1st partCLIA 

Primary Aldosteronism

Sergio Bernardini

Sofia, January 14th

2nd partANA Assays

Shenzhen New Industries Biomedical Engineering (SNIBE)

ABEI

CLIA

Chemiluminescence (CLIA, cLEIA, ECLI)Enzyme-linked immunosorbent assay

(ELISA)

Monoclonal Antibodies

Immune colloidal gold technique

Radio immunoassay (RIA)

Fluorescent antibody technique 1940s

1960s

1970s

1990santigen

signal

mAbSolomon Berson and Rosalyn Yalow

Georg Kohler  & Cesar Milstein

Chemiluminescence (CL) is defined as the emission of electromagnetic radiation caused by a chemical 

reaction to produce light.

Chemiluminescenceimmunoassay (CLIA) 

is an assay that combine 

chemiluminescence technique with immunochemical 

reactions. 

Type of Label Example Measurement Principal Limit of Detection

Radiolabels I125 Radioactive delay 10‐13 M

Fluorescent

Fluorescein,Rhodamine Fluorescence 10‐10 M

Rare earth chelates Time‐resolved fluorescence 10‐13 M

Enzymatic Horse radish peroxidase

Formation of colored product by enzyme

10‐11 M

Chemiluminescent Acridinium esters, luminol

Light production by chemical reaction

10‐13 M

Enzyme Catalyzed Light Emission Reaction

it is an enzyme linked immunoassay that uses luminescent chemical as substrate instead of chromogen

425nm 470nm

(1)

Enzyme DetectionLimit

Linearity Kinetics Half life

HRP 25 amol ‐/+ 1 min 1 h

XOD 3‐18 amol + 5 min 30 h

AP 3‐13 amol ‐/+ 30 min 5h

Enzyme label reagents are not stable and are easily affected by the change of storageconditions. 

Matrix components can interfere with the enzymatic reaction. Solid phase are better for reactions!

Nevertheless they produce a long stable signal in the steady state of the reaction and the reaction can be set up outside the luminometer

The automation and dispensing instrumentsare less costly

Label Chemical Directly Involved in the Light Emission Reaction

Exposure of an acridinium ester label to an alkaline hydrogen peroxide solution  triggers a flash of light

(2)

The equipment to measure chemiluminescence  is strongly dependent from the kind of label.

The transient labels (acridinium esters and luminol) have  very fast kinetics (seconds), the enzyme labels (HRP, AP, XO) steady state kinetics (minutes , hours)

The addition of an enhancer(ferrocyanide,metallic ions) leads to extremely elevated analytic sensitivity (mol‐16per litre)

Fig 1. The effects of enhancers on the CL intensity on the luminol—H2O2–HRP system.

Yang L, Jin M, Du P, Chen G, Zhang C, et al. (2015) Study on Enhancement Principle and Stabilization for the Luminol‐H2O2‐HRP Chemiluminescence System. PLOS ONE 10(7): e0131193. https://doi.org/10.1371/journal.pone.0131193https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131193

Please update the clinicians !

Larger potential dynamic ranges

Greater sensitivity than RIA and ELISA

Accurate detection of elevated concentrations

High stability of reagents and their conjugates

Low consumption of reagents and sample

Shorter TAT

Application in Total Lab Automation

No “batch”;  planning and executing the analytical procedure for each individual test (reflex test possible)

Random Access and Increased productivity (60‐70 test/h)

Multi‐analytes test 

hormones

drugs

vitamins

tumour markers

infectious disease

myocardial damage

autoantibodies

Primary Aldosteronism

Primary aldosteronism (PA) is 

a commoncurable form of 

arterial hypertension characterized by low levels of plasma 

renin andhigh plasma levels of 

aldosterone

Resistant or difficult‐to‐control hypertension is a problem that may affect as many as 13% of all 

persons with hypertension

Non‐adherence to medications 

Suboptimal treatment regimens

Unidentified secondary causes

salt‐retaining hormone> Na< K

renin via angiotensin IIelevated serum potassium concentrationACTHObesity 

SETTING No.PATIENTS COUNTRY PREVALENCE %

Unselected/primary care hypertension

Loh KC, 2000 350 Singapore 5

Fogari R, 2007 3000 Italy 5,9

Omura M, 2004 1020 Japan 6,0

Referred to specialty clinic/resistant hypertension

Rossi GP, 2006 1125 Italy 11,2

Lim PO, 2000 465 UK 9,2

Amdelhamid S, 1996 3900 Germany 6,6

Stowasser M, 2003 300 Australia 18

Yin G, 2012 313 China 12,5

Matrozova JA, 2010 376 Bulgaria 6,9

Strauch B, 2003 402 Czech Repuplic 19

Prevalence of Primary Aldosteronism

Primary Aldosteronism

SURGICALLY NOT CURABLE

• BAH• Aldosterone producing cell clusters (APCCs)

• Familial Hyperaldosteronism type I (glucocorticoid remediable aldosteronism)

• Familial Hyperaldosteronism type II‐IV

SURGICALLY CURABLE

• APA (uni o bilateral)• Primary multinodular unilateraladrenocortical hyperplasia

• Ovary aldosterone‐secreting tumor• APA or BAH with concomitantpheochromocytoma

• Aldosterone producing carcinoma

HE Korah, 2015

L Lenzini, 2019

Direct end‐organ damage•Development of CKD (CS Fox, 2006, Rossi GP, 2006)

• x 4 times risk for stroke; x 6.5 times risk for myocardial infarction, x 12 times risk for atrial fibrillation compared to general hypertension (MilliezP, 2005)

•Higher severity of hypertension

PAPY (Primary Aldosteronism Prevalence in Hypertension) study 2006

WHO Screen? Endocrine Society Clinical Practice Guideline

• patients with resistant hypertension to 3 conventional antihypertensivedrugs

• hypertension and hypokalemia (spontaneous or diuretic induced)

• hypertension and an adrenal incidentaloma

• hypertension and a family history of early‐onset hypertension or cerebrovascular accident at age <40 years

• hypertension and sleep apnea

WHO Screen? Endocrine Society Clinical Practice Guidelines

• patients with sustained blood pressure > 150/100 mmHg

• all first‐degree relatives of patients with PA 

• In total, around 50% of the hypertensive population should be screened.

• It has been suggested that all patients with hypertension should be screened for PA

Diagnosis according the Endocrine Society Guidelines

•Screening (ARR)•Confirmatory testing•Subtype differentiation

ounilateral aldosterone producing adenoma (APA): unilateral adrenalectomy

obilateral adrenal hyperplasia (BAH):  medicaltherapy with Mineralcorticoid ReceptorAntagonists

Correct use of ARR as a screening Test for PA  (before 10:00 a.m.) 1981

Serum K+ Hypokalemia lowers aldosterone level: correct hypokalemia if present before

Plasma aldosterone concentration

A low salt intake or diuretic agents can lead to high PAC . Measure 24‐h urinary Na+ excretion and withdraw diuretic agents 4 weeks before testing, they can rise renin and lower K+

Renin Fix the lowest level of renin to be used in the ARR at 0.2 ng(ml/h for PRA; at 2 mUI/l for DRC.

Patient position atthe bloodsampling

Resting supine or sitting 60 minutes before sampling considering the halflife of 15 minfor renin and aldosterone

Handling of bloodsampling

DRC handled plasma at room temperature; PRA handled in icewater to blockangiotensin‐I generation and angiotensinogen consumption

Drugs and Diet to avoid false‐negative results for the test, patients must refrain from taking spironolactonefor at least four to six weeks prior and supplemented with K+ (taken orally) if hypokalemic. Ideally, patients should follow an ad lib or high‐salt diet for two days before testing 

PG Rossi, 2019

Aldosterone

Plasma Renin Activity

Plasma

Inhibitors of Ang‐I    ‐degrading enzymesPRA

PRC

Plasma

Inhibitors of Ang‐I    ‐degrading enzymes

Saturating quantities of human angiotensinogen

or plasma from 

nephrectomized sheep 

DRCPlasma

ReninABEI labeled with another anti-Renin

m o n o c l o n a l antibody

Magnetic microbeads coated with one anti-Renin monoclonal antibody

A.H. Jan Danser, 2005

Renin

Magnetic microbeads coatedwith one anti-Renin

monoclonal antibody

ABEI labeled with another anti-Reninmonoc lonal antibody

• Automated CLIA• Quantitative PrinciplePrinciple

• 0.5 IU/ml SensitivitySensitivity

• EDTA PlasmaSample TypeSample Type

• 100 μlSample volumeSample volume

• 38 minTest timeTest time

• 0,5-1000 IU/ml Dynamic rangeDynamic range

• 100 T availablePackage Package

Direct Renin CLIA

ADDR

Maching learning 

application in APA diagnosis

N Lazzarini, 2015

PAPY study which contains 1124 patients29 features (demographic and 

biochemical data) 

Confirmatory tests

oral sodium loading test

saline infusion test

captopril challenge test (ACE inhib)

fludrocortisone with salt loading test

Confirmatory Testing

Y Yang , 2019

Subtyping

ADRENAL VEIN SAMPLING