Method%20Selection%20and%20Evaluation

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Method Selection and Evaluation

• D. Kefaya EL- Sayed Mohamed
• Prof. Of Clinical Pathology (Clinical Chemistry),
  Mansoura University

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• Before a new test or methodogy is introduced into
  the laboratory.
• both managerial and techinal information must be
  compiled and carefully considered.
• The information should be collected from many
  different sources,including manufacturer
• sales representatives,colleagues,scientific
  presentations,and the scientific literature.

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Linear range
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• O.D
• 1.1
• 1.0
• 0.9
• 0.8
• 0.7
• 0.6
• 0.5
• 0.4
• 0.3
• 0.2
• 0.1
• 0.0 200 400 600
  800
• Glucose concentration (mg/dl)

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• Observed value
  True value

X
µ
RE
SE
TE
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• Inaccuracy
• The difference between a measured value and its
  true value.
• Is due to the presence of systematic analytical
  error (S.E.)
• S.E. can be constant or proportional .

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• Test Values
  

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Constant E
Ideal
Proprtional E
0
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Reference Values
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• Inaccuracy
• Can be estimated from three studies
• recovery .
• interference .
• a comparison-of-methods study

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• Example of a recovery study
• Sample preparation
• Sample 12.0 ml serum 0.1 ml H2O
• Sample 22.0 ml serum 0.1 ml 20 mg /dl calcium
  standard
• Sample 32.0 ml serum 0.1 ml 50 mg /dl calcium
  standard
• 
  Concentration
• Calcium measured added
  Recovered Recovery
• Sample 1 7.50mg/dl
• Sample 2 8.35mg/dl 0.95mg/dl
  0.85mg/dl 89
• Sample 3 9.79mg/dl 2.38mg/dl
  2.29mg/dl 96

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• Calculation of recovery
• Concentration added standard concentration x Ml
  standard
• 
  Ml standard ml serum
  
• 20 x 0.1
  20x 1 20 0.95 (Sample2)
• 20.1
  2.1x10 21
• conc. recovered conc. ( diluted test) conc. (
  baseline)
• 8.35-7.50.85
• recovery Conc. Recovered x100
  
• Conc. added
• 0.85 x 100 89 (
  sample 2)
• 0.95

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• ( b )Interference
• The interference experiment is used to measure
  systematic errors caused by substances other than
  the analyte

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• 
  Interference
• An interfering material can cause systematic
  errors in one of two ways
• The material itself may react with the analytical
  reagents.
• Or it may alter the reaction between the analyte
  and the analytical reagents

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• 
  Interference
• The interference likes recovery except that the
  substance suspected of interference is added to
  the patient sample.
• The concentration of the potentially interfering
  material should be in the maximally elevated
  range.
• If an effect is observed its concentration should
  be lowered to discover the concentration at which
  test resultes are frist invalidated

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• Example of an Interference study
• Sample preparation
• Sample 1 1.0 ml serum 0.1 ml H2O (base line)
• Sample 2 1.0 ml serum 0.1 ml of 10 mg/dl
  magnesium standard
• Sample 3 1.0 ml serum 0.1 ml of 20 mg/dl
  magnesium standard
• Calcium
  Measured Magnesium added
  Interference
• Sample 1 9.80mg/dl
• Sample 2 10.53 mg/dl
  0.91mg/dl
  0.73mg/dl
• Sample 2 11.48 mg/dl
  1.81mg/dl
  1.68mg/dl

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• Calculation of interference
• Concentration added standard concentration x
• 10x 0.1 10
  0..91mg/dl (sample2)
• 
  1.1 11
• Interference conc. ( diluted ) conc. (
  baseline)
• 10.53 9.8 0.73 (sample2)

ml standard ml standard ml serum
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• Common interferences (eg., hemoglobin,
  lipids,bilirubin,anticoagulants preservatives,
  and so on)also should be tested.
• Glick and Ryder have presented " interferographs
  " for various chemistry instruments these are
  graphs relating analyte concentration measured
  versus interferent concentration

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• ( C ) comparison of methouds Experiment
• The best comparative method that can be used is
  the reference method , which is a method with
  negligible inaccuracy in comparison with its
  imprecision .

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• Reference methods may be laborious and time-
  consuming Because most laboratories are not
  staffed and equipped to perform reference
  methods, the results of the test method are
  usually compared with those of the method
  routinely in use.

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• Westgard et al27 and the NCCLS35 recommend that
  at least 40 sample and preferably 100 samples, be
  run by botli methods.
• span the clinical range
• represent many different pathologic conditions.
• Duplicate analyses of each sample by each method.

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• If 40 specimens are compared , two to five
  patient specimens should be analyzed daily for a
  minimum of 8 days.
• If 100 specimens are compared the comparison
  study should be carried out during the 20-day
  replication study.

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• Test method
  

Regression line
Reference
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Example of Method selection and evaluation
(Glucose in Serum)

• Analytical Needs -
• Rapid procedure is needed at time out side the
  regular working hours.
• Sample Volume of 0.2 ml or less
• Analytical range of 0.0 to 500.0 mg/dl.
• Turn arround time 30 min.or less is needed

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• Quality goals-
• The medical decision levels of interest are
• 50 (hypoglycemia)
• 200 mg/dl (hyperglycemia)
• The decision level for screening is 140mg/dl is
  not needed.
• Precision goals are 1.5 mg/dl at 50
  mg/dl
• 
  5.0mg/dl at 200 mg/dl
• Total error goals (TEA) are 6.0 mg/dl
  at 50.0mg/dl
• 
  20.0mg/dl at 200.0 mg/dl

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• Method selection -
• In kit form
• Can be set up on an existing laboratory
  instrument
• Requirements-
• Primary reference solutions are prepared for
  calibration
• Control materials
• Randomly selected clinical specimen.

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• Within Run Precision
• Analyze 20 aliquots of low abnormal control mean
  SD 56.5 mg/dl 0.7
• Analyze 20 aliquots of moderatly high control
  mean SD 182.6 2.1 mg/dl
• SDs are less than the allowable error in non
  automated procedure .

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• Day to Day precision
• Two Q.C pools are analyzed for 20 days mean SD
• SD 1.5 mg /50 mg glucose
• 5.0 mg/200mg glucose

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• Analytical Range -
• Glucose solutions prepared from a stock glucose
  reference solution of 1000mg/dl
• Duplicate analyses
• The plot shows excellent linearity to 600mg/dl.
  which meets the specification for linearity to
  500mg/dl.
• The absorbance at zero glucose concentration is
  the reagent blank value

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• O.D
• 1.1
• 1.0
• 0.9
• 0.8
• 0.7
• 0.6
• 0.5
• 0.4
• 0.3
• 0.2
• 0.1
• 0.0 200 400 600
  800
• Glucose concentration (mg/dl)

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• Recovery-
• Two pooled sera are perpared as baseline
  specimens 61mg/dl ,171mg/dl
• concentrated glucose solution is prepared
  (10,000mg/dl)
• two different amounts of glucose are added to 9.6
  ml of pooled sera

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Recovery-
171mg/dl 61mg/dl Total Nacl 0.15mol/l Glucese solution 10,000mg/dl
b b b a a a 10.0mL 10.0mL 10.0mL 400.0ul 300.0ul 0.0ul 0.0ul 100.0ul 400.0ul
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• Quadruplicate assays of A and B are performed and
  take the mean
• Subtract the original (basal) glucose levels
  (61,171) from the mean of each quadruplicate
• Recovered
  glucose
• Recovery
• Proportional error 100- recovery

Recovered glucose Glucose added X100
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Recovery-
Glucose Recovered mg/dl Glucose found Glucose added Glucose added Pooled sera
Glucose Recovered mg/dl Glucose found mg/dl ml/10ml Pooled sera
--- 159-6198 369 --- 97 391 61 159 457 171 268 562 0.0 100 400 0 100 400 0.0 100 400 0 100 400 A A A B B B
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• Interference
• The potential interference of visible abnormal
  specimens is tested by
• A analyzing a series of icteric , turbid,and
  hemolyzed sera using the new method and an
  established method know to be free of such
  interferenc

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• The difference ( ) the two methods are 1
  4 mg which is less than the allowable TEA.
• B add a small amount of highly concentrated
  solutions to pooled sera e.g. Ascorbic acid ,
  sod. Salicylate, Trisodium
  citrate, Heparin, disod. EDTA.
• The difference must be lt TEA.

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Interference
METHOD FOR GLUCOSE METHOD FOR GLUCOSE INTERFERENCE INTERFERENCE
Test Comparative (mg/dl) Concentration Type ( Serum)
1135.6 976.4 1207.6 1407.9 1124.4 985.3 1223.2 1445.5 --- 6.5mg/dl 96mg/dl Turbid Normal Bil.(Icteric) Hb.(Hemo.) A660 0.45