Basophil ActivationTest 1 - Confex

Basophil  Activation  Test

Marta Ferrer, MD, PhD Clinica Universidad de Navarra

Medical School

Hausmann  OV  et  al  Immunol  Allergy  Clin  North  Am  2009;29:555-­‐‑66

CCR3+/CD3- CD123+/HLA-DR- IgE+/CD203+

CD63 CD203 p38 MAPK CD300

Detect  basophil  activation  

Identification  markers Activation  markers

Method

Basal

Alternatives: Buffy coat leukocytes Leukocyte enriched plasma Whole Blood

Isolation Buffer+IL3 prewarming

Stimulation Anti-IgE CD123 HLA-DR CCR3

Buffer+IL3 prewarming

Basal Anti-IgE

Anti-IgE CD123 HLA-DR CCR3

Buffer+IL3 prewarming

Basal Anti-IgE

Stimulation Allergen, drug

Allergen drug

15-­‐‑40  min  37°C

centrifugation

supernatant pellet

BAT

Anti-IgE CD123 HLA-DR CCR3

Buffer+IL3 prewarming

Basal Anti-IgE

Allergen, drug

Allergen drug

Basal Anti-IgE Allergens drug

Alternative: Anti-­‐‑CD63-­‐‑PE Anti-­‐‑CD123-­‐‑PerCP Anti-­‐‑DR-­‐‑FITC Anti  CD203c,aCD45 CCR3,CRTH2...

anti-CD63 PE anti-IgE, CD123

Basophil  labeling

R1

Region 1: Lymphocytes

R2

Region 2: Cells with anti-IgE receptor

Software  CellQuest:  50000  cells

Flow  cytometer  analysis

Negative  control

1.6%

75.% Drug

84%

Positive  control  anti-­‐‑IgE

Steps  and  differences 1.  Blood sample preparation 50-100 µl (whole heparinized blood vs. leukocyte

isolation) 2.  Preincubation with IL-3 vs. prewarming of blood 3.  Samples and reagents to 37C, allergen is added, and basophils are

incubated for 15-30 minutess at 37C in the water bath. 4.  Range concentration 5.  Staining with monoclonal antibodies coupled to fluorochromes and lysis of

contaminating erythrocytes. 6.  Select basophils (anti-CCR3/anti-CD3, anti-CD123/anti-HLA-DR). 7.  Activation markers CD63, CD203c, or P-p38MAPK

8.  Minimal number of basophils 200 per test tube but it is preferred that 600 to 1000 of results.

9.  Include positive control (anti-IgE or anti-FcεRI) 10.  Thresholds for positivity vs. ROC

CD63 CD203c

Basophil  activation  markers CD63 CD203c LAMP  family tetraspanin

Ectoenzyme

Expressed  only  upon  activation Depends  upon   P38-­‐‑MAPK  phosphorilation

Expressed  under  resting  conditions  and  up-­‐‑regulated  if  activation

Tmax:  25-­‐‑30  min

Tmax:  10-­‐‑20  min

Basophils,  monocites,  platelets

Specific  for  basophils  and  mast  cells

IL-­‐‑3  -­‐‑ IL3  + Ebo  DG  et  al.  Allergy,  2006

MacGlashan  D  Jr.  Clin  Exp  Allergy.  2010

CD63  vs.  CD203c

0.8%

65.9%

71.2%

31.6%

74.4%

94.4%

Negative  control

Negative  control

Anti  IgE

Anti  IgE

Parietaria

Allergen

CD

203c

PE

CD

63 P

E CD63

CD203c

De  Weck  Al,  Sanz  ML.  Clin  Exp  Allergy  2003

CD63  vs.  CD203c

Hausmann  OV  et  al  Immunol  Allergy  Clin  North  Am  2009

p38-­‐‑MAPK

Allow  conjugates

Steiner  M.  J  Vis  Exp.  2011

Conjugates

Steiner  M.  J  Vis  Exp.  2011

European  BAT  Interest  group  (Eurobat)

•  Optimal drug concentration •  Positivity thresholds •  Minimal basophil number •  Drug solubility •  Blood storage sample and preparation •  Whole blood vs. Isolated leukocytes •  Preincubation conditions:

o  IL3 or prewarming o  Time from 10 to 30 minutes

Betalactams

Sanz  ML  et  al.  Clin  Exp  Allergy  2002

CAP:  38%  and  87% BAT:  50%  and  94% CAP+BAT:  65.5% 34.5%  with  positive  skin  test  were  negative  for  both  techniques

Pat. Cntrl. Test   Sensitivity Specificity Sensitivity  CAP+BAT

Ref.

58 30 CAP,  ST 53 93.3 65.5% Sanz,  2002

70 40 CAP,  RAST,  ST,  DBPT

49 91 65.7% Torres,  2004

27 14 ST,  DBPT CD63:  33 CD203c:  67

CD63:  79 CD203c:  100

Abuaf  2008  

Group n Positive  BAT

ST  positive 53 27 ST  negative  CAP/RASTpositive

10 6  

ST  and  CAP/RAST  negative 7 1

Cephalosporin 77%

Torres  MJ  et  al.  Cl  Exp  Allergy  2004

Torres  MJ  et  al.  Cl  Exp  Allergy  2004

Neuromuscular  blocking  agents

Pat. Ctrls. Tests Sensibility Specificity Ref

NMBA 41 23 ST 64 93 Abuaf  1999

NBMA 50 17 ST 54 100 Monneret,  2002

NMBA 21 10 ST,  HRT CD63:  79 CD203c:  36

100 Sudheer,  2005

NMBA 47 40 ST,  DBPT 36-­‐‑86 93.3 Kvedariene,  2006

Rocuronium

14 8 ST 91.7 100 Ebo,  2006

NMBA 26 23 ST,  SIgE 68 100 Sainte-­‐‑Laudy,  2008

Ebo  DG  et  al  Allergy.  2006

BAT  sensitivity  and  time  interval

Kvedariene  V  et  al.  Allergy.  2006  

NSAIDs •  Low sensitivity (15 % to 55%) •  Positive in healthy controls •  Useful hypersensitivity to an isolated NSAID:

pyrazolones Drug Patients Controls Test

s Sensibility Specificity Ref

Metamizol 26 30 ST 42.3 100 Gamboa,  2002

Pyrazolones 51 56 ST 54.9 86 Gomez,  2009

Discussion  of  clinical  cases

BAT: ü  Complementary  technique  for  immediate  drug  reactions   ü  Sensitivity:  54%  muscle  relaxants  ,  50%  beta-­‐‑lactams   ü  Specificity  80-­‐‑90%,  confirmed  by  several  studies ü  Specially  high  for  cephalosporine ü  Combined  with  skin  tests  and  sIgE  increases  sensitivity   ü  Allows  cross  reactivity  studies  among  muscle  relaxants ü  Useful  when  no  alternative  in  vitro  diagnosis  test  is  available ü  Early  diagnosis  is  crucial ü  Needs  to  standardize  the  protocol ü  There  is  a  need  of  large  multicenter  studies  to  validate  the  

technique  and  provide  a  definitive  assessment  of  its  sensitivity.