RHD genotyping from cell-free fetal DNA circulating in pregnant women peripheral blood and sensitivity assessment of innovated diagnostic approaches for introduction into the clinical practice


Authors: J. Böhmová 1;  R. Vodička 1;  M. Lubušký 1,2;  M. Studničková 3;  I. Holusková 3;  R. Vrtěl 1;  R. Kratochvílová 1;  M. Frydrychová 1;  E. Krejčiříková 1;  H. Filipová 1
Authors‘ workplace: Ústav lékařské genetiky a fetální medicíny FN a LF UP, Olomouc, přednosta prof. MUDr. J. Šantavý, Ph. D. 1;  Porodnicko-gynekologická klinika FN a LF UP, Olomouc, přednosta prof. MUDr. R. Pilka, Ph. D. 2;  Transfuzní oddělení FN a LF UP, Olomouc, vedoucí MUDr. D. Galuszková, Ph. D., MBA 3
Published in: Čes. Gynek.2013, 78, č. 1 s. 32-40

Overview

Objective:
Introduction of fetal RHD genotyping from cell-free fetal DNA circulating in the peripheral blood of pregnant women to clinical practice. Sensitivity assessment of innovated method using range of dilution series and internal control of amplification.

Design:
Procedure creating of noninvasive determination of fetal RHD genotyping from blood plasma of pregnant women. Detection of limit of minority representation RHD+/- sample in the RHD-/- sample.

Setting:
University Hospital Olomouc, Institute of Medical Genetics and Fetal Medicine, Clinic of Obstetrics and Gynecology, Transfusion Department.

Methods:

  1. TaqMan Real-Time PCR without an internal amplification controls.
  2. Optimization and calibration of RHD genotyping using RHD multiplex by TaqMan Real-Time PCR with an internal amplification control and by minisequencing (Snapshot – multiplex) with an internal amplification controls.

Results:

  1. RHD positive or negative fetuses were determined by amplification curves from Real-Time PCR system that matches the parameters for the evaluation of the output data using series of amplification and contamination parallel controls.
  2. TaqMan based Real-Time PCR and minisequencing (SNaPshot) based quantification were able to detect 0.22% of artificial RHD+/- sample diluted in RHD-/- sample. In addition, SNaPshot assay is suitable for heterozygozity and homozygozity recognition.

Conclusion:
Current established and routinely used procedure is based on the detection of exon 7 of the RHD gene and on the series of parallel amplification and contamination controls. Both newly developed methods could be, after validation of the larger set of control samples, introduced into clinical practice.

Keywords:
RHD genotyping – cell-free fetal DNA – maternal plasma – TaqMan Real-Time PCR – SNaPshot – minisequencing


Sources

1. Gautier, E., Benachi, A., Giovangrandi, Y., et al. Fetal RhD genotyping by maternal serum analysis: a two-year experience. Am J Obstet Gynecol, 2005, 192, 3, p. 666–669.

2. Chinen, PA., Nardozza, LM., Martinhago, CD., et al. Noninvasive determination of fetal rh blood group, D antigen status by cell-free DNA analysis in maternal plasma: experience in a Brazilian population. Am J Perinatol, 2010, 27, 10, p. 759–762.

3. Innan, H. A two-locus gene conversion model with selection and its application to the human RHCE and RHD genes. Proc Natl Acad Sci U S A, 2003, 100, 15, p. 8793–8798.

4. Lázár, L., Nagy, B., Bán, Z., et al. Non invasive detection of fetal Rh using real-time PCR method. Orv Hetil, 2007, 148, 11, p. 497–500.

5. Macher, HC., Noguerol, P., Medrano-Campillo, P., et al. Standardization non-invasive fetal RHD and SRY determination into clinical routine using a new multiplex RT-PCR assay for fetal cell-free DNA in pregnant women plasma: results in clinical benefits and cost saving. Clin Chim Acta, 2012, 413, 3–4, p. 480–484.

6. Müller, SP., Bartels, I., Stein, W., et al. The determination of the fetal D status from maternal plasma for decision making on Rh prophylaxis is feasible. Transfusion, 2008, 48, 11, p. 2292–2301.

7. Palomaki, GE., Kloza, EM., Lambert-Messerlian, GM., et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med, 2011, 13, 11, p. 913–920.

8. Sedrak, M., Hashad, D., Adel, H., et al. Use of free fetal DNA in prenatal noninvasive detection of fetal RhD status and fetal gender by molecular analysis of maternal plasma. Genet Test Mol Biomarkers, 2011, 15, 9, p. 627–631.

9. Scheffer, PG., van der Schoot, CE., Page-Christiaens, GC., et al. Noninvasive fetal blood group genotyping of rhesus D, c, E and of K in alloimmunised pregnant women: evaluation of a 7-year clinical experience. BJOG, 2011, 118, 11, p. 1340–1348.

10. Silvy, M., Simon, S., Gouvitsos, J., et al. Weak D and DEL alleles detected by routine SNaPshot genotyping: identification of four novel RHD alleles. Transfusion, 2011, 51, 2, p. 401–411.

11. Silvy, M., Chapel-Fernandes, S., Callebaut, I., et al. Characterization of novel RHD alleles: relationship between phenotype, genotype, and trimeric architecture. Transfusion, 2012, elektronické verze před tiskem doi: 10.1111/j.1537-2995.2011.03544.x.

12. Wang, XD., Wang, BL., Ye, SL., et al. Non-invasive foetal RHD genotyping via real-time PCR of foetal DNA from Chinese RhD-negative maternal plasma. Eur J Clin Invest, 2009, 39, 7, p. 607–617.

Labels
Paediatric gynaecology Gynaecology and obstetrics Reproduction medicine

Article was published in

Czech Gynaecology

Issue 1

2013 Issue 1

Most read in this issue

This topic is also in:


Login
Forgotten password

Don‘t have an account?  Create new account

Forgotten password

Enter the email address that you registered with. We will send you instructions on how to set a new password.

Login

Don‘t have an account?  Create new account