Reference intervals Establishing and understanding reference intervals Changing reference intervals

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Reference intervals- Establishing and
understanding reference intervals- Changing
reference intervals role of all actors in a
reference interval change and impact in the
clinical laboratory

• Ferruccio Ceriotti
• Chair IFCC committee on Reference Intervals and
  Decision Limits
• Diagnostica e Ricerca San Raffaele, Milano, Italy

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Reference Intervals interested parties

• 1. Manufactures
• DIRECTIVE 98/79/EC on in vitro diagnostic medical
  devices
• ANNEX I
• ESSENTIAL REQUIREMENTS
• B. DESIGN AND MANUFACTURING REQUIREMENTS
• 8. Information supplied by the manufacturer
• 8.7. Where appropriate, the instructions for use
  must contain the following particulars
• (l) the reference intervals for the quantities
  being determined, including a description of the
  appropriate reference population

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Reference Intervals interested parties

• 2. Clinical Laboratories
• ISO 15189 Medical laboratories Particular
  requirements for quality and competence
• 5 Technical requirements
• 5.5.5 Biological reference intervals shall be
  periodically reviewed. If the laboratory has
  reason to believe that a particular interval is
  no longer appropriate for the reference
  population, then an investigation shall be
  undertaken, followed, if necessary, by corrective
  action. A review of biological reference
  intervals shall also take place when the
  laboratory changes an examination procedure or
  pre-examination procedure, if appropriate.

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Reference Intervals interested parties

• 3. Clinicians
• ? different reference intervals from different
  laboratories? confusion between Reference
  Intervals and Decision Limits.
• 4. Patients
• ? same value can be considered normal or
  abnormal in different laboratories.

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R.I. Traditional Method

• Determine biological variables analytical
  interferences
• Determine selection/exclusion/partitioning
  criteria
• Obtain written consent and completed
  questionnaire
• Categorize reference individuals
• Exclude individuals as determined a priori
• Insure an adequate number of reference
  individuals
• Prepare reference individuals for sample
  collection
• Collect samples
• Analyze samples
• Inspect frequency distribution of data
• Identify data errors and outliers
• Determine reference intervals (and confidence
  limits)
• Document all of the previously mentioned steps
  and procedures.

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Number Needed

• for non-parametric technique,
• n (100/P)-1
• for p 2.5 (central 95), n (100/2.5) - 1
  39
• problem with using n 39 is two-fold
• Impossible to define confidence intervals
• The lower and the upper values represent 2.5 and
  97.5 percentile overly sensitive to extreme
  values
• To calculate confidence intervals, minimum n is
  120 (per partition)

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R.I. present situation some confusion

• Reasons
• The requirement that each laboratory defines its
  own R.I. method-dependent results
• Changes in methodology not accompanied by R.I.
  modification
• Adoption of R.I. proposed by the manufactures
  (sources often not declared)
• Adoption of literature data (without any critical
  appraisal)

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Inorganic phosphate
Laboratories
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ALT (IFCC method)Reference intervals (61 labs)
System A
System B
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Possible solution

• Common Reference Intervals defined
  internationally through multicenter trials

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Rationale for common reference intervals

• With a good level of standardization only
  population differences justify different R.I.
• The use of the old reference intervals with the
  new methods can impair the clinical
  interpretation of the results
• The absence of reliable reference intervals for
  the newly standardized methods hampers their
  adoption

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Are common R.I. applicable?

• Limitations
• Biology population differences
• Do they exist?
• How big are the effects due to ethnicity or
  different life habits?
• Pre-analytical and analytical
• Standardization
• Traceability

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Ethnicity of CK reference values
Harris EK et al Clin Chem 1991371580-2
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Specific protein
Myron Johnson A et al. Clin Chem Lab Med
200442792-9
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Analytes with geographical (ethnical?) differences
Ichihara K. et al. Clin Chem 200854356
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Analytes with and without geographical differences

• With
• LDH, Total protein, Globulin, Urea, C3, C4, IgG,
  CRP, Na, K, Cl, Inorganic phosphate

• Without
• AST, ALT, GGT, CK, Amylase, Albumin, Creatinine,
  Uric acid, Ca, IgA, IgM

From Ichihara K. et al. Clin Chem 200854356
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Serum Creatinine reference intervals
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Analytical requirements for the use of common R.I.
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Clinical requirements for the use of common R.I.
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Well defined reference interval (multicenter or
not)
Significant population differences
YES
NO
?
Application of the reference interval
Validation of the reference interval
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Validation of an existing R.I.

• A priori selection of 20 reference subjects
• Formal outlier test
• Apply one of the following techniques
• Binomial test (insensitive if skewed population)
• Kolmogorov-Smirnov test (but you need all the
  data)
• Use Horn and Pesce robust technique to calculate
  R.I, if the proposed R.I. are inside the
  confidence limits you can accept them

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Common reference intervals multicenter studies
possible approaches

• Each center, properly standardized, analyzes its
  own fresh samples.
• The samples are collected in the different
  centers, frozen and shipped to a central
  laboratory where all the analyses are performed.

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Pros and cons of the 2 approaches

• Approach 1 requires a rather complex preliminary
  phase, but uses native samples and the obtained
  results include the inter-laboratory variability
  it helps to improve the quality of participating
  laboratories.
• Approach 2 is simpler, allows a better control of
  the analytical phase, but uses frozen samples
  thus introducing a variable not typical for the
  routine laboratories.

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Multicenter reference interval studiesTwo examples

• IFCC experiment for AST, ALT and GGT reference
  values
• Asian project for common reference intervals

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IFCC Multicenter enzyme Reference Intervals Study

• It was a mix of the two approaches
• part of the data were obtained by clinical
  laboratories using methods for which the
  traceability of the produced results to primary
  reference methods was experimentally verified
  (approach 1)
• part of the data were obtained in one centralized
  laboratory analyzing frozen samples collected in
  other centers.

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Standardization phase

• Preparation of the material for trueness
  traceability control
• 3 serum pools at borderline concentrations,
  minimally processed (filtering, freezing at
  -80C)
• value assignment with a primary reference method
  by a network of three reference laboratories
  according to a defined protocol (three batches,
  two replicate measurements per batch)

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Standardization phase

• 2. Verification of trueness and precision of the
  participating laboratories
• 3 replicate measurements in 5 different runs (if
  relevant with different calibrations) (total of
  15 results per laboratory)

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Analytical performances during the preliminary
phase
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Multicenter R.I. study contribution of the
different centers ()
() after application of the exclusion
criteria () frozen samples
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Multicenter R.I. study AST, ALT, GGT reference
population
In total 823 subjects, 389 males and 434 females,
682 Caucasians and 141 Asians
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Exclusion criteria

• Diabetes mellitus type 1 and type 2
• Myopathies
• Burns and muscle traumas
• Hypothyroidism
• Chronic nephropathies
• Acute and chronic infection
• Hepato-biliary diseases
• Therapeutic drugs with influence on serum and
  plasma enzyme concentration (e.g. warfarin,
  antiepileptics, diphenylhydantoin, aminopyrin,
  antidepressants, analgesics).
• Antibiotics
• Pregnancy
• BMI gt30
• Heavy exercise in the previous days
• Alcohol gt 30 grams per day

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Exclusion criteria laboratory tests

• Fasting Glucose gt 126 mg/dl (7.0 mmol/L)
• Creatinine gt 0.2 mg/dl above URL
• CK gt 300 U/L
• CRP gt 12 mg/L
• UA gt 8.0 mg/dl (475 µmol/L)
• TG gt 200 mg/dl (2.26 mmol/L)
• Chol gt 260 mg/dl (6.7 mmol/L)
• Albumin lt 32 g/L
• Erys (RBC) lt 4.0 gt 5.5 mil/µL (males), lt 3.4
  gt 5.2 mil/µL (females)
• Hb lt 13 g/dl (130 g/L)(males), lt 11 g/dl (110
  g/L) (females)
• WBC lt 3000/?L gt 12000 /?L
• PLT lt 100 /nL
• Hematocrit (HCT) lt 42 gt 52 (males), lt 37 gt
  47 (females)
• MCV lt 80 gt 96 fL
• HB surface antigen Positive
• IgG antibodies anti HB core antigen Positive
• Anti HCV antibodies Positive

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ALT males

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ALT males
130
97.5
ALT (U/L)
65
32.5
0
Nordic countries
Turkey (Bursa)
Beijing
Italy (Milan)
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ALT malescorrelation with age
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Asian project

• Exclusion criteria
• BMIgt28,
• consumption of ethanol gt70g/day,
• cigarette smoking gt20 pieces,
• under regular medications for chronic diseases
  (DM, hypertension, hyperlipidemia, allergic
  disorder, depression, etc),
• recent (lt2 weeks) recovery from acute illness or
  surgery,
• in pregnancy or within 1 year after childbirth.
• A health-status questionnaire survey will be
  conducted to evaluate these factors and to obtain
  information regarding sources of regional
  differences in test results.

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References for traceability (1)
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References for traceability (2)
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Reference intervals a shared responsibility

• Manufactures provide reliable methods, able to
  provide traceable results support common
  reference intervals studies
• International organizations and Scientific
  Societies organize multicenter studies provide
  guidelines on how to develop and apply reference
  intervals
• Clinical laboratories implement and control
  carefully their methods verify and apply the
  suggested reference intervals

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Conclusions

• The possibility of providing common reference
  intervals applicable by any laboratory (able to
  produce results traceable to the reference
  measurement procedure) seems quite realistic, but
  probably not for all the analytes.
• Common reference intervals have to be applied
  carefully, after adequate checks
• A large amount of data is still missing, but
  efforts are in place to close these gaps, at
  least for the more common analytes.

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Thank you!