Proves diagnòstiques bàsiques per al diagnòstic de les IDPOferim al pacient el benefici del coneixement més avançat Proves diagnòstiques bàsiques per al diagnòstic de les IDP

Oferim al pacient el benefici del coneixement més avançat

Proves diagnòstiques bàsiques per al diagnòstic de les IDP

V Curs d’Immunodeficiències Primàries

Dra. Martínez Gallo20 Abril 2015


Immunology Diagnostic Laboratory

§ Immunochemistry

§ Immunoallergy

§ Autoimmunity

§ Cellular Immunology

§ Immunogenetics0 102 103 104 105

0

102

103

104

10510.7 24.8

4.9459.6


•Diagnostic tools in humoral defects

•Diagnostic tools in cellular defects

•Diagnostic criteria of FHLH


Diagnostic tools in humoral defectsLevel 1Concentration of immunoglobulins in serumAntibody titer against specific antigens


Concentration of immunoglobulins in serum



Level 2 B lymphocytes (absolute values, immunophenotype)Antibody response to immunization (Type T-dep, T-indep)


B lymphocytes immunophenotype


Antibody titer against specific antigens



Level 2 B lymphocytes (absolute values, immunophenotype)Antibody response to immunization (Type T-dep, T-indep)

Level 3Protein expression: intracellular Btk, CD40, CD154, CD79a, etc.Immunophenotype B in bone marrowGenetic study of BTK, IGHM, CD179B, BLNK, CD81, etc


Btk

Protein expression: intracellular Btk


Protein expression: WB Btk


Genetic study of BTK, IGHM, CD179B, CD40LDisorder B Lymphocytes Immunoglobul

insMechanism

X-linked agammaglobulinemia

Absent/low Low IgG, IgA, and IgM

Failure of B-lymphocyte

differentiationCommon variable immunodeficiency (CVID)

≥1% Low IgG and IgA with

normal or elevated IgM

Failure of B-lymphocyte

function

Hyper IgM syndromes Normal Increased IgM with low IgG

and IgA

Failure of T-lymphocyte cooperation

Selective IgG subclass deficiency with or without IgA deficiency

Normal Normal Unknown

Specific antibody deficiencies

Normal Normal Unknown


Diagnostic tools in cellular defectsLevel 1Hemogram or complete blood count (CBC)Lymphocyte Subset Panels: T-cells, B-cells, and NK-cells

• Congenital neutropenias

• Severe lymphopenias

• Thrombocytopenias

• Specific defects with pancitopenias

• Others…


Flow Cytometry


Cellular immunologyC

OM

PLEX

ITY

SIZE FSC

SSC

Granulocitos

Monocitos

Linfocitos


• Fluorescent labeling of antibodies is to enhance the sensitivity and

specificity of immunological tests.

• Fluorescent antibodies can be used to stain proteins from patient

serum or tissue sections fixed on a slide or live cells in suspension.

• Fluorescent antibodies can be detected with a fluorescent

microscope or a flow cell sorter.

Fluorescent Antibodies


CD3+CD19+

CD3+CD19-

CD3+CD16++56+

CD3+CD16-+56-

Analysis:% lymphocyte populations

Linfocitos B

Linfocitos T

Linfocitos NKT+ B+ NK @ 100%


Diagnostic tools in cellular defectsLevel 1Hemogram or complete blood count (CBC)

Level 2 Lymphocyte Subset Panels: T-cells, B-cells, and NK-cellsNaïve (CD4RA+, CD8RA+) or memory cells (CD4RO+, CD8RO+)Activation DR+ expression Lymphocyte proliferation to mitogens/antibodies


0 102 103 104 105

0

102

103

104

105 79.8 1.46

12.76.07

0 102 103 104 105

0

102

103

104

105 0.93 0.46

97.90.7

CD8

CD4

CD45RO

CD45

RA

ADA Syndrome


100 101 102 103 104

APC-A: CD45RO APC-A

100

101

102

103

104P

E-C

y7-A

: CD

45R

A P

E-C

y7-A

25.1 7.52

66.11.29

100 101 102 103 104

PE-A: TCRgd PE-A

100

101

102

103

104

FITC

-A: T

CR

ab F

ITC

-A

88.1 0.53

6.754.58

CD45RO

TCRgd

TCRabCD45RA

Omenn Syndrome


0 102 103 104 105

0

102

103

104

105

30.1

48.2

0 102 103 104 105

0

102

103

104

105

9.48

0 102 103 104 105

0

102

103

104

105

3.79

CD3

CD8

CD3

HLA-

DR

Lymphocyte activation


Cellular immunology

PATIENT

CONTROL


0 10 2 10 3 10 4 10 5

0

10 2

10 3

10 4

10 50 69.4

30.60

0 10 2 10 3 10 4 10 5

0

10 2

10 3

10 4

10 50 0.61

99.40

CD4

CD69

a-CD3Ø

0 102 103 104 105

0

102

103

104

1050 3.85

96.10

CD3

CD69

0 102 103 104 105

0

102

103

104

1057.5 78.2

131.35

PHAØ

Activation markers after in vitro stimulation


Activation markers after in vitro stimulation


Diagnostic tools in cellular defectsLevel 1Hemogram or complete blood count (CBC)Lymphocyte Subset Panels: T-cells, B-cells, and NK-cells

Level 2 Naïve (CD4RA+, CD8RA+) or memory cells (CD4RO+, CD8RO+)Activation DR+ expression Upregulation of activation protein expression after stimulus

Level 3Analysis of signaling and functional responsesLymphocyte proliferation to mitogens/antibodiesIn vitro cytokine production


Jak1 Jak3

IL -2

ISRE/GAS

EXPRESIÓN GÉNICA

RESPUESTA BIOLÓGICA

Stat5a/b

Stat5a/b

P

P

IL2 signaling pathway

Stat5 a/b

P

b

a

g

8 8 . 3

àSTAT5-P


39.4 88.376.4

13.2 30.1 62

IL2 [100 U/mL] IL2 [5000 U/mL]IL2 [1000 U/mL]

Patient

HD

àSTAT5-P


Lymphocyte proliferation assays

3H-Timidina

The patient's peripheral blood mononuclear cells are incubated in vitro for 3–5 days with mitogens

Thymidine is incorporated into the DNA of dividing cells.

Radioactivity of the cell culture is measured after 24 hours and is directly proportional to the degree of induced cellular proliferation.

Results are affected by:-immunosuppressive drugs, -severe nutritional deficiencies-intercurrent illness and these factors must be considered when interpreting results.

mitogens


IL2

FOSJUN

intracellular

membrane



IL2

FOSJUN

intracellular

membrane

Anti CD3 Entrecruzamiento de distintos ligandos de superficie por su unión a carbohidratos

PMA

Ionomicina

Anti- CD28SEB /SEAAntígenos

PHA








Defects of Respiratory Burst


Defects of Respiratory Burst

NBT testNitroblue of Tetrazolium Test

100 101 102 103 104

FITC-A

0

20

40

60

80

100

% o

f Max

100 101 102 103 104

FITC-A

0

20

40

60

80

100

% o

f Max

HD p47phox Deficiency

Burst Test


Hemophagocytic lymphohistiocytosis (HLH)

HLH

GENETIC

Familial HLH (FHLH)- FHL1: Cr9q

- FHL2: Perforin mutations

- FHL3: UNC13D mutations

- FHL4: STX11 mutations

- FHL5: STXBP2 mutations

Immune deficiencies- Chediak-Higashi: LYST mutations

- Griscellis type 2: Rab27 mutations

- Hermansky-Pudlak type 2: AP3B1 mutations

- XLP: SAP/XIAP mutations

ACQUIRED

Infections

Sepsis

MASMalignant diseases

Others


Clinical criteria

1-Fever2-Splenomegaly

Laboratory findings

3-Cytopenias (≥ 2 cell lines)

Hemoglobin (<90 g/L), Platelets (<100 109/L), Neutrophils (<1.0 109/L)

4-Hyperferritinemia >500µg/mL

5-Hypertriglyceridemia (>3.0 mmol/L) and/or Hypofibrinogenemia (<1.5 g/L)

6-Low or absent NK-cell activity

7-sIL-2R >2400UI/mLHistopathological criteria

8-Hemophagocytosis in bone marrow, spleen, or lymph nodes

Diagnostic criteria of FHLH

To establish the diagnosis, the patient must fulfilled 5 out of 8 criteria or a molecular defect have to be demonstrated.

Pediatr Blood Cancer. 2007;48:124–131


British Journal of Haematology, 2013, 160, 275–287

Diagnostic criteria of FHLH


HLA-I+

-

K562

NK

- NK

Target Cell Effector Cell


Ensayo citotoxicidad NK

0

10

20

30

40

50

60

70

80

90

BASAL 3:1 6:1 12:1 25:1 50:1

Ratio Efector: Diana

% m

uer

te c

elu

lar

control sano

PACIENTE 1

control patológico


HEALTHY DONOR

ISOTYPECONTROL

0 102 103 104 105

<FITC-A>: PERFORINA

0

102

103

104

105

<AP

C-A

>: C

D3

42.4 0.22

0.1157.3

0 102 103 104 105

<FITC-A>: PERFORINA

0

102

103

104

105

<AP

C-A

>: C

D3

42.2 45.1

9.962.79

PERFORIN

CD

3

0 102 103 104 105

<FITC-A>: PERFORINA

0

102

103

104

105

<AP

C-A

>: C

D3

34 7.66

29.329.1

Mut PRF1Pro459Leu

Mut PRF1p.Gly477fs àX479

100 101 102 103 104FITC-A

100

101

102

103

104

AP

C-A

76.6 2.6

0.919.9

NK

CD8

PACIENT 1 PACIENT 2


- UNC13D mutation- FHLH type 3

- Rab27a mutation- Griscelli type 2

Cytotoxic cellCytotoxic cell

Cytotoxic cell

Cytotoxic cell

Case report: Degranulation assay à%NK expressing CD107a


-Actividad citotóxica

-Ensayo de degranulación NK y CD8

-Expresión de perforina

1Sd. Hemofagocíticos

-Chediak Higashi Sd-Griscelli Sd-Hermansky Pudlak Sd

-FHL3 (UNC13-D)-FHL4 (STX11)-FHL5 (STXBP2)

-FHL2 (PRF1)

(En general se realizan las tres determinaciones a la vez para aprovechar la disponibilidad de muestra)

2 -Actividad citotóxica

-Ensayo de degranulación NK y CD8

(Dependiendo del caso, se puede encontrar resultadosausentes, o normales)

-Estudio genético


Pediatrics International (2012)

-SAP/XIAP intracellular expression

-B cell subpopulation (Def. CD27)

EBV-associated lymphoproliferative disorders


-SAP/XIAP intracellular expression

-B cell subpopulation (Def. CD27)

EBV-associated lymphoproliferative disorders



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