2nd part ANA Assays - BSCL · 2020. 1. 30. · Sergio Bernardini Sofia, January14th 2ndpart ANA...
Transcript of 2nd part ANA Assays - BSCL · 2020. 1. 30. · Sergio Bernardini Sofia, January14th 2ndpart ANA...
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