AOAC Official Methods of

AnalysisSM
Darryl Sullivan, Covance Laboratories and Past President, AOAC INTERNATIONAL
On March 28, 2011, the AOAC INTERNATIONAL
Board of Directors approved an alternative path to
achieve an Official Method (Official First Action status)
for methods selected and reviewed using the AOAC
volunteer consensus standards development
processes.

How this change came about…

Rationale for Change
•AOAC’s ability to validate fully collaboratively
studied methods has been steadily on the decline,
approving only three in 2010.
•AOAC was repeatedly disappointing customers
and communities who needed methods to solve
problems.
•AOAC already had a reputation of being slow and
old with cumbersome processes – was potentially
facing decline in the confidence of our brand.
Rationale for Change
•AOAC has evolved and now acts as a problem solver
through a broader process of consensus building and
standards development.

•AOAC is trying to meet community/customer needs

by gathering the world’s authorities to articulate and
evaluate methods needs through expertise and
judgment.
Rationale for Change
•AOAC decided to find a way to give proper weight to
the confidence we have in the judgment and collective
knowledge of our experts.

•AOAC decided to align our brand and method output

closer to our proven standards development processes

The “new” or “alternate” path…

 How it Works – At a Glance

Funded Stakeholder Panel

Working Groups to establish Standard

Method Performance Requirements
(SMPRs)

Expert Review Panels to adopt

methods as Official First Action based
upon performance against SMPRs
 How it Works: The Details
Expert Review Panels
 Must be supported by relevant stakeholder body

 Membership is carefully managed and properly vetted by

the AOAC Official Methods Board

 Holds transparent public meetings only

 Remains in force to monitor methods as long as method is

in First Action Status.
 How it Works: The Details
Official First Action Status Decision

 Method adopted by ERP must perform adequately against

the SMPR set forth by the stakeholders

 Method becomes Official First Action on date when ERP

decision is made.
 How it Works: The Details
Official First Action Status Decision

 Methods to be drafted into AOAC format by a

knowledgeable AOAC staff member or designee in
collaboration with the ERP and method author.

 Report of decision complete with ERP report regarding

decision including scientific background (references etc) to
be published concurrently with method in traditional
AOAC publication venues
 How it Works: The Details
Transition to Final Action Status

 ERP will monitor performance and data submitted for two

years

 Further data indicative of adequate method reproducibility

performance to be collected. Data may be collected via a
collaborative study or by proficiency or other testing data
of similar magnitude
 How it Works: The Details
Transition to Final Action Status

 Removed from Official First Action and OMA if no evidence

of method use or no data indicative of adequate method
reproducibility is forthcoming

 ERP makes recommendations to the Official Methods Board

(OMB)

 OMB renders decision on transition to Final Action Status

 Expected Benefits
 More Official Methods of Analysis generated

 We can provide solutions faster and take full advantage of

collective expertise of AOAC members

 Methods can be put into regular use right away – generating

more useable data to evaluate performance
 Expected Benefits

 OMA can be more flexible – if a method is not performing

up to ERP expectations it is removed

 The transition from first to final action becomes more

meaningful and dynamic, more credence is given to final
action methods.
 Time for Change

 AOAC will convene its first Expert Review Panel charged

with adopting Official First Action Methods of Analysis this
afternoon.

 Many more ERPs will follow, developing out of the many

stakeholder activities going on at AOAC
Questions and Comments?

Thank you!
 Standard Method Performance
Requirements [SMPR] Guideline

Gaithersburg, Maryland, USA

Thursday June 30, 2011
Background

• First written in 2010.

• First used for the Endocrine Disruptor
Compounds (EDC) project in 2010.
• Reviewed and revised by Official Methods
Board in 2010.
• Still in review, but also in use.
Background
• Resulted when AOAC staff started a project
create a standard SMPR format.
• It was realized that:
• If the SMPR format required certain
parameters (i.e. “recovery”) then a definition
was needed.
• If we defined parameters then we needed to
offer guidance on how to collect data.
Background
• If we defined parameters then we needed to
offer guidance on how to collect data.
• If we offered guidance on how to collect data
then we needed to provide guidance on
acceptance criteria.
• If we offer guidance on acceptance criteria
then we needed to explain the concept.

• So 2 pages turned into . . .

Background

. . . into 13 pages with 14 pages of

appendices.
But . . . it a single, comprehensive document
with good information all in one place for
many different kinds of methods.
Philosophical Direction
• An attempt to bring to together several
different AOAC technical documents.
• OMB Guidelines
• Microbiology Methods Guideline (Appd. X)
• AOAC Single Laboratory Guideline
• BTAM Guideline
• Best Practices for Microbiological Method
(BPMM) Validation
Philosophical Direction
• An attempt to bring to together several
different types of methods:
• Chemistry
• Microbiology
• Qualitative
• Quantitative
• Identity
Components of the Guideline
1. SMPR Format
2. Recommended Performance Requirement
Parameters
3. Definitions
4. Recommendations for Evaluations
5. Explanations
6. Appendices
Architecture of Performance
Requirements in SMPR Guideline
Classification of methods
Quantitative / Qualitative
Main component / trace (contaminant)
Identification method.
Type of data
single laboratory
independent
collaborative study
Big Notes!

• No distinction made between microbiology

and chemistry!
• Not intended to require
SLV → independent lab → collaborative
• Identification methods separated from
qualitative methods,
 Classifications of Methods9
Quantitative
Quantitative Method Qualitative Method
Method Qualitative Method Identification
(trace or (trace or
( main (main component1) Method
contaminant2) contaminant2)
component1)
Single laboratory
validation
Parameters

pendent
Inde-
Collaborative
Study
 Classifications of Methods9
Qualitative Method
Qualitative Method
(trace or Identification Method
(main component1)
contaminant2)

Reference Method Comparison Reference Method Comparison Reference Method Comparison

Inclusivity/Selectivity Inclusivity/Selectivity Inclusivity /Selectivity
Laboratory
validation

Exclusivity/Specificity Exclusivity/Specificity Exclusivity/Specificity

Single

Environmental Interference Environmental Interference Precision

Laboratory Variance Laboratory Variance Environmental
Bias Bias Interference
Probability of Detection Probability of Detection (POD) at the Bias
Parameters

AMDL
Collabora- Indepen-
dent

Probability of Detection (POD)

TBD5 Bias
at the AMD

POD (0)
POD (0) POD (0)
Study

POD (c)
tive

POD (c) POD (c)

Laboratory Probability of
Laboratory Probability of Laboratory Probability of Detection
Detection
Detection8
Parameters

• Reference Method Comparison

• Inclusivity/Selectivity
• Exclusivity/Specificity
• Environmental Interference
• Laboratory Variance
• Bias
• Probability of Detection (POD)
Inclusivity/Selectivity
• Definition: Strains or isolates or variants of
the target agent(s) that the methodcan
detect.
• Recommendation: Analyze one test
portion containing a specified
concentration of one inclusivity panel
member. More replicates can be used.
Exclusivity/Specificity
• Definition: Strains or isolates or variants of
the target agent(s) that the method must
not detect.
• Recommendation: Analyze one test portion
containing a specified concentration of one
exclusivity panel member. More
Bias
• Definition: The difference between the
expectation of the test results and an
accepted reference value. Bias is the total
systematic error as contrasted to random
error. There may be one or more
systematic error components contributing
to the bias.
• No recommendations.
Probability of Detection (POD)

• Definition: The proportion of positive

analytical outcomes for a qualitative
method for a given matrix at a given
analyte level or concentration. Already
discussed.
Probability of Detection (POD)

• Recommendations: Determine the desired

Probability of Detection at a critical
concentration. Consult with table 7 to
determine the number of test portions
required to demonstrate the desired
Probability of Detection.
No definitions or recommendations

• Reference Method Comparison

• Environmental Interference
• Laboratory Variance
Summary
• SMPR summarized a variety of AOAC
guidelines.
• SMPR is comprehensive, but not detailed.
• SMPR includes chemistry and microbiology;
quantitative and qualitative.
• Work in progress.
 Chi-Square Statistics in Method
Validation

ISPAM Microbiology and Chemistry Working Groups

30 June, 2011
Dan Tholen, M.S.
Chi-Square and Related Issues
Different designs
Statistical estimators vs. statistical tests
McNemar Chi-Square test in ISO 16140
Related estimators and tests
Relationship to POD and dPOD
Estimators vs. Hypothesis Tests
Estimators provide best estimates of
parameters of interest, based on design
– Accuracy, Sensitivity, Specificity, POD, etc.
Hypothesis tests provide advice on
whether differences in estimators could
have occurred by chance
– Assumes a statistical distribution
– Requires consideration of Type 1 and 2 error
– Requires decision level
Estimators vs. Hypothesis Tests
Estimators should be accompanied by
confidence intervals that show a range of
values that could be the „correct‟ value
– Similar to measurement uncertainty
Hypothesis tests usually come with a
„reject‟ or „do not reject‟ decision, and
perhaps with a „p value‟ which is a
likelihood for the evidence if H0 is not true
– Not as informative as C.I. or MU
 Chi-Square Analysis
Recommened in ISO 16140, and in
proposed CD 16140:2011
– And in protocols influenced by ISO 16140
Used for testing equivalence of methods
by looking at discordant results
Very powerful technique, often based on
only a few discordances out of hundreds
of agreements (that is, small differences
between methods can be significant)
Comparative Accuracy - 16140
Separate study for several categories of
food (up to 5 categories)
Select at least 3 types of food from each
category
Select at least 20 samples representative
of each type
– Independent samples, not replicates
– Ideally 10 negative, 10 positive
Test each sample with both methods
 Two Way Designs - 16140
Unpaired: from the same sample, but
separate test portions

Paired: from the same sample and shared

first step in the enrichment procedure
From same enrichment medium (microbiology)
From the same extraction (chemistry)
 2x2 Layout – Paired
(and ISO 16140 unpaired)

Method B Total
(Reference)

Method A Present Absent

(Alternati
ve)
Present a b NA+

Absent c d NA-

Total NB+ NB- N

Estimators - ISO 16140 Paired
Relative accuracy: AC = (a+d)/N
Relative specificity: SP = (d)/NB-
Relative sensitivity: SE = (a)/NB+
Sensitivity (altern): SEalt = (a+b)/(a+b+c)
Sensitivity (refrnc): SEref = (a+c)/(a+b+c)
Alternative method results confirmed for
reference method negatives (cells b & d)
Estimators – 16140 Unpaired
Relative accuracy: AC = (a+d)/N
Relative specificity: SP = (d)/NB-
Relative sensitivity: SE = (a)/NB+
– These estimators are same as for paired
All alternative method results are
confirmed, estimators are listed separately
for confirmed and unconfirmed results
Chi-Square Test – ISO 16140
Only McNemar test is discussed
2 = |b-c|2 / (b+c) (1 degree of freedom)
Considers only discordant results
Other estimators not tested, McNemar is
considered the most sensitive test to rule
out differences due to random error
Requires minimum size, b and c (b+c>22)
Often the exact test (Binomial) is used for
small size samples, or in all cases
Chi-Square Test – ISO 16140
Test for significant difference in proportion
of positives
– P+A = (a+b)/N
– P+B = (a+c)/N
Since P+A and P+B both use a, the
proportions are correlated
Most sensitive test is for whether b and c
are statistically different
– Binomial, p=0.5 n=b+c
Equivalence with POD Concept
P+A: PODA = (a+b)/N
P+B: PODB = (a+c)/N
P+A - P+B = dPOD
Test of dPOD using the Binomial (for H0:
dPOD=0) is the same as the McNemar
– Large and small numbers of tests
– Both paired and unpaired
– For single lab or multi-laboratory studies
 Note on Nordval
Nordval (May 2010), for Qual. Chemistry
Uses same 2x2 layout, for paired data
Does not use Chi-Square.
Uses Kappa, a measure of agreement that
“corrects” for random agreement
The extent to which agreement exceeds
chance agreement is a measure of
concordance
Nordval recommends agreement > 80%
 Note on Nordval
Uses Kappa, a measure of agreement that
“corrects” for random agreement
– That is, if methods A and B are totally
unrelated, there is a likelihood that they will
agree on a lot of results, just by chance
e.g., if A reports 80% positive and B reports 70%
positive, then we expect them to agree 56% of the
time, just by chance
(0.8x0.7 = 0.56)
So the extent to which the methods agree in excess
of 56%, is a measure of concordance
 Classic Unpaired Design
When two methods are used on unpaired
samples
For example, drug studies on “treatment”
and “placebo” groups
This is a classic design, not done in
method validation studies
Mentioned in ISO 16140, but not used
 2x2 Layout – Unpaired
(not used in ISO 16140)

Result Total

Method Present Absent

Method A e f NA

Method B g h NB

Total N+ N- N
 Estimators
Assume random samples from same
population, randomly assigned to A or B
Proportion Positive A = (e)/NA (= PODA)
Proportion Negative A = (f)/NA
Proportion Positive B = (g)/NB (= PODB)
Proportion Negative B = (h)/NB
Accuracy, sensitivity, specificity not
defined unless all results are confirmed
 Chi-Square and POD
P+A - P+B = dPOD
Chi-Square test is the same as a Binomial
test of null hypothesis:
H0: dPOD = 0
 Chi-Square Test
Checks only for differences between
observed and expected numbers of results
in each cell
“Expected” based on random assignment
of subjects to A or B, so expect same
proportion of positives in A and B (and
same proportion of negatives)
“Expected” calculated from marginal
frequencies
 Estimators POD Concept
P+A: PODA = (e)/NA
P+B: PODB = (g)/NB
P+A - P+B = dPOD
Chi-Square test is the same as a Binomial
test of null hypothesis:
H0: dPOD = 0
Thank you
For the Validation of Qualitative
Methods
Paul Wehling
June 30, 2011

1
Qualitative (Binary) Methods
 Methods that are restricted to 2 possible
outcomes:
 Positive or Negative
 Pass or Fail
 Heads or Tails
 1 or 0
 Yes or No
 Presence or Absence
 Identified or Not Identified
2
POD
 Parameter – Probability of Detection
 General – Designed to be used by any Qualitative (Binary)
Method
 Microbiological
 Chemical
 Bio Threat Agent Methods
 Botanical Identification
 Allergens

3
POD Parameter
 Method Parameter that describes and
predicts method behavior
 Probability of Detection or POD
 The probability of getting a positive
result at a given concentration of
analyte.
 POD is a function of concentration
4
POD Curve

5
POD Curve

6
POD
 A simple descriptive statistic that describes the
method performance at a given concentration.
 It is a calculation of proportion of observed positive
outcomes per total trials.
 This simple statistic is inherent in all other systems,
such as Chi-Square, LOD, RLOD.
 The “POD Concept” is only new in that it recognizes
the POD as a key parameter and plots a graph of POD
vs concentration.

7
WHY PLOT POD?
 Plot of POD Curves are intended to assist
method users
 To assist users in selecting best method for intended
use.
 Understanding POD Curve is crucial for
interpretation of results.
 The POD curve can be an indicator of the
“usefulness” of the method.
 If POD were constant across all concentrations, the
method would not be useful.

8
VALIDATION
 The task of validating a qualitative (binary)
method is characterizing the POD curve at
critical concentration points.
1. Make up a series of test materials at concentrations
of interest.
2. Analyze with replication
3. Calculate the proportion of positive responses at
the concentrations.
4. Plot observed proportions as POD curve by
concentration.
9
10
Example POD Response Curve

11
A BIT ABOUT POD
 POD is a combination of sensitivity,
specificity, false positives, false
negatives.
 Where did they all go?

12
“Where’s my False Negative?”
POD Response vs Concentration
1
“False Negative at 1 ppm”
0.9
1-POD(1 ppm)
0.8

0.7 “Specificity”

1-POD(0)
0.6
POD

“Sensitivity at 1 ppm”
0.5
POD(1 ppm)
0.4

0.3

0.2
“False Positive”
0.1
POD(0)
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

Concentration (ppm)
13
Some Statistics
 To do Classical Collab Statistics, Code Results
 “Positive” = 1
 “Negative” = 0

Use AOAC Calculations from Quantitative Stats to estimate

 Mean = POD
 Reproducibility Standard Deviation
 Repeatability Standard Deviation
 Laboratory Standard Deviation

14
POD = Mean
LAB1 LAB2 LAB3
Trial1 1 1 0
Trial2 0 1 1
Trial3 1 1 1
Trial4 0 0 0
Trial5 1 0 0
Trial6 1 0 1
Trial7 0 0 1
Trial8 0 1 0
Trial9 1 1 0
Trial10 0 1 0
Mean 0.5 0.6 0.4 LPOD = 0.50
15
Analogous Parameters
Quantitative Quantitative Qualitative
Method Attribute Qualitative Estimate
Parameter Estimate Parameter
General Mean or
Expectation
Mean, μ Mean, x POD POD or LPOD
Repeatability
Variance
 r2 sr2  r2 sr2
Reproducibility
Variance
 R2 sR2  R2 sR2
Laboratory Variance
 L2 sL2  L2 sL2
Expected difference
between Two Methods*
Bias, B x1  x2 dPOD POD1  POD2

16
Difference Between Methods -
dPOD
 Compare any two methods by comparing
POD values at a given concentration.
 Difference by subtraction

 dPOD = PODc – PODr

 dPOD is always dependent on concentration

17
 dPOD(3.5) = -0.10

dPOD (2) = -0.27

dPOD (c = 0.5) = -0.30

18
dPOD Curve vs Concentration

19
Big Ideas
 Combine sensitivity, specificity, false
positive, false negative into 1 parameter –
Probability of Detection or ‘POD’
 Graph POD vs. Concentration with
Confidence Intervals
 Compare methods by difference of POD at
same concentration
 Use the classic statistical model and
descriptive stats for quantitative methods as
the tool for calculating qualitative stats.
20
POD Concept
 Works for single lab and Multilab experiments.
 Works for paired and unpaired designs.
 Provides harmonization across
qualitative/quantitative methodologies.
 Does comparisons and hypothesis tests via confidence
interval analysis – equivalent to chi-squared tests.
 POD Curve plots mean response and uncertainty on
the same graph.

21
Qualitative Method Validation
Studies for Quantal Data:
LOD, dPOD, PRE = RLOD and ω

Robert A LaBudde, BS, MS, PhD,

ChDipl ACAFS, PAS

AOAC Statistical Advisor

Least Cost Formulations, Ltd.

Old Dominion University
Copyright 2011 by Robert A LaBudde 1
 Summary
• Example POD vs. Concentration curves
• Ideal POD vs. Concentration curve
• Transition range models
• Method performance requirements I
• Method performance requirements II
• Limit of Detection (‘LOD’)
• The ‘Concentration Fallacy’ for micro

Copyright 2011 by Robert A LaBudde 2

 Summary (cont’d)
• Method Performance Parameters III
• Difference in POD: dPOD(C,R)
• Odds ratio ω
• Poisson Efficiency Ratio (‘PRE’)
• Relative LOD (‘RLOD’)
• Examples: Micro

Copyright 2011 by Robert A LaBudde 3

 Summary (cont’d.)
• Non-micro methods
• Choice of metamer
• Warning for micro studies
• Conclusions & Recommendations

Copyright 2011 by Robert A LaBudde 4

 Example POD vs.
Concentration curves

Copyright 2011 by Robert A LaBudde 5

 Ideal Response vs.
Concentration curve
• POD = ‘Probability of Detection’
= # Positive / # Trials
= mean of 0 or 1 data
• The ideal test method gives POD = 0 at
Concentration = 0, and POD = 1 for all
concentrations > 0.
• For real methods, there is a transition from POD =
0 to POD = 1 over a range of Concentration.

Copyright 2011 by Robert A LaBudde 6

 Transition range models
• True shape of transition curve depends upon
underlying model of what happens in test method.
• Symmetric distribution threshold crossing: Probit
and Logit (historically these have been most
commonly used).
• Asymmetric distribution threshold crossing: can
be concave or convex shape.
• ‘Hormesis’: drop-off at high concentrations.

Copyright 2011 by Robert A LaBudde 7

 Transition range models
(cont’d)
• There are a dozen or more possible model
forms in common use.
• Choice of a model form is subjective and
subject to controversy.
• Some curves convex, some curves concave,
some symmetric.
• Logit and probit are traditionally used as
middle ground when true shape is unknown.
Copyright 2011 by Robert A LaBudde 8
 Transition range models
(cont’d)
• Advantage over individual POD values may
be improved precision by pooling across
concentrations.
• If model form is incorrect, may have worse
precision than individual POD values.
• Generally requires Concentration be known
accurately.

Copyright 2011 by Robert A LaBudde 9

 Method Performance
Requirements I: Confirmation
• At the most basic level, a qualitative method is
meant to discriminate between the presence and
absence of an analyte.
• At zero concentration, POD < PODmax with 95%
confidence. (Control false positive fraction.)
• At moderate concentration, POD > PODmin with
95% confidence. (Control false negative fraction.)
• Attainment of these two requirements validates the
method as a ‘confirmation’ or ‘identification’
method in testing.
Copyright 2011 by Robert A LaBudde 10
 Method Performance
Requirements I (cont’d)
• No real method, despite claims, has POD =
0 at zero concentration or POD = 1.0 even
at high concentration, due to various error
sources, including human-in-the loop .
• One method is better than another if it has
lower POD (FPF) at zero concentration and
higher POD (lower FNF) at moderate
concentration.

Copyright 2011 by Robert A LaBudde 11

 Method Performance Requirements
II: Transition region
• The ‘I’ set of requirements does not speak to the
transition range of the POD vs. Concentration
curve.
• A method which satisfies the PODmin performance
requirement at lower Concentration is ‘better’ than
another method does so at higher Concentration.
• A method which has POD < 1 may still be useful
in repeated testing if no better method is available
(e.g., outbreak investigations for micro).

Copyright 2011 by Robert A LaBudde 12

 Limit of Detection ‘LOD’
• One way commonly used to characterize a method in the
transitional range is to estimate the concentration at which
a particular POD is attained.
• ‘LOD50’: Concentration for which POD = 0.50
• ‘LOD90’: Concentration for which POD = 0.90
• Various techniques for estimation, including non-
parametric ones, such as linear interpolation and
Spearman-Kaerber (POD-based), or assumed models.
• Requires several points (at least 2, preferably more) in the
transitional or ‘fractional’ range.
• Requires accurately known concentrations!

Copyright 2011 by Robert A LaBudde 13

LOD50

Copyright 2011 by Robert A LaBudde 14

The ‘Concentration Fallacy’ for
Micro Methods
• The transition region for qualitative methods for micro
testing typically occurs below 10 CFU/test portion and so
cannot be quantified by plate count methods, particularly
with other flora present. Instead a ‘MPN’ method is used,
based on a reference qualitative method.
• So Concentration is determined from POD (not v.v.), and
typically has large error limits (e.g., + 60% or much
worse). POD is known more accurately than
Concentration.
• Models fitting POD using Concentration as a predictor are
invalid.
• LOD50 will be imprecise and unknown to a multiplicative
factor (bias) due to clumping of cells.
Copyright 2011 by Robert A LaBudde 15
Micro: 1-Hit-Poisson Model

Copyright 2011 by Robert A LaBudde 16

 Method Performance Requirements
III: Comparison of Methods
• Two methods which both satisfy the ‘I’
requirements equally can only be discriminated if
one or the other has data in its transition range.
• There are a number of measures of effect in
common use to compare a candidate method ‘C’
to a reference method ‘R’ (or any two methods) to
each other, based on measured POD values at
different concentrations in the transition range
(i.e., fractional POD range).

Copyright 2011 by Robert A LaBudde 17

 Difference in POD:
dPOD(C,R)
• The most basic comparison between a reference
method ‘R’ and a candidate method ‘C’ is the
difference in their POD values at a fixed
concentration
dPOD(C,R) = POD(C) – POD(R)
• Non-constant for difference concentrations.
• Expected difference in # positives easily estimated
as n x dPOD(C,R).
• Requires no assumptions, applicable in all cases.
Copyright 2011 by Robert A LaBudde 18
 Odds ratio ω
• The most common measure of effect used to
compare two binary methods in scientific
research is the ‘odds ratio’ or ‘ω’
POD(R)/[1-POD(R)]
ω = ---------------------------
POD(C)/[1-POD(C)]
• If a Logit model is appropriate, the ‘odds
ratio’ is a constant across concentrations.
Copyright 2011 by Robert A LaBudde 19
 Poisson Relative Efficiency
‘PRE’ or ‘R’
• LaBudde, R.A. (2006). Statistical analysis of interlaboratory validation
studies. X. Poisson-plot and Poisson relative efficiency to compare the
dose-response curves of two presence-absence methods. TR239. Least
Cost Formulations, Ltd., Virginia Beach, VA.
ln [ 1 – POD(C) ] γR
• R= ---------------------- = ----
ln [ 1 – POD(R) ] γC

where the one-hit Poisson model ‘1HPM’ is assumed to hold.

Copyright 2011 by Robert A LaBudde 20

 PRE (cont’d)
• If both the reference and candidate methods
obey the 1HPM model with cluster sizes γR
and γC, resp., the R = γR / γC is the ratio of
the two cluster sizes needed. If the reference
method is ‘better’ (has a lower γ or
LOD50), then R < 1.0.
• If the 1HPM is valid, R will be constant
across different concentrations.
• Generally applicable to micro studies only.
Copyright 2011 by Robert A LaBudde 21
 Relative LOD ‘RLOD’
• Anon. (2008). ISO 16140.
• If a 1HPM model assumption is made for the
mathematical form of POD, and
log(Concentration) is used as the metamer in the
model, a ‘complementary-log-log’ model results.
• For the complementary-log-log model, RLOD =
R, and γR and γC are the factor coefficients for the
‘Method’ term in the regression model.
• ‘RLOD’ is the same value as ‘PRE’ = ‘R’.
Copyright 2011 by Robert A LaBudde 22
 R vs. ω vs. POD
• If POD(C) and POD(R) are both small,
R ~ ω ~ POD(C) / POD(R)
• If POD(C) and POD(R) are both large,
R ~ ω ~ POD(C) / POD(R)
• If POD(C) ~ POD(R),
R ~ ω ~ POD(C) / POD(R) ~ 1
• Differ more otherwise.

Copyright 2011 by Robert A LaBudde 23

 Examples: Micro
Analyte Matrix Study Level MPN(R) dPOD(C,R) w(C,R) R(C,R)
Salmonella Raw ground turkey Unpaired H 3.66 -0.04 0.38 0.75

Salmonella Raw ground beef #1 Unpaired H 2.18 -0.13 0.40 0.65

H 2.33 -0.10 0.45 0.70

Salmonella Raw ground beef #2 Unpaired H 2.11 -0.11 0.47 0.70

L 0.58 -0.23 0.34 0.41

Salmonella Dried whole egg Paired H 2.58 -0.05 0.59 0.82

L 1.05 -0.03 0.88 0.92

Salmonella Milk chocolate #2 Paired H 1.55 -0.03 0.84 0.91

L 0.48 -0.03 0.88 0.90

Salmonella Dry dog food Paired H 1.10 -0.03 0.86 0.91

L 0.27 -0.02 0.91 0.92

E. coli O157:H7 Raw ground beef Unpaired H 3.18 0.72 74.41 11.80
L 0.84 0.46 11.61 7.93

Copyright 2011 by Robert A LaBudde 24

 Method Performance
Requirements III
Possible performance requirements:

|dPOD(C,R)| < dPODmax with 95% confidence

ω(R,C) > ω0 with 95% confidence

R(C,R) > Rmin with 95% confidence

Copyright 2011 by Robert A LaBudde 25

 Non-Micro Methods
• Toxins, residues, chemicals, allergens, botanicals.
• There is a large literature associated with POD vs.
Concentration modeling and fitting for toxicology.
• Complementary-log-log is not typically a good
match for non-micro methods, typically logit and
probit have been used successfully.
• None of the standard regression models work for
botanical identification methods where complex
thresholding occurs.
Copyright 2011 by Robert A LaBudde 26
 Choice of metamer
• Most models use either Concentration directly or
log(Concentration) as a predictor.
• The transform of Concentration to a new
independent variable is called the ‘metamer’ of
Concentration.
• It should be noted that linear models using
Concentration as metamer and linear models using
log(Concentration) as metamer cannot both be
correct.

Copyright 2011 by Robert A LaBudde 27

 Warning for micro studies
• In the case of micro methods, cells are indivisible,
and CFUs finitely divisible, so sampling error
dominates at low concentrations. Method
differences are obscured, if they have low
LOD50’s.
• The 1HPM or complementary-log-log model will
appear to fit the data very well, and this is
fallacious because Concentration is determined
assuming 1HPM in the MPN method. Micro study
modeling should not use numerical values of
Concentration!
Copyright 2011 by Robert A LaBudde 28
 Conclusions
• Models involving Concentration are flawed at
inception for micro methods. (This applies to
Method Performance Requirements III in the
transition region and to LOD50.)
• Serial dilution should be considered as a possible
method to achieve a POD-independent
Concentration estimate.
• Many alternative models exist, each with their
own history and literature.
• PRE (aka RLOD or R) depends on the assumption
that the 1HPM is correct, which it often is not.
Copyright 2011 by Robert A LaBudde 29
 Conclusions (cont’d)
• LOD50 requires Concentration be known
reasonably accurately, problematic for micro.
• LOD50 can be nonparametrically estimated from
POD, with no model assumptions.
• The choice of statistic used to characterize the
transition region of POD vs. Concentration should
be made based on the scientific validity of the
model assumptions and the ease and usefulness of
interpretation of the result.
• Chemical-based methods have accurate
Concentrations; Micro studies do not.
Copyright 2011 by Robert A LaBudde 30
 Conclusions (cont’d)
• Use of the wrong model form will may give
poorer results than using the POD vs.
Concentration curve directly, and comparing
methods by POD difference (‘dPOD’).
• Model forms that work for one analyte or matrix
may not be appropriate for another, even in the
same scientific method area.
• Nonparametric methods (POD included) that are
distribution and model assumption-free are
preferred to unvalidated model assumptions.

Copyright 2011 by Robert A LaBudde 31

 Working Group on Statistics
• Provide advisory guidance to Micro and Chem
Working groups on aspects of statistical
methodologies.
• Advise on strengths, weaknesses and applicability of
various models.
• Advise on power of various validation experiment
designs.
• Look for potential areas of agreement and encourage
flow of ideas across Chem/Micro working groups.
 Working Group on Statistics
• Develop scientific consensus on the best
statistical techniques to use for validating
qualitative methods.
Microbiological Harmonization
June 29/30, 2011
Comparison of Method Validation
Schemes
 Worldwide Validation Schemes

• ISO 16140: internationally accepted standard for

microbiological method validation
• AOAC Microbiology Guidelines
• Health Canada Part 4
• NordVal (essentially 16140 w/o collaborative)
• FDA Draft Guidelines
• USDA/FSIS Draft Guidelines
 Comparison of Elements
• Comprehensive table constructed
• Six schemes compared
• Qualitative: 30 topics
• Quantitative: 19 topics
• Initial effort on Qualitative
• 5 of 6 schemes are either new or under
revision
 Areas of Divergence
• Microbiology Working Group (WG) had 2
teleconferences
• Identified the 5 most significant areas of
divergence among the 6 schemes.
• Nominated a Project Group (PG) with
representative from each organization
• ISO/NordVal both use 16140 so only 1
representative
 Significant Topics
• From 30 topics 5 were chosen as most critical:
• Reference method choice
• Food/Sample Matrix Applicability Table.
Selection of Food/Category
• # of levels/ # of samples/sample size/ # of
laboratories: Method Comparison &
Collaborative
• Definition of fractional positive recovery
• Data analysis (Chi square, RLOD, LOD, POD) &
performance parameters reported
 Today
• 8 page comparative summary table
prepared for the 5 significant topics
• PG will hold inaugural meeting to share
ideas on harmonization
 ISO 16140 AOAC OMA Health Canada NordVal FDA Draft USDA/FSIS
QUALITATIVE ISO 16140 AOAC OMA Health Canada NordVal FDA Guidelines Draft USDA/FSIS
methods Doc N 1199 Draft revision document Draft Part 4 dated March, 2011 Protocol for the validation FDA’s Qualitative Guidelines
(ISO CD 16140-2)PIV dated 3/24/11 of alternative
Microbiology Methods Disclaimer: The use of the
C2011-04-06 microbiological methods
Pending revision of Part 2 March 2009 Validation (ORA-LAB.7 term “validation” is not
version 1.2), pending intended to have any
revision (proposed revision application to the
marked in red). implementation of 9 CFR
417.4(a)(1) on initial
validation of HACCP plans.
The Draft FSIS Guidelines
deals exclusively with the
evaluation of pathogen test
kit methods.

Pre-
Collaborative
Phase(s)

-Reference -Defined in ISO -Can be various pre- -Acceptable Ref published ISO, CEN, NMKL, -Must be BAM, unless there For FSIS regulated products,
Method 16140-1 existing recognized by HC (Part 1) BAM, etc. It is up to is no BAM reference the current FSIS method,
-1st priority is ISO analytical methods -May include any methods the applicant; method. which is found in the
method, 2nd priority is e.g. AOAC OMA, from methods organizations, however, as the EU -If these is no BAM Microbiology Laboratory
CEN method, if ISO, FDA BAM, such as AOAC, BAM, regulation in EC reference method, but if Guidebook (MLG), is the
neither exists, then 3rd FSIS MLG and APHA, ICMSF, IDF, ISO 2073/2005 there is a most appropriate reference
priority is other Health Canada etc. Microbiological nationally/internationally cultural method for validating
recognized methods -If no appropriate Ref -Where no Ref exists, MMC criteria states EN ISO recognized reference methods used by FSIS-
can indicate “NA” in assess on case by case basis methods, these are method, then FSIS MLG, regulated establishments.
Note: definition still summary tables for most frequently used. AOAC, ISO, and Health FDA BAM, or methods
under discussion at POD Canada are all potential referenced by ISO or Codex
ISO level to open up reference methods. APHA, Alimentarius may be
for non ISO/CEN ICMSF, and IDF methods appropriate. Non-cultural
methods (PIV) also may be used as methods applicable in some
reference methods. circumstances.

Page 1 of 8
 ISO 16140 AOAC OMA Health Canada NordVal FDA Draft USDA/FSIS
-Selection of RA SLV RA, SE, SP, Kappa The selection of foods is Matrices commonly sampled
food -5 categories for all -All claimed matrices -5 categories for all foods -5 categories for all determined by FDA’s in FSIS regulated
foods applications, 3 must be included in applications, 3 food types foods applications, 3 regulatory needs. establishments: meat,
food types per the study, in other per category (Table 1). food types per poultry, and egg products,
category (see below) words, no defined -Environmental samples is category (see below) and environmental samples
-Feed, environmental categories, and “all additional category -Feed, environmental (sponges, swabs, brines)
samples and primary foods” claim not samples are
production samples applicable additional categories All claimed matrices must be
(PIV)are additional -Environmental LOD included in the study.
categories surfaces claim Same, except 1 food Contains proposal to create
RLOD require 3-7 different type per category (if matrix categories based on
Same, except 1 food surfaces (# possible) a different intrinsic properties. “All
type per category (if required is under food type Foods” claim not applicable
possible) a different review (RF)
food type IV
At least 1 matrix that
was tested in the
SLV. For every 5
foods claimed, 1 food
matrix must be
included

Page 2 of 8
 ISO 16140 AOAC OMA Health Canada NordVal FDA Draft USDA/FSIS
- Food Each Food type can be Only one single food Each food type can be made Each Food type can Currently, foods are
category/type/ made of various item is accepted to of various relevant food be made of various validated individually and
item relevant food items. meet the sample size items. Table 1. relevant food items. there are no category
Annex B provides requirement of a food At NordVal’s claims. There are no “All
guidance ( not type, i.e. 20 CLARIFICATION homepage Foods” claims.
mandatory) replicates. NEEDED (www.nmkl.org)
These are then Can these be grouped provides a list of food
grouped together to together to meet the sample categories
meet the sample number requirement of a These are then
number requirement of food type, in this case 20 grouped together to
a food type, i.e. 20 samples? meet the sample
samples. Yes, they can be group number requirement
together to meet the sample of a food type, i.e. 20
This is allow for the number requirement. This samples.
use of naturally notion has been introduce to
contaminated samples allow for heterogeneity with
(BL) in a food type. Products in a
type may vary greatly in
origin, composition,
preparation processes,
natural background; all
those small variabilities
could have an influence on
the detectability of the target
organism. (I.I.)

Page 3 of 8
 ISO 16140 AOAC OMA Health Canada NordVal FDA Draft USDA/FSIS
-No. of RA SLV and IV 3 levels: RA Level 1, 6 replicates/level, For each matrix and analyte:
levels/samples 20 samples per food 3 levels: -negative controls =5 20 samples per food single level 1) minimum 60 samples
type or 60 samples per -negative controls =5 samples type or 60 samples Level 2, 6 replicates/level, 1 inoculated at fractional
category samples -1 level with fractional per category inoculated level + 1 recovery level per
RLOD -1 level with positive results = 20 LOD uninoculated level (5 alternative and reference
3 levels fractional positive samples 3 levels replicates) method
-negative controls =5 results = 20 samples -Another level up to 1 log -negative controls =5 Level 3, 10 replicates/level, 2) 5-10 uninoculated
samples -Another (high) level higher= 20 samples samples 1 inoculated level + 1 samples per alternative
-1 level ( theoretical = 5 or 20 samples -1 level ( theoretical uninoculated level (5 and reference method
LOD, with fractional (under review (RF) LOD) = 20 samples replicates)
positive results (BL)) -Another level = at Level 4, 20 replicates/level,
= 20 samples least 5 samples 1 inoculated level + 1
-Another level = at uninoculated level (5
least 5 samples replicates)
It is proposed that each of
the 4 levels use 20 replicate
test portions and that all
levels have a negative
control.

-Sample size Undefined Standard is 25 g or 25 -25 g, but larger sample Undefined 25 g unless otherwise Application dependent.
MicroVal: Is specified mL, unless Ref sizes are permitted specified. Portions should not be made
in the reference method specified -Sample size must be the larger without validation.
method, other (larger) larger sample size same for alternate and Ref Validation study conclusions
samples size is methods, consult MMC if from larger portions
allowed but specified testing composite samples applicable to smaller
in the certificate. portions.
(PIV)
-Fractional Can be achieved by Can be achieved by Can be achieved by either Can be achieved by Yes, one or both methods defined as a range of 20-80%
positive either alternate or Ref. either alternate or alternate or Ref. either alternate or must give 40 – 90% positive confirmed positive results
- All samples should Ref. -proportion of positives Ref. results. It is proposed that using reference method
not be all positive or -proportion of 25% to 75%, - All samples should the percentage positive
all negative. positives 25% to not be all positive or results be changed to 25 –
-Ideal is 10 positive 75%, ideal is approx all negative. 75%.
and 10 negative (50%) 50% -Ideal is 10 positive
but any fractional (10% to 90% is under and 10 negative
results is acceptable review (RF) (50%) but any
fractional results is
acceptable

Page 4 of 8
 ISO 16140 AOAC OMA Health Canada NordVal FDA Draft USDA/FSIS
-Results analysis RA - by level and by Method Equivalence: RA By level/individual Performed for each matrix.
and criteria -By type and by matrix -POD -By type and by experiment for each matrix. Unpaired study: One sided
category - by POD Probability -one-tailed POD 95% category Per AOAC Microbiology chi-square test with alpha =
-Relative accuracy of Detection 95% confidence interval (I.I.) -Relative accuracy guidelines, McNemar Chi 0.05. Criterion:
AC, relative confidence interval AC, Square statistics are used. indistinguishable or better
specificity SP, relative for the alternate the Performance parameters: -Relative specificity performance than reference
sensitivity SE Ref and presumptive - by level and by food, but SP, method. Paired study:
-First by unconfirmed and confirmed results only calculated for those -Relative sensitivity Evaluate sensitivity with
results, again by -then by difference that passed POD SE minimum 29 confirmed
confirmed results between POD successfully - Kappa positive results. Zero false
-McNemar test as alternate and POD For Unpaired : -First by unconfirmed negative results from 29
criteria, (for paired Ref, confidence level -Performance parameters is results, again by confirmed positives would be
and unpaired) with must contain zero for the comparison of confirmed results consistent with a test having
caveats i.e. really not method to be presumptive vs. confirmed a sensitivity that met or
suitable for unpaired considered not results of the alternate Criteria: exceeded 90% and zero
and “never be different at 95% method ( not the Ref SE ≥ 95% negative results from 50
interpreted by only confidence method results) Kappa ≤ 0.80 confirmed positives would be
the McNemar test” - Chi Square is not -Specificity is based on LOD: fit for purpose consistent with a test with a
RLOD required but presumptive results sensitivity that met or
-by category “interesting” -Sensitivity is based on final exceeded 94%. Criterion:
-LOD of alternate ( confirmed) results none proposed
method divided by - Equivalence of alternate
LOD of Ref method and Ref can only be
For paired, no lower determined by the number
limit, but LOD of true positives in both sets,
alternate might not be done by POD method
> 2 times the LOD Ref
For unpaired samples, For Paired:
no lower limit, the Use “absolute” results
LOD alternate might where Ref can have FN
not be >3 times the
LOD Ref (In the Criteria:
ISO/CD 16140-2 Sensitivity 98%
version, I don’t find Specificity 90.4%
any acceptability False negative rate < 2%
limit settled for False positive rate ≤ 9.6%
unpaired samples, Efficacy 94%
only specified for LOD must be comparable or
paired samples BL) exceed the lower LOD of
the Ref
The values of 2
(paired) and 3
(unpaired) are still
tentative values!!!!!
(PIV)

Page 5 of 8
 ISO 16140 AOAC OMA Health Canada NordVal FDA Draft USDA/FSIS
Inter- Applicable to alternative
laboratory methods with a major
Study modification, defined as any
significant change in the
design or the component
reagents for a screening test,
for example, the introduction
of a new antibody or
oligonucleotide primer.

Follow guidance provided by

the AOAC International
Official Methods of Analysis
Program
- minimum no. -10; defined as -10 valid lab data sets Minimum of 8 labs -10; defined as 2 for a Level 2 study, 3 for a
of valid data individuals working required reporting valid data, labs individuals working Level 3 study, and 10 for a
sets/collaborators independently using - Specifies that 12 should be accredited per independently using level 4 study.
different sets of labs should start 17025 or demonstrate is different sets of
samples; from a min. functioning under samples; from a min.
of 5 different equivalent quality system of 5 different
organizations, organizations,
including organizing including organizing
lab and different lab and different
locations from same locations from same
company company

-Sample size NA Standard is 25 g or 25 CLARIFICATION NA 25 g unless otherwise

mL, unless Ref NEEDED specified.
Is defined by the method specified Consistent with Pre-
protocol of the larger sample size collaborative?
reference method Sample size is 25g unless
(PIV) otherwise specified by the
method or need for larger
size ( to achieve enhance
detectability, regulatory
purpose or compositing)
(I.I.)

Page 6 of 8
 ISO 16140 AOAC OMA Health Canada NordVal FDA Draft USDA/FSIS
- number of 1; relevant food item, 1 At least 1 1; relevant food One or more.
foods inoculated with target, item, inoculated with
using a challenging target, using a
enrichment protocol challenging
enrichment protocol

- number of 3; negative control, 3; negative control, 3; negative control, one 3; negative control, 2 for a Level 2 study (1
levels one level which one level which level which produce one level which inoculated and 1
produce fractional produce fractional fractional positive and produce fractional uninoculated.
positive and another positive and another another level about 10 times positive and another 3 for Levels 3 & 4 (high,
level level greater than the detection level low, and uninoculated.
level

- number of 8; per level of 12 per level of 8 per level 8 laboratories; 6

replicates contamination contamination - min of 24 results per - 3 levels in
-minimum of 48 - 72 results per collaborator ( 8 x3 levels ) duplicates
results per collaborator collaborator = 12 per method
= 8 replicates x 3 replicates x 3 levels x 8 labs x 3 levels x 2
levels x 2 methods 2 methods = 72 replicates x 2
-minimum of 480 - minimum of 720 methods
results (48 from each results ( 360 per
collaborator) = ( 240 method) for statistical
per method) for analysis
statistical analysis

-Confirmation for Paired, only Matched or Confirm all samples for Paired, only Yes.
confirm the + Alt/- unmatched, confirm confirm the + Alt/-
Ref, for Unpaired, all samples Ref, for Unpaired,
confirm all confirm all
enrichments enrichments

Page 7 of 8
 ISO 16140 AOAC OMA Health Canada NordVal FDA Draft USDA/FSIS
- Comparisons Analyzed two ways: By level and by CLARIFICATION Alternative to reference
1.Unconfirmed matrix analyzed and NEEDED method (if available).
Alternate method reported separately Consistent with Pre-
results vs. confirmed collaborative?
Ref By level and by matrix, all
2. Confirmed result confirmed
Alternate method Confirmed alternate method
results vs. confirmed results vs reference (I.I.)
Ref

-Parameters Specificity ( only for Cross Lab Probability CLARIFICATION Rel Specificity Per AOAC guidelines,
Calculated Neg controls) of Detection (LPOD) NEEDED Rel Sensitivity Sensitivity, Specificity,
Sensitivity ( only for Difference between Consistent with Pre- Rel Accuracy False Negative, and False
inoculated levels ) Alternate LPOD and collaborative? Kappa Positive Rates.
Relative Accuracy Ref LPOD Yes,
(%of agreements ) POD , dPOD determined
RLOD of the for each matrix-level . All
different participants dPOD data is then used to
(BL) assess the comparative
performance of both
methods
All 5 method
parameter(specificity,
selectivity, FP, FN and
method efficacy) calculated
in one of two ways,,
depending if sample is
paired or un paired. (I.I.)
- Interpretation
McNemar test (chi If confidence interval CLARIFICATION Criteria: Per AOAC guidelines,
square) of dLPOD does not NEEDED SE ≥ 95% McNemar Chi Square
RLOD is for contain zero, then the Consistent with Pre- Kappa ≤ 0.80 statistics.
information only diff is statistically collaborative? [LOD: fit for its
: analysis of deviance significant Yes , dPOD one-tailed and purpose]
test to assess the method parameter
laboratory effect on requirement must be met.
RLOD then (I.I.)
acceptability of
RLOD global value
(BL)

Page 8 of 8
 International Stakeholder Panel on Alternative Methods
Microbiology Working Group for Harmonized Matrix Comparison

Thursday, June 30, 2011 at 1:00pm – 3:00pm

Twinbrook
HILTON WASHINGTON D.C./ROCKVILLE EXECUTIVE MEETING CENTER

DRAFT MATRIX TABLES FROM:

EN ISO 1614:2008 – NORDVAL - AOAC BPMM –

AOAC BPMM WORKING GROUP MATRIX EXTENTION DRAFT

ISPAM

1) EN ISO 16140:2008 E

2) NORDVAL MATRIX TABLES

a. Salmonella
b. Listeria
c. Campylobacter
d. E.coli O157

3) Annex A AOAC OMA Microbiological Guidelines

4) Appendix B – BPMM AOAC Microbiological Working Group for

Matrix Extension

AOAC ISPAM Small Group Micro Working Groups Meeting Agenda nlm PRE-DECISIONAL Page 1
EN ISO 16140:2008 (E) nlm
EN ISO 16140:2008 (E) nlm
Matrix for Salmonella (NORDVAL)
Matrix group

Matrix Food examples

1. Meat 1.1 Raw red meat Minced meat, offal
1.2 Raw white meat Chicken, turkey, duck
1.3 Raw smoked salted products Bacon
1.4 Heat treated products Sliced meat and poultry products
1.5 Fermented products Salami
2. Fish 2.1 Raw fish and shelfish Raw two-shelled mollusc, raw
shrimps
2.3 Heat treated fish products Heat treated shrimps
and shelfish
3. Milk 3.1 Milk Raw milk
3.5 Desserts, ice-cream Ice-cream

3.6 Dry milk products Milk powder

4. Eggs 4.1 Raw egg Whole egg
4.2 Egg products Manufactured egg
4.3 Dried products Dried whole eggs
5. Vegetable products 5.1 Raw vegetables Sprouts
5.2 Dried products Spices
5.4 Fatty products Chocolate, mayonnaisesalads

7. Environment tests 7.1 Environment tests Swab tests

8. Feed 8.1 Animal feed Meat bone meal, fish meal, fish food

9. Animal faeces
10. Miscellaneous

NORDVALnlm
Matrix for Listeria (NORDVAL)
Matrix group Matrix Food Examples
1. Meat 1.1 Raw red meat Minced meat, (tatar – type)
1.3 Raw smoked salted Bacon, smoked filet
meat-products
1.4 Heat treated products Sliced meat and poultry products
1.5 Fermented products Salami
2. Fish 2.1 Raw fish, shelfish and Cold smoked salmon
Fish products
2.3 Heat treated fish products Heat treated shrimps
3. Milk 3.1 Milk Raw milk
3.4.1 Firm cheese Yellow cheese

3.4.2 Soft cheese Mould cheese

3.5 Desserts, ice-cream Ice-cream

4. Eggs 4.1 Raw egg Whole egg

5. Vegetable products 5.1 Raw vegetables Cut salads, sprouts
7. Environment tests 7.1 Environment tests Swab tests, Cleaning water
10. Miscellaneous
Matrix for Campylobacter (NORDVAL)
Matrix group Matrix Food Examples
1. Meat 1.1 Raw red meat Minced meat, offal
1.2 Raw white meat Chicken, turkey, duck
1.3 Raw smoked salted products Sliced smoked turkey meat
1.4 Heat treated products Sliced poultry meat
2. Fish 2.1 Raw fish and shelfish Raw two-shelled mollusc, raw shrimps
3. Milk 3.1 Milk Raw milk
4. Eggs 4.1 Raw egg Whole egg
4.2 Egg products Manufactured eggs
5. Vegetable products 5.1 Raw vegetables Sprouts
7. Environment tests 7.1 Environment tests Swab tests
9. Animal faeces
10. Miscellaneous

Matrix for E. coli O 157 (NORDVAL)

Matrix group Matrix Food Examples
1. Meat 1.1 Raw red meat Minced meat, cut meat, offal
1.3 Raw smoked salted Bacon, smoked filet
meat-products
1.4 Heat treated products, ready Sliced meat and poultry products,
to eat smoked products smoked turkey filet

1.5 Fermented products Salami

3. Milk 3.1 Milk Raw milk
3.2 Sour milk products Yoghurt with fruit
3.4 Cheese Mould cheese
3.5 Desserts/ice-cream Ice cream
5. Vegetable products 5.1 Raw vegetables Cut salads, sprouts
5.4 Fatty products Mayonaise-salads
7. Environment tests 7.1 Environment tests Swab tests
9. Animal feces
10. Miscellaneous
Annex A. OMA Microbiology Guidelines AOAC INTERNATIONAL
Annex A. OMA Microbiology Guidelines AOAC INTERNATIONAL