advanced clinical chemistry for medical lab

clinical chemisty

1. Biomarker for Disease
2.3. The Phosphatases
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2. Learning Objectives
After accomplishing the lectures and completing the
exercises, the student will be able to:
Describe the biochemical theory & metabolic pathways,
and physicochemical properties of alkaline phosphatase
(ALP) and acid phosphatase (ACP)
Discuss the normal & abnormal states affecting levels of
ALP and ACP
Describe the principles of alkaline phosphatase, analysis
in terms of key reagents and their role.
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3. Learning Objectives
After listening to the lectures and completing the exercises, the
student will be able to:
Describe the principles of acid phosphatase, analysis in terms of key
reagents and their role.
Differentiate causes of common preanalytical, analytical and
postanalytical errors in alkaline phosphatase and acid phosphatase
analysis.
Interpret results of ALP and ACP compared to reference ranges.
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4. Outline of Lecture: ALP, ALP Isoenzymes
and ACP
Introduction
Source
Clinical Significance
Methods of Analysis
Calculations
Specimens
Quality Control
Interpretation of Results
Sources of Errors
Reporting and Documentation
Summary
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5. Introduction to Phosphatase
The phosphatases include:
Alkaline phosphatase (ALP)
Acid phosphatase (ACP)
Red cell phosphatase
Phosphatases catalyze the following:
Organic phosphate monoester + water  Alcohol
+ Phosphate ion
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6. Alkaline Phosphatase: Biochemical Theory and
Metabolic Pathway
Hydrolase enzyme catalyzes dephosphorylation reactions:
Removal of phosphate groups from nucleotides, proteins, and
alkaloids
Alkaline pH improves activity.
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7. Sources of Alkaline Phosphatase
Hepato-
cellular
Hepato-
biliary
Osteo-
blasts
(Bone)
Intestinal
Mucosa
Placenta
NA ALP ALP ALP ALP
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8. Isoenzymes
Alkaline phosphatase has two main forms:
Bone sources
Intestinal and liver sources
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9. Alkaline Phosphatase
Isoenzyme Characteristics
Name of Isoenzyme Hepatic Bone
Heat StabilityStable at 56
0
C for 30
minutes
Labile: disappears
at 56
0
C within 10
minutes
Electrophoretic Order Most anodic Intermediate
Chemical InhibitionModerate inhibition by
urea but low inhibition
by l-phenylalanine
Strong inhibition by
urea but low inhibition
by l-phenylalanine
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10. Alkaline Phosphatase
Isoenzyme Characteristics
Intestinal Placental Other
Intermediate labile:
disappears at 56
0
C
within 15 minutes
Stable at 56
0
C for 30
minutes
Regan isoenzyme:
most stable
Cathodic -bone
fraction
Migrates with hepatic
or bone forms
Renal isoenzyme: rare
but most cathodic
Strong inhibition by l-
phenylalanine.
Resistance to urea but
Strong inhibition by l-
phenylalanine.
Regan isoenzyme:
Strong inhibition by l-
phenylalanine.
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11. Clinical Significance of ALP Results
High concentration of ALP in hepatobiliary cells
Biliary inflammation or ductal obstruction
Cellular inflammation and necrosis
Increased with bone diseases of
osteoblastic activity
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12. Cholestasis and ALP
Release of ALP into the circulation.
Cholestasis may cause ALP increased 3-10 X the normal levels.
Serum total and direct bilirubin are increased.
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13. Test Methodology: Alkaline
Phosphatase
Analysis by the Bessey Lowry and Brock ALP method
p-nitrophenyl phosphate + H
2O –(ALP, glycine buffer, Mg
2+
, pH
10.5) p-nitrophenol + PO4
3+
-> yellow quininoid chromagen
measure increase in Abs. at 400 nm at 37
0
C
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14. Test Methodology: Alkaline Phosphatase
Analysis of alkaline phosphatase: Bowers and McComb modified
method:
4-nitrophenyl phosphate + H
2O –(ALP, Mg
2+
, pH 10.3) 4-
nitrophenoxide
Increase in Abs. at 405 nm at 37
0
C
Photometer used for two-point analysis.
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15. Calculating Absorbance Per Minute
In ALP the absorbance increases over time
The result is A X Factor
Min
Where F405 = positive number
So final activity is a positive number U/L
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16. Analysis Results Example
The following results for ALP were determined

Are the results progressing in the expected direction?
Time
(min)
Absorbance
0 1.250
1 1.350
2 1.449
3 1.551
Answer: yes, they are increasing
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17. ALP analysis
What is the  Abs for each minute?
Answer: 0.100/min; 0.099/min; and 0.102 /min
Are the results consistent?
Answer: Yes, absorbance increases
consistently
Average  Abs = 0.100 /min
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18. Calculating Absorbance Per
Minute from the Example
F = 2040
Calculate the final activity in U/L for this test result.
Answer:  Abs = 0.100 /min x 2040 = 204
U/L
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19. Calculation of Activity
ALP is reported as U/L activity.
What does U/L mean?
Answer: Activity is the amount of enzyme able to
convert 1 micromole of substrate to product per minute
per liter. U/L.
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20. Specimens for Alkaline
Phosphatase Analysis
Non-hemolyzed serum
heparinized plasma
Fresh or refrigerated
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21. Interpretation of Alkaline Phosphatase
Reference ranges vary with method used:
 53 -128 U/L
2x or more increases in serum or plasma:
Bone cancer, bone disease (such as Paget’s)
Hepatobiliary disease such as cholestasis, cholelithiasis
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22. Quality Control
A normal & abnormal quality control sample should be
analyzed along with patient samples, using Westgard or
other quality control rules for acceptance or rejection of the
analytical run.
Assayed known samples
Commercially manufactured (Humastar)
Validate patient results
 Detects analytical errors.
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23. Sources of Error for Alkaline
Phosphatase Methods
Pre-analytic Errors
Anticoagulants that remove Ca or Mg
Not Fresh Sample
False increased activity over time due to increasing pH of the
sample
Hemolysis:
Poor sample collection
Poor processing
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24. Sources of Errors in
Alkaline Phosphatase
Analytic Errors
Substances that absorb light at 405 nm:
Lipids (lipemia)
Bilirubin
Hemoglobin
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25. Sources of Errors
Alkaline Phosphatase
Too Acidic
Substrate exhaustion from excessively elevated enzyme levels
Unstable temperature
Unstable photometer
readings
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26. Reporting and Documentation
Alkaline Phosphatase
Results should be carefully review before reporting to clinicians.
Documentation of occurrences patient and quality control results
in logbooks is necessary.
Avoid common Post-analytic Errors:
Wrong name
Incorrect units or reference range
Transcribing wrong number
Report too late
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27. Problem-solving results
The following results for ALP were determined:
Are the results progressing in the expected direction?
Time (min)Absorbance
0 1.350
1 1.369
2 1.350
3 1.401
Answer: No, they are increasing and then decreasing
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28. Problem-solving:
Fluctuating Results
What might cause unstable absorbance readings?
Answer: Unstable temperature or photometer
readings.
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29. Problem-solving Results
A patient serum alkaline phosphatase result printed from the
analyzer as NL: nonlinear due to substrate exhaustion. 20
microliters (uL) of serum is mixed with 40 uL of diluent and the
sample was analyzed again. Results on the next screen.
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30. Problem-solving Results:
Dilution for ALP Analysis
The diluted result printed out as 550 U/L and a manual calculation
is required.
What is the actual ALP activity?
Answer: 3 x 550 = 1650 U/L
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31. Cirrhosis and ALP
These results were obtained from a patient suspected of having cirrhosis, causing
chronic scarring of the liver and loss of liver function.  
Describe what you observe regarding the liver enzymes.
Test Result Reference
T. Bilirubin3.1 0.0-2.0 mg/dL
Dir. Bilirubin0.5 0.0-0.2 mg/dL
ALT 65 <34 U/L
ALP 805 53 -128 U/L
Answer: ALP, the biliary enzyme is 8x the normal level and
ALT, the hepatocellular is almost 2x the normal level.
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32. Sources of Acid Phosphatase (ACP)
All cells except RBCs
Largest amounts in:
Prostate gland (semen)
Liver
Spleen
Breast milk
Platelets
Bone marrow
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33. Acid Phosphatase: Biochemical Theory and
Metabolic Pathway
Hydrolase enzyme catalyzes dephosphorylation of phosphoric
monoester
Acid pH improves activity.
Found in many tissues
prostatic tissues and seminal fluid
Non-prostatic sources
Analysis is directed toward specific source of ACP eg. prostatic
ACP or non-prostatic ACP
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34. Clinical Significance of ACP
Prostatic diseases
Metastatic prostatic cancer
Forensic investigation of rape victims
Bone disease
Metastatic bone cancer
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35. Two-point photometric analysis of ACP
The Bessey-Lowry and Brock (BLB) method
 determination of total ACP
P-Nitrophenyl phosphate (PNPP) + H
2O p-Nitrophenol +
→
Phosphate ion (Colorless) -> (Yellow chromagen) at
410nm
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36. Two-point photometric analysis of ACP
Determination of prostatic ACP
PNPP procedure repeated with tartarate solution to measure
only the 'tartarate-stable' or non-prostatic enzyme activity.
Prostatic ACP activity = Total ACT activity – Nonprostatic
ACP activity
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37. Assay of Prostatic ACP
Immunological Methods
Radioimmunoassay (RIA)
Chemiluminescence immunoassay
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38. Specimens for ACP
Serum
Separated immediately from whole blood
Add 5 mol/L acetic acid per mL of serum or sodium citrate to
preserve
Store up to 1 week in refrigerator
Vaginal Swab
Forensic
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39. Interpretation of Acid
Phosphatase:
Serum or plasma reference ranges vary with method
For example: 0.0 – 4.3 U/L male Total ACP
Prostatic cancer causes 2x or more increases in serum or plasma
0-0.6 U/L male Prostatic ACP
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40. Quality Control
A normal & abnormal quality control sample should be
analyzed along with patient samples, using Westgard or
other quality control rules for acceptance or rejection of the
analytical run.
Assayed known samples
Commercially manufactured (Humastar)
Validate patient results
 Detects analytical errors.
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41. Sources of Error in ACP
Hemolysis
Serum not separated immediately from whole blood
Nonacidified serum
Lipemia
Serum > 1 week in refrigerator
Anticoagulants (other than citrate)
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42. Summary
This lecture provided information about:
Specific biochemical enzyme-based markers of disease
Hepatocellular versus biliary conditions
The effects of enzymatic activity upon chemical reactions
The principles of alkaline phosphatase analysis in terms of key reagents
and their role
The assessment of serum enzymes and liver pathologies
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43. References
Burtis, Carl A., and Ashwood, Edward R.. Tietz: Fundamentals
of Clinical Chemistry. Philadelphia, 2001
Arneson, W and J Brickell: Clinical Chemistry: A Laboratory
Perspective 1
st
ed. 2007 FA Davis
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