Validation study of CG cut-off and a retrospective study – the Italian experience

1. Validation study of CG cut-off and a retrospective study – the Italian experience Antonio Pecoraro PhDStudent in Clinical and Experimental Medicine Division of Allergy and ClinicalImmunology Department of TranslationalMedical Science University of Naples Federico II Center for Basic and Clinical Immunology Research (CISI) St Thomas’ Hospital, York Road Education Centre, London Friday 7 September 2018

3. Diagnostic delay in antibodydeficiency: results from the European Society for Immunodeficiencies (ESID) 2014 1746 patients with common variableimmunodeficiency (CVID) Meandiagnostic delay: 4.5 years Gathmann et al. JACI. 2014

4. Diagnostic delay in antibodydeficiency: results from an Italiancohort 224 patients with common variableimmunodeficiency Meandiagnostic delay: 8.9 years Age atdiagnosis Years of diagnostic delay • Quinti et al. J ClinImmunol. 2007

5. Causes of diagnostic delay in antibody deficiency • Heterogeneity of the clinicalpicture • Poor education and awareness in bothprimary and secondary care • Lack of screening tests

6. Calculated globulin as screening test for antibody deficiency: the Wales experience Perspectivestudy: 826 samples with low CG detected in 9 hospitals in Wales in 1 year Pilotstudy to assess the correlationbetweenvalues of IgG and CG • Jolles et al. ClinExpImmunol. 2014

7. Delay in diagnosis affects the clinical outcome in CVID patients with marked IgA deficiency 75 CVID patientsenrolledat the University of Naples Federico II An higher age at the diagnosis of CVID due to diagnostic delay was significantly associated with a reduction of patients survival if stratified per median of IgA (less than 8.00 mg/dl) • Graziano et al. ClinImmunol. 2017

8. Validation of Calculated Globulin (CG) as a Screening Test for Antibody Deficiency in an Italian University Hospital Study design: Selection of 200 anonymizedserumsamples (25 samples for eachvalue of CG between 15 and 22 g/l) BiochemistryLaboratory Dosage of IgGserumlevels for each sample ClinicalPathology Laboratory Division of Allergy and ClinicalImmunology Statistical analysis (correlationIgG vs CG, ROC curve analysis) Division of Allergy and ClinicalImmunology Retrospectiveanalysis of the correlationbetween CG and IgGvalues in 38 CVID patients

9. Results: Correlation of CG values with IgG serum levels

10. Results: ROC curve analysis of CG as independent discriminative value to detect hypogammaglobulinemia IgG 600 mg/dl IgG 500 mg/dl IgG 400 mg/dl IgG 300 mg/dl

11. Results: Comparison of sensitivity and specificity of different calculated globulin (CG) cutoffs for IgG serum levels below 600, 500, 400 and 300 mg/dl.

12. Results: Retrospective analysis of the correlation between CG values and IgG serum levels at diagnosis in 38 CVID patients

13. Validation of Calculated Globulin (CG) as a Screening Test for Antibody Deficiency in an Italian University Hospital: Summary of the findings • CG cut-off of 19 g/l detected patients with IgG ˂than 600 mg/dl with a sensitivity of 70% and a specificity of 75% • Results are consistent with the findings of the study on CG by Jolles et al., where a cut-off of 18 g/l, with a sensitivity of 66% and a specificity of 78% for samples with IgG<500 mg/dl • In the retrospective analysis, our CG cut-off of 19 g/l detected antibody deficiency in 97.3% (37/38) of the subjects of the cohort of patients diagnosed with CVID at our Centre

14. Next step….Perspective study Study design: Phase 1: Identification and collection of the serum samples with a CG level lower than or equal to 19 g/l from adults (age > 18 years) during a period of 1 year BiochemistryLaboratory Phase 2: For each sample selected: Dosage of IgG, IgA, IgM Serumelectrophoresis Serumimmunofixation (when appropriate) ClinicalPathology Laboratory Division of Allergy and ClinicalImmunology Phase 3: Collection of laboratoryresults and clinical data (age, gender, clinical speciality of source, and reason for the test) and Statistical Analysis

15. Perspective study: Timeline

16. Primary Objectives of the Perspective study • To assess the sensitivity, the specificity and the positive predictive value of the CG cut-off to detect hypogammaglobulinemia • To assess the ratio between the new diagnosed immunodeficiency cases (using CG screening) and the total population analyzed • To determine the source (in terms of clinical specialties) of the hypogammaglobulinemic patients

17. Calculated Globulin (CG) as a Screening Test for Antibody Deficiency: Open Questions • Sensitivity and Specificity of CG are potentially limited by the fact that IgG accounts for only about 30% of total serum globulin fraction • Acute inflammation may cause an increase of alpha and beta globulins and potentially mask low gamma-globulin levels • No validation for children, whose the normal IgG values vary according to age • Costs ?

18. Costs of Diagnosed vs Undiagnosed Immunodeficiency • Modellet al. Immunol Res. 2011

19. Conclusions The results of this study confirm the potential utility of a screening test based on CG as a tool to: • Reduce diagnostic delay of antibody deficiency • Improve long-term prognosis of patients living with antibody deficiency • Reduce the healthcare costs of antibody deficiency.

20. Aknowledgements Immmunodeficiency Centre for Wales Stephen Jolles Mark Ponsford Department of Biochemistry SohaZouwail Division of Allergy and ClinicalImmunology Giuseppe Spadaro Arturo Genovese Ludovica Crescenzi BiochemistryLaboratory Marcella Savoia Antonio Del Rio ClinicalPathologyLaboratory Margaret Ricciardone