This blog post will serve as a guide to help you estimate the quantity of a protein called serum Alkaline Phosphatase (ALP) which is a significant enzyme discovered in the liver, bone and intestine. ALP enzymes or Activity levels in the blood are some important diagnostic parameters that can be used for determining the presence of liver injury, bone pathology and some other diseases.
Importance of ALP Measurement
ALP
measurement is a vital diagnostic tool in various medical contexts, including:
1. Liver Disease
Because Alkaline Phosphatase (ALP) is a sensitive indicator of dysfunction in the liver, it is a necessary test for diagnosing and monitoring liver diseases like hepatitis, cirrhosis and liver cancer.
2. Bone Disorders
Alkaline Phosphatase (ALP) is a valuable marker for bone metabolism and remodeling, helping diagnose and
manage conditions like osteoporosis, Paget's disease and bone cancer.
3. Intestinal Disorders
Alkaline Phosphatase (ALP) measurement can aid in diagnosing intestinal obstruction, inflammation and
ischemia.
4. Cancer Diagnosis
Alkaline Phosphatase (ALP) is a useful tumor marker for detecting liver, bone and pancreatic cancers.
5. Genetic Disorders
Alkaline Phosphatase (ALP) measurement can help diagnose genetic conditions like hypophosphatasia (ALP
deficiency).
6. Pediatric Care
Alkaline Phosphatase (ALP) levels can indicate bone growth and development issues in children.
7. Geriatric Care
Alkaline Phosphatase (ALP) levels can indicate age-related bone loss or osteoporosis.
8. Pregnancy Care
Alkaline Phosphatase (ALP) levels can indicate liver or bone issues related to pregnancy.
9. Medication Monitoring
Medication or toxins can cause potential damage to the liver or bones, which can be detected by measuring Alkaline Phosphatase (ALP).
10. Research and Diagnostic Labs
Alkaline Phosphatase (ALP) measurement is used as a biomarker in research studies and Diagnostic Labs to
evaluate the efficacy of new treatments for various diseases.
Methods
1. Colorimetric Assays
Take note of the color shift that occurs when Alkaline Phosphatase (ALP) reacts with a substrate, such as p-nitrophenyl phosphate (pNPP).
2. Enzyme-Linked Immunosorbent Assays (ELISA)
Use of antibodies specific to Alkaline Phosphatase (ALP) measures the enzyme's activity.
3. Chemiluminescent Assays
Measure
the light emitted during the reaction of Alkaline Phosphatase (ALP) with a substrate, such as luminol.
4. Electrophoresis
Separate
and measure Alkaline Phosphatase (ALP) isoenzymes based on their charge and size.
5. Kinetic Assays
Measure the rate of reaction between Alkaline Phosphatase (ALP) and a substrate, such as p-nitrophenyl phosphate pNPP.
6. Fluorometric Assays
Measure
the concentration of fluorescence emitted during the reaction of Alkaline Phosphatase (ALP) with a specific substrate, such as
4-methylumbelliferyl phosphate (MUP).
7. Radio Immunological Assays (RIA)
Use
radioactively labeled antibodies to measure Alkaline Phosphatase (ALP) concentration.
8. Enzyme-Linked Immunofluorescent Assays (ELIFA)
Combines ELISA and fluorometric techniques to measure Alkaline Phosphatase (ALP).
9. Microfluidic Assays
Use
microfluidic devices to measure Alkaline Phosphatase (ALP) activity.
10. Point of Care Testing (POCT)
Use of portable devices to measure Alkaline Phosphatase (ALP) at the home.
Materials Needed
- Blood Specimen
- Clotting Tubes
- Microcentrifuge tubes
- Centrifuge
- ALP assay kit
- Pipettes
- Spectrophotometer
- Calculator
Tips and Considerations
- Handle samples and reagents
carefully to avoid contamination.
- Follow the manufacturer's
instructions for assay kits and equipment.
- Use appropriate controls and
calibrators.
- Consider interfering substances
and hemolysis.
Step-by-Step Procedure
1. Sample Preparation
- Collect specimen in appropriate
tubes
- Centrifuge the specimen for
5-10 mnts at low speed.
- Carefully pour the serum into
microcentrifuge tubes.
2. Reagent Preparation
- Prepare working reagent for ALP
assay reagents as per the instructions provided by the manufacturer.
3. Assay Procedure
- Add a specified volume of serum to the assay
mixture as per instructed ratio.
- Incubate the
mixture at a specified temperature for a recommended time.
- Read the
absorbance of assay at 405 nm or as per manufacturer guidelines.
4. Calculation
- To Calculate the levels of Alkaline Phosphatase (ALP),
follow the manufacturer's instructions.
The
calculations for estimating serum Alkaline Phosphatase (ALP) levels requires the absorbance
values measured from the assay procedure.
Here's
a step-by-step explanation for calculation:
4.1. Absorbance Measurement
Measure
the absorbance of the sample and blank (can be a control) using a spectrophotometer.
4.2. Calculate Delta Absorbance
Subtract
the blank absorbance from the sample absorbance to get the delta absorbance
(ΔA).
4.3. Calculate Enzyme Activity
Use
the manufacturer's formula to calculate the enzyme activity (EA) from the delta
absorbance. The formula typically includes a factor for the molar absorptivity
(ε) of the enzyme:
EA
(U/L) = (ΔA x ε x TV) / (t x V)
Where:
- EA = Enzyme activity (U/L)
- ΔA = Delta absorbance
- ε = Molar absorptivity
(L/mol/cm)
- TV = Total volume (L)
- t = Incubation time (min)
- V = Sample volume (L)
4. Calculate ALP Levels
- Convert the enzyme activity
to Alkaline Phosphatase (ALP) levels (U/L) using a predetermined conversion
factor or calibration curve.
Example Calculation
- Absorbance (Sample): 0.5
- Absorbance (Blank): 0.1
- Delta Absorbance (ΔA): 0.4 (0.5
- 0.1)
- Enzyme Activity (EA): 100 U/L
(using manufacturer's formula)
- ALT/SGPT Level: 50 IU/L (using
conversion factor or calibration curve)
Note: The exact
calculations and formulas may vary depending on the specific assay kit and
manufacturer's instructions.
Reference Ranges
Typical reference ranges for ALP are:
Adults
Male:
40-120 U/L
Female:
50-140 U/L
Children
Newborns:
100-250 U/L
Infants
(1-12 months): 80-200 U/L
Children
(1-12 years): 60-180 U/L
Pregnant women
First
trimester: 60-180 U/L
Second
trimester: 70-200 U/L
Third
trimester: 80-220 U/L
High ALP
High Alkaline Phosphatase levels can indicate various health issues, including:
As some of the conditions associated with this gene; Bone disorders, Kidney disease, Intestinal Obstruction, Liver cancer and Thyroid disorders. Hepatitis, liver cirrhosis and liver cancer are few medical disorders that may affect the working of liver as Jaundice and bone deformities. They also observe that some bone diseases, including osteoporosis and osteomalacia, are directly linked with bone cancer. These conditions can also be caused by other factors such as intestinal obstruction, inflammation and cancerous tissues.
Associated conditions that may lead to increased ALP include Kidney disease, Thyroid disorders and Sarcoidosis.
Low ALP
Low Alkaline Phosphatase levels can indicate various health issues, including:
Thyroid disorders, intrauterine growth retardation, hypophosphatasia, malnutrition, liver disease, renal failure and thyroid disorders are known to cause low levels of ALP in newborns. Conditions that affect the liver will lead to decreased production of ALP it includes liver failure or cirrhosis diseases. Genetic disorders which include osteoprotegerin also known as bone morbidity factor can also lead to low ALP level especially if complications like osteoporosis or osteopenia are also present. Renal dysfunction, impaired kidney function and it can also cause reduction in ALP levels and it is also known as kidney diseases.
Another disease that has low ALP levels are panhypopituitarism which is a known condition characterized by an under-active pituitary gland that fails to secrete necessary hormones into the bloodstream. These cause- low ALP levels can also be noticed during pregnancy and in newborns.
Conclusion
It is highly significant in diagnosing the health disorder in a healthcare setup as it is useful in showing liver abnormalities, bone disorders, among other findings several techniques are employed by healthcare professionals to estimate serum ALP concentration, and these include enzymatic assays, the colorimetric method, immunoenzymometric assay, ELISA, chemiluminescence immunoassay, and electrophoresis. The adaptation is done depending on the clinical scenario and the laboratory facilities available. Other practicalities include manipulation of samples, storage and interference substances.
Reference ranges are set and accepted values of a particular biomarker or laboratory test just as ALP that gives one a clear indication of the typical acceptable or standard deviation (SD) of whatever factor in a given population or age group. These ranges allow a healthcare professionals to determine the inherent meaning of the test results and possibly, detect some underlying health complications. Though ALP is elevated, it is not exclusive to any particular disease; and therefore further investigations have to be carried out in order to know the cause. ALP levels are utilised by healthcare professionals alongside other diagnostic tests, patients’ histories and physical assessments in defining and administering different diseases and disorders.
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