Diagnostic Assay Developed for Unclassified Severe Combined Immune Deficiency

Severe combined immune deficiency (SCID) is a group of rare hereditary genetic disorders, and is characterized by a total absence of immune system function, including an absence of T-lymphocytes, the white blood cells that play a crucial role in the body's immune defense.

Without appropriate treatment, this disorder is fatal during the first months of life in the majority of cases. Newborn screening has led to an increased incidence of patients diagnosed with SCID. Although many SCID-causing genes have been identified, clinicians may face a patient without any abnormal gene identified even with advanced sequencing technologies.


A team of scientists from The Centre Hospitalier Universitaire Sainte-Justine (Montreal, QC, Canada) isolated a very small number of stem cells from patients using a limited amount of blood (3mL to 5 mL). A test with a 3-dimensional (3D) culture that mimics the function of a human thymus is used to test this small number of cells, and a response is obtained in less than five weeks. If the results are normal, thymus transplantation is recommended, but if they are abnormal, then a bone marrow transplant is preferred.

For 3D culture, CD34+ cells were purified from mononuclear cells purified by Ficoll from either fresh or frozen and thawed umbilical cord blood (CB) or peripheral blood (PB) using the MACS kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD34+ cells were mixed with trypsin-harvested OP9-DLL4 cells (1:23 mix) to form a 2.5-µL cell pellet, which was placed on a dry 25-mm polycarbonate culture insert. This insert was transferred in a 6-well dish containing 1.5 mL media. Media was changed every three or four days. Cells were harvested from the insert for fluorescence-activated cell sorter analysis.

The investigators applied the in vitro 3D T-cell differentiation assay to verify whether they could discern intrinsic from extrinsic hematopoietic stem cell (HSC) differentiation defects using limiting quantities of peripheral blood from young SCID patients. As a demonstration of an intrinsic defect of differentiation, they showed that PB-CD34+ HSCs from an IL2RG/γc patient could not differentiate into CD34−CD7+CD1a+ double-negative (DN) cells, CD4+CD8+ double-positive (DP) cells, or CD3+ cells after three weeks of culture, although CD34+CD7+ pro-T cells were abundantly present.

On the other hand, PB-CD34+ HSCs from a SCID patient with complete RAG2 deficiency (null mutation) advanced normally to the CD34−CD7+CD1a+ DN stage, with scarce presence of CD4+CD8+ DP cells (0.72% versus 22.7% for control) and no CD3+ cells after five weeks of culture. An additional RAG1 hypomorph patient presenting with Omenn syndrome could however differentiate efficiently up to the CD4+CD8+ DP stage (39.5% versus 12.36% for control) after five weeks of culture, but did not show evidence of CD3+ cell presence.

The authors concluded that they have presented a proof-of-principle for an assay using cells obtained from a minimal volume of PB to inform the physician about the approximate level of deficiency (hematopoietic stem and progenitor cell versus thymus defect) in unclassified SCID. The study was published on June 17, 2020 in the journal Blood Advances.

Related Links:
The Centre Hospitalier Universitaire Sainte-Justine
Miltenyi Biotec