Molecular Testing, Cellular Therapy and Advanced Applications
Molecular Testing
Where molecular testing fits
Platforms include real-time PCR, multiplex PCR, transcription-mediated amplification, Sanger sequencing, next-generation sequencing, microarray and digital PCR. In donor screening, nucleic acid testing shrinks the window period for HIV, HCV, HBV, WNV and other agents, using minipools or individual-donation testing. In immunohematology, genotyping resolves RHD zygosity and weak/partial D, ABO subgroups, extended antigen profiles for chronically transfused patients (especially sickle cell disease, who need phenotypically matched units), HPA and HLA typing, fetal cell-free DNA genotyping (RhD, c, K), chimerism/engraftment monitoring, and donor-derived cell-free DNA after transplant.
Limits you must know
Genotype is not phenotype. Molecular testing reports only what was probed; silencing mutations, hybrid RHD-CE-D genes, cis/trans arrangements and regulatory variants can mislead. Serologic weak D phenotypes warrant RHD genotyping: weak D types 1, 2 and 3 can safely be managed as D-positive, whereas partial D types (such as 4.0, 4.1, 11, 15) and DAAR alleles should be managed as D-negative and candidates for RhIG. Molecular testing also cannot determine whether a patient has formed an antibody - serology remains the reference.
Traps
Do not treat a negative molecular result as proof of absent antigen expression, and do not assume PCR results are quantitative. Contamination control (unidirectional workflow, UNG, separate rooms) is a favorite technical question.
Worked example
A pregnant woman of African ancestry types as serologic weak D. RHD genotyping shows a partial D allele (e.g., type 4.0). Manage her as D-negative: she requires RhIG prophylaxis and D-negative RBCs.
Cellular Therapy and Advanced Applications
Products and standards
Cellular therapy products include HPC-A (apheresis), HPC-M (marrow), HPC-C (cord blood), and manipulated or engineered products such as CD34-selected grafts, T-cell–depleted grafts, mesenchymal stromal cells, tumor-infiltrating lymphocytes and CAR-T cells. Typical thresholds are a CD34+ dose of roughly 2–5 × 10^6/kg recipient for HPC-A, and for cord blood about 2.5 × 10^7 total nucleated cells/kg with a CD34+ dose near 1.7 × 10^5/kg. Governing requirements include 21 CFR 1271 (good tissue practices) plus FACT and AABB standards. Chain of identity and chain of custody are the highest-risk controls; a mislabeled product is a catastrophic error.
Handling and infusion
Cryopreservation uses 5–10% DMSO with controlled-rate freezing near −1 °C/min and storage in liquid nitrogen vapor. Products are thawed rapidly at 37 °C, sometimes washed to remove DMSO, and infused with monitoring for DMSO toxicity (nausea, hypotension, bradycardia, respiratory symptoms) and for febrile reactions.
ABO incompatibility
Major incompatibility means recipient isohemagglutinins would attack donor red cells - the graft is red-cell depleted or the recipient plasma exchanged. Minor incompatibility means donor plasma antibodies would attack recipient red cells - the graft is plasma depleted. CAR-T therapy brings cytokine release syndrome and neurotoxicity, managed with tocilizumab and corticosteroids.
Traps
Questions invert major and minor incompatibility, or confuse total nucleated cell dose with CD34+ dose. Viability and sterility results must be reviewed before release.
Worked example
A group A donor marrow is infused into a group O recipient. Recipient anti-A and anti-B would hemolyze donor red cells - a major incompatibility requiring red cell depletion or recipient plasma exchange.