د.احمد دلال باشي

الدكتور الصيدلاني احمد يحيى دلال باشي استاذ في الكيمياء الحياتيه الطبيةرئيس فرع الكيمياء الحياتيه كلية الطب/ جامعة الموصلمدير وحدة التعليم الطبي في زاخو

TYPES OF SPECIMENS

Blood
Blood is a suspension of cells in a protein-salt matrix. The non-cellular portion of blood contains proteins, some of which are involved in the coagulation process. This fluid is called plasma. When the coagulation process is allowed to proceed to completion, the non-cellular fluid, which can be separated from the clotted material, is termed serum.
Plasma – clotting factors (Fibrinogen) = Serum
Blood used for biochemical analysis is collected from the veins, arteries or capillaries. The venous blood is utilized for most testing because of ease of collection.
For a limited number of analytes such as blood gases and lactate, significant difference arises between arterial and venous samples.

Most testing is performed on serum fraction of the blood that has been allowed to clot. The assumption is made that the distribution of constituents between cellular and extracellular compartments of blood is roughly equal. For some analytes it may be necessary to prevent the blood clotting process using an anticoagulant and the separated liquid (plasma) will be then used for analysis.
Blood is usually drawn from a patient by a syringe, and transferred immediately to a clean plain tube (or a tube containing an anticoagulant) after removal of the needle to prevent breakdown of RBCs. The tube is then centrifuged after clot formation which takes about 10-15 min. (for serum) or immediately (for plasma).

Hemolysis

During sample collection and until the serum or plasma are isolated from RBCs, care must be taken to minimize hemolysis. Hemolysis may arise because of the use of too small needle, moisture in a syringe, vigorous mixing of the blood, etc.
Whatever the cause, hemolysis may interfere with a number of chemical procedures and should be avoided. Some constituents are present in high concentrations within the erythrocytes, and so hemolysis will falsely increase the value for those substances in serum such as potassium and enzymes.

On the other hand, for those substances that exist at lower concentration in the red cells than outside, hemolysis will result in a dilution effect on the serum constituents such as sodium and chloride, so falsely low result will be obtained.
In addition, hemoglobin may directly interfere in a chemical determination by inhibiting an enzyme such as lipase, by interfering with a reaction such as the reaction of diazo with bilirubin, or by yielding a significant color and thus interfering with a colorimetric analysis.
Glucose changes most rapidly of all cells constituents when serum or plasma are left in contact with RBCs. Since glycolysis is an enzyme catalysed reaction, sodium flouride has to be added to the blood sample for glucose estimation to inhibit the enzyme enolase that is involved in glycolysis.


Anticoagulants
Heparin: a-mucoitin polysulphuric acid inhibits the formation of thrombin from prothrombin. It is usually available as the Na, K, NH4 and Li salts.
EDTA (Ethylene diamine tetra acetate): It chelates calcium ions which are essential for clotting mechanism. Its dipotassium and dilithium salts are most often used.
Oxalate and citrate: Oxalate acts by precipitating the calcium. Potassium oxalate is the most soluble and so it is most commonly used.
Sodium citrate does not precipitate calcium but converts it into a non-ionised form.
Sodium flouride: It is also considered as an anticoagulant, but since larger amounts are needed, it is rather used as a preservative for glucose determination by inhibiting red cell metabolism, glycolysis and bacterial action.

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Collection of Urine Specimens

Single specimen of urine is used for general urine examination and for most qualitative tests. For quantitative work, 24 hour specimen is best employed.
For collecting a 24h sample, the patient empties the bladder first and the urine is discarded. All specimens passed thereafter during the day and during the following night are saved and the specimen obtained by emptying the bladder at the same time the following morning is added to them. The sample is collected in a clean covered container and kept in a cool place preferably in the refrigerator.
If urine has to be kept, it may be necessary to prevent the effect of bacteria by adding a proper preservative such as hydrochloric acid, chloroform or formalin to the urine.

Other Body Fluids

The laboratory is also perform a variety of testing on cerebrospinal fluid, amniotic fluid and other body fluids. In all instances, it is essential to ensure that the proper specimen is collected and contamination is avoided. In addition, biochemical analysis may be performed on stool samples, gastric aspiration, and renal or biliary calculi. Stool samples are usually collected over 48-72 h periods. Renal calculi (stones) collection may be a random event depending on when the stone is passed.

URINE

(General Analysis)
Urine is one of the biological fluids that is responsible for the removal of toxic substances from the body. The normal quantity of urine that is usually passed daily varies widely from (700-2500) ml depending on the fluid intake and weather. Normally, more urine is excreted during the day than during the night.
A pathological increase in urine output is called (polyuria) in which urinary output becomes more than 2500 ml/day which occurs in:
Diabetes mellitus.
Diabetes insipidus.
Renal failure.


A pathological decrease in the urine output is called (oliguria) in which urine output becomes less than 400 ml/day which occurs in:
Dehydration (diarrhoea, vomiting, fever, severe haemorrhage).
Sudden lowering of blood pressure (hypotension).
Severe heart failure.
Urine examination should include:
I Physical examination
Colour
Appearance
Reaction (pH)
Specific gravity
II Biochemical examination
Glucose
Protein
Ketone bodies
Bilirubin and urobilinogen
III- Examination of Urinary Deposits

I- Physical Examination

1. Colour
2. Appearance
Urochrome and uroerythrin are pigments which give normal urine its characteristic colour (light yellow), small quantities of blood give urine a smoky appearance with larger quantities make it brownish or red.
Bile pigments cause the urine to appear brown with the production of yellow froth when the urine is shaken in a test tube.
Drugs may also lead to discolouration of urine. Normal urine is quite transparent when freshly passed but it may be opalescent from the presence of various substances in suspension, of which the most important are pus, bacteria, and phosphates.
Record the colour and appearance of your sample.


3. Reaction
Normal urine is slightly acidic with pH range between (6-7).
With the help of pH paper, determine and record
the approximate pH of the urine provided.

4. Specific gravity

Fill a suitable sized cylinder with the urine then place a hydrometer in the fluid, taking care that it floats and does not touch the sides of the cylinder. The normal specific gravity of urine varies from 1.010-1.025. Specific gravity of urine increases in case of dehydration, heavy proteinuria and glucosuria, and it decreased in case of diabetes insipidus.

CONSTITUENTS OF NORMAL URINE

Among the inorganic substances present are chlorides, phosphates, and sulphates of sodium, potassium, calcium, and magnesium.

1. Uric acid
This constituent of urine is derived partly from the metabolism of food proteins and tissue proteins.
Follin test for uric acid
Take 2 ml of urine and add few drops of (Follin phosphotungstic acid reagent) and a small amount of anhydrous sodium carbonate, mix. A deep blue colour is produced.

2. Creatinine

It arises almost entirely from the breakdown of muscle creatine.
Jaffe’s test for creatinine
To 1 ml of saturated picric acid add 0.5 ml of 10% NaOH, then add 3 ml of urine, mix. A deep reddish-orange colour indicates the formation of creatinine-picrate complex.
3. Ammonium salts
Take 2 ml of urine, boil for 3 min then put a moist red litmus paper at the neck of the test tube, ammonia liberated due to the effect of boiling will change the colour of litmus paper to blue.
An average sample of urine has about 4% solids dissolved in it. Of these, approximately 2% is urea, 1% is sodium chloride and all other organic and inorganic constituents make up the remaining 1%. Urea, uric acid, creatinine and ammonium salts are the principal nitrogenous substances.


ABNORMAL CONSTITUENTS OF URINE
Glucose and reducing sugars
A sample of urine which reduces benedict reagent indicates the presence of reducing carbohydrate or substances. These substances are glucose, galactose, fructose, lactose, maltose, ascorbic acid (Vit C), acetylsalicylic acid (aspirin) or homogentisic acid.
Benedict’s Test
To (5) ml of benedict reagent add (0.5) ml (about 8 drops) of urine, boil for about (5) min, allow to cool, turbidity or precipitate indicates the presence of reducing sugar in the following manner:
- Light green turbidity (+) 0.1 – 0.5%
- Green precipitate (++) 0.5 – 1.0%
- Yellow (+++) 1.0 – 2.0%
- Red or reddish brown (++++) 2% and over

However a more specific & sensitive qualitative test for glucose is the clinistix method. It is specific for glucose. The reagent strip is dipped in the urine, and the colour of the test area is compared with the marker’s colour chart (10) seconds later.

Protein
Before doing the test for protein, it is essential that the urine should be clear and it may be, therefore, necessary to filter or centrifuge it.
Sulphosalicylic acid test (SSA)
It is a specific test for protein that is reliable, simple and does not require heat.
Take (5) ml of urine in a test tube then add 20% S.S.A drop by drop, the presence of protein is indicated by cloudy precipitate.

b. Boiling test (heat test)

Take (5) ml of urine in a test tube, incline the tube at an angle, boil the top 2 cm over a flame while holding the bottom of the tube, cloudiness indicates the presence of protein or phosphate then add few drops of acetic acid, if the cloudiness disappears, it is due to phosphate; if it persists protein is present.
If proteinuria is detected the presence of Bence-Jones proteinuria should be suspected.
This is confirmed by heating the urine sample to 50 C° and the formation of precipitate which disappears on boiling and reappears on cooling may indicate the presence of Bence-Jones protein. This is an abnormal protein that is excreted in urine of patients with multiple myeloma.


Ketone bodies
These are acetoacetic acid, B-hydroxybutyric acid and acetone. They may appear in the urine of patients with:
Severe diabetes mellitus.
After starvation and prolonged vomiting.
Lestradate test
One drop of fresh urine is allowed to fall on the filter paper containing lestradate powder, a purple coloration of the powder that appears (30) seconds later indicates the presence of ketone bodies.
Bilirubin and urobilinogen
In health, bilirubin is not found in the urine. The finding of bilirubinuria in a jaundiced patient suggests that the jaundice is due to the appearance of conjugated bilirubin in the plasma which could be due to either hepatocellular damage or hepatic obstruction.

Tests for bilirubin

Hay’s test:
Take small amount of urine in a test tube and sprinkle finely powdered dry sulphur over the surface from a height.
If bile salts are present, sulphur will sink, if bile salts are absent, sulphur will float on the surface.
Clinical significance
Bilirubin is found in urine of patients with obstructive jaundice. Also bilirubinuria may be found in early stages of viral hepatitis.