WHO/BS/10.2149 ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 18 to 22 October 2010
A Proposed 1
stWHO Reference Reagent RHD/SRY Plasma DNA sensitivity standard
07/222
Paul Metcalfe1, Peter Rigsby2, Evelyn Tait3, Stan Urbaniak3
1National Institute for Biological Standards and Control Potters Bar, Hertfordshire, UK
2University of Aberdeen, Regional Transfusion Centre, Aberdeen, UK
1Biotherapeutics Group, 2Biostatistics, NIBSC, Blanche Lane, South Mimms, Herts UK, EN6 3QG 3University of Aberdeen, Regional Transfusion Centre, Aberdeen, UK
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WHO/BS/10.2149 Page 2
Summary
Nineteen laboratories participated in an international collaborative study to assess the suitability of a freeze-dried plasma preparation (code number 07/222) as a WHO Reference Reagent for the detection of RHD and SRY genes using PCR. The participants evaluated dilutions of the material in their own in-house routine assays and recorded the highest dilution where the genes could be detected.
The material is intended for use as a minimum sensitivity standard which laboratories can use to assess the sensitivity of their own techniques. Conclusions from this study indicated that a dilution of 1 in 2 would be a suitable minimum dilution at which both RHD and SRY should be detected. All of the study participants approved this recommendation.
Introduction
The determination of fetal RHD genotype using fetal DNA contained in the maternal circulation is increasingly used to manage pregnancies at risk for haemolytic disease of the newborn (HDN) caused by maternal anti-D. The test offers significant advantages over previous procedures which relied on fetal DNA extracted from amniotic fluid or chorionic villi, obtained from invasive procedures. The detection of RHD sequences is usually carried out by real-time PCR techniques and a positive result is used to predict fetal D status. At the same time laboratories usually test for sequences unique to the Y chromosome, where any positive result indicates that (male) fetal DNA has been extracted and was present in the sample. However, there is no confirmatory test for female fetuses.
The test is becoming increasingly reliable and routine clinical services have been established in some centres. However, laboratories currently have no materials with which to determine the performance of their tests. We have therefore produced a material that can be used as a
minimum sensitivity reagent and could be replaced from the same source if required. It will be of paramount importance for laboratories setting up new in-house methods or commercial techniques for the detection of RHD sequences, and for validating existing techniques after a change of reagents, operator or equipment.
Aims of study
The aim of this international collaborative study was to test the suitability of the proposed material as a WHO Reference Reagent (minimum potency). The results of the study allowed us to establish a dilution at which most laboratories can be expected to detect the RHD gene.
WHO/BS/10.2149 Page 3
Participants
Thirty-five laboratories were invited to take part in the study, 21 laboratories agreed to participate and results were returned by 19 laboratories (Appendix 1). Seventeen different countries were represented among participants returning results. Each laboratory has been assigned a code number which does not reflect the order of listing in the table.
Materials
The candidate material 07/222 is RhD positive male plasma diluted in an excess of RhD negative female plasma, collected from blood donors attending Scottish National Blood and Transfusion Service (SNBTS) Aberdeen who both gave specific consent for this use. No other stabilisers or bulking agents were added. Both plasmas were tested and found negative for antibodies against HIV1, HCV, and HBV. PCR tests for HCV and HIV on both plasmas were also negative. The RhD phenotype of both donors was confirmed by serological methods and the genotype was confirmed by PCR. 1mL aliquots of the pooled plasma were freeze-dried in glass ampoules for long-term stability and the material has performed well in pilot studies. Table 1 shows the product summary for the material.
Table 1. Product summary of the candidate material.
NIBSC Code 07/222
Date filled 07.09.07.
Coefficient of variation of the fill (%) (n=81) 0.17 Residual moisture after lyophilisation (%) (n=6) 0.28
Mean dry weight (g) (n=6) 0.0703
Mean residual oxygen% (n=6) 0.21
No of ampoules available 1797
Presentation Sealed, glass 3mL DIN ampoules
Excipient none
Address of facility where material was processed NIBSC, Potters Bar, Herts, UK
Present custodian NIBSC, Potters Bar, Herts, UK
Storage temperature -20oC
This standard is intended to be used in the in vitro diagnostics field and it relates to BS EN ISO 17511:2003 Section 5.6.
Stability studies
Accelerated degradation studies [5] were carried out on samples stored at elevated temperatures compared with others stored at -70°C (see table 2 below) using real-time quantitative PCR (Q- PCR) with two RHD targets (exons 5 & 7) and SRY.
WHO/BS/10.2149 Page 4
Table 2. Samples tested in accelerated degradation studies.
Storage temperature Duration of storage
-70oC -20oC +4oC +20oC +37oC +45oC +56oC
4 months
8 months
15 months
23 months
To date, no degradation has been detected in any of the samples stored at elevated temperatures, compared to samples stored at -70°C, however, testing will continue at regular intervals. It was not possible to test samples stored at +56oC and +45oC after 4 months and 8 months respectively, because freeze dried plasma stored at these temperatures for more than a short time becomes increasingly difficult to reconstitute fully. Samples stored at lower temperatures were not tested at these relatively early time-points because no degradation could be detected at significantly higher temperatures and also, previous has shown DNA to be extremely stable when freeze- dried.
Study design
Eight ampoules of the material coded 07/222 were sent to each laboratory with instructions for reconstitution and storage. Participants were asked to test the material using the PCR assays that they currently use for routine clinical testing and that they should use the same criteria usually used for deciding whether the RHD gene, and the control gene if applicable, is present or absent.
On Day 1, up to four ampoules were reconstituted and after extraction the DNA was pooled before subsequent dilution and testing. The number of ampoules used depended on the number of RHD exons and control genes routinely tested and, consequently, the amount of extracted DNA required. Enough material was required to make doubling dilutions of the pooled DNA in nuclease-free water for all the tests to be carried out in triplicate. The dilutions were tested in the same tests as the undiluted sample. On another day (Day 2) the test was repeated in the same way with the remaining ampoules and different test reagents (and a different operator if possible).
The RHD-positive signal was already fairly weak and we did not expect any positive reactions to be seen beyond the 1 in 8 dilution, but laboratories were advised that if they could detect the RHD or control genes at this dilution then the dilution series should be continued.
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Laboratories using real-time PCR were asked to report the cycle threshold (CT) or crossing point (CP) in whole numbers. For each dilution and for each of the targets tested, labs were asked to interpret their data and indicate whether they considered the target to be present or absent. They were also asked to consider the results from all the individual targets and make an overall
interpretation of whether the RHD gene and the control gene was present or absent. The CTs/CPs and an interpretation of the results were also requested for all tests involving targets on the Y chromosome.
Methods
The methods used by the participants are shown in Table 2. Seventeen laboratories used in- house real-time PCR assays and one laboratory used a multiplex endpoint-PCR method. No two methods were entirely identical and some of the critical elements are shown in Table 3.
Table 3. Methods used by participants.
Lab no.
DNA extraction method
Volume of plasma extracted
µL
Elution volume
µL
Volume of eluate used/PCR
µL
Total PCR volume
µL
RHD detection method [reference]
1 Q. DSP virus kit 1000 55 9 20 In-house QPCR
2 R. MagNA Pure 1000 50 5 20 In-house QPCR
3 Q. MinElute virus kit 1000 60 7.5 25 In-house QPCR
4 Q. DSP Blood Mini kit 800 60 5 25 In-house QPCR
5 Chemagic viral kit 1000 100 5 - 15 50 In-house QPCR 6 Q. Blood Mini kit 1000 60 3 - 9 25-50 In-house QPCR
7 Q. Blood kit 800 55 5 25 In-house QPCR
8 R. MagNA Pure
Compact 1000 50 5 20 In-house QPCR
9 Q. MinElute virus kit 1000 55 5 25 QPCR based on [1, 2]
10 Q. DSP virus kit 1000 60 5 25 QPCR [2]
11 Q. EZ1 virus kit v2 400 60 5 20 In-house QPCR and [2]
12 Q. DNA Blood kit 200 60 17.5 25 QPCR SAFE protocol
based on [3]
13 Q. EZ1 forensic card
tissue kit 200 50 6 25 In-house QPCR
14 B. easyMAG platform 1000 25 10 25 In-house QPCR
15 Q. Blood Mini kit 800 50 8 20 In-house QPCR
16 Q. Blood Mini kit 800-2000 55-60 2.5 12.5 (none supplied)
17 Q. DSP virus kit 500 50 15 50 Multiplex PCR
(not QPCR)
18 Q. Blood Mini kit 1000 40 9 - 12 25 QPCR [4]
19 Q. DSP virus kit 1000 60 7.5 - 15 25 - 50 In-house QPCR Key. QPCR: real-time PCR Q: Qiagen R: Roche B: bioMérieux
The targets used to detect the RHD and control genes are shown in Table 4. Laboratories were asked to list the targets that they use for the investigation of individual clinical cases and for
WHO/BS/10.2149 Page 6
routine antenatal screening, if applicable, as in some cases the selection is different. Six
different targets were used to detect the RHD gene and three different targets were used to detect DNA sequences on the Y chromosome.
Table 4. RHD and control genes used for analysis.
RHD target
Exon Intron
Y chromosome Specific target Lab.
no.
3 4 5 7 10 4 SRY AMELY DYS14 1 D
S
2 D
3 D
4 D S
5 D
S
6 D
7 D
8 D S
9 D
10 D
11 D S
12 S
13 D S
14 D
15 D
16 D
17 D
18 D
19 D S
Key. D: markers used in diagnostic testing S: markers used in routine antenatal screening SRY: Sex-determining Region Y AMELY: Amelogenin-Y
DYS14: Testes-specific Y-encoded protein1
Results
All laboratories were able to generate useful results from the samples; the raw data is shown in Appendix II along with the laboratory’s interpretation of their own results. Lab. 9 did not interpret the results of individual targets but did make an overall conclusion for each dilution.
Lab. 17 was the only to use end-point PCR rather than real-time PCR, so only their interpretation
WHO/BS/10.2149 Page 7
of the results is shown. Six laboratories continued the dilution series beyond 1 in 8 and their results are shown in Appendix III.
The overall interpretation of tests for the RHD gene in each dilution, tested on two separate occasions, is shown in figure 1 below. Two laboratories (10 and 16) were unable to conclude that there were any RHD sequences present, even in the undiluted material. Reproducibility within laboratories was good; 11 labs achieved the same result on both days, 6 labs were only one dilution apart and only 2 labs reported results two dilutions apart.
Figure 1. Overall interpretation, last dilution reported as RHD gene present
14 14 13 11
11 17
8 15 17
13 7 9 19
12 7 8 18
16 12 4 6 18
16 9 2 6 15
10 4 1 3 5
10 3 1 2 5 19
No +ve neat 1 in 2 1 in 4 1 in 8 1in 16 1in 32 Key. Numbers in boxes indicate laboratory number
No +ve: no overall RHD positive conclusions for any dilution.
Figure 2 shows the laboratories interpretation of their individual targets for the RHD gene. The distribution of results is different to figure 1 because there were several instances where
laboratories could detect RHD sequences with one target at a particular dilution, but their overall conclusion was negative. The relative sensitivity of the different RHD targets was compared by analysis of variance of the geometric mean dilution for each lab/target. The results showed no significant differences between targets (p=0.670). Where enough laboratories had used more than one target the relative sensitivities of the techniques were compared using a paired t-test.
This was possible for Exon 5 vs Exon 7 (n=8 labs; not significant, p=0.351), Exon 7 vs Exon 10 (n=7 labs; not significant, p=0.411), Exon 5 vs Exon 10 (n=7 labs; significant, p=0.023). The final (significant) p-value may suggest that the Exon 5 assays are more sensitive than the Exon 10 assays.
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Figure 2. Interpretation of individual RHD targets, last dilution reported as RHD gene present.
14 7 7 17 17 19
15 Exon 3
2 10 14 Exon 4
2 10 14 Exon 5
14 15 5 Exon 7
4 13 5 Exon 10
12 3 19 Intron 4
11 3 8
11 13 6
1 12 6
1 11 5
11 13 10 3 19
11 11 3 18 6
16 4 10 17 18 6
16 13 8 17 15 19
16 12 14 15 7 7
16 12 14 8 7 7
16 13 13 8 2 5
16 11 11 4 2 4
10 10 4 1 1 4
No +ve neat 1 in 2 1 in 4 1 in 8 1in 16 1in 32 Key. Numbers in boxes indicate laboratory number.
No +ve: no RHD target present in any dilution.
Figure 3 shows the laboratories interpretation of their individual targets for the Y chromosome. It was not possible to compare the relative sensitivity of the different targets because of the small number of labs using a target other than SRY. Reproducibility within laboratories was not as good as with RHD detection; 5 labs achieved the same result on both days, 10 labs were only one dilution apart, 2 labs were two dilutions apart and 1 lab, which used two targets, reported results three and four dilutions apart.
WHO/BS/10.2149 Page 9
When the results for overall RHD interpretation (figure 1) were compared to Y chromosome interpretation (figure 3) using an unpaired t-test, no significant difference in sensitivity (p=0.722) was demonstrated.
Figure 3. Interpretation of individual Y chromosome-specific genes, last dilution reported as target present.
19 DYS14
17 AMELY
2 15 SRY
12 15
12 13
14 13
11 11
4 14 9 9 2
19 10 8 8 13
16 5 6 7 19
16 4 5 7 17
10 1 1 6 18 13 19
No +ve neat 1 in 2 1 in 4 1 in 8 1in 16 1 in 32 1 in 64 1 in 128 Key. Numbers in boxes indicate laboratory number
No +ve: no Y chromosome target present in any dilution.
Participant Responses
Participants were offered the chance to comment on the materials supplied and the way the study was organised. Apart from agreeing with the proposal, most made no comment, but three labs were very positive about the materials and the study design. One laboratory commented that the reconstituted material could not be extracted from the ampoules with their routinely used pipettes. The ampoules used for this material were specifically designed to allow the bottom of the ampoule to be in reach of a standard 200µL tip and this was the only comment of a similar nature that was received. The same laboratory commented that the SRY signal was much weaker than the RHD signals, but this was not the case in most laboratories.
Discussion
Genetic testing is widespread but few reference materials exist. The first International Reference Panel was approved by WHO in November 2004 and comprised a panel of three freeze-dried
WHO/BS/10.2149 Page 10
preparations for the genetic diagnosis of Factor V Leiden. Since then WHO has established genetic reference materials for the Prothrombin Mutation G20210A, Haemophilia A Intron 22 inversion, Fragile X syndrome, Prader-Willi and Angelman syndromes, and the quantitation of BCR-ABL translocation in leukaemia.
Non-invasive pre-natal diagnosis (NIPD) which utilises the fetal DNA circulating in the maternal blood is being used increasingly in several situations, these include: the determination of fetal sex in families at risk of a sex-linked disease, the detection of specific single gene disorders, the detection of fetal aneuploidy (particularly Down’s syndrome), and the determination of fetal RhD status. Currently, laboratories carry out fetal RHD genotyping in two situations, diagnostic testing and routine antenatal screening. Diagnostic testing involves those women at high risk for HDN, either because of a rising anti-D titre or a previously affected pregnancy. Routine
antenatal screening of all D-negative women booking in for antenatal care is a more recent development and the introduction of such a service has been shown to be feasible in the Netherlands, Germany and the UK. Accurate fetal RHD genotyping at an early stage of
pregnancy could avoid invasive testing in the first group and unnecessary administration of anti- D to women carrying D-negative fetuses in the second group. Currently, laboratories setting up such tests have no certified reference materials with which to determine their performance.
NIBSC was approached by the Special Non-invasive Advances in Fetal and Neonatal Evaluation (SAFE) Network of Excellence (funded by the EU framework 6 programme 2004-2009) to make a stable material for use in RHD testing and validate it in a large scale study. It would not be possible to obtain sufficient plasma from an individual pregnant woman and pooled samples would not allow the use of insertion/deletion or SNP genotyping controls which are being tested by some labs as a means of identifying fetal DNA. Instead, RhD positive male plasma was diluted in an excess of RhD negative female plasma to make the reference material. Both
samples were obtained from adult blood donors. Adult cell-free DNA (cfDNA) is similar to fetal cfDNA, though the plasma concentration is higher and the fragments are larger. It would be possible to obtain more materials from the same source in the future should the present preparation be used up.
Nineteen laboratories participated in the current international collaborative study to assess the suitability of a freeze-dried plasma preparation as a WHO Reference Reagent for the detection of RHD and SRY genes using PCR. The study was designed to determine how well the material performed in a large number of laboratories, using a variety of methods, and which dilution was the most appropriate to set as the minimum dilution that routine laboratories could expect to detect both genes. Laboratories were asked to use their routine techniques, rather than a prescribed protocol, as these would be the tests utilised for routine patient testing.
Four out of 19 laboratories could not detect RHD or SRY on one or both occasions, even in the undiluted material, clearly demonstrating the need for improvements in sensitivity in some laboratories. However, overall the study showed that the materials are suitable for use as a reference material and we propose that the minimum sensitivity required should be set at a dilution of 1 in 2. This proposal was endorsed by all participants in the study and the
WHO/BS/10.2149 Page 11
International Society for Blood Transfusion (ISBT) Working Party on Red Cell Immunohaematology and Blood Group Terminology, see Appendix V.
Conclusions and Proposal
The results of this international multi-centre study show that the material 07/222 is suitable for use as a sensitivity standard for the measurement of DNA in plasma and it is proposed that the above material be established as the WHO 1st International Reference Reagent, RHD/SRY Plasma DNA sensitivity standard.
Acknowledgements
The authors would like to thank SNBTS Aberdeen for supplying the materials and the participants of the study for taking part and returning data.
References
1. Finning, K, Martin, P. Daniels, G. A Clinical Service in the UK to Predict Fetal Rh (Rhesus) D Blood Group Using Free Fetal DNA in Maternal Plasma. Ann N Y Acad Sci 2004;1022:119-23.
2. Finning K, Martin P, Soothill PW, Avent ND. Prediction of fetal D status from maternal plasma: introduction of a new non-invasive fetal RHD genotyping service. Transfusion 2002; 42:1079-85.
3. Legler, T, Liu Z, Mavrou, A et al. Workshop report on the extraction of foetal DNA from maternal plasma. Prenat Diagn 2007;27:824–29.
4. Lo, YM, Magnus Hjelm N, Fidler C, et al. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med 1998;339:1734-38.
5. Kirkwood TBL. Predicting the stability of biological standards and products. Biometrics 1977;33:736-742.
WHO/BS/10.2149 Page 12
Appendix I. List of Participants.
Name Institute Country
Annie Levy-Mozziconacci Université de la Méditerranée, and Assistance
Publique des Hôpitaux de Marseille, Marseille France Aggeliki Kolialexi,
Ariadni Mavrou, Georgia Tounta
Athens University School of Medicine, Athens Greece
Andrea Doescher German Red Cross Blood Transfusion Service,
Oldenburg Germany
Åsa Hellberg Clinical Immunology and Transfusion Medicine, Laboratory Medicine Skåne
Sweden Catherine Hyland,
Helen O’Brien, Glenda Millard
Australian Red Cross Blood Service, Brisbane Australia
Evelyn Tait,
Stan Urbaniak University of Aberdeen, Aberdeen UK
Ewa Brojer, Katarzyna Guz, Agnieska Orzinska
Institute of Haematology & Transfusion Medicine,
Warsaw Poland
Geoff Daniels,
Peter Martin International Blood Group Reference Laboratory,
Bristol UK
Giuliana Coata Ospedale “S. Maria della Misericordia”, Centro Didattico-Università degli Studi di Perugia
Italy Glenice Cheetham Institute of Medical and Veterinary Science, Adelaide Australia Janet Carvalho Pereira Centro Hospitalar de Coimbra, Coimbra Portugal
Masja de Haas Sanquin, Amsterdam Netherlands
Mette Christiansen Århus University Hospital, Skejby Denmark Miryam Martinetti Servizio di Immunoematologia e Medicina
Transfusionale, Pavia
Italy
Nuria Nogues Banc de Sang i Teixits, Barcelona Spain
Reena Ray Clinical Biochemistry & Genetics Diagnostic Services of Manitoba
Canada I C Verma, Renu Saxena
Sudha Kohli Sir Ganga Ram Hospital, New Delhi India
Eduardo Levi , Silvano Wendel
Hospital Sírio Libanês, São Paulo Brazil Tadeja Dovc Drnovsek Blood Transfusion Centre of Slovenia, Ljubljana Slovenia Tobias J. Legler,
Susanne Holzhausen, Manuela Peter
University Medical Center, Göttingen Germany
Ziyan Zhu Shanghai Blood Center, Shanghai China
WHO/BS/10.2149 Page 13
Appendix II. Real-time PCR results expressed as cycle threshold (CT) and interpretation of results (in bold).
Dilution of 07/222 Lab.
No. Target Day Neat 1 in 2 1 in 4 1 in 8
1 RHD Exon 7
RHD Exon 10
RHD Exon 7 RHD Exon 10
SRY SRY
1 1 1 2 2 2 1 2
36, 36, 37 P 34, 34, 35 P RHD Positive 36, 36, 36 P 36, 36, 36 P RHD Positive 35, 35, U M 37, 38, 37 M
35, 35, 36 P 34, 35, 35 P RHD Positive 36, 36, 38 P 36, 35, 37 P RHD Positive U, U, 36 - 38, 37, 36 M
U, U, U - 36, U, 36 P inconclusive 35, U, U - 36, 36, 36 P inconclusive U, U, U, - U, U, 36 -
U, 35, U - 36, U, 36 P inconclusive U, U, U - U, U, 37 - RHD negative U, U, 37 - 36, U, U -
2 RHD Exon 3
RHD Exon 10
RHD Exon 3 RHD Exon 10
AMG AMG
1 1 1 2 2 2 1 2
34, 35, 35 P 37, 38, 38 P RHD Positive 34, 33, 35 P 37, 36, 38 P RHD Positive 36, 36, 36 M 37, 37, 36 M
36, 35, 35 P 38, 37, 37 P RHD Positive 34, 36, 35 P 40, 38, 38 P RHD Positive 37, 38, 38 M 39, 38, 37 M
35, >40, >40 ? 37, 38, 36 P RHD Positive 39, >40, >40 ? 39, 39, 39 ? no interpretation 38, 37, 38 M 38, >40, >40 -
35, >40, >40 ? 38, 38, 39 P inconclusive 36, >40, >40 ? 38, 37, 38 P no interpretation 38, 42, 38 M
>40, >40, >40 -
3 RHD Exon 5
RHD Exon 7
RHD Exon 5 RHD Exon 7
1 1 1 2 2 2
34, 38, 36 P 38, 36, 39 P RHD Positive 44, 38, 36 P 37, 38, 38 P RHD Positive
36, 37, 37 P 38, 38, 39 P RHD Positive 37, 37, U P U,U,U - RHD negative
38, 36, 36 P 39, U, 39 P RHD Positive U, U, 38 - U, 39, 39 P RHD negative
U, 47, U - 40, U, U - RHD negative 38, U, 38 P U,U,U - RHD negative
4 RHD Exon 4
RHD Exon 5 RHD Exon 10
RHD Exon 4 RHD Exon 5 RHD Exon 10
SRY SRY
1 1 1 1 2 2 2 2 1 2
44, 42, 42, 42 P 37, 40, 38, 38 P 37, U, 38, 37 P RHD Positive 41, 41, 41, 41 P 38, 38, 38, 38 P 40, 40, 40, 40 P RHD Positive U, 38, 37, 36 M 42, 44, 45, 43 -
41, 41, 42, 44 P 39, 38, U, 41 P 37, 37, 38, U P RHD Positive 41, 42, U, U, - U, 38, 39, 40 P 40, 41, U, U, P inconclusive U, U, U, U - U, 42, U, 43 -
U, U, 43, 42 - 39, 42, 40, 40 P 37, U, U, 38 P inconclusive 44, 44, U, 42, - U, 40, 40, 38 P U, U, 43, 42 - inconclusive U, U, U, 36 - U, 43, 42, U, -
U, 44, U, 43 - 39, 40, 41, 40 P U, U, 39, U - inconclusive U, 44, 44, 42, - 40, 40, U, U P U, U, 43, U - inconclusive U, U, U, U - U, U, U, U -
5 RHD Exon 5
RHD Exon 7
RHD Exon 5 RHD Exon 7
SRY SRY
1 1 1 2 2 2 1 2
38, 37, 37 P 38, 37, 38 P RHD Positive 37, 38, 38 P 37, 36, 37 P RHD Positive 39, 39, 50 M 38, 38, 38 M
39, 39, 38 P 38, 38, 38 P RHD Positive 39, 38, 39 P 39, 39, 38 P RHD Positive 50, 50, 40 - 40, 40, 50 M
43, 39, 42 P 38, 41, 39 P RHD Positive 40, 39, 40 P 38, 40, 40 P RHD Positive 47, 50, 50 - 40, 50, 50 -
40, 40, 50 P 40, 40, 41 P RHD Positive 39, 39, 41 P 41, 50, 41 P RHD Positive 40, 50, 50 - 50, 50, 50 - Key. P:RHD target present -: target absent M: male fetal DNA present U: undetected ?:inconclusive
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Appendix II (continued). Real-time PCR results expressed as cycle threshold (CT) and interpretation of results (in bold).
Dilution of 07/222 Lab. Target Day
neat 1 in 2 1 in 4 1 in 8
6 RHD Exon 5
RHD Exon 7
RHD Exon 5 RHD Exon 7
SRY SRY
1 1 1 2 2 2 1 2
36, 36, 36 P 36, 38, 36 P RHD Positive 37, 36, 36 P 36, 36, 36 P RHD Positive 40, 37, 45 M 37, 37, 38 M
39, 38, 39 P 37, 40, 38 P RHD Positive 37, 38, 37 P 37, 37, 36 P RHD Positive 37, 38, 50 M 38, 39, 50 M
38, 50, 38 P 38, 39, 50 P RHD Positive 38, 38, 37 P 50, 37, 38 P RHD Positive 38, 50, 50 - 50, 40, 39 M
40, 39, 50 P 50, 45, 37 ? Inconclusive 38, 38, 50 P 50, 40, 50 - Inconclusive 39, 50, 50 - 50, 50, 39 -
7 RHD Exon 4
RHD Exon 5 RHD Exon 10
RHD Exon 4 RHD Exon 5 RHD Exon 10
SRY SRY
1 1 1 1 2 2 2 2 1 2
39, 38, 39, 40 P 36, 35, 35, 35 P 39, 38, 38, 40 P RHD Positive 38, 38, 39, 37 P 39, 35, 35, 36 P 39, 39, 38, 40 P RHD Positive 38, 38, 38, 36 M 37, 36, 41, 39 M
40, 39, 40, 41 P 37, 37, 38, 38 P 38, 39, 39, 40 P RHD Positive 39, 38, 39, 39 P 37, U, 36, 37 P 39, 37, 39, 39 P RHD Positive 38, 39, 39, U M 36, 37, 36, 37 M
39, 41, U, U P 37, 37, 38, U P 38, 39, U, U P RHD negative 38, 40, U, 38 P 37, U, U, 37 P 40, U, U, 38 P RHD negative 37, 38, U, U M 39, U, 38, 37 M
39, 39, U, U P 37, U, U, U - 39, 39, U, U P RHD negative 43, 40, 39, 39 P 38, U, U, U - 39, U, U, 39 P RHD negative 37, U, U, U - 39, U, U, U -
8 RHD Exon 5
RHD Exon 10
RHD Exon 5 RHD Exon 10
SRY SRY
1 1 1 2 2 2 1 2
36, 36, 36 P 36, 37, 34 P RHD Positive 38, 36, 36 P 38, 36, 39 P RHD Positive 35, 35, 37 M 37, 35, 37 M
38, 37, 36 P 37, 36, 37 P RHD Positive 36, 38, 37 P 38, 37, U P RHD Positive 38, 38, M 38, 38, U M
U, U, U - 36, 37, U P Inconclusive 38, 38, U P 37, 39, U P RHD Positive 40, 38, U M 39, U, U ?
38, U, U ? 39, U, U ? Inconclusive 39, 38, 39 P U, U, U - Inconclusive U, U, U - U, U, U -
9 RHD Exon 4
RHD Exon 5 RHD Exon 10
RHD Exon 4 RHD Exon 5 RHD Exon 10
SRY SRY
1 1 1 1 2 2 2 2 1 2
36, U, 39, 39 34, 35, 40, 34 36, 35, 35, 35 RHD Positive 39, 38, 37, 40 31, 32, 32, 33 34, 35, 35, 34 RHD Positive 38, 39, 38, 36 M 34, 34, 35, 34 M
U, U, U, 38 U, 35, 34, 34 35, 36, 39, 38 Inconclusive 37, 39, 36, 38 34, 33, 33, 32 36, 37, 35,36 RHD Positive U, 38, U, 39 M 37, U, U, 36 M
U, U, 39, U 34, 34, U, 35 U, U, U, U Inconclusive 37, 39, 38, 38 33, 35, 33, 33 35, 35, 36, 36 RHD Positive U, U, 37, U ? 37, 35, 36, U M
40, U, U, U U, U, 34, 35 U, U, 36, 37 Inconclusive 38, 38, U, U U, U, U, 34 38, U, U, U Inconclusive U, U, U, 40 ? U, U, 37, U ?
9 RHD Exon 4
RHD Exon 5 RHD Exon 10
RHD Exon 4 RHD Exon 5 RHD Exon 10
SRY SRY
1 1 1 1 2 2 2 2 1 2
36, U, 39, 39 34, 35, 40, 34 36, 35, 35, 35 RHD Positive 39, 38, 37, 40 31, 32, 32, 33 34, 35, 35, 34 RHD Positive 38, 39, 38, 36 M 34, 34, 35, 34 M
U, U, U, 38 U, 35, 34, 34 35, 36, 39, 38 Inconclusive 37, 39, 36, 38 34, 33, 33, 32 36, 37, 35,36 RHD Positive U, 38, U, 39 M 37, U, U, 36 M
U, U, 39, U 34, 34, U, 35 U, U, U, U Inconclusive 37, 39, 38, 38 33, 35, 33, 33 35, 35, 36, 36 RHD Positive U, U, 37, U ? 37, 35, 36, U M
40, U, U, U U, U, 34, 35 U, U, 36, 37 Inconclusive 38, 38, U, U U, U, U, 34 38, U, U, U Inconclusive U, U, U, 40 ? U, U, 37, U ?
WHO/BS/10.2149 Page 15
Appendix II (continued). Real-time PCR results expressed as cycle threshold (CT) and interpretation of results (in bold).
Dilution of 07/222 Lab. Target Day
neat 1 in 2 1 in 4 1 in 8
10 RHD Exon 4
RHD Exon 5 RHD Exon 10
RHD Exon 4 RHD Exon 5 RHD Exon 10
SRY SRY
1 1 1 1 2 2 2 2 1 2
42, 40, 41, 39 P 38, 38, 38, 39 P 44, 43, 41 - Inconclusive 39 - 39, 37, 38, 38 P 41, 41, 41 P Inconclusive 38 - 39,41,41 M
41, 39 P 38 P 43 - Inconclusive 42,43,41 P 39, 38, 38, 39 P 41 - Inconclusive 40 - 39 -
41, 40, 41 P 41, 39, 39 P 43 - Inconclusive 40, 41, 43 P 40 - 41, 43 - Inconclusive 41 - U -
44 - U - 41, 42 P Inconclusive 41 - 39 - U - Inconclusive 39 - 40, 41 M
11 RHD Exon 5
RHD Exon 7 RHD Exon 10 RHD intron 4
RHD Exon 5 RHD Exon 7 RHD Exon 10 RHD intron 4
SRY SRY
1 1 1 1 1 2 2 2 2 2 1 2
38, 38, 38 P U, U, U - 38, U, 39 P 41, 41, 40 P RHD Positive 38, U, 37 P U, U, 38 - U, 38, U - 40, 38, U P RHD Positive U, 37, 40 M U, 39, 39 M
U, 39, 39 P 38, U, 38 P U, U, U - 41, U, 39 - RHD Positive 38, 38, 39 P 38, 38, U P U, 40, 40 P U, U, 40 - RHD Positive 39, 39, U M 38, 39, 39 M
39, 39, U P U, U, U - U, U, U - U, U, 39 - RHD negative 38, U, U - 38, U, U - U, 39, U - 40, U, U - RHD negative 39, U, 38 M U, 39 -
U, U, U - U, 38, U - 45, 40, U - U, U, U - RHD negative U, 39, U - 38, U, U - U, U, 41 - 40, U, U, - RHD negative 39, U, U - U, U, U -
12 RHD Exon 5
RHD Exon 7
RHD Exon 5 RHD Exon 7
DYS14 DYS14
1 1 1 2 2 2 1 2
36, 37, 38 P 38, 40, U P RHD Positive 37, 39, 39 P 37, 38, 37 P RHD Positive 35, 35, U, 35 M 36, 35, 36, 36 M
U, 37, U - 38, U, 38 P Inconclusive 39, U, U - 36, U, U - RHD negative 35, U, 37, 37 M 35, 36, 37, U M
U, 38, 38 P U, U, 38 - Inconclusive U, U, U - U, U, 38 - Inconclusive U, U, 36, 38 ? U, 36, 38, U ?
U, U, U - U, U, U - Inconclusive U, U, 38 ? U, U, 38 ? Inconclusive U, U, U, U - U, U, U, U -
13 RHD Exon 5
RHD Exon 7 RHD Exon 10
RHD Exon 5 RHD Exon 7 RHD Exon 10
SRY DYS14 SRY DYS14
1 1 1 1 2 2 2 2 1 1 2 2
U, 37, 36 P 36, 37, 36 P 37, U, U P RHD Positive 42, U, 37 P U, 36, U P U, U, 38 P RHD Positive U, U, 39 M 32, 32, 32 M 37, U, U M 33, 32, 31 M
U, 36, 37 P 37, U, 38 P U, U, 37 P RHD Positive U, U, 36 P U, U, U - U, U, U - Inconclusive U, U, U - 33, 34, 33 M 37, U, U M 33, 33, 33 M
U, U, U - 37, U, U P U, U, U - Inconclusive U, 38, U P U, U, U - U, U, U - Inconclusive U, 37, U M 34, 34, 35 M U, 38, U M 34, 35, 34 M
U, U, U - U, U, U - U, U, U - Inconclusive U, U, U - U, U, U - U, U, U - Inconclusive U, U, U - 37, 37, 37 M U, U, U - 37, 35, 36 M Key. P:RHD target present -: target absent M: male fetal DNA present U: undetected ?:inconclusive
WHO/BS/10.2149 Page 16
Appendix II (continued). Real-time PCR results expressed as cycle threshold (CT) and interpretation of results (in bold).
Dilution of 07/222 Lab. Target Day
neat 1 in 2 1 in 4 1 in 8
14 RHD Exon 7
RHD Exon 10 RHD intron 4
RHD Exon 7 RHD Exon 10 RHD intron 4
SRY SRY
1 1 1 1 2 2 2 2 1 2
33, 33 P 34, 34 P 32, 33 P RHD Positive 33, 32 P 33, 34 P 33, 34 P RHD Positive 36, 35 M 35, 35 M
33, 33 P 35, 35 P 34, 35 P RHD Positive 34, 34 P 34, 35 P 34, 34 P RHD Positive 37, 37 ? 36, 37 M
35, 35 P 38, 35 ? 38, 37 ? Inconclusive 35, 35 P 39, 36 ? 36, 37 P Inconclusive 38, 42 ? 38, 38 ?
36, 36 P 37, U ? U, U ? Inconclusive 35, 35 P U, 37 ? 36, 37 P Inconclusive 39, U ? 37, 39 ?
15 RHD Exon 7
RHD Exon 10
RHD Exon 7 RHD Exon 10
SRY SRY
1 1 1 2 2 2 1 2
34, 34 P 34, 35, 35 P RHD Positive 36, U ? 35, 36, 37 P RHD Positive 36, 36, 37 M 36, 37, 37 M
35, 36 P 35, 36, 36 P RHD Positive 39, 40 P 36, 37, 37 P RHD Positive 37, 38, U M 38, 38, 39 M
37, 38 P 37, 37, 37 P RHD Positive 38, 38 P 37, 37, 38 P RHD Positive 38, 38, 39 M 38, 38, U M
37, 38 P 37, 38, U P RHD Positive 37, U - 37, U, U - RHD negative 39, U, U - 39, U, U -
16 RHD Exon 5
RHD Exon 7 RHD Exon 10
RHD Exon 5 RHD Exon 7 RHD Exon 10
SRY SRY
1 1 1 1 2 2 2 2 1 2
U, U, U - U, U, 38 - 38, U, U - Inconclusive U, U, 38 - U, U, U - U, U, U - RHD negative U, U, U - U, U, U -
U, U, U - U, 38, U - U, U, U - RHD negative U, U, U - U, U, U - U, U, 38 - RHD negative U, U, U - U, 41, U -
U, U, U - U, U, U - U, U, U - RHD negative 37, U, U - U, U, U - U, U, 38 - Inconclusive U, U, U - U, U, U -
U, U, U - U, U, U - U, U, U - RHD negative U, U, U - U, U, U - U, U, U - RHD negative U, U, U - U, U, U -
18 RHD Exon 10
RHD Exon 10
SRY SRY
1 1 2 2 1 2
34, 42 P RHD Positive 33, U P RHD Positive U, U ? Not tested
U, U ? Inconclusive 33, 34 P RHD Positive 34, 34 M Not tested
35, 41 P RHD Positive 44, 35 P RHD Positive 33, 34 M Not tested
43, 34 P RHD Positive 43, U P RHD Positive 34, 35 M Not tested Key. P:RHD target present -: target absent M: male fetal DNA present U: undetected ?:inconclusive
WHO/BS/10.2149 Page 17
Appendix II (continued). Real-time PCR results expressed as cycle threshold (CT) and interpretation of results (in bold).
Dilution of 07/222 Lab. Target Day
neat 1 in 2 1 in 4 1 in 8
19 RHD Exon 5
RHD Exon 7
RHD Exon 5 RHD Exon 7
SRY DYS14 Albumin SRY DYS14 Albumin
1 1 1 2 2 2 1 1 1 2 2 2
37, 37, 37 P 37, 38, 37 P RHD Positive 39, 37, 38 P 36, 36, 36 P RHD Positive U, U, U - 32, 32, 32 M 33, 33, 33 D 39, 42, 37 M 31, 31, 31 M 33, 33, 32, 34 D
38, 38, 38 P 36, 38, 38 P RHD Positive 39, 39, 39 P 38, 38, 37 P RHD Positive U, U, U - 33, 33, 32 M 34, 34, 34 D 37, 37, 37 M 32, 33, U M 35, 34, 34, 35 D
48, 39, 38 ? 38, 38, 40 P RHD Positive 45, 42, 42 ? 38, 38, 38 P RHD Positive 42, U, 40 - 33, 33, 34 M 35, 35, 34 D 38, 38, 39 M 33, 33, 33 M 25, 36, 35, 37 D
38, 46, 39 P 40, 39, 38 P RHD Positive 41, 46, 40 ? 39, 37, 45 P RHD Positive 40, 40, U - 34, 34, 34 M 35, 35, 35 D 40, U, 43 ? 34, 34, 34 M 38, 37, 36, 37 D
Laboratory 17 results (multiplex endpoint PCR, not real-time PCR) interpretation only
17 RHD Exon 7
RHD Exon 10
RHD Exon 7 RHD Exon 10
SRY SRY
1 1 1 2 2 2 1 2
P P
RHD Positive P
P
RHD Positive M
M
P P
RHD Positive P
P
RHD Positive M
M
P P
RHD Positive P
P
RHD Positive M
M
P -
RHD Positive P
-
RHD Positive M
-
Key. P:RHD target present -: target absent M: male fetal DNA present U: undetected ?:inconclusive
WHO/BS/10.2149 Page 18
Appendix III. Extended titres, Real-time PCR results expressed as cycle threshold (CT) and interpretation of results (in bold).
Dilution of 07/222 Lab.
No. Target Day 1 in 16 1 in 32 1 in 64 1 in 128
5 RHD Exon 5
RHD Exon 7
RHD Exon 5 RHD Exon 7
SRY SRY
1 1 1 2 2 2 1 2
50, 50, 50 - 41, 50, 50 - RHD negative 42, 50, 40 P 39, 50, 50 - Inconclusive 50, 50, 50 - 50, 50, 50 -
50, 50, 50 - 50, 50, 50 - RHD negative 38, 50, 50 - 50, 50, 50 - RHD negative 50, 50, 50 - 50, 50, 50 -
50, 50, 50 - 50, 50, 50 - RHD negative 50, 50, 50 - 50, 40, 50 - RHD negative 50, 50, 50 - 50, 50, 50 -
50, 50, 50 - 50, 50, 50 - RHD negative 50, 50, 50 - 50, 50, 50 - RHD negative 50, 50, 50 - 50, 50, 50 -
6 RHD Exon 5
RHD Exon 7
RHD Exon 5 RHD Exon 7
SRY SRY
1 1 1 2 2 2 1 2
50, 50, 50 - 37, 40, 50 P Inconclusive 50, 50, 42 - 39, 50, 39 P Inconclusive 50, 50, 50 - 40, 50, 50 -
50, 50, 39 - 38, 50, 50 - RHD negative 50, 50, 50 -
NOT TESTED NOT TESTED
7 RHD Exon 4
RHD Exon 5 RHD Exon 10
RHD Exon 4 RHD Exon 5 RHD Exon 10
SRY SRY
1 1 1 1 2 2 2 2 1 2
U, U, U, U - 41, 40, U, U P U, U, U, U - RHD negative 40, U, U, U - 38, 38, 39, U P 39, U, U, U - RHD negative U, U, U, U - U, U, U, U -
NOT TESTED NOT TESTED NOT TESTED
9 RHD Exon 4
RHD Exon 5 RHD Exon 10
RHD Exon 4 RHD Exon 5 RHD Exon 10
SRY SRY
1 1 1 1 2 2 2 2 1 2
U, U, U, 40 U, 34, U, 36 U, U, U, U - Inconclusive U, 39, U, U U, U, 35, U 38, 38, U, U Inconclusive U, U, U, U - U, U, 36, U
U, U, U, U - U, U, 34, U U, U, 36, U Inconclusive U, U, 39, U 36, U, U, U 37, U, U, 36 Inconclusive U, U, 37, U U, U, 37, U
U, U, U, U - U, U, U, U - U, U, U, U - RHD negative U, U, U, U - U, U, U, U - U, U, 36, U RHD negative U, U, U, U - U, U, U, U -
U, U, U, U - U, U, U, U - U, U, U, U - RHD negative U, U, U, U - 34, U, U, U 37, U, U, U Inconclusive U, U, U, U - U, U, U, U - Key. P:RHD target present -: target absent M: male fetal DNA present U: undetected ?:inconclusive
