 1 anti-Kp^a, 3 anti-Jk^a, 1 anti-Jk^b, 2 anti-Fy^a, 1 anti-Fy^b, and 3 anti-S). The negative samples were evaluated with the two panels and the positive samples with one of the panels according to the known specificity (57 with the first panel and 47 with the second). The tests were carried out using the method defined by the instrument, which required interpretation of the results by the operator. In the case of discrepancy with the previous result, the specificity involved was verified with the manual methods used in the laboratory. Complete correspondence with known results (Table I) was observed in non-immunised subjects and in 91 (87.5%) of the 104 samples with red cell antibodies. In 11 of these, 12 additional antibodies were found, of which 9 were identified with the first panel (2 anti-c, 1 anti-E, 1 anti-Jk^a, 1 anti-K, 1 anti-C^w, 1 anti-N and 2 autoantibodies reacting only in the solid-phase) and three with the second panel (2 antibodies against low incidence antigens and 1 autoantibody reacting only in the solid-phase).

Table I.

Results of the identification of red cell antibodies carried out with a completely automated instrument

Type of sample	Complete agreement (n.)	Additional antibodies detected by the instrument (n.)	Antibodies not detected by the instrument (n.)
Immunised subjects	9 1	11 samples 12 antibodies*	2 IgM antibodies (anti-K, anti-M)
Non-immunised subjects	6 1	0	0

^*2 anti-c, 1 anti-E, 1 anti-Jk^a, 1 anti-K, 1 anti-C^w, 1 anti-N, 3 autoantibodies, 2 antibodies against low incidence antigens

Two class IgM antibodies were not detected by the first panel (1 anti-K) and by the second panel (1 anti-M).

3) Selecting platelet concentrates for patients with immunological refractoriness

The condition known as ‘immunological refractoriness to transfusion of standard platelet concentrates' is one of the most important complications in subjects requiring transfusion support and indicates repeated, poor increments in post-transfusion platelet count (three consecutive platelet counts below 5 x 10⁹/L). This condition, which can be associated with severe clinical complications, is caused by the presence of class I antibodies against human leucocyte antigens (HLA) and is observed in 13–14% of patients with leukaemia transfused with standard blood components and in 3–4% of subjects transfused with leucocyte-depleted blood components. The traditional transfusion approach is based on choosing HLA identical or compatible donors or on selecting appropriate donors through tests of platelet compatibility. In our laboratory we have chosen the latter approach by using the platelet concentrates (from buffy-coats or obtained through apheresis products) present in the daily inventory of the units and a completely automated instrument based on the solid-phase technology.

Over a period of 33 months, post-transfusion platelet count increments were evaluated in 40 refractory subjects (27 women, 13 men) transfused with platelet concentrates selected using this procedure^11,12 and the increments were related to known detrimental clinical factors and to the post-transfusion increments (at 1 hour after the transfusion), observed after the last three transfusions with standard platelet concentrates (not selected by platelet cross-match). Within 48 hours from starting the selection procedure, the subjects under consideration had been transfused with 569 platelet concentrates (median value 8 concentrates/ patient, containing 202 ± 71 x 10⁹ platelets), obtained from buffy-coats or by apheresis procedures. The median pre-transfusion platelet count was 7.7 ± 5.5 x 10⁹/L and the post-transfusion platelet increments exceeded 10,000 platelets/ μL in 68% of the cases (Figure 1).

Figure 1.

Pre- and post-transfusion platelet counts in 40 refractory patients (median, 25^th and 75^th percentiles)

The post-transfusion counts in subjects with detrimental factors were lower (28.9 ± 20.3 x 10⁹ platelets/ μL at 1 hour after the end of the transfusion) than those observed in subjects without such factors (35.9 ± 21.2 x 10 ³ platelets/μL at 1 hour after the end of the transfusion).

The investigation of autoimmune haemolytic disorders

Among the many red cell immunhaematology problems, one of the most difficult to manage is autoimmune haemolytic disease with a negative direct antiglobulin test (DAT). In order to resolve the diagnostic problem in these cases, a battery of investigations must be used, carried out with different methods (agglutination tests, solid phase tests, ELISA, flow cytometry, immunoradiometric tests, evaluation of complement consumption). One particularly useful test in the study of these complications is the mitogen stimulation test (MS-DAT), designed by Barcellini^{13, 14} and colleagues, which is used to evaluate the prevalence of positive results in subjects with autoimmune haemolytic anaemia (AIHA) in clinical remission or in an active phase of the disease. The MS-DAT test is carried out by stimulating whole blood cultures from the investigated subjects with mitogen (phytohaemagglutinin – PHA; phorbol-12-myristate-13-acetate – PMA; or pokeweed mitogen-PWM); the production of antibodies in the culture after stimulation is evaluated by a competitive ELISA in solid phase. An agglutination DAT, using the standard test-tube method, a DAT with red cells washed in low ionic strength solution or cold physiological saline, and a solid phase test for immunoadherence were carried out in parallel in all subjects ^4–6. Using this technique, 33 subjects with AIHA were studied (of whom 27 in an active phase of disease with a positive DAT, and 6 subjects with previous AIHA who had become DAT negative) and 7 subjects with DAT-negative AIHA, whose disease had been diagnosed on the basis of exclusion of all other causes of haemolysis and on the response to steroid therapy. Furthermore, we studied 69 subjects with chronic B-cell lymphocytic leukaemia (B-CLL), a disease associated with a high prevalence of autoimmunity against red blood cells, with or without a positive DAT. Mitogenic stimulation caused an increase in the quantity of IgG that adhered to red cells in culture in patients with AIHA and B-CLL, but not in controls (Table II). The MS-DAT was positive in 6 patients with previous AIHA who had become DAT-negative (Table III, subjects 1–6) and in 7 subjects with DAT-negative haemolytic disease was diagnosed as having an autoimmune nature by exclusion (Table III, subjects 7–13). Finally, MS-DAT was positive in one third of the subjects affected by B-CLL without signs of haemolysis. The MS-DAT was also carried out during clinical monitoring of patients with AIHA, with results strictly related to the clinical course of the disease, to the changes in haemolytic parameters and to the response to therapy (Figure 2).

Table II.

The effect of mitogenic stimulation in the presence of antibodies against autologous red blood cells in whole blood culture

	Not stimulated	PHA#	PMA$	PWM&
Patients with AIHA	322±49*	623±122**	465±55***	635±134**
Patients with B-CLL	134±15**	207±29*	182±37**	183±25*
Controls	75±7	75±9	70±6	76±14

^#Phytohaemagglutinin;

^$Phorbol-12-myristate-13-acetate;

^&Pokeweed mitogen;

^*p ≤ 0.001;

^**p ≤ 0.01;

^***p ≤ 0.05

The values (mean ± SD) are expressed as IgG ng/mL [33 patients with autoimmune haemolytic anaemia (AIHA), 69 with B-cell chronic lymphatic leukaemia (B-CLL) and 81 controls]

Table III.

Clinical and laboratory characteristics of patients with DAT-negative and MS-DAT positive AIHA

N.	Sex	Hb g/dL	Total bilirubin (indirect) μmol/L	Reticulocytes %	Haptoglobin mg/L	LDH U/L	Steorid therapy	DAT*	MS-DAT IgG ng/mL
1	F	13.3	8 (5)	1.4	1,200	420	Yes	neg	279
2	F	12.4	13.7 (12)	0.8	2,290	296	Yes	neg	321
3	F	12.3	10.3 (7)	0.7	1,830	377	No	neg	433
4	M	13.1	10 (9)	1.8	1,200	472	No	neg	302
5	M	1 2	10 (8)	0.9	690	303	Yes	neg	256
6	M	1 5	13.7 (8)	0.2	870	291	No	neg	322
7	M	1 2	27 (22)	3.7	200	480	No	neg	813
8	F	10.9	28 (22)	4.4	200	420	No	neg	433
9	M	11.1	20 (18)	1.1	200	550	No	neg	1,230
10	F	5.3	20 (19)	6.9	200	232	No	neg	1,660
11	M	8.8	n.d.	4.3	200	520	Yes	neg	516
12	M	10.8	40 (27)	15.9	200	470	No	neg	856
13	F	11.1	9 (8)	0.9	500	338	No	neg	314
Normal range		Women 11.5-15 Men 14-16.5	0-17 (0–12)	<2%	600–3,000	230–460

^*The DAT was carried out by agglutination, using the standard test-tube method, with washed red blood cells in low ionic strength solution or cold physiological saline, and in solid phase by immunoadherence.

Figure 2.

MS-DAT in the clinical follow-up of AIHA. The levels of haemoglobin (♦), haptoglobin (▪) and MS-DAT (▴) are expressed as percentages of the values at the start of the follow-up. For the first patient (figure on the left) these values were: Hb=5.8 g/dL, haptoglobin=200 mg/L, MS-DAT=4,121 ng/mL IgG. For the second patient (figure on the right) they were: Hb=13.6 g/dL, haptoglobin=590 mg/L, MS-DAT=41 ng/mL IgG.

Molecular techniques

The advances made over the last 20 years in defining the molecular nature of red cell and platelet antigens has enabled the use of molecular techniques in various fields of immunohaematology, particularly in some situations in which the traditional methods are difficult or impossible to apply^15–21. The most relevant of these include:

1. determination of the antigen profile in subjects:

   • - recently transfused or with a positive DAT,

   • - with suspected variants, weak expression or null phenotype of antigens or with discrepancies in blood group determination that cannot be resolved with the traditional serological techniques;

2. antenatal investigations to evaluate:

   • - the risk of haemolytic disease of the newborn,

   • - the risk of neonatal alloimmune thrombocytopenia,

   • - RHD zygosity;

3. large scale typing of blood donors for red cell and platelet antigens, when typing antisera are not available or difficult to obtain.

Molecular characterisation of antigens is essential in transfused immunised patients, in order to select compatible units of red blood cells and in pregnant women, in order to decide whether to administer RhD prophylaxis.

Over the course of about 2 years, molecular techniques were used to study 28 blood donors (5 suspected ABO variants, 5 discrepancies in Rh determination, 3 discrepancies in the typing of other red cell antigens and 15 donors negative for high incidence antigens) and 40 patients sent for immunohaematological investigations (6 with ABO discrepancies and 4 with Rh discrepancies in the agglutination techniques, 11 transfused subjects for bone marrow transplantation or thalassaemia or malignancy, 10 DAT-positive transfused subjects, 3 immunised pregnant women and their partners, whose offspring were suspected to be affected by foetal or neonatal alloimmune thrombocytopenia, 1 foetus with suspected haemolytic disease, 4 subjects with haemolytic transfusion reactions and 1 with platelet-specific alloimmunisation). Molecular typing was carried out using DNA, at a concentration of 5–10 μg, extracted by from peripheral blood in EDTA, using the salting out method²². The commercial kits used were based on the Polymerase Chain Reaction Sequence-Specific Primers (PCR-SSP) method, and prepared according to international knowledge in the relevant field ^23–42. The findings were 3 ABO variants (A_el), 4 D variants (1 DFR, 1 Rh33, 1 Dweak type 1 and 1 Dweak type 5), 14 cases of absence of a high incidence antigen (3 k, 2 Lu^b, 1 Co^a, 7 Fy null, 1 HPA-1a); in another 6 cases (5 donors and 1 patient) genomic typing revealed the presence of antigens that the serological techniques had not detected (3 Fy^bweak, 2 Lu^b and 1 antigen of the KEL system).

A second study was carried out to identify donors of platelets negative for human platelet antigens (HPA).

The anti-HPA alloantibodies and relative antigens were involved in cases of post-transfusion purpura, in immunological refractoriness to standard platelet transfusions and in cases of foeto-neonatal alloimmune thrombocytopenia. The availability of donors of known platelet type is essential in order to ensure effective transfusions of platelet concentrates in these subjects. However, it is difficult to identify such subjects because of the scarcity of specific typing antisera to use in the classical methods (in ELISA, in flow cytometry or in solid phase). Genomic DNA from 149 Caucasian, group O blood donors was analysed in order to determine the genotype of the HPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-5a and HPA-5b antigens. The PCR-SSP method and a commercial kit were used. The allelic, phenotypic and genotypic frequencies observed in this population of blood donors were compatible with those reported in other European studies^43–48 (Table IV).

Table IV.

Results of HPA typing and a comparison with frequencies reported in other European countries

Allele frequency (%)
HPA antigen	Observed1	Italy (Fratellanza 2005)	the Netherlands (Simsek 1993)	Finland (Kekomaki 1995)	Wales (Sellers 1999)	Germany (Kiefel 1993)	Austria (Holensteiner 1995)
HPA-1a	0.832	0.838	0.846	0.86	0.825	0.834	0.852
HPA-1b	0.167	0.162	0.154	0.14	0.175	0.166	0.148
HPA-2a	0.879	0.85	0.934	0.91	0.902	0.940	0.918
HPA-2b	0.12	0.15	0.066	0.09	0.098	0.060	0.082
HPA-3a	0.701	0.658	0.555	0.59	0.607	0.616	0.612
HPA-3b	0.298	0.342	0.445	0.41	0.393	0.384	0.388
HPA-5a	0.859	0.920	0.902	0.95	0.903	0.889	0.892
HPA-5b	0.14	0.080	0.098	0.05	0.097	0.111	0.108

¹Centro Trasfusionale e di Immunoematologia, Dipartimento di Medicina Rigenerativa, Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Istituto di Ricovero e Cura a Carattere Scientifico, Milano (Italy)

Conclusions

In this article we have presented our recent experience with some of the new technologies internationally applied to the field of red cell and platelet immunohaematology^49–54 to reduce the risk of incompatibility between donors and recipients and, therefore, to improve transfusion safety.

The use of automation, in particular, seems to be a valid approach for reducing the risk related to human error and guaranteeing the traceability of all the operative phases in all critical processes involving large numbers, such as extended red cell typing, identification of antibodies in multiply immunised subjects and in the transfusion therapy of patients affected by immunological refractoriness to standard platelet concentrates.

The use of molecular techniques has become indispensable, in combination with agglutination methods, for defining the red cell antigen profile in recently transfused and immunised subjects or in cases in which discrepancies cannot be resolved with traditional serological techniques and in the consequent selection of compatible red blood cell units. These techniques represent the only method available for characterising the platelet antigen profile, which cannot be determined otherwise because of the lack of suitable typing reagents, and in antenatal investigations to evaluate the risk of haemolytic disease of the newborn, the risk of alloimmune thrombocytopenia and the antigen profile of the foetus. The mitogen stimulation test is a clinically important assay in the management of cases of suspected AIHA, that gives negative results in the traditional test.

Finally, it should be emphasised that transfusion safety depends on a series of processes which must be improved and monitored over time and that the use of new technologies is only one element in the transfusion procedure.

It is, therefore, essential to use new technologies within a carefully defined process including all the phases between selection of the donor, the transfusion of blood components and the follow-up of the patients.