Prevalence of adsorbed A antigen onto donor-derived group O red cells in children following stem cell transplantation: A single-centre evaluation.

BACKGROUND AND OBJECTIVES: A group AB D-positive child presented 1 year after haematopoietic stem cell transplant (HSCT) from a group O D-negative donor as group A D-negative. Engraftment remained at 100% in white cell lineages. The reason for the unusual result was explored, and the scarcely reported phenomenon of adsorption of secreted antigen was considered. This study also investigated the prevalence of secreted antigen adsorbed onto donor-derived group O red blood cells (RBCs) in children after HSCT and defined a process for laboratory management. MATERIALS AND METHODS: Retrospective data analysis of HSCTs carried out over 19 months at Great Ormond Street Hospital was conducted to identify cases of adsorbed A antigen after HSCT. Investigation of RBC reactions with different clones of anti-A and in vitro experiments was performed to recreate adsorption. RESULTS: Nineteen A to O HSCTs were conducted over 19 months, of which six (31%) displayed weak A antigen on RBCs despite full myeloid engraftment. Negative reactions with anti-A were obtained when run on an alternative clone. Laboratory protocols for the future management of these cases have been developed. CONCLUSION: Passive adsorption of secreted antigen is responsible for these results and is more widespread than previously reported, as a third of A to O HSCTs at our centre demonstrated this phenomenon. A process has been implemented into the laboratory to manage this cohort, ensuring component groups compatible with both donor and recipient are given, and the shared care centres are aware of these requirements.

The significance of mixed chimaerism and cell lineage chimaerism monitoring in paediatric patients post haematopoietic stem cell transplant.

Haematopoietic stem cell transplants (HSCTs) are carried out across the world to treat haematological and immunological diseases which would otherwise prove fatal. Certain diseases are predominantly encountered in paediatric patients, such severe primary immunodeficiencies (PID) and diseases of inborn errors of metabolism (IEM). Chimaerism testing for these disorders has different considerations compared to adult diseases. This review focuses on the importance of cell-lineage-specific chimaerism testing and examines the appropriate cell populations to be assessed in individual paediatric patient groups. By analysing disease-associated subpopulations, abnormalities are identified significantly earlier than in whole samples and targeted clinical decisions can be made. Chimaerism methods have evolved over time and lead to an ever-increasing level of sensitivity and biomarker arrays to distinguish between recipient and donor cells. Short tandem repeat (STR) is still the gold standard for routine chimaerism assessment, and hypersensitive methods such as quantitative and digital polymerase chain reaction (PCR) are leading the forefront of microchimaerism testing. The rise of molecular methods operating with minute DNA amounts has been hugely beneficial to chimaerism testing of paediatric samples. As HSCTs are becoming increasingly personalised and risk-adjusted towards a child's individual needs, chimaerism testing needs to adapt alongside these medical advances ensuring the best possible care.

Screening of Neonatal UK Dried Blood Spots Using a Duplex SMN1 Screening Assay.

Spinal muscular atrophy (SMA) is an autosomal inherited neuromuscular genetic disease caused, in 95% of cases, by homozygous deletions involving the SMN1 gene exon 7. It remains the leading cause of death in children under 2 years of age. New treatments have been developed and adopted for use in many countries, including the UK. Success of these treatments depends on early diagnosis and intervention in newborn babies, and many countries have implemented a newborn screening (NBS) or pilot NBS program to detect SMN1 exon 7 deletions on dried blood spots. In the UK, there is no current NBS program for SMA, and no pilot studies have commenced. For consideration of adoption of NBS for a new condition, numerous criteria must be satisfied, including critical assessment of a working methodology. This study uses a commercially available real-time PCR assay to simultaneously detect two different DNA segments (SMN1 exon 7 and control gene RPP30) using DNA extracted from a dried blood spot. This study was carried out in a routine clinical laboratory to determine the specificity, sensitivity, and feasibility of SMA screening in a UK NBS lab setting. Just under 5000 normal DBSs were used alongside 43 known SMA positive DBSs. Study results demonstrate that NBS for SMA using real-time PCR is feasible within the current UK NBS Laboratory infrastructure using the proposed algorithm.

Chimerism Analysis in the Pediatric Setting: Direct PCR from Bone Marrow, Whole Blood, and Cell Fractions.

Certain blood components and anticoagulants interfere with the PCR process and subsequent analysis. Here we demonstrate that reliable test results can be obtained for chimerism analysis despite omitting a DNA-extraction step and performing PCR and fragment analysis directly on bone marrow, whole blood, and individual cell fractions. For chimerism analysis, direct-tissue PCR is possible with the use of a robust, commercially available PCR mix containing a DNA polymerase capable of DNA amplification directly from the sample without the need for pretreatment. A total of 178 chimerism samples were processed directly, and results were compared to those obtained from the corresponding DNA sample. No differences were observed between the two sets of results. For the cell fraction-purity assessment, commercially available PCR kits were used directly on T and B cells without the use of any additional lysing agent. A total of 53 purity samples and their corresponding DNA samples were analyzed and showed a correlation similar to that obtained for the chimerism samples. The results show that chimerism testing and associated cell fraction-purity assessment can be performed reliably without the need for prior DNA extraction and that this method can easily be integrated into existing routine laboratory procedures.