Bio Medical Principles of Analysis

8/8/2019 Bio Medical Principles of Analysis

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Analytical Principals and Methods

- A Clinical Chemists View


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Performance Characteristics of

Biomedical AnalysesReliability Criteria

a. Sensitivity can it measure low enough levels ?

b. Specificity can it distinguish the analyte from other

substances ?

c. Bias (Accuracy) does it give the correct value ?

d. Precision does it always give the same answer ?


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Precision and Bias

High accuracy

and precision

Poor precision,

high accuracy

Poor accuracy,

high precision

Bias


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Performance Characteristics of

Biochemical AnalysesPracticability Criteria

a. Speed turn around time

b. Cost

c. Analytical Skill

d. Dependability equipment reliability

e. Safety


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Methodological Aims

Analytical Accuracy

Measure of the agreement between a measured quantity

and the true value

Analytical Imprecision

Measure of the agreement between replicates

These will vary depending on the analyte, methodology etc.


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Methodological Aims (Cont.)

The analytical aims will vary according to the clinicalneed.

For following the course of a disease or monitoringtherapy, imprecision (reproducibility) is important.

For diagnosis, accuracy is important as the measuredvalue is compared with reference data.


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In screening for a disease, accuracy and imprecision

must be acceptable, if not :

- May generate further unnecessary tests

- May produce false positives

- May produce false negatives

- May cause analyses to be repeated


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Internal and external quality control mechanisms help

assess accuracy and imprecision on a batch to batch,day to day, month to month basis.

QC samples containing known amounts of analyte must

be routinely analysed and results reported.


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Quality Control Material

Similar matrix to patients samples

Have a reasonable degree of stability

Available in a range of analyte concentrations

Exhibit minimal between-sample variability

May have assigned or approximate values


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InternalQC

3 analytical ranges: H,M,L (within analytical range)

Introduce samples into run on x number of occasions e.g. every

20 samples

Record data calculate mean+/- S.D.

Compare with assigned value or derived value

Act on results e.g. accept or reject


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InternalQC (Cont.)

May also have 2 or more analysers performing the

same analyses, hence must be able to compare :

1. Patient samples

2. Internal QC data

3. ExternalQA results


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ExternalQC

Organised by various external bodies e.g. NEQAS

Process is voluntary, but necessary for accreditation

Regular dispatch of samples from these centres

Analyses required and returns within a set time period

Reports issues, grouped according to method / instrumentation


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ExternalQC (Cont.)

Indication of continuing performance given

Warnings issued to consistently poor performers. If trend continues,

then advisory panel notified

Action limitsT+/- 3SDs

Target value

Warning limits

T +/- 2SDs

Shewart Chart

Time

Measured

variable


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Levey Jennings QC Chart


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Westgard Rules

1 control exceeds = +/- 3 SDs

2 consecutive controls exceed = +/- 2 SDs

1 control exceeds the + 2 SD limit and a second control

exceeds the - 2 SD limit.

4 consecutive controls exceed +/- 1 SD


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Reference Ranges

Reference ranges established by measuring the concentration of aparticular analyte in a normal healthy population andfrom the calculation of the mean value and S.D.


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Defining the Reference Range

n

-2 SD 2SD

Mean

Test Value

Normal Subjects


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Reference Ranges (Cont.)

Usually, defined as the mean +/- 2SDs as shown previously.

But this excludes 5% of the healthy population.

1 in 20 will have abnormal values.

May require separate reference ranges for certain analytes

age, sex, ethnic differences.

A hospitalised population may have a different reference range !


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Reference values

Dependent on:

Selection of subjects

Assessment of the state of health

Characteristics of population, age and sex

Specimen collection and storage

Analytical technique performance characteristics

Data handling techniques


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Definitions (IFCC)

A reference individualis an individual selected

using defined criteria

A reference population consists of all possiblereference individuals

A reference value is the value obtained by

measurement of a particular quantity on areference individual


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Factors affecting reference

values Endogenous factors

age (Igs, phosphate)

sex (oestradiol/testosterone) body mass

circadian (eg cortisol / testost)

menstrual (LH/FSH/Oest / Prog),

seasonal (vitamin D),

pregnancy (hCG, urea, creatinine)


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0

100

200

300

400

500

600

700

800

900

1000

0 4 8 12 16 20 24

Time of Day (hours)

Cortisol

(mmol/L)

Cortisol


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values Preanalytical factors

Food intake (fasting/nonfasting/trigs)

alcohol intake posture (renin/aldosterone)

Immobilization

previous medical and surgical care

stress (cortisol)

exercise (growth hormone)

drug administration


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values Laboratory factors Specimen collection

transport

storage

e.g serum/plasma

Analytical technique magnitude of imprecision

Genetic factors

Ethnicity E.g. increased dyslipidaemia in Asians who settle in the

US


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Reference ranges

Two approaches:

Select a small number of individuals. Those who pass

selection criteria have samples collected and analysed under

controlled conditions. A reference range is generated.

Select a large number of people and collect samples from all

of them. After analysis the data is examined and exclusioncriteria applied.


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Problems with reference ranges

Reference values cover 95% of population

1 in 20 (5%) normals will have a test resultoutside the reference interval

2.5% individuals will have results lower than thelower ref limit and 2.5% will have results higherthan the higher ref limit


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No of tests % chance of a result

outside ref range

1 5

2 10

3 14

4 19

5 23

10 40


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No of tests

2-7

8-16

17-28

29-40

41-52

No of tests results

outside ref. range

1

2

3

4

5


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Biological variation of individuals around theirhomeostatic setting points

Multiple results for an individual for a single testmay be -

Always within the ref range

Both within and outside the interval Always outside the interval


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Is test value abnormal?

Compare with reference range

Does result differ significantly from previousresult?

Difference between 2 results on same patient isunlikely (1/20) to be due to analytical variation if>2.7 x analytical SD.

Significant differences could be due to biologicalvariation eg ALP


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Other ranges which aid interpretation

Action Limits

cholesterol

Paracetamol

TherapeuticRanges - for drugs

lithium

digoxin

Used to trigger therapeutic / investigative actions


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Action Limits for Cholesterol

Ideal

5.2

7.8

6.5

Rela ive isk H

0

100

200

300

400

500

0 2.5 5 7.5 10 12.5

les e l l/

Rela ive

Risk


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Action Limits for Paracetamol

10

100

1000

0 4 8 12 16

Tim ince ingestion (hours)

Paracetamol

(mmol/L)

Severe Liver

Damage Likely

No Treatment

Needed

Too Early

to Tell

Liver

DamagePossible -

Treatment

Desirable


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The Ideal Diagnostic Test

Norm

No false positives ornegatives

Diseased

Test Valuen

[Analyte]


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Ideal Tests

Rarely available in routine practice

High sensitivity and specificity rarely coexist

Increased sensitivity traded for decreased specificity

and vice versa


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Specificity Vs Sensitivity

the necessary compromise

Normal Diseased

Test Valuen

False Negatives False Positives

[Analyte]


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Sensitivity

Measure of the incidence of positive results in

patients with the disease

T

RUEP

OSITIV

ES

TP

TP + FN


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Specificity

Is a measure of the incidence of negative results

in people who do not have the disease

T

RUEN

EGATIV

ES

TN

FP + TN


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Positive Predictive Value

Proportion of patients with a positive test who

are in fact correctly diagnosed.

True Positives

Total Positive Tests


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ROC curves

Receiver operator curves.

Plot of sensitivity against 1- specificity Best test is that in the upper left hand corner.


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1-Specificity

Sensitivity

ROC Plot


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True negatives

TruePositives

ROC Plot


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References

Clinical Investigation and Statistics in Lab. MedicineR.Jones & B.Payne ISBN 0902429213

Interpretation of Clin Chem Lab Data. C.Fraser ISBN

0632015799 Tietz3rdEdition Chapter 13 pg 320-335

www.aacc.org

J. Automatic Chem. Vol 6 No 3 1984 122-141

Minimum Acceptability Performance Evaluation of ClinChem Methods NCCLS DocumentEP10-P Vol 6 No 3

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Bio Medical Principles of Analysis