CERULOPLASMIN Test. lab information about test and diagnosis of diseases

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Ceruloplasmin Test. Lab information about test and diagnosis of diseases. 

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Ceruloplasmin Test – Manual of Medical Laboratory Techniques

Purpose:
To measure the amount of ceruloplasmin in human serum. Low levels are reported in Wilson’s disease and nephrotic syndrome, and it is valuable in differentiating chronic liver diseases from Wilson’s disease.

Principle:
Ceruloplasmin, a ferro-oxidase enzyme, catalyzes the oxidation of certain polyamines. Its activity on p-phenylenediamine is measured to determine the amount of ceruloplasmin in serum.

Performance Specifications:

• Linearity: up to 60 mg/dL
• Measurement range: 16–60 mg/dL
• Sensitivity: 16 mg/dL

Primary Sample:

• Use serum as the specimen.
• Collect 4 mL of venous blood in a plain red vacutainer.
• Allow to clot for 30 minutes, then centrifuge at 2500 rpm for 10 minutes.
• Avoid hemolyzed or turbid samples.
• Analyze within 3 hours of collection, or store serum at –20°C for up to 7 days.

Reagents and Consumables:

• p-Phenylenediamine hydrochloride: 5 g in 1 L distilled water (purified and crystallized).
• Acetic acid (1 M): 60 mL glacial acetic acid diluted to 1 L.
• Sodium acetate (1 M): 136 g per liter of water.
• Acetate buffer (400 mM, pH 5.5): Prepared by mixing acetic acid and sodium acetate.
• Sodium azide or sodium fluoride: Preservatives (5 g/L or 20 g/L).

Instrument:
Spectrophotometer (read at 530 nm).

Procedure:

Calculation:

Reference Range:

• Normal: 20 – 40 mg/dL

Critical/Alert Values:

• Not applicable.

Potential Sources of Error:

• Hemolyzed serum may give falsely elevated values.
• Samples not analyzed promptly must be stored at –20°C to maintain accuracy.

References:
Ramakrishnan, Manual of Medical Laboratory Techniques, pp. 25–27.
King, J. Practical Clinical Enzymology, 1965.

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Produce by : Dr. Yousef AL-Adbai. 

ALBUMIN Test. lab information about test and diagnosis of diseases.

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ALBUMIN Test. Lab information about test and diagnosis of diseases. 

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Albumin Test – Manual of Medical Laboratory Techniques

Purpose:
Quantitative estimation of albumin in human serum by photometric method using bromocresol green (BCG) dye binding. Elevated serum albumin levels are associated with dehydration. Low serum albumin levels indicate potential malnutrition, liver diseases, kidney disorders (especially nephrotic syndrome), and rheumatoid arthritis.

Principle:
Albumin acts as a cation at pH 3.8 and selectively binds to the anionic dye bromocresol green, forming a green-colored complex. The intensity of the color, measured at 630 nm, is directly proportional to the concentration of albumin in the sample.

Performance Specifications:

• Linearity: up to 6.0 g/dL
• Measurement range: 0.5–6.0 g/dL
• Sensitivity: 0.5 g/dL
• Specificity: certain drugs (e.g., Ampicillin) may interfere with dye-binding properties. Only human albumin standards should be used.

Primary Sample:

• Use serum (fasting sample recommended).
• Avoid venostasis during collection to prevent hemoconcentration.
• Collect 4 mL venous blood in a plain red vacutainer; allow to clot for 30 minutes.
• Centrifuge at 2500 rpm for 10 minutes.
• Avoid hemolyzed or turbid samples.
• Analyze within 3 hours or store serum at 2–8 °C (up to 30 days).

Reagents and Equipment:

• Reagent: Bromocresol Green (BCG) buffer, pH 3.68
• Standard: Bovine albumin fraction V (5 g/dL)
• Instrument: Semi-auto analyzer (e.g., RA 50)
• Wavelength: 628 nm
• Temperature: 37 °C
• Incubation time: 10 minutes

Procedure:

1. Run reagent blank with distilled water.
2. Process standard, then patient samples.
3. Measure absorbance at 628 nm.

Calculation:

Reference Range:

• Adults: 3.5 – 5.0 g/dL

Critical Values:

• Below 2.0 g/dL indicates severe hypoalbuminemia.

Interpretation:

• Low albumin: liver disease, nephrotic syndrome, malnutrition, chronic illness, or pregnancy.
• High albumin: dehydration.

Potential Sources of Error:

• Hemolyzed serum may give false high values.
• Non-human albumin standards or contaminated reagents may cause inaccurate results.

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References:
Ramakrishnan, Manual of Medical Laboratory Techniques, pp. 23–25.

Produce by :Dr. Yousef AL-Adbai

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TOTAL Protein. Information about test and diagnosis of diseases

                     TOTAL PROTEINS.

Information about test and diagnosis of diseases 

1. Purpose:

Estimation of total protein in serum/body fluids by Biuret method. Low protein levels are observed in malnutrition, acute or chronic liver diseases, nephrotic syndrome, water intoxication, salt retention syndromes, and massive intravenous infusions. Elevated protein levels are observed in dehydration due to vomiting, diarrhea, Addison’s disease and diabetic ketoacidosis. High protein levels of over 2 g/dL in body fluids are suggestive of inflammation or malignancy and are called exudates. 

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2. Principle: 

Peptide bonds of proteins in serum react with cupric ions in alkaline solutions to form a blue colored complex, the absorbance of which is measured at 578 nm. The intensity of the blue color is proportional to the amount of protein present. The reaction sequence employed in the assay of total proteins is as follows:

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3. Performance specifications: 

3.1. Linearity: Up to 12 g/dL 

3.2. Measurement range: This method has a measurement range of 5.3–8.4 g/dL of total protein in serum and body fluids.

3.3. Sensitivity: The minimum detection limit by the kit is 5.3 g/dL. 

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4. Primary sample:

4.1. Use serum/body fluids (Pleural, Pericardial, Ascitic fluid) as specimen for the test. 

4.2. Collect blood sample in a red color vacutainer tube, separate serum within 30 minutes of collection. 

4.3. Process the sample on the same day within 1 hour of collection. If analysis is done on the next day, separate the serum and store it at 2–8°C for up to 30 days.

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5. Type of container and additive:

Use plain vacutainer tubes for collecting samples. No additive/Preservative is needed to be added.

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6. Reagents/Consumables: 

6.1. Biuret reagent. 

6.2. Total protein standard.

7. Instrument: Semi-autoanalyzer

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8. Procedure:

8.1 Switch on the machine and press “FLUSH “button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 

8.2. Press “PROC”. Different test procedures will be displayed. 

8.3. Select the test to be processed by entering its number and then press “ENTER” key. 

8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 

8.5. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.150, if the absorbance of the ‘blank is more than 0.150’ discard the reagent at 546 nm. 

8.6. Then feed the test samples and record the values. 

8.7. Check whether the sample is hemolyzed or icteric before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10.

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Assay type: End point,

Wavelength: 546 nm.

Sample volume: 10 μL, 

Reagent volume: 1000 μL

Incubation time: 20 min at RT, Temperature: 37oC

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9. Interferences:

9.1. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.150, If the absorbance of the ‘blank is more than 0.150’ discard the reagent at 546 nm. 

9.2. Keep the reconstituted reagent at 2–8ºC. Discard the same if it develops precipitate. 

9.3. Highly hemolytic or icteric samples, prepare sample blank by adding 1 mL of 0–9% saline to 10 microliter samples. The value of the blank is subtracted from the corresponding sample value.

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10. Calculating results:

11. Biological reference range:

Adults: 6.6–8.4 g/dl.

12. Critical/Alert values:

Below 5.0 g/dL and above 9.0 g/dl.

13. Laboratory interpretation: 

Increase of proteins in dehydration, multiple myeloma and chronic infections (gammopathy); 

decrease in malnutrition, liver diseases, nephrotic syndrome.

14. Potential sources of variability: 

14.1. The reagent is linear to 12.0 g/dL. Samples with values above 10 g/dL should be diluted 1:1 with 0.9% saline, re-run, and the result multiplied by two (2) 

14.2 The biuret procedure is not sensitive at low ranges (< 1 g/dL). Do not use for urine or spinal fluid.

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BIBLIOGRAPHY

1. Henry J, Winkelman JW. Clinical Chemistry Principles and Technique. Harper and Row, 2nd edn 1974. 

2. Stricklad RD, Freeman ML, Gurule FF. Copper Binding by proteins in alkaline solution. Anal Chem 1961;33.

Reference: Manual of Medical Laboratory Techniques

Produce by: Dr. Yousef Al Adbai

Laboratorist. 

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triacylglycerols (triglycerides). Information about test and diagnosis of diseases

TRIACYLGLYCEROLS (TRIGLYCERIDES)

Information about test and interpretation about Result 

1. Purpose: 

Quantitative estimation of triacylglycerols in human serum by enzymatic method using Glycerol -3 Phosphate Oxidase (GPO) Measurement of triglycerides in conjunction with other lipid assays is used in screening the lipid status of an individual to detect atherosclerotic risks and in monitoring the response to lipid lowering measures triglyceride determinations when performed are useful in the diagnosis of primary and secondary hyperlipoproteinemia. They are also of interest in following the course of diabetes mellitus, nephrotic syndrome, biliary obstruction and various metabolic abnormalities due to endocrine disturbances.

 2. Principle:

 The procedure involves hydrolysis of triglycerides by lipoprotein lipase. The glycerol concentration is then determined by enzymatic assay coupled with Trinder reaction that terminates in the formation of a quinoneimine dye which is generated from 4-aminoantipyrine and 4-chlorophenol by hydrogen peroxide under the catalytic action of peroxidase. The amount of the dye formed, determined by its absorption at 500 nm, is directly proportional to the concentration of triglycerides in the sample.

3. Performance specifications:

3.1. Linearity: Up to 1000 mg/dL of serum 

3.2. Measurement range: 1–1000 mg/dL of cholesterol in serum

3.3. Sensitivity: The minimum detection limit by this kit is 1 mg/dL.

4. Primary sample:

4.1. Use only fasting serum as specimen 

4.2. Collect blood sample after an overnight fast of 12–14 hours when testing is a part of lipid profile 

4.3. Collect 4 mL of venous blood in a plain vacutainer tube. 

4.4. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10 minutes 

4.5. Do not use lysed serum for testing as it may give very high results 

4.6. Do not use contaminated/turbid samples for testing 

4.7. Process the sample on the same day within 3 hours of collection. 

4.8. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 20–25 °C for up to 2 days or at 4–8 °C for up to 7 days

5. Type of container and additive:

Use plain vacutainer tubes for collecting samples. No additive/preservative is needed to be added.

6. Reagents/Consumables:

Lipoprotein lipase, magnesium acetate, 4 aminoantipyrine, glycerol-3-phosphate oxidase, glycerol kinase, peroxidase, triglyceride standard 200 mg/dL triglycerides as triolein. 

7. Instrument: Semi-autoanalyzer.

8. Procedure:

8.1. Switch on the machine and press “FLUSH “button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 

8.2. Press “PROC”. Different test procedures will be displayed. 

8.3. Select the test to be processed by entering its number and then press “ENTER” key. 

8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 

8.5. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.300 at 520 nm if the absorbance of the ‘blank is more than 0.300, discard the reagent. 

8.6. Then feed the test samples and record the values. 

8.7. Check whether the sample is hemolyzed, icteric before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or highly lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10.

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Assay: End point.    

Reagent volume: 1000 μL

Wavelength: 546 nm

Sample volume: 10 μL

Temperature: 30°C

Zero setting with distilled water

Incubation time: 5 minutes

Conc. of standard: 200 mg/dL

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9. Calculating results:

10. Biological reference range:

Male: 60–165 mg/dL 

Female: 40–140 mg/dL

11. Critical/Alert level values: 400 mg/dL

12. Laboratory interpretation: Triacylglycerolemia is a risk factor for myocardial infarction.

 TGA is phenomenally increased in an eye disease lipemic retinalis.

13. Potential sources of variability: 

13.1. Lysed serum specimens may give falsely elevated values 

13.2. Do not use if the reagent is turbid as it indicates contamination of the reagent and if the absorbance of the blank reagent is more than 0.300.

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BIBLIOGRAPHY:

1. Annoni G, Bottasso BM. Ciaci D, Donato MF Tripoli A, Lab JJ. Res Lab Med 1982;9:115. 

2. Buccolo G, David M. Clin. Chem 1973;19:476. 

3. Werner M, Gabrielson DG, Estman G. Clin Chem 1981;21:268.

Reference: Manual of Medical Laboratory Techniques

Produce by: laboratorist

Dr. Yousef Al Adbai