Basic Laboratory Test

Urine Analysis

Routine urine analysis is done for 2 purposes.
- To find out metabolic or endocrine disturbances of the body
- To determine intrinsic conditions that may affect urinary tract or the kidneys

The first part of urine analysis is direct visual observation or physical examination of urine, and the second is the chemical examination. Normal urine has the following properties.

Volume : 60 - 2500 ml / 24 hrs
Specific gravity : 1.003 - 1.030
Reaction : Acidic

Physical examination

• The various aspects include
• Volume
• Colour
• Appearance
• Sediment formation
• Odour
• Reaction in pH
• Specific Gravity

Colour

Normal urine varies from pale yellow to dark amber, depending on the concentration of urochrome. The pH of urine also influences color. Very pale urine can result from high fluid composition diuretic drugs, etc. Dark yellow urine indicates symptoms of jaundice. It can be detected in urine by urine bile salt and bile pigment. A red or red-brown (abnormal) color could be from a food dye, eating fresh beets, a drug, or the presence of either hemoglobin or myoglobin. If the sample contained many red blood cells, it would be cloudy as well as red.

Appearance

Normal urine is usually clear. The appearance may be cloudy if amorphous phosphates are present; they form a white precipitate, which dissolves when acid is added. Urine may also appear cloudy or turbid due to leukocytes, or epithelial cells.

Sediment formation

If urine has amorphous phosphates, or urates then on standing for sometime, sediment formation occurs at the bottom.

Odour

The presence of ketone bodies gives a sweet smell to urine. Contaminated urine with bacteria usually gives a pungent smell.

Reaction and pH

Fresh urine is slightly acidic. A high protein intake or urinary tract infections also produce acidic urine. Urinary tract infections by Proteus and Pseudomonas may cause alkaline urine formation.

Specific gravity

It is the ratios of weight of volume of urine to the weight of same volume of distilled water. It is used to measure the concentration and diluting capacity of the kidneys. It varies from 1.003 to 1.030.

Chemical examination

The routine chemical analysis includes the estimation of protein and glucose, both of which are qualitative tests.

Determination of urine protein

Normally only a small amount of low molecular weight protein is filtered at the glomerulus. Renal tubules secrete a mucoprotein called Tam-Horsfull protein. This is normally excreted in urine. The presence of protein in urine is the first sign of a serious problem.

Determination of urine glucose

Only a small amount of glucose (2 - 20 mg/dl) may be present in fasting urine, which is not detectable by chemical methods. The presence of chemically detectable amount of glucose in urine is called glycosuria. This may be an indicator of diabetic mellitus and the blood sugar has to be treated. The quantity of glucose is dependent on:

1. Blood glucose level
2. Rate of filtration
3. Rate of tubular reabsorption

Normal renal threshold level for Glucose is 150 - 170 mg/dl. When this level is exceeded renal glycosuria occurs.

Practical skill

Testing for urine albumin (Protein)

It is easy to identify the function of the kidneys and to identify the presence or absence of salt. For this test early morning sample is suitable.

The normal urine has protein less than 50 mg per 24 h. The level deviates in certain undesirable conditions such as in irregular reabsorption.

Testing for albumin (Protein)

Method : Qualitative test

Principle : The test is based on the principle of precipitation of protein by chemical agents (acids) or coagulation by heat.

Application : This test is used in the determination of glomerular proteinuria, glomerulonephritis, hypertension etc.

Sampling instructions

• Collect urine in clean container (need not be sterile)
• Reagents / Consumables
• Reagent I: Acetic acid (glacial) 3% Sulphosalicylic acid

Procedure

• Transfer 3-4ml of urine in to a small test tube.
• Add 2-3 drops of sulphosalicylic acid on the top of the specimen.
• Observe for turbidity after 5 min.

Interpretation

• No formation of turbidity at the upper portion of urine-protein absent
• Formation of turbidity - protein present
• If protein are present grade the result according to the degree of turbidity as +, ++, +++,+++++.

Testing for urine glucose:

This test is used to identify presence or absence of glucose in urine. Glucose present in urine is called glycosuria. This is an easy test to identify the diabetic patient.

1. Method
      - Benedict 's qualitative test
2. Principle
      - Benedict's reagent (5 ml) is heated with 8 drops of urine (0.5 ml). Glucose present in urine
reduces cupric ions present in the reagent to cuprousions. The blue color of the reagent changes to green, orange, yellow or red according to the concentration of glucose present in urine.
3. Application
      - This test is used in the determination of glycosuria and diabetes mellitus and also in cases of endocrine hyperactivities. Non-pathological cases of hyperglycemia can occur as in pregnancy, stress and anxiety.
4. Sampling instructions
5. Collect urine in clean container
6. Reagents / Consumables
   - Reagent I: Benedict's reagent
7. Procedure
   • Pipette 5.0ml of Benedict's reagent in the test tube.
   • By using a Pasteur pipette add 8 drops of urine.
   • Heat carefully on the flame of a gas burner or spirit lamp for 5-10 min.
   • Cool under tap water.
8. Reference range
   - Blue +, Green ++, Yellow +++, Red ++++

Blood Hemoglobin (Hb) Sahli's method

The routine hematological test include Hb concentration, total RBC, total WBC etc. The hematological values are affected by various factors in individuals apparently in normal health. They include:

• Sex, age, weight, height and environment.
• Physiological conditions under which specimens are obtained.
• The technique and timing of specimen collection, transport and storage.

A decrease in haemoglobin below the normal range is an indication of anemia, whereas an increase usually occurs in haemoconcentration due to loss of body fluids in severe diarrhoea and vomiting. High values are also observed in congenital heart diseases.

Practical skill

This test is used to identify whether patient is anaemic or polycythemic. Haemoglobin decrease is known as anaemia. Increase in haemoglobin known as polycythemia.

Method:	Sahli's method
Principle:	When blood is added to 0.1 N HCL, haemoglobin is converted to brown colored acid hematin. The resulting colour after dilution is compared with a standard brown glass reference blocks of Sahli's haemoglobinometer.
Application:	This test is useful in the diagnosis of anemia, polycythemia.
Limitations:	The method gives an error up to 1g/dl and estimation of haemoglobin is not useful in diagnosing the cause of anemia.

Equipment

• Sahli heamoglobinometer
• Hb pipette
• A graduated tube

Consumables

• 0.1 N Hcl
• Distilled water
• Pasteur pipettes

Procedure

• Using a Pasteur pipette add 0.1 N Hcl in the tube up to lowest mark (20% mark)
• Draw blood up to 20ml mark in Hb pipette. Adjust the blood column carefully without bubbles
• Wipe off excess blood on the sides of the pipette by using a piece of dry cotton
• Transfer blood to the acid in the graduated tube
• Rinse the pipette well, mix the reaction mixture and allow the tube to stand for at least 10 minutes
• Dilute the solution with distilled water by adding few drops at a time carefully and by mixing the reaction mixture until colour matches the glass plate in the comparator
• The matching should be done only in natural light

Sampling instructions

• Use whole blood as sample
• Collect 1 - 2 ml of venous blood in a Lavender topped EDTA vacutainer tube
• When capillary blood is used, collect 20 µ/dl of blood directly in to Hb pipette without adding any anticoagulant or preservative

Sampling, handling instruction & storage

• Process all samples for haemoglobin estimation within 1 hour
• Do not store any sample for more than 1 hour
• Care should be taken as all samples are potentially infectious
• Wear gloves while collecting and processing specimens

Precautions

• Immediately after use, rinse the Hb pipette by using tap water in a beaker
• Ensure sample is at room temperature in case of stored specimens

Reference range

Men : 12 - 15 g/dl

Women : 11 - 15 g/dl attitude

1. Communicate the necessary information to the patient before testing
2. Perform tests with utmost care to avoid any false positive result
3. Communicate the necessary information immediately to the ophthalmologist in case of any abnormal results

Summary

The OAs are perform many clinical procedures. It is important that they know the fundamental concepts of biochemistry in connection with ocular problems. It is also important that they have a good understanding of the best method of taking samples and interpretation of the lab results. They should counsel the patient while taking samples and make them aware about their body conditions. In the case of disabled, mentally ill or old patients, they should help them get in wheelchairs and help the patient cooperate while taking samples. They should be intelligent and active in the laboratory. They must be clear about what tests need to be done for the patient. They should not change the samples and interpret wrong results in the laboratory. They should also be tactful in the time of emergencies due to hyper / hypoglycemia in a patient. The role of the OA in the biochemistry lab is crucial, requiring intelligence and good attitude.

Key points to remember

• The container used for collection should be clean, dry. Bottles need not be sterile.
• For qualitative tests, morning urine is best.
• Always perform urine analysis on fresh samples In case of time delay; it should be stored at 2-8 degree centigrade
• Do not reuse containers; dispose of them in a bin of sodium hypochlorite.
• Venous blood is preferred for most hematology examination.
• Capillary blood should be used only when it is not possible to collect venous blood as it may give erroneous results.

Student exercise

A. Answer the following

1. What is meant by glycosuria?
2. What test is used to find bilirubin in urine?
3. Which method is used to find whether urine is acidic or alkaline?
4. Which value is renal threshold level?
5. Which test is used to find out salt in urine?

B. Choose the correct answer

1. In which condition will haemoglobin decrease?

• Anaemic
• Polycythemic
• Diabetes mellitus
• Kidney failure

2) In which condition will haemoglobin increase?

• Anaemic
• Polycythemic
• Diabetes mellitus
• Kidney failure

3) Which test is used to evaluate the function of kidney?

• Urine glucose
• Urine albumin
• Urine bile salt
• Urine bile pigment

4) Which test is used to find jaundice?

• Urine glucose
• Urine albumin
• Urine bile salt & urine bile pigment
• Urine pH

C. Fill in the blanks

1. Normal value of haemoglobin for man is__________.
2. Renal threshold level is _____________.
3. Dark yellow urine indicates the presence of _____________.
4. Name of method to identify the hemoglobin is ________________.
5. Name of the method to identify the presence of glucose in urine is __________.

Practical skill

1. Preparation of patient before blood collection
2. Interpretation of the reaction from the Sahli's scale
3. Maintenance of Sahli pipette
4. Demonstration of blood collection procedure before performing the test