Abstract
Background
The aim of this study was to compare the routine use of two automated systems (OrthoAutoVue Innova, microcolumn, and Immucor Galileo, solid phase) for the screening and identification of irregular red blood cell alloantibodies in samples, analysed in our Transfusion Service during 6 months of normal activity. The study focused particularly on an evaluation of the repeatability of the screening tests, the identification of antibody specificities and the identification of antibodies in samples showing discordant results.
Materials and methods
Overall 2,229 samples from potential blood donors (A), multiply transfused patients with blood disorders (DH), potential transfusion recipients (TS), and external cases (E) were studied. The protocols were carried out according to the manufacturers recommendations.
Results
The screening tests detected 78 samples that were positive with both systems, while 18 were positive only with Immucor and 11 only with Ortho (thus, overall, Immucor detected 96 positive samples and Ortho 89 positive samples). The use of the respective identification panels enabled us to identify the antibodies in 65 samples with Immucor and in 61 samples with the Ortho system; 74 antibodies were identified with Immucor (55 with a single specificity and 19 with mixed specificities) and 68 antibodies with Ortho (51 and 17, respectively). In the remaining cases (31 samples for Immucor and 28 for Ortho), the antibody specificity was not identified. The two systems were found to be essentially similar. The Immucor system revealed a greater number of antibodies, mainly because of its greater sensitivity at detecting anti-D antibodies.
Conclusions
Both systems showed a repeatability of over 85%, demonstrating that automation of immunohaematological tests is advantageous. The specificity of the antibody was identified in 68% of the samples. Furthermore, using the two systems led to the identification of ten new antibodies (6 anti-D, 2 anti-E, 1 anti Lea, and 1 anti-Vel), which would not have been detected had only one of the two methods been used.
Keywords: red blood cell antibodies, antibody screening and identification, automated methods
Introduction
There are two phases of the immunological reaction that leads to red cell agglutination: in the first phase the antibody attaches to the red cell antigen, while in the subsequent phase, a lattice of red blood cells, united by antibody bridges, is formed and manifested as visible agglutination.
In order to evaluate the attachment of the antibody to the antigen in the first phase of the immunological reaction more directly and to improve the reading of the agglutination further, already by the end of the 1980s, techniques based on agglutination on columns and reactions in solid phase had been introduced into immunohaematology laboratories1. Alongside this evolution in techniques, automated systems for the preparation and performance of red cell immunohaematology tests became used routinely in order to obtain a complete guarantee of the tests performance and standardisation of the procedures2.
The purpose of this study was to compare, as in other published studies3–8, the routine use of two automated systems for the screening and identification of irregular antibodies in a large number of serum samples, analysed in our Service of Immunohaematology and Transfusion Medicine (SIMT) in 6 months of normal activity. There were numerous parts of this study (agreed with the manufacturers of the automated systems). In detail, we evaluated the repeatability of the screening tests, the results achieved in different categories of subjects, the number and the validity of the panels of test red blood cells used to identify the antibody specificity, the identification of discordant specificities in samples that were positive according to both methods and the antibody specificity when only one of the systems gave a positive result.
Materials and methods
Between 22 August 2005 and 28 February 2006, we analysed 2,299 samples from the following categories of subjects: 560 potential blood donors (A), 260 patients with blood disorders or haemoglobinopathies, followed in the Haematology and Thalassaemia Units of the SIMT of Ragusa (DH), 1,181 inpatients in surgery wards in our hospital, who were candidates for transfusion therapy (TS) and 298 external subjects undergoing indirect antiglobulin tests for various reasons, including Rh-negative pregnant women, being monitored for anti-D prophylaxis (E).
All the samples were tested, in a completely automated manner, with both the systems used in our Service, that is, with the Immucor Galileo (Immucor Italia srl, Opera, MI, Italy), which uses solid phase technology, and Ortho AutoVue Innova (Ortho-Clinical Diagnostics, Milan, Italy) which uses microcolumn technology. The aim was to compare the performance of the two systems used in a routine context.
It should be highlighted that the operative protocol (reported in diagram 1) was agreed upon with both the companies, which provided the reagents for carrying out the double testing. All the tests were carried out following their instructions scrupulously.
The samples were taken at the blood collection centre of the SIMT of Ragusa or in the wards of the Maria P aternò Arezzo Civil Hospital of Ragusa and analysed according to the agreed protocol.
The screening tests were carried out with red blood cell panels from the Immucor company (four-cell Capture-R Ready Screen, for the Immucor Galileo analyser) and from Ortho (three-cell Surgiscreen at 0.8±0.2%, column agglutination, for the OrthoAutoVue Innova analyser). The specificities of the antibodies were investigated using, respectively, the appropriate panels from Immucor (first panel: 13-cell Capture-R Ready-Id; second panel: 14-cell Extend I, D-positive; third panel: 14-cell Capture-R Ready-Id Extend II, D negative panel) and those from Ortho (first panel: Resolve Panel C Untreated; second panel: Resolve Panel B; third panel: Resolve Panel C Ficin Treated, all with 11 cells at 0.8±0.2%).
The search for irregular antibodies and the reading of the results were conducted using only these two automated methods; the data were interpreted by at least two different people.
The indirect antiglobulin test and the "type and screen" were carried out on the same day with the two automated systems, according to routine methodology in the SIMT.
Tests that gave positive or doubtful results were repeated the same day with both methods and the results reported on the registration forms. The samples, that were confirmed to be positive or that gave doubtful results in the screening tests, were stored at 4 °C for identification of the antibody specificity in the following days. A doubtful result was one, whose result in the automated screening was given as "NTD " by Immucor and as "?" by the Ortho system.
Results
Table I reports the total number of samples that were positive according to both screening tests, those positive only with the Immucor system and those positive only with the Ortho system.
Table I.
Number of samples found to be positive by the screening tests
| Samples positive with both systems | 78 |
| Samples positive only with Immucor | 18 |
| Samples positive only with Ortho | 11 |
| Total n. of samples positive with Immucor | 96 |
| Total n. of samples positive with Ortho | 89 |
Repeatability of the tests
The repeatability (or not) of the tests, expressed in absolute values and as percentages, is reported in table II. As can be seen, the repeatability was almost identical for the two systems.
Table II.
Results and repeatability of the screening tests with both systems
| Screening panels used | Positive, confirmed reactive (a) | Doubtful, confirmed reactive (b) | Doubtful, not confirmed reactive (c) | Repeatability (a)+(b) (a)+(b)+(c) |
|---|---|---|---|---|
| Immucor Capture-R Ready-Screen | 96/2,299 (4.2%) | 3/2,299 (0.13%) | 15/2,299 (0.6%) | 86.8% |
| Ortho Surgiscreen | 89/2,299 (3.9%) | 2/2,299 (0.08%) | 17/2,299 (0.7%) | 84.2% |
Results of the screening tests divided by system and category of samples
Table III presents the results obtained with the screening tests, divided according to the category of the samples examined and the red cell panels used for the screening tests.
Table III.
Results of the screening tests divided by category of samples and type of panels
| Results | A | DH | TS | E | Total |
|---|---|---|---|---|---|
| Immucor Capture-R Ready Screen | |||||
| Positive, confirmed reactive | 4/560
(0.7%) |
34/260
(13.1%) |
22/1,181
(1.9%) |
36/298
(12%) |
96/2,299
(4.2%) |
| Doubtful, confirmed reactive | 1/560
(0.2%) |
2/260
(0.8%) |
0/1,181
- |
0/298
- |
3/2,299
(0.13%) |
| Doubtful, not confirmed | 5/560
(0.9%) |
1/260
(0.4%) |
8/1,181
(0.7%) |
1/298
(0.3%) |
15/2,299
(0.6%) |
| Ortho Surgiscreen | |||||
| Positive, confirmed reactive | 2/560
(0.3%) |
34/260
(13.1%) |
18/1,181
(1.5%) |
35/298
(11.7%) |
89/2,999
(3.9%) |
| Doubtful, confirmed reactive | 1/560
(0.2%) |
1/260
(0.4%) |
0/1,181
- |
0/298
- |
2/2,299
(0.08%) |
| Doubtful, not confirmed | 2/560
(0.3%) |
5/260
(1.9%) |
10/1,181
(0.8%) |
0/298
- |
17/2,299
(0.7%) |
The 32 samples whose results were doubtful results and not confirmed, that is 15 samples examined with the Immucor system and 17 samples examined with the Ortho system, were considered negative and therefore identification of the antibody specificity was not undertaken. Only one of these 32 samples gave doubtful results not subsequently confirmed according to both the Immucor and Ortho screening tests.
Table IV reports the number of panels used to identify the antibodies, with the data divided according to the category of samples and the system used. Identification with the first panel was attributed only when the antibody specificity was certain.
Table IV.
Samples divided according to category, system used and number of panels necessary to reach a certain identification of the antibodies
| Number of panels used | A | DH | TS | E | Total |
|---|---|---|---|---|---|
| Immucor panels | |||||
| 1 panel | 1/2 | 10/21 | 9/15 | 17/27 | 37/65 (57%) |
| 3 panels | 1/2 | 11/21 | 6/15 | 10/27 | 28/65 (43%) |
| Ortho panels | |||||
| 1 panel | 1/2 | 10/20 | 7/13 | 13/26 | 31/61 (51%) |
| 3 panels | 1/2 | 10/20 | 6/13 | 13/26 | 30/61 (49%) |
Identification with "1 panel" means that the antibody specificity was determined using the Capture-R Ready- ID panel (for Immucor) or the Resolve Panel C panel (for Ortho), whereas identification with "3 panels" means that the antibody specificity was determined using, for Immucor, also the Capture-R Ready ID Extend I and Extend II panels or, for Ortho, also the Resolve Panel-C treated with ficin and Resolve Panel B.
Results of the panels for antibody identification
The use of the antibody identification panels, tested on samples that were positive according to either of the two systems, as defined above, gave the results shown in table V.
Table V.
Results of the use of panels for identifying the antibodies present in samples that gave positive results, subdivided by the testing system and outcome (Ab: antibodies, Id: identified)
| Panels used | Samples with Ab Id | Samples with Ab not Id | Panagglutinated samples |
|---|---|---|---|
| Immucor Panels | 65/96 (67.7%) | 19/96 (19.8%) | 12/96 (12.5%) |
| Ortho Panels | 61/89 (68.5%) | 22/89 (24.7%) | 6/89 (6.7%) |
The samples with unidentified antibodies reacted with only some cells of the panels used.
Despite this, it was not possible to reach a certain identification of the antibodies.
The panagglutinated samples were positive with all the cells of the panels used, including the screening panels. This made automated identification of the antibodies impossible.
Table VI reports the number of samples in which antibodies were identified, divided by category of sample and the system used.
Table VI.
Samples in which the antibodies were identified, divided by the system used and category of sample.
| Panels used | A | DH | TS | E | Total |
|---|---|---|---|---|---|
| Panels Immucor | 2/4 (50%) | 21/34 (61.8%) | 15/22 (68.2%) | 27/36 (75%) | 65/96 (67.7%) |
| Panels Ortho | 2/2 (100%) | 20/34 (58.8%) | 13/18 (72.2%) | 26/35 (74.3%) | 61/89 (68.5%) |
Overall, the use of the pertinent panels enabled the identification of 74 antibodies with the Immucor system and 68 antibodies with the Ortho system. These totals obviously include the samples with mixtures of antibodies (that is, with multiple specificities, table VII).
Table VII.
Antibodies identified, divided by the system and number of panels (in brackets) used to reach certain identification of the antibodies
| anti -D | anti -Cw | anti c | anti-E | anti-V | anti-K | anti-Kpa | anti-Jkb | anti-M | anti-Lea | Total | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Immucor panels | |||||||||||
| 28/39 (1); 11/39 (3) | 1/1 (3) | 1/1 (1) | 4/7 (1)
3/7 (3) |
1/1 (3) | 2/2 (1) | 1/2
1/2 |
(1)
(3) |
1/1 (3) | 1/1 (3) | 55 | |
| Multiple antibodies: 1: anti D+C (1), 1: anti D+V (3), 2: anti E+Cw (3); 1: anti Kpa+ 1 not identified (3); 1:anti K+ Kpa+ 1 not identified (3); 1: anti Jka+ Kpa (3); 1: anti E+ c (3); 2: anti c+ Jkb (3) | 19 | ||||||||||
| Total | 74 | ||||||||||
| Ortho panels | |||||||||||
| 18/34 (1); 16/34 (3) | 1/1 (1) | 2/2 (1) | 2/5 (1)
3/5 (3) |
1/2 (1)
1/2 (3) |
1/1 (1) | 2/2 | (3) | 1/1 (3) | 1/2 (1)
1/ 2 (3) |
1/1 (1) | 51 |
| Multiple antibodies: 2: anti D+C: 2/2 (1), 1: anti E+K (3), 2: anti E+Cw (1/2: 1; 1/2: 3), 1: anti K+ 1 not identified (3), 1: Jka+Kpa (3), 1: anti E+c (3), 1: anti c+anti Jkb (3) | 17 | ||||||||||
| Total | 68 | ||||||||||
Differences found in the specificities of the alloantibodies in samples positive according to both the screening methods
As shown in table I, 78 samples were positive according to both screening tests. Using the identification panels of the two systems the same antibody specificity was found in 51 out of 55 samples, whereas, in the other four samples, different specificities were found.
Table VIII reports the data on the positive samples, divided by category and results (concordant and discordant), whereas table IX presents the data on the four samples that gave discordant results, in relation to the type of panel used.
Table VIII.
Differences found in the specificities of antibodies (Ab), divided by category of the samples
| Category of samples | Positive samples with concordant Ab (%) | Positive samples with discordant Ab (%) |
| A | 2/2 (100%) | 0/2 (0%) |
| DH | 16/19 (84.2%) | 3/19 (15.8%) |
| TS | 11/11 (100%) | 0/11 (0%) |
| E | 22/23 (95.6%) | 1/23 (4.4%) |
| Total | 51/55 (92.7%) | 4/55 (7.3%) |
Table IX.
Antibodies (Ab) detected by the two systems in positive samples showing discordant specificities (the category of the samples is shown in brackets)
| Positive samples with discordant Ab | Immucor | Ortho |
|---|---|---|
| 1 (DH) | anti E | anti E+K |
| 2 (DH) | anti K+Kpa | anti K |
| 3 (DH) | anti D+Vel | anti D |
| 4 (E) | anti c+Jkb | anti c |
Positive screening results with only one of the methods used and identification of the antibodies
The comparison of the two systems showed that 18 out of 2,299 (0.78%) samples were positive with the Immucor screening test and negative with the Ortho screening test and 11 out of 2,299 (0.47%) samples were positive with the Ortho screening test and negative with the Immucor screening test (see also table I).
The panels used to identify the antibodies in these cases provided the results and specificities shown in table X.
Table X.
Antibodies (Ab) identified in the samples reacting positive in only one of the two screening tests used
| Panels used | Samples with unidentified Ab | Panagglutinated samples | Samples with identified Ab | Ab specificity | ||
|---|---|---|---|---|---|---|
| Immucor panels | 6/18
(33.3%) |
5/18
(27.7%) |
7/18
(38.9%) |
anti D:
5/18 |
anti E:
2/18 |
|
| Ortho panels | 7/11
(63.6%) |
1/11
(9.1%) |
3/11
(27.2%) |
anti D:
1/11 |
anti Lea:
1/11 |
anti Vel:
1/11 |
In conclusion, the use of the Immucor system led to the identification of 81 antibodies, while the Ortho system identified 71 antibodies.
Discussion
The screening tests for the presence of antibodies found 78 samples that were positive according to both systems. The Immucor system detected another 18 different samples that were positive and the Ortho system another 11 samples. Overall, therefore, the Immucor system detected 96 positive samples and the Ortho system 89 positive samples.
The repeatability of the tests with the Immucor Galileo system was 86.8%, whereas that of the Ortho AutoVue Innova system was 84.2%. The performance of the two systems was, therefore, essentially similar. Likewise, the two systems differed only slightly in the number of positive samples repeatedly reactive (4.2% for Immucor and 3.9% for Ortho).
It should be underscored again that the testing in this study was carried out completely as routine practice in our Service. Importantly, all the samples were handled in the same way and by the same staff, therefore eliminating the variability inherent in multicentre studies.
The considerable percentage of samples found to be positive in the screening tests is due to the large number of patients in our series with blood disorders (mostly thalassaemia), who have received multiple transfusions (DH): among these patients, the rate of positive results was 13.1%, and was identical according to both systems. The rate of positivity was slightly lower in the samples from external patients (E): 12% according to Immucor and 11.7% according to Ortho. It should be noted that these external subjects included pregnant women, whose positivity was usually due to antenatal administration of anti-D immunoglobulin for prophylactic purposes. The incidence of positive samples was clearly lower among the subjects undergoing surgery (TS), among whom the rate of positivity was 1.9% according to Immucor and 1.5% according to Ortho, and even lower among potential blood donors (A), whose rates of positivity ranged from 0.7% (Immucor system) to 0.3% (Ortho system).
The attempt to identify the antibodies not only in those samples positive in the screening test and confirmed as such in a second test, but also in those with doubtful results at screening and doubtful results again in the second test, enabled a thorough comparison of the two systems.
In the samples that were positive or doubtful in the screening tests, the use of the respective identification panels determined the antibody specificity in 65 out of 96 samples (67.7%) with the Immucor system and in 61 out of 89 samples (68.5%) with the Ortho system, these percentages being essentially the same.
On the other hand, in 32.3% of the cases investigated with the Immucor system and in 31.5% of these examined by the Ortho system, it was not possible to determine the specificity of the antibodies detected: the Immucor identification panels and the Ortho panels were unable to identify the antibodies in, respectively, 19 of 96 samples (19.8%) and 22 of 89 (24.7%), while the Immucor system showed panagglutination in 12 of the 96 samples (12.5%) and the Ortho system in 6 of 89 cases (6.7%).
The evaluation of the number of panels used to identify the antibodies showed a slight advantage for Immucor, which enabled the identification of 57% of the cases using only one panel, compared to 51% for Ortho. To the best of our knowledge, a comparison of this parameter has never previously been published.
As far as concerns the specificity of the antibodies, the Immucor system detected a higher number of antibodies than did the Ortho screening test (74 versus 68). The most frequent specificities were those of the Rh system, in particular anti-D, in direct relationship with antenatal prophylaxis against haemolytic disease of the newborn.
Investigations of the 78 samples that were positive according to both screening tests failed to identify the antibody specificity in 23 cases; in the other 55 cases the specificities were determined (although in four cases the results were not concordant).
Finally it should be noted that of the 2,999 samples screened, 18 (0.78%) were positive only according to the Immucor system and 11 (0.48%) only according to the Ortho system. Attempts to identify these antibodies were successful in 38.9% of the cases using Immucor and in 27.2% using the Ortho system.
Conclusions
The performances of the two systems were essentially similar.
The Immucor system detected a greater number of antibodies, mainly due to its greater sensitivity for anti-D antibodies, while maintaining a good specificity.
Testing with both systems was shown to be repeatable (in over 85% of the cases), bearing witness to the advantage of using automation in immunohaematological tests.
Both systems were able to identify the antibody, in a completely automated manner, in about 68% of the positive samples. The capacity of both systems to identify the antibodies decreased in direct relation to the complexity of the patients immunological profile.
The use of both technologies identified ten additional antibodies (specificities: 6 anti-D, 2 anti-E, 1 anti-Lea, 1 anti-Vel), that would not have been detected and identified, had only one of the methods been used. Having two technologies is an undoubted advantage, although systematic testing of all samples with two systems, as done in our study, is certainly not feasible.
Although the level of automation achieved was high, there is room for further improvement of this aspect, both in the management of the analytic phase and in the interpretation of the results.
Finally, it is to be hoped that the companies ensure consistency between the antigens present on the red blood cells used in the screening tests and those present in the identification panels, in order to avoid the occurrence of positive screening tests and negative results with the identification panels, because of the absence of some antigens from the relevant panels.
Acknowledgements
We thank the Ortho and Immucor companies for their kind collaboration and for having provided the reagents necessary for duplicating the tests.
We also thank the laboratory staff of the SIMT of Ragusa, who made this study possible, by doubling the number of tests carried out over the whole period of 6 months.
References
- 1.Molaro GL. L evoluzione tecnologica in immunoematologia eritrocitaria. The Ligand Quarterly. 1993;12:466–74. [Google Scholar]
- 2.Bunker LB, Thomas CL, Geyer SJ. Optimizing pretransfusion antibody detection and identification: a parallel, blinded comparison of tube PEG, solid-phase, and automated methods. Transfusion. 2001;41:621–6. doi: 10.1046/j.1537-2995.2001.41050621.x. [DOI] [PubMed] [Google Scholar]
- 3.Poole GD, Evans RG, Voak D, et al. Evaluation of six systems for the detection of red cell antibodies. In: Chapman J, Grgicevic D, Dubrovnik, editors. Quality Assurance in Transfusion Medicine; Proceedings of the ESTM residential course; 23–26 April 1998; pp. 153–200. [Google Scholar]
- 4.Rossi D, Tasca R, Borzini P, et al. Indagine multicentrica sull opportunità di impiegare un panello eritrocitario recante antigeni rari nell ambito del Type and Screen (T&S), gel sedimentazione versus fase solida. Atti del XXXII Convegno Nazionale di Studi di Medicina Trasfusionale; Taormina. 25–29 May 1996; pp. 140–2. [Google Scholar]
- 5.Stute R, Patek A. Comparison of three different methods of antibody screening and identification (P410). Proceedings of ISBT Congress; Paris. 15–18/07/2001. [Google Scholar]
- 6.Weisbach V, Kophnhauser T, Strasser E, et al. Comparison of the performance of seven different test systems in the detection of red cell alloantibodies. Transf Med Hemother. 2003;30:117–22. [Google Scholar]
- 7.Tenali EL, Watson CR. The evaluation of solid phase and gel technology in antibody screening and identification: a comparative study (P1488). Proceedings of the ISBT Congress; Oslo. 27/6–2/7/1998. [Google Scholar]
- 8.Chabert T, Rodrigues M, Brilhante D, et al. Comparative multicentric study between two antibody screening and identification technologies (P1498). Proceedings of the ISBT Congress; Oslo. 27/6–2/7/1998. [Google Scholar]
- 9.Garozzo G, Miceli A, Bonomo P. Ricerca di anticorpi irregolari in 992 emoglobinopatici politrasfusi: confronto tra due sistemi di screening e di identificazione. La Trasf del Sangue. 2001;46:384–94. [Google Scholar]
- 10.Schonewille H, Haak HL, van Zijl AM. Alloimmunization after blood transfusion in patients with hematologic and oncologic diseases. Transfusion. 1999;39:763–71. doi: 10.1046/j.1537-2995.1999.39070763.x. [DOI] [PubMed] [Google Scholar]
- 11.Sallander S, Shanwell A, Aqvist M. Evaluation of a solid-phase test for erythrocyte antibody screening of pregnant women, patients and blood donors. Vox Sang. 1996;71:211–5. doi: 10.1046/j.1423-0410.1996.7140221.x. [DOI] [PubMed] [Google Scholar]