This document discusses molecular testing for neonatal alloimmune thrombocytopenia (NAIT). NAIT occurs when a fetus inherits a platelet antigen from the father that is absent in the mother, causing the mother to produce antibodies against the antigen. These antibodies cross the placenta and destroy the fetus's platelets. Molecular testing can identify incompatibilities by genotyping the mother, father, and fetus for platelet antigens. It has advantages over serological methods as it doesn't require fresh platelets and can detect rare antigens. Various techniques are described like PCR-SSP, PCR-RFLP, TaqMan PCR, and high-throughput methods. Molecular testing is useful for diagnosing NAIT after

1. NAIT
& the role of molecular testing
Dr. G.D.A. Samaranayaka

3. Pathogenesis - NAIT
• Fetus has platelet antigen that is inherited
from the fetus’s father & absent on maternal
platelets
• Antigen positive fetal platelets pass into
circulation of antigen negative mother
• This prompts maternal production of IgG
antibodies against ‘foreign’ antigen
• Maternal IgG antibodies cross placenta and
enter the fetal circulation
• Within the fetal circulation maternal
antibodies bind to fetal platelets and cause
destruction by phagocytes in the RE system
• Fetal thrombocytopenia results

4. Clinical presentation
• Fetal thrombocytopenia (< 150,000 platelets/μL)
• Most cases are mild - widespread petechiae and other skin lesions.
• Severe cases - intracranial hemorrhage (ICH)
• Leading cause of severe thrombocytopenia in the fetus & neonate
• Leading cause of intracranial hemorrhage in the full term infant
• Incidence estimated between 7 - 25%
• Unlike erythrocyte alloimmunization
• NAIT may appear during first pregnancies
• high recurrence rate
• often with progressively more severe manifestations in subsequent pregnancies

5. Antigens implicated in NAIT
• Platelet-specific (HPA) antigens
• ABO antigens
• Glycoprotein IV (CD36, Nak)
• Human leucocyte antigen (HLA) antigens

6. Human Platelet Alloantigens
• 28 HPAs have been completely characterized
• HPA-1a most common in Caucasians
• Located on GPIIIa platelet glycoprotein
• Responsible for >80% NAIT cases
• HPA-5b 2nd most common in Caucasians
• HPA-4 is the most common in Asians
• The allelic forms of HPA are as a result of single nucleotide
polymorphisms (SNPs) in the genes encoding the relevant platelet
proteins.
• Platelet-specific alloantigens inheritance - autosomal dominant manner.

7. Diagnosis
• Based on demonstrating HPA incompatibility between the biologic
mother and father
• Identification of a maternal antibody specific for the incompatible
antigen.

9. Molecular testing in NAIT

10. Indications
• Platelet antigen phenotyping
• Diagnosis of NAIT after birth
• Maternal & paternal blood samples
• Buccal smears or blood samples – from the baby
• Screening for NAIT for at risk pregnancies
• Amniocentesis
• Cell free fetal DNA

11. Advantages
• Fresh platelets are not required.
• More sensitive and specific than serological methods.
• Genomic DNA can be obtained from multiple sources including
white blood cells, amniocytes, and buccal smears.
• Typing for low-frequency HPAs for which antisera is unavailable is
possible.
• Automated and multiplex methods are available - decreasing the
risk of error and time needed to perform these assays.
• Platelet genotyping is the gold standard method for HPA typing.

12. Disadvantages
• DNA must be free of contamination in a sufficient quantity and
quality.
• Precautions -prevent contamination with DNA from other sources, as false
positive results may occur.
• Important when typing fetal cells - amniocentesis or percutaneous umbilical
blood sampling
• Molecular results must be interpreted carefully
• Unknown polymorphisms near the SNP of interest can affect primer
annealing and hybridization leading to false negative results
• Point mutations leading to the expression of rare low-frequency HPA are
difficult to identify

13. Platelet genotyping
• Sequence-specific primer-polymerase chain reaction (PCR-SSP)
• Restriction fragment length polymorphism-PCR (PCR-RFLP)
• TaqMan real-time PCR (Applied Biosystems,Foster City, CA)
• High-throughput methods

14. Sequence-specific primer-polymerase chain
reaction (PCR-SSP)
• AKA allele-specific PCR
• Uses two reactions with two sets of primers
• one primer is specific for each allele (allele-specific primer)
• paired with a second common primer to control for PCR efficiency
• This method is a relatively simple and inexpensive procedure for
HPA genotyping.
• The basis - reduction in the efficiency of Taq polymerase to
amplify DNA when there is a 3’ terminal nucleotide mismatch
between the target DNA and the allele-specific primer.

15. • The HPA genotype is identified by the presence or absence of DNA
bands after gel electrophoresis of the PCR products
product
Amplification
controls
Allele-specific
SSP= Sequence-specific primer
No
amplification
SSP
SSP
Amplification
SSP matches allele
SSP does not match allele

16. Advantages and disadvantages - PCR-SSP
Advantages Disadvantages
Technically simple Requires precise primer design
Relatively inexpensive Requires two reactions per assay
sample
Difficult to automate
Subjective interpretation

17. Restriction fragment length polymorphism
PCR (PCR-RFPL)
• PCR-RFLP relies on the loss or gain of
a restriction enzyme recognition site
at the polymorphic site in the target
gene
• The region of the gene encoding the
polymorphism is amplified by PCR and
then subjected to digestion with a
specific restriction enzyme
• Recognize and cut DNA wherever a
specific short sequence occurs – AKA
restriction digest.

18. • Resulting DNA fragments are separated according to their lengths by gel
electrophoresis.
• After gel electrophoresis, the use of a UV transilluminator allows
visualization of the DNA and fragment pattern interpretation.

19. Advantages and disadvantages PCR-RFLP
• Potential disadvantage - smaller fragments produced by the
restriction enzyme will produce only faint bands with
electrophoresis - can be avoided by careful choice of primers.
Advantages Disadvantages
Technically simple SNP must create an allele-
specific digestion site
Relatively inexpensive Requires additional digestion
step
Easier primer design Cannot be automated
Less strict PCR reaction
parameters

20. TaqMan Real-Time PCR
• Quantification or identification of the
PCR amplification product in real time.
• Based on 5' nuclease chemistry - uses a
fluorogenic probe to enable the
detection of a specific PCR product as it
accumulates during PCR.
• SSP (TaqMan probe) that binds to the SNP
of interest
• reporter dye (fluorophore) attached to the
5’ end
• quencher attached to the 3’ end - prevents
the reporter dye from fluorescing
• The SSP binds to the DNA

21. • Taq DNA polymerase synthesizes new strands
using the unlabeled primers and the
template.
• When the polymerase reaches a TaqMan
probe, its endogenous 5' nuclease activity
cleaves the probe, separating the dye
(reporter) from the quencher - reporter
fluoresce due to the decreased proximity to
the quencher.
• Fluorescence detection is directly
proportional to the release of the reporter
and the amount of the target DNA present.
• Each cycle of PCR will increase the
fluorescent signal detected allowing for
quantification of the amount of PCR product
produced

22. Advantages and disadvantages - TaqMan
Advantages Disadvantages
Process automation Probes - expensive
Detect homozygosity and heterozygosity -
using two different allele-specific probes
with different reporter dyes.
Does not require additional handling after
amplification

23. High-Throughput Methods
• AKA - Next-generation sequencing
• Detects the whole ATGC sequences of the DNA
…ACGTGACTGAGGACCGTG
CGACTGAGACTGACTGGGT
CTAGCTAGACTACGTTTTA
TATATATATACGTCGTCGT
ACTGATGACTAGATTACAG
ACTGATTTAGATACCTGAC
TGATTTTAAAAAAATATT…

25. Bead arrays
• Multiplex high-throughput platforms
• HPA typing of known antigens using capture allele-
specific probes affixed to beads labeled with
fluorescent dyes.
• In some assays, multiple beads may be used at one
time each targeting a different SNP.
• Target DNA fragments are allowed to anneal to the
capture probes.

26. • Annealed target DNAs are then elongated using fluorescent labeled
nucleotides.
• The beads are either affixed to a microchip or analyzed by flow
cytometry
• Fluorescence patterns are analyzed to determine HPA type

27. Advantages and disadvantages
Advantages Disadvantages
Relatively automated Expensive
Can be multiplexed Requires test-specific technical
expertise
Relatively fast

28. References
• Determination of human platelet antigen typing by molecular methods: Importance in
diagnosis and early treatment of neonatal alloimmunethrombocytopenia; Suzanne A.
Arinsburg, Beth H. Shaz, Connie Westhoff, Melissa M. Cushing; Am. J. Hematol. 87:525–528,
2012.
• Neonatal alloimmune thrombocytopenia: pathogenesis, diagnosis and management; Julie
A. Peterson,1 Janice G. McFarland, Brian R. Curtis, and Richard H. Aster
• Prenatal Fetal DNA Testing for Predicting HDFN, FNAIT, and RhIG Candidacy ; C. Ellen van
der Schoot, Florentine Thurik, Peter G. Scheffer, Frithjofna Abbink, van der Ploeg P.B.
Catharina, Barbera Veldhuisen and Masja de Haas; Blood 2013 122:SCI-51
• https://en.wikipedia.org/wiki/DNA_sequencing#High-throughput_methods

29. Thank You