Table 5.

Strengths and limitations of the different techniques for MSMD diagnosis.

Procedure	Test	Strengths	Limitations	MSMD defect detected (published)
Culture and cytokine determination	Basal IFN-γ in plasma	Easy and cheap to perform, when established, high levels of IFN-γ are indicative of IFN-γ receptor defect	Differences between kits, normalization of IFN-γ levels after acute phase of the infection	AR IFN-γ receptors defects
	WB culture with BCG stimulation	Most similar condition to reality	Partial defects can be occult, some MSMD defects have normal results; big variability in healthy controls, use of BCG difficult a lot ISO regulations acceptance Needs to be performed in fresh blood (max. 48h after extraction)	AR IFN-γ receptors, IL-12Rβ1, IL-12p40, ISG15/TYK2 defects
	PBMCs culture with BCG stimulation	Can be performed in cryopreserved cells	Partial defects can be occult, some MSMD defects have normal results; big variability in healthy controls, use of BCG difficult a lot ISO regulations acceptance
	WB culture with mitogen stimulation	NO use of BCG	Mycobacterial-specific immunity is not tested Needs to be performed in fresh blood (max. 48h after extraction)	AR IFN-γR1, IL-12Rβ1, IL-12p40 defects NEMO deficiency performed in PBMCs
	PBMCs culture with mitogen stimulation	NO use of BCG. Can be performed in cryopreserved cells	Mycobacterial-specific immunity is not tested
Cytometry (in primary cells)	IFN-γ binding	Useful to detect defects in IFN-γR1 when it is expressed in the membrane	There are some defects that bind IFN-γ but have no functional IFN-γR1	IFN-γR1 defects
	IFN-γR1 determination	Easy to perform, rapid diagnosis of some forms of IFN-γR1deficiency.	Some forms of IFN-γR1 deficiency present a non-functional form of IFN-γR1 in the membrane, so presence of the receptor does not exclude the defect.	IFN-γR1 defects
	IFN-γR2 determination	Easy to perform, rapid diagnosis of some forms of IFNGR2 deficiency	Some forms of IFNGR2 present a non-functional form of IFN-γR2 in the membrane, so presence of the receptor does not exclude the defect.	IFN-γR2 defects
	STAT1 phosphorylation determination	Rapid test that evaluates the function of both IFN-γR1/IFN-γR2 and can detect some forms of STAT1 deficiency	Partial forms of IFN-γ R1/2 defects can be occult if only high levels of IFN-γ are used, some STAT1 defects present normal STAT1 phosphorylation after IFN-γ stimulation	IFN-γR1/2 defects, STAT1, TYK2 (help)
	IL-12Rβ1 determination	Rapid/easy test; all but one mutation in IL12Rβ1 present a lack of IL-12Rβ1 in the membrane.	There is a form of IL-12Rβ1 defect that present IL-12Rβ1 in the membrane of act lymphocytes, so it’s present does not exclude the defect	IL-12Rβ1 defects
	STAT4 determination	Evaluates both IL-12Rβ1 and Tyk2 function.	Long technique; no STAT4 antibody to detect total STAT4.	IL-12Rβ1, TYK2 defects
Genetics	Sanger	When the number of candidate genes is small, it can be easy, rapid and cheap. Easy to analyze and interpret	Slow and expensive if there is not a clear orientation of the defect. Not useful for the discovery of new genes.	All known defects
	Next generation sequencing	Indicated when there is no clear candidate gene, study of all known defects at once	Expensive, difficult to analyze and interpret results due to the huge amount of generated data. Especially with whole genome sequencing, gene panels are more affordable and easy to interpret.	All known defects, discovery of new genes or new manifestations of known genes
		Useful for new genes discovery; detection of mutations in non-coding/regulatory regions with whole genome sequencing (not whole exome sequencing)

PBMC: peripheral mononuclear cells; WB: whole blood.