PID/Flow markers

Question 1

Basophil activation test by flow - what is the principle of the assay?

Answer 1

Basophils gate
Granulocytes, then
CD123+/HLA DR-

Stimulate with allergen, or control (anti IgE, anti FcERI)

Activation markers
CD63, 203a

Question 2

LAD type 1- briefly what is the pathophysiology of this condition and what test would you do?

Answer 2

Leukocyte adhesion deficiency

Type 1 most common and is AR

Children have delayed wound healing, cord falling, absent pus, bacterial infection and some fungal/opportunitist

Type 1 is defect/deficiency of b2 integrin impairing neutrophilic migration

Flow CD18/ (CD11a? check) expression on neutrophils/monocytes/ CHECK

Question 3

MSMD/ defect of Th1/IL 12 axis- what is the presentation and what tests can be done for this?

Answer 3

Weakly virulent non tuberculos environmental mycobacteria, IC org salmonella/ viral.

Possible defect

• IL12/23 p40 subunit
• • dec IFNg
• • BCG stim PBMC no IFNg but restored when IL12 added
• IL12Rb1
• • low exp by flow
• • dec STAT4 phos on stim by IL12
• IFNg autoab
• • ELISA
• -no response mitogen response
• • PBMC washed STAT1 phos
• IFNgRi/ii defect*
• inc IFNg in IGRA tube unstim
• dec STAT1 phos on IFNg stim; if partial then higher IFNg dose can overcome
• expression
• fail IL12 secretion when stim IFNg, if partial the reduced IL12

OR
STAT4 LOF
STAT1 LOF

STAT- phosphorylation assay by flow
Cytokine production measurement after stimulation by ELISA or multiplex

Question 4

STAT1 LOF - what presentation does this lead to and what kind of lab testing can be performed?

Answer 4

STAT3 GOF like effects

STAT1 in type 1/2 cytokines,
- anti MB anti viral affected

complete or partial

Question 5

STAT1 GOF- what presentation and what kind of lab testing?

Answer 5

enhanced STAT1, STAT3 LOF like function= CMC phenotype,, IPEX like disease

Question 6

Contrast STAT 3 GOF and LOF

Answer 6

STAT3 phosphorylation less useful

Question 7

STAT 5

Answer 7

IL2- STAT5b

Question 8

CMC- presentation, possible defects and lab tests- outline

Answer 8

recurrent mucocutaneous candidial infections

Primary vs secondary
Primary isolated vs syndromic SCID/CID

Primary

• dectin
• CARD9 - invasive fungal
• DOCK8- eczema, FA, viral, malignancy
• Nfkb - CVID like
• STAT3 LOF HIES
• STAT1 GOF autoimmunity/viral/ fungal/polyendocrinopathy
• IL17F AD
• IL17A/F, IL12 - autoanb AIRE APECED
• IL17Ra mutation epithelial barrier dysfunction

Question 9

Hyper IgM- outline principle of disease, subtypes, and testing done for these

Answer 9

Commonest CD40L X linked, CD40 AR, AID/UNG AR

failure of antibody production, isotype switching and somatic hypermutation due to role of CD40L -40 interaction and also cellular defects as CD40 on DC/macrophages

Bacterial, fungal, opportunitist, some IBD, malignancy

AID/UNG only impaired CSR/SHM so only humeral immunity, autoimmunity

Lack of polysacch vaccine response
Dec CD27+ memory B cells/switched

CD40L on T cells
CD40 on mono/B cells via flow

Activate T cells with mitogens PMA/ionomycin, maB CD40L, 45/3/8/40L (154)/69/25

gate on CD8 neg cells bcs CD4 expression downreg with in vitro activation, cd69/25 activation markers

Question 10

Primary HLH genetic causes - list them

Answer 10

FHL - think about albinism/other syndromes associated

All AR
FHL2 perforin- 107a normal /low perforin
FHL3 munc 13-4 granule maturation
FHL4 syntaxin 11 docking
RHL5 munc 18-2 docking
rab27a griscelli- albinism docking granules to membrane
chediak higashi lyst- lysosomal trafficking, late, albinism

All defective 107a degran

XLP- X linked
XLP1 SH2D1a gene
XLP2 BIRC4 gene

reduced SAP eps NK/CD8 cells , EBV driven

Question 11

What are the diagnostic criteria for HLH?

Answer 11

Either 1 or 2

1. molecular diagnosis
2. 5/8
   - fever
   - splenomegaly
   - cytopenia
   - hyperferritinemia
   - hyper tryglyceridemia
   - hemophagocytosis BM/tissue spleen /Ln
   - low/absent nk activity
   - s C25> 2400

Question 12

Outline how compensation is set up

Answer 12

Compensation to avoid spectral overlap

Single stained controls with unstained/unlabelled controls for neg population
Cells stained singularly with each Ab fluorochrome conjugate in the panel

Pairs of fluorochrome analysed one pair at a time
Compensation adjusted- until median of pos pop directly in line with median of neg pop and parallel to the axis

compensation reflect % signal that is subtracted from the channel

• same fluorochrome
• same antibodies
• as bright as on the cells as in sample