BILIRUBIN TEST. Information about test and diagnosis of diseases

Dr. Yousef Al Adbai. 

              BILIRUBIN TEST 

1. Purpose: Quantitative estimation of serum bilirubin (Total and Direct) by Jendrassik and Grof Method. Measurement of total bilirubin is useful in the diagnosis of jaundice due to any cause and is an indicator of liver function.

2. Principle: Bilirubin reacts with diazotized sulfanilic acid to form an azo dye which is red in neutral and blue in alkaline solution. Whereas the water-soluble bilirubin glucuronides react “directly” (the free bilirubin). “Indirect” bilirubin reacts only in the presence of an accelerator. The total bilirubin in serum or plasma is determined using by coupling with diazotized sulfanilic acid after the addition of caffeine, sodium benzoate and sodium acetate. A blue azobilirubin is formed in alkaline Fehling solution II. This blue compound can also be determined selectively in the presence of yellow byproducts (green mixed coloration) by photometry at 578 nm. The direct bilirubin is measured as the red azo dye at 546 nm using the method of Schellong and Wende without the addition of alkali.

3. Performance specifications:

3.1. Linearity: Up to 20 mg/dL.

3.2. Measurement range: As low as 0.05 mg in serum.

3.3.  Sensitivity: Lower detection limit is 0.05 mg/dL.

4. Primary sample:

4.1. Use only serum as specimen for the test.

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

4.3. Do not expose samples for serum bilirubin estimation to tube light/sunlight.

4.4. Do not use hemolyzed, contaminated or lipemic sera.

4.5. Separate serum as soon as possible; Store the serum at −10°C until required, for a maximum up to one month.

5. Type of container and additive: Use plain vacutainer tubes for collecting samples.

6. Reagents/Consumables:

6.1. Sulfanilic acid

6.2. Accelerator: Caffeine, sodium benzoate, sodium acetate

6.3. Sodium nitrite

6.4. Fehling solution II: 930 mmol/L Potassium sodium tartrate, 1.9 mol/L sodium hydroxide solution.

7. Instrument: Semi-autoanalyzer.

8. Procedure:

8.1. Switch on the machine and press “FLUSH” button by keeping the tubing in 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.

8.6. Then feed the standard followed by test samples and record the values.

   Assay: End point

· Sample volume: 50 µL

· Reagent volume: 500 µL

· No. of readings: 3

· Incubation time: 15 minutes

Total Bilirubin Procedure:

Measure the absorbance of the sample against distilled water or if necessary against the blank.

Direct Bilirubin Procedure:

Measure the absorbance of the sample against distilled water or if necessary against the blank.

9. Interferences: Turbid lipemic and lysed sera. In patients taking heavy dose of B complex, riboflavin and the folate in it may interfere by giving yellow color to the blood and urine.

10. Calculating results: For measurements against a blank: Total bilirubin concentration = A × 10.5 mg/dL.

11. Biological Reference Range:

    *   Total Bilirubin– up to 1.0 mg/dL

    *   Indirect or unconjugated bilirubin– 0.1 to 0.6 mg/dL

    *   Direct or conjugated bilirubin– up to 0.3 mg/dL

12. Critical/Alert level values: 3.0 mg/dL

13. Laboratory interpretation: Increase of bilirubin suggests jaundice; increase of both total and direct bilirubin suggests obstructive/hepato-cellular jaundice. Increase of total bilirubin alone with normal direct bilirubin suggests hemolytic jaundice. A level of 0.4 mg/dL or more of direct bilirubin suggests liver involvement.

14. Potential sources of variability:

    *   Lysed serum specimens may give falsely elevated values.

    *   Dilute the specimen if the bilirubin value is> 10 mg/dL suitable dilution can be done with normal saline. In such a case, the results obtained should be multiplied by dilution factor to be obtained correct bilirubin value.

                  Reference:

 Manual of Medical Laboratory Techniques

CREATININE TEST. Information about test and diagnosis of diseases

Dr. Yousef Al Adbai. 

                    Creatinine test

1. Purpose: Quantitative estimation of creatinine in human plasma by modified Jaffe’s method (initial rate or fixed time method) Measurement of plasma creatinine is useful in the diagnosis, treatment and follow-up of renal diseases/renal failure. Increase of serum creatinine indicates a definite damage of renal tissue.

2. Principle: Creatinine reacts with picric acid in alkaline medium to form an orange-yellow colored complex of creatinine picrate. This colored complex absorbs light at 492 nm the rate of increase in absorbance is directly proportional to the creatinine concentration in the sample.

3. Performance specifications:

3.1. Linearity: Up to 24 mg/dL in plasma.

3.2. Measurement range: 0.1–24 mg/dL of creatinine in plasma.

3.3. Sensitivity: The minimum detection limit by this kit is 1.0 mg/dL.

3.4. Specificity: This method measures a number of other non-creatinine substances also other than creatinine.

4. Primary sample:

4.1. Use only plasma as specimen for the test.

4.2. Collect 4 mL of venous blood in a heparin vacutainer tube.

4.3. Do not use lysed plasma for testing as it may give very high results.

4.4. Do not use contaminated/turbid samples for testing.

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

5. Type of container and additive: Use heparin vacutainer tubes for collecting blood samples.

6. Instrument: Semi-autoanalyzer

7. Reagents/Consumables:

7.1. Creatinine reagent: Picric acid 8.73 mmol/L

7.2. Buffer solution: 200 mmol/L of sodium hydroxide

7.3. 25 mmol/L of phosphate

7.4. Creatinine standard (2 mg/dL) solution containing creatinine in hydrochloric acid with preservative.

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 blank with each batch and ensure the absorbance of reagent blank to zero

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

Assay: Fixed time (Initial rate) Reagent volume:500μL/1000μL

Wavelength: 490 nm Sample volume: 25μL/50μL

Temperature: 37°C Zero setting with distilled water

No. of readings: 2 Time: 60 sec

Concentration of Std: 2 mg/dL

9. Calculating results:

Sample absorbance× Concentration of standard = Sample concentration

Standard absorbance

10. Biological reference Range:

Male: 0.7 –1.4 mg/dL

Female: 0.6 –1.2 mg/dL

11. Critical/Mert level values: More than 30 mg/dL.

12. Laboratory interpretation: Increase of creatinine in blood suggests kidney damage.

Example: Chronic glomerulonephritis.

13. Potential sources of variability:

13.1. Lysed plasma specimens may give falsely elevated values

13.2. Creatinine remains stable in plasma for up to 2 days.

13.3. Number of substances other than creatinine interfere with the assay.

               Reference :

 Manual of Medical Laboratory Techniques

Glucose Test. Information about test and diagnosis of diseases

 Dr. Yousef Al Adbai. 

                 Glucose Test

1. Purpose: Quantitative estimation of glucose in human serum or plasma or Cerebrospinal fluid (CSF) or other body fluids by enzymatic method (GOD-POD). Plasma Glucose determinations are useful in the diagnosis and treatment of diabetes mellitus and in monitoring the response to treatment of diabetes mellitus with insulin or oral hypoglycemic agents. Elevated glucose levels may be associated with pituitary or thyroid dysfunction, renal failure and liver disease, whereas low glucose levels may be associated with insulinoma, hypopituitary neoplasms, or insulin induced hypoglycemia. CSF and fluids have increased glucose in diabetic condition.

2. Principle: Glucose oxidase (GOD) converts glucose to gluconic acid. Hydrogen peroxide formed in this reaction, in the presence of peroxidase (POD), oxidatively couples with 4 - aminoantipyrine (AAP) and phenol to produce red quinone-imine dye. This dye has absorbance maximum at 505 nm. The intensity of color complex is directly proportional to the concentration of glucose in specimen.

3. Performance specifications 

3.1. Linearity: Up to 500 mg/dL of plasma. 

3.2. Measurement range: 40–500 mg/dl

3.3. Sensitivity: The minimum detection limit by this kit is 40 mg/dL

4. Primary sample

4.1. Use only plasma as specimen for the test

4.2. Collect 2 mL of venous blood in a Fluoride—EDTA mixture tube Heparin vacutainer tube.

4.3. Do not use lysed plasma for testing as it may give very high results 

4.4. Do not use contaminated/turbid samples for testing 

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

4.6. Type of container and additive: Fluoride—EDTA mixture tube. 

5. Equipment: Semi-autoanalyzer

6. Reagents: Phosphate buffer; pH 7.5; glucose oxidase; peroxidase; 4 aminoantipyrine; phenol

7. Procedure: 

7.1. 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

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

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

7.4. 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. 

7.5. Feed the blank with each batch and ensure the absorbance of the blank is less than 0.15. If the absorbance of the ‘blank is more than 0.15’ discard the reagent.

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

7.7. Check whether the sample is hemolyzed, icteric or lipemic before processing. If the sample is lysed, collect another sample and proceed.

Assay: 

End point 

Reagent volume: 1.0 mL

Wavelength: 505 nm (500–550)

Sample volume: 10 μL

Temperature: 37°C

Zero setting with Reagent blank

Incubation: 5 minutes

8. Interference: Turbid, lipemic, hemolyzed samples, high levels of ascorbic acid, and plasma bilirubin will interfere. Oxalate and fluoride do not interfere.

9. Calculating results:

 10. Biological reference range: 

Glucose Fasting is 60–110 mg/dL. Glucose PP is 90–140 mg/dL Glucose Random is 60–130 mg/dL

11. Critical/Alert level values: 

Below 60 mg/dL 

Above 400 mg/dL

12. Laboratory interpretation: 

Increase of blood glucose usually in diabetes mellitus, decrease in insulinoma. Decrease of CSF sugar in infection. Increase of CSF sugar in hyperglycemia.

13. Potential sources of variability:

13.1. Do not use if the absorbance of the blank reagent is greater than 0.15 at 500 nm as it indicates deterioration of the reagent. 

13.2. Check if the patient has followed the instructions regarding preparation before collecting samples for fasting/post- prandial, plasma glucose/glucose tolerance test.

13.3. The periodic update on the reference ranges needs to be made note of.

BIBLIOGRAPHY

1. Trinder P. Ann Clin Biochem 1969; 6: 24.

Reference➵ Manual of Medical Laboratory Techniques

Urea test. Information about test and diagnosis of diseases

Dr. Yousef Al Adbai. 

                          UREA

1. Purpose: Quantitative estimation of urea in human serum by Urease-GLDH/UV kinetic method. Determination of serum urea nitrogen is an important index of kidney function. Impaired renal function or increased tissue protein breakdown is associated with increased urea nitrogen levels, whereas liver damage or pregnancy is associated with decreased levels.

2. Principle: Urea is hydrolyzed by urease to form ammonium carbonate. In the second reaction 2-oxoglutarate reacts with ammonium ion in the presence of glutamate dehydrogenase (GLDH) and the coenzyme NADH to produce L-glutamate. In this reaction two moles of NADH are oxidized to NAD^+ for each mole of urea hydrolyzed. The rate of decrease in the NADH concentration is directly proportional to the urea concentration in the specimen. It is determined by measuring the absorbance at 340 nm.

   

3. Performance specifications:

   · 3.1. Linearity: Up to 240 mg/dL of serum

   · 3.2. Measurement range: 2–240 mg/dL

   · 3.3. Sensitivity: Lower limit of detection is 2 mg/dL

4. Primary sample:

   · 4.1. Use plasma

   · 4.2. Collect 2 mL of venous blood from a peripheral vein in a heparin vasculature tube

   · 4.3. Do not use hemolyzed/contaminated plasma for testing

5. Type of container and additive

   · Use heparin/plain vasculature tubes for collecting samples; do not use hemolyzed/contaminated plasma for testing

6. Instrument: Semi-automatizer

7. Reagents/Consumables: The reconstituted reagent contains the following:

   · 7.1. TRIS pH 7.8, 2-Oxoglutarate, ADP, Urease, GLDH

   · 7.2. NADH

   · 7.3. Urea (50 mg/dL)

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. Run the standard with each batch of patient sample.

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

  · Assay: 2-point Kinetic

  · Sample volume: 10 μL

  · Wavelength: 340 nm

  · Reagent volume: 400 μL

  · Start reagent 100 μL

  · Temperature: 37 °C

9. Biological reference range: 15–38.5 mg/dL

10. Alert/Critical values: Above 80.0 mg/dL

11. Laboratory interpretation: Increase suggests impaired renal function, acute nephritis, chronic glomerulonephritis.

12. Potential sources of variability

   · 12.1. Use of only clear, unhemolyzed plasma separated from the erythrocytes as soon as possible. Lysed plasma specimens may give falsely elevated values

   · 12.2. On storage, the working reagent may develop a pink color which makes the use of reagent blanks necessary with every run.

   · 12.3. This method is recommended to perform only on mechanized equipment. It is difficult to include all samples and reagent blank exactly for the same intervals.

   · 12.4. The scheme may use for adaptation purpose for instruments with no specific adaptation sheet.

BIBLIOGRAPHY

1. Kassiter JP. New Eng J Med 1971;285:385.

2. Mackay EM, Mackay LL. Clin.Invest 1927;4:295.

3. Talke HN, Schubert, GE Kin. Wschr 1965;42:174.

Glucose tolerance Test. Information about test and diagnosis of diseases

Dr. Yousef Al Adbai. 

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GLUCOSE TOLERANCE TEST

1. Purpose:

To recognize milder cases of diabetes and renal glycosuria. Quantitative estimation of glucose in human plasma by enzymatic method (GOD-POD). Plasma glucose determinations are useful in the diagnosis and treatment of diabetes mellitus and in monitoring the response to treatment of diabetes mellitus with insulin or oral hypoglycemic agents. Elevated glucose levels may be associated with pituitary or thyroid dysfunction, renal failure, and liver disease, whereas low glucose levels may be associated with insulinoma, hypopituitarism, or insulin-induced hypoglycemia. CSF and fluids have increased glucose in diabetic condition.

2. Principle:

Glucose oxidase (GOD) converts glucose to gluconic acid. Hydrogen peroxide formed in this reaction in the presence of peroxidase (POD) oxidatively couples with 4-aminoantipyrine and phenol to produce red quinoneimine dye. This dye has absorbance maximum at 505 nm. The intensity of color complex is directly proportional to the concentration of glucose in specimen.

3. Performance specifications:

3.1. Linearity: Up to 600 mg/dL of plasma.
3.2. Measurement range: 25–600 mg/dL.
3.3. Sensitivity: The minimum detection limit 40 mg/dL.

4. Primary sample:

4.1. Use only plasma as specimen for the test.
4.2. Collect 2 mL of venous blood in a fluoride–oxalate mixture tube.
4.3. Do not use lysed plasma for testing as it may give false results.
4.4. Do not use contaminated/turbid samples for testing.
4.5. Process the sample on the same day within 3 hours of collection.

5. Type of container and additive:

Fluoride—EDTA mixture tube.

6. Reagents/Consumables:

For patient use, commercially available glucose (75 g) mixed with water.

7. Instrument:

Semi-autoanalyzer.

8. Procedures:

Instructions to be given to the patient: These instructions should be given to the patients by previous day of the investigation.

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Biochemistry

8.1. The patient should not take any food after 9 pm the previous night till the test is performed.
8.2. The subject should have normal diet for at least 3 days prior to the test.
8.3. He/she should not have taken drugs which affect blood sugar.
8.4. In exceptional cases, when the patient has to come from a distant place, light tea without sugar may be allowed (2 hours before collection).

Method: Upon arrival of the patient, the following should be done:
8.5. Body weight should be noted down.
8.6. Fasting blood sample should be collected and glucose estimation should be performed.
8.7. Specimen of fasting urine is collected and test for glucose, albumin, and acetone to be done.
8.8. 75 g of glucose dissolved in 300 mL of water should be given orally.
8.9. Blood and urine samples will be collected for every half an hour interval for 2 hours after the glucose has been taken.
8.10. It is not always possible to collect urine at every half hour interval. In such cases, urine sample can be collected for every 1 hour interval.

Glucose estimation: As per the method given in this manual.
Urine sugar: As per the method given in this manual, with the standard curves.

Normal responses:
Fasting glucose within normal limit. Maximum blood glucose is reached either half or one hour after taking the glucose. The blood glucose then returns rapidly to the normal fasting limits, which are often reached in one and a half hour and almost always at two hours. There should be no sugar in any of the urine specimens.

9. Reference:

The GTT curve will be interpreted with the standard curves.

10. Critical/Alert level values:

Below 40 mg/dL, above 400 mg/dL.

11. Potential sources of variability:

11.1. Do not use if the absorbance of the blank reagent is greater than 0.150 at 500 nm as it indicates deterioration of the reagent.
11.2. Check if the patient has followed the instructions regarding preparation before collecting samples.

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BIBLIOGRAPHY:

1. Harold Varley. Practical Clinical Biochemistry, 5th ed., 1980;1:406–10.
2. Trinder P. Ann Clin Biochem 1969; 6: 24.

The Hidden World Inside a Drop of Blood

🧠 Title: The Hidden World Inside a Drop of Blood

Have you ever looked at a drop of blood and wondered what secrets it holds?

To the naked eye, it’s just red liquid—but under a microscope, it’s a living universe.

Inside that single drop, millions of red blood cells rush to carry oxygen, while white blood cells act like tiny soldiers, protecting your body from invisible invaders.

And then, the platelets—the quiet heroes—stand ready to stop bleeding the moment you get a cut.

Every time you take a blood test, the lab reveals stories your body can’t tell on its own.

From your sugar levels to liver function, each result whispers a clue about your health.

So next time you see a drop of blood, remember — it’s not just blood.

It’s a microscopic city working non-stop to keep you alive. 💉✨