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Blood Bank

Molecular Techniques

Molecular Techniques is a relatively newer frontier compared to the century-old serology we’ve been discussing, but it’s becoming increasingly vital. It’s a powerful addition to our toolkit, offering solutions where traditional serology sometimes struggles

Think of it this way: Serology looks at the finished product (the antigens expressed on the cell surface or the antibodies in the plasma). Molecular techniques look at the genetic blueprint (the DNA sequences that code for those antigens)

The Core Purpose: Analyzing the Genes

Molecular testing in immunohematology focuses on analyzing the genes that encode blood group antigens. Instead of using antibodies to detect the antigen protein or carbohydrate on the cell surface, we look directly at the DNA sequence to predict which antigens should be expressed

Why is this useful? It helps us overcome several limitations of traditional serologic methods:

  • Positive Direct Antiglobulin Test (DAT): When a patient’s cells are coated with antibody in vivo, serologic typing can be difficult or impossible because the bound antibody blocks the antigen sites or causes spontaneous agglutination. Molecular testing isn’t affected by antibody coating the cells
  • Recent Transfusion: Patients recently transfused have a mixture of their own cells and donor cells, making accurate serologic phenotyping unreliable. Molecular testing uses DNA (usually from white blood cells), which reflects the patient’s own genetic makeup, regardless of transfused red cells
  • Limited Antisera: Some blood group antigens have no available reliable serologic typing reagents (especially rare antigens). Molecular methods can identify the alleles responsible for these antigens
  • Weak Antigen Expression: Some individuals express antigens very weakly (like certain Weak D types or other variants), making serologic detection challenging. Molecular testing can identify the underlying gene variant
  • High-Throughput Donor Typing: Molecular platforms allow for rapid screening of blood donors for a large number of clinically significant antigens simultaneously, helping find rare antigen-negative units efficiently
  • Fetal Genotyping: Determining the fetus’s blood type for antigens implicated in HDFN (like D, K, C, c, E, e, Fya, Fyb, Jka, Jkb) using fetal DNA circulating in the mother’s plasma. This is non-invasive and crucial for risk assessment

Key Applications in Blood Banking

  • Predictive Phenotyping for Patients
    • Patients with positive DATs
    • Recently transfused patients
    • Patients with suspected antigen variants (e.g., Weak D, partial D)
    • Patients requiring extensive antigen matching (e.g., sickle cell disease, thalassemia) where multiple antibodies or lack of antisera complicate serology
  • Predictive Phenotyping for Donors
    • Screening donors for multiple antigens simultaneously using high-throughput platforms
    • Identifying donors with rare antigen profiles
  • Fetal Genotyping for HDFN Risk Assessment
    • Testing cell-free fetal DNA (cffDNA) from maternal plasma to predict fetal RhD type (most common) and other significant antigens (K, RhCcEe, Duffy, Kidd)
    • Helps guide management decisions during pregnancy (e.g., need for Rh Immune Globulin, intensity of fetal monitoring)
  • Resolving Serologic Discrepancies: When ABO or Rh typing results are unclear or conflicting
  • Zygosity Determination: Determining if a person is homozygous or heterozygous for certain alleles (e.g., is the father homozygous D/D or heterozygous D/d?). Important for predicting HDFN risk

Common Molecular Techniques Used

While the underlying technologies can be complex, the basic approach involves obtaining DNA and analyzing specific gene sequences:

  • DNA Source: Usually extracted from nucleated cells (WBCs) in an EDTA whole blood sample. For fetal testing, cffDNA is extracted from maternal plasma
  • PCR (Polymerase Chain Reaction): The cornerstone technique. Used to amplify (make millions of copies of) the specific DNA region (gene or part of a gene) of interest
  • Detection Methods (How we see the result)
    • Sequence-Specific Primers (PCR-SSP): PCR primers are designed to only bind and amplify DNA if a specific allele sequence is present. The result is often visualized by running the PCR product on an agarose gel – the presence or absence of a specific sized band indicates the presence or absence of the allele
    • Real-Time PCR (qPCR) with Allele-Specific Probes: Uses fluorescent probes that bind preferentially to one allele sequence over another during the PCR process. Fluorescence is monitored in real-time. Often combined with Melt Curve Analysis, where the temperature at which the probe detaches from the DNA differs depending on how perfectly it matches the allele sequence
    • BeadChip Technology / Microarrays: A high-throughput method. Short DNA probes corresponding to different blood group alleles are attached to microscopic beads or fixed spots on a microarray chip. The patient’s amplified DNA is labeled (e.g., with a fluorescent tag) and allowed to hybridize (bind) to the probes on the array. Specialized readers detect where binding occurred, revealing which alleles are present. Allows for simultaneous testing of dozens or hundreds of alleles
    • DNA Sequencing (Sanger or Next-Generation Sequencing - NGS): Determines the exact nucleotide sequence of the amplified DNA region. Provides the most detailed information, essential for identifying new alleles or complex variants. NGS allows for massive parallel sequencing of many genes at once. More often used in reference labs or research

Advantages of Molecular Testing

  • Overcomes limitations of serology (positive DAT, recent transfusion)
  • Can predict phenotype when antisera are unavailable or antigens are weakly expressed
  • Provides detailed information about alleles and variants
  • High-throughput capability for donor screening
  • Enables non-invasive fetal genotyping

Limitations and Considerations

  • Genotype vs. Phenotype Discrepancy: Molecular testing predicts the phenotype based on the DNA sequence, but gene expression can be complex! Rare “silent” or “null” alleles exist where the gene is present but doesn’t produce a functional antigen on the cell surface. Serology detects the actual antigen expression, so the two methods are complementary
  • Doesn’t Detect Antibodies: Molecular testing tells you about the patient’s antigens (or potential antigens), not about any antibodies they may have formed. Antibody screening/ID remains essential serologic work
  • Cost and Expertise: Requires specialized equipment and trained personnel
  • Turnaround Time: Can sometimes be longer than routine serology, although high-throughput platforms are fast
  • Discovery of Variants of Unknown Significance (VUS): Sequencing can sometimes identify new genetic variations whose effect on antigen expression is unknown

Key Terms

  • Molecular Techniques (Genotyping): Laboratory methods that analyze DNA sequences to predict blood group antigen phenotypes
  • Gene: A segment of DNA that codes for a protein or functional RNA molecule
  • Allele: A specific variant form of a gene
  • Genotype: The specific combination of alleles an individual possesses for a particular gene
  • Phenotype: The observable characteristics resulting from the genotype (e.g., the antigens actually expressed on the red blood cell surface)
  • PCR (Polymerase Chain Reaction): A technique to amplify specific DNA sequences exponentially
  • cffDNA (Cell-Free Fetal DNA): Small fragments of fetal DNA circulating in the maternal bloodstream, used for non-invasive prenatal testing
  • PCR-SSP (Sequence-Specific Primers): PCR method using primers that only amplify specific alleles
  • Real-Time PCR (qPCR): PCR method that monitors DNA amplification using fluorescence during the reaction
  • Melt Curve Analysis: Technique used after qPCR to identify specific DNA sequences based on the temperature at which double-stranded DNA dissociates
  • Microarray / BeadChip: High-throughput platforms with immobilized DNA probes used to detect multiple alleles simultaneously via hybridization
  • Hybridization: The process where complementary single strands of DNA (or DNA/RNA) bind together
  • DNA Sequencing: Determining the precise order of nucleotides (A, T, C, G) in a DNA segment
  • Null Allele / Silent Allele: A gene variant that is present in the DNA but fails to produce a functional protein product (antigen) on the cell surface
  • Zygosity: The state of having identical (homozygous) or different (heterozygous) alleles for a particular gene