pcr in Transfusion medicine

1. POLYMERASE CHAIN REACTION ITS TYPE AND IMPORTANCE IN TRANSFUSION MEDICINE DR LAXMIPRIYA BEHERA FIRST YR PG RESIDENT DEPARTMENT OF TRANSFUSION MEDICINE SCB MCH

2. Contents • Introduction • Components of PCR • Principle • Steps • Thermocycler • Type of PCR • Application in Transfusion Medicine • Summary • Reference

3. Introduction: • PCR is a in-vitro molecular technique used to amplify specific DNA sequences of interest. • Invented by Kary Mullis in 1985. • PCR has become an essential tool in various fields such as medical diagnostics, forensic science, genetic research, and biotechnology.

4. ComponentsofPCR • Template DNA - strand to be replicated ---each newly formed DNA strand act as template for next cycle--- Chain reaction • Primers (forward and reverse) –synthetic oligonucleotide –15-20 nucleotide long and flank the target region *Designed to anneal to complimentary DNA sequence at 3’ end of each strand. • DNA Polymerase (usually thermostable Taq polymerase,extracted from bacterium thermusaquaticus ,which lives in hot springs)– for replication of DNA • dNTPs (nucleotides)—dATP,dTTP,dCTP,dGTP • Buffer solution – maintaining PH • Magnesium (enzyme cofactor)

5. PRNCIPLEofPCR : • DENATURATION • ANNEALING • EXTENSION

6. Steps: • Denaturation: • First step in PCR cycle. • DNA strands are separated at (94–98°C) for 30 sec • Due to high temp, Hydrogen bond will be broken. • Double helix single strand. • Thermostability of Taq Polymerase is very important, as it is heat denaturation. • Denaturation is repeated in every cycle to allow continuous DNA amplification.

7. Annealing: • Second step in PCR cycle. • Primers bind to flanking sequence of both the strand by cooling to (50–65°C) for 20-40 sec • promote renaturation(reestablishment of hydrogen bond) • Exact optimum temperature determined by no. of G&C(Guanine and Cytosine) residue….. • The more GC more hydrogen bond will break higher melting point. • Formation of short DNA-DNA hybrid start as starting points for DNA synthesis • Precise annealing is crucial for the specificity of PCR.

8. Extension: • Third step in PCR cycle. • Also called elongation. • Thermostable DNA polymerase synthesizes new DNA by adding DNTP to primers at (72°C) . • EXTENSION TIME depends upon DNA polymerase as well as length of Nucleotides to be amplified. • typically about 1000 base pairs are added per one minute.

10. THERMOCYCLER • PCR is carried out in programmable heating and cooling machine ---THEMOCYCLER. • The instrument contains a heating block built from materials like silver which has high thermal conductivity • It allow for rapid changes of temperatures • It shortens time between the steps of each cycle. • This thermal built with holes where tubes holding reaction mixture inserted.

11. CONTINUED … • Repeated cycles (20–40) to amplify the target DNA • If it goes n number of cycle, then 2n number of product produced • In each cycle, an original template strand is copied to generate a complementary strand, which begins at the 5′ end of the primer and ends wherever the thermostable polymerase ceases to function. • After the second cycle, DNA is synthesized, using the newly copied strands as templates. • In this case, synthesis stops when it reaches the end of the molecule defined by the primer. • By the end of the third cycle, a new blunt-ended double-stranded product is formed, with its 5′ and 3′ ends precisely coinciding with the primers. • These blunt-ended fragments accumulate exponentially during subsequent rounds of amplification.

13. TypesofPCR : • Conventional PCR • Real-Time PCR (qPCR) • Reverse Transcriptase PCR (RT-PCR) • PCR-SSP • PCR–RFLP • TMA • Multiplex PCR

14. Conventional PCR : • Principle: Amplifies DNA sequences using thermal cycling (denaturation, annealing, extension). • Output: End-point detection via gel electrophoresis. • Use in TM: • Basic infectious disease screening, • presence/absence of specific genes (e.g., pathogen detection).

15. Real-Time PCR (qPCR) • Principle: Amplifies and quantifies DNA in real-time using fluorescent dye(SYBR green dye) or probe(TaqMan or molecular beacons or FRET probes – emit light when bind to newly synthesized amplicon). • Conducted in light cycler(special type thermocycler) • Output: Quantitative data shown in amplification curves ---melting curve analysis. • Single sharp peak at specific Tm = specific product • Multiple peak =possible contamination ,primers-dimers, or nonspecific amplification. • Use in TM: • NAT Testing Viral load estimation (HIV, HCV, HBV) • donor TTI screening • Detecting bacterial DNA in platelet units • Genotyping rare or weak blood group • detection of factor V Leiden mutation in patients with inherited coagulopathy • Gene expression analysis

17. Advantage over conventional PCR : • Quantitative Results • Measures the exact amount of DNA or RNA in real time, unlike conventional PCR which is qualitative. • High Sensitivity • Can detect even very small amounts of target DNA or RNA, useful for early detection of infections. • High Specificity • Uses fluorescent probes or dyes that ensure specific binding to the target sequence, reducing false positives. • Speed and Efficiency • Faster than traditional PCR because there’s no need for post-PCR gel electrophoresis. • Reduced Risk of Contamination • Closed-tube system minimizes the chance of cross-contamination, making results more reliable. • Real-Time Monitoring • Tracks DNA amplification cycle by cycle, allowing early result interpretation. • Versatility

18. Reverse Transcriptase PCR (RT-PCR) • Principle: • Converts RNA to cDNA using reverse transcriptase, then amplifies DNA. • Detects gene expression through the synthesis of cDNA. • Amount of amplified fragments produced proportional to amount of target mRNA in original sample. • The first RT-PCR assays developed by National Genetics Institute (UltraQual HIV-1) and by Roche (COBAS AmpliScreen HIV-1) were designed to detect these pathogens in donors’ blood and were approved by the FDA in 2001 and 2003. • One-Step RT-PCR • Reverse transcription and PCR amplification occur in a single reaction tube. • Faster and reduces handling, lowering the risk of contamination. • Commonly used for routine diagnostics like virus detection (e.g., COVID-19, HIV). • Two-Step RT-PCR • Reverse transcription and PCR are done in two separate steps : • RNA is first converted to cDNA. • cDNA is then used for PCR amplification.

19. Uses in TM: • Detection of RNA Viruses — like HIV, HCV, SARS-CoV-2, Influenza, etc. • Nucleic Acid Testing (NAT) — for blood donor screening to detect viral RNA during the window period. • Gene Expression Analysis — to study mRNA levels in cells for research and diagnostics. • Viral Load Monitoring — to measure the amount of virus in patient samples, especially in chronic infections. • Cancer Diagnostics — detection of fusion genes, mutations, or abnormal gene expression. • Genetic Disease Diagnosis — used in identifying gene mutations linked to inherited diseases.

20. Advantage : • Highly sensitive — detects even low levels of RNA. • Specific — uses target-specific primers. • Allows early diagnosis during the window period. • Quantitative (qRT-PCR) — measures viral load accurately. Limitations : • RNA is unstable — easily degraded. • Requires expensive reagents and equipment. • Needs skilled handling to avoid contamination. • Two-step process — reverse transcription + amplification takes time. • Contamination risk can cause false positives.

21. PCR–SSP (Polymerase Chain Reaction-Sequence Specific Primers) • PCR-SSP (Polymerase Chain Reaction - Sequence Specific Primers) is a molecular technique used to detect specific DNA sequences or genetic variations, like single nucleotide polymorphisms (SNPs) or HLA typing. • In this method, primers are designed to match only the target DNA sequence. • If the target sequence is present, the primers bind and DNA amplification occurs. • If the target is absent, no amplification happens.

22. Applications: • Blood group genotyping — especially for Rh, Kell, Duffy, Kidd, and MNS blood group systems. • HLA typing — useful in transplant compatibility testing. • Identifying weak or variant antigens in multi-transfused or serologically challenging cases • Advantages of PCR-SSP: • High specificity. • Speed and simplicity. • Cost-effective. • No need for sequencing. • Reliable for low DNA quantity.

23. PCR-RFLP (Polymerase Chain Reaction – Restriction Fragment Length Polymorphism) • A specific DNA segment is first amplified using PCR. • The PCR product is then treated with a restriction enzyme that cuts DNA at a specific sequence. • If a genetic variant (mutation or polymorphism) alters the restriction site, the enzyme will either cut or not cut the DNA, resulting in different fragment patterns. • These fragments are separated and visualized using gel electrophoresis. • Applications: • Blood Group Genotyping. • Mutation Detection • Donor and Recipient Matching

24. Advantages of PCR-RFLP : • High specificity and sensitivity for detecting known polymorphisms. • Cost-effective and does not require complex equipment beyond a PCR machine and basic gel electrophoresis setup. • ⁠Reliable and reproducible results for genotyping and mutation detection. • ⁠Useful for confirming serological discrepancies in blood group typing, especially in multi-transfused or hemolytic patients. • Limitations: • Can only detect mutations at restriction enzyme recognition sites. • Requires prior knowledge of the sequence and appropriate restriction enzyme selection. • Labor-intensive compared to newer genotyping technologies like real-time PCR or sequencing

25. Transcription Mediated Amplification • TMA is an isothermal nucleic acid amplification technique that amplifies RNA or DNA targets without using thermal cycling (unlike PCR). • Key Features: Isothermal Reaction: Occurs at a constant temperature (around 42°C). • The starting material in this assay is RNA. • Enzymes Used: Reverse Transcriptase — converts RNA to complementary DNA (cDNA), which is bound to the RNA by hydro gen bonds to form an RNA/DNA hybrid. • After enzymatic(Rnase H) removal----RNA Polymerase — produces multiple RNA copies from cDNA. • multiple RNA molecules go through another reverse transcription process to produce more RNA/DNA hybrids, and the whole process is repeated many times.

27. Applications : • Used for NAT testing (Nucleic Acid Testing) to screen donated blood for: • HIV RNA • HCV RNA • HBV DNA • Helps detect infections earlier than serological methods, reducing the window period. Advantages: • Faster and more sensitive for RNA viruses. • Works at a single temperature — no need for a thermal cycler. • High amplification efficiency, making it ideal for clinical screening.

28. Multiplex PCR • Multiplex PCR is a variation of the standard Polymerase Chain Reaction (PCR) in which multiple sets of primers are used within a single reaction tube to simultaneously amplify two or more DNA targets in one PCR run. • In multiplex PCR, each primer pair is designed to target a specific DNA sequence. • During thermal cycling, all target sequences are amplified at the same time, provided the primers are compatible and the reaction conditions are optimized.

29. Applications: • Simultaneous detection of transfusion-transmitted infections (HIV, HBV, HCV, Parvovirus B19, etc.) from a single donor sample. • Blood group genotyping — detecting multiple blood group antigens in one reaction. • HLA typing for transplant compatibility. • Pathogen detection in cases of suspected transfusion reactions. • Advantages: • Time Saving • Cost Effectiveness • Require less sample volume • Reduced risk of contamination • Allows simultaneous detection of multiple target • Limitation: • Primer design complexity • Risk of non specific amplification • Detection difficulty

30. Applications of PCR in Transfusion Medicine : • Infectious Disease Screening: • Detection of HIV, HBV, HCV, malaria, CMV, etc are done by NAT Testing (Nucleic Acid Testing) • Reduces risk of transfusion-transmitted infections (TTIs) after transfusion • Blood group Genotyping • Identification of blood grouping antigen at DNA level • Useful when serological methods are inconclusive (e.g. recent transfusion, autoantibodies) • Also useful in multi-transfused patients and prenatal testing. • Prevents alloimmunization • Molecular genotyping of weak or variant blood group antigens (e.g., weak D, Kell, Kidd)

31. CONTINUED … • Fetal Genotyping • Non-invasive detection of fetal antigens (RhD, K) as PCR detects fetal DNA in maternal plasma • Guides management of HDFN. • Avoids unnecessary use of anti-D immunoglobulin if the fetus is RhD-negative. • HLA Typing • PCR based typing for HLA class I and I • Essential for platelet transfusion refractoriness • Essential for Hematopoietic stem cell transplantation compatibility.

32. CONTINUED … • Microchimerism Detection • Detects small populations of donor cells post-transfusion • Important in transfusion associated Graft-versus-Host Disease (TA-GvHD) surveillance. • Also in Monitoring post-transplant chimerism • Pathogen Reduction and Quality control • Testing for residual viral nucleic acid post-pathogen inactivation • PCR helps confirm effective pathogen reduction in blood components.

33. Summary • PCR is a molecular technique used to amplify specific DNA sequence. • PCR is carried out in THERMOCYCLER. • Principle are Denaturation ,Annealing and Extension . • One cycle produces 2 copies of the target DNA. Multiple cycles (typically 30–40) exponentially amplify the DNA segment. • End point detection is by gel Electrophoresis. • Types of PCR are Conventional PCR ,Real-Time PCR (qPCR),Multiplex PCR ,Reverse Transcription PCR (RT-PCR) ,TMA ,PCR-SSP ,PCR-RFLP .. • In TM ,real time PCR,RT-PCR,TMA are important in Transfusion Medicine. • NAT testing is done by these three technique. • PCR helps in infectious disease (TTI)screening, fetal typing, HLA typing ,micro chimerism detection, pathogen detection and quality control.

34. PCR helps in infectious disease (TTI)screening, fetal typing, HLA typing ,micro chimerism detection, pathogen detection and quality control. • PCR has High sensitivity & specificity, Faster and more accurate . • It helps in Rapid detection, Early diagnosis reduces TTIs. • It Helps in personalized transfusion strategies, hence reduces transfusion risks. • Enhances blood safety and patient management. • Limitationsare very costly, require technical expertise. • It is Not always suitable for resource-limited settings.

35. References : • Transfusion medicine technical manual (DGHS) • Harmening 7th Edition • AABB Technical Manual 21st Edition • Journal articles on PCR in transfusion medicine

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