BASIC LABORATORY PRINCIPLES AND PROCEDURE

INTRODUCTION: -

Pathology is a branch of biological science which deals with the nature of disease, through a study of its causes, its processes and its effects, with associated structural and functional alteration. When a person becomes ill, his symptoms are due to disturbances in the normal functions of the affected cell of his body. Pathology deals with the study of these disturbed function, how they arise, how they and how they affects other cell system, pathology also consider the factor which return the function of the affected cell to normalcy.

CLINICAL PATHOLOGY: -

It is the application of laboratory techniques to find out the cause of disease. Clinical laboratory testing is regarded as one of the several important sources of clinical data essential for diagnosis, prognosis and treatment of disease. Clinical laboratory medicine involve all aspects of medicine ranging from field of biochemistry, microbiology, hematology, immunology, clinical microscopy, biophysics, cytogenetic to is topology etc..

THE PHYSICIAN: -

Orders laboratory test in the order to arrive at a diagnosis when a patient approaches the laboratory with a requisition form, requesting pathological tests, the pathologist, with the help of other laboratory staff carries out the following duties –

Ø Instructing the patient

Ø Collecting the specimen

Ø Preserving the specimen

Ø Analyzing the specimen

Ø Preparing the reports and

Ø Dispatching of the typed and signed reports

The test reports are useful to the physician to-

Ø Establish a diagnosis

Ø Confirm a clinical interpretation

Ø Monitor therapy

Ø Establish prognosis

Ø Detect disease

The functional components of the clinical laboratory can be listed as follows-

Ø Clinical pathology

Ø Hematology and Blood bank

Ø Clinical biochemistry

Ø Clinical microbiology and serology

Ø Histology and cytology

THE RESPONSIBILITY OF THE LABORATIRY WORKER

Ø The laboratory workers play an important role to find out the cause of disease by providing the physician the required laboratory test results.

Ø The laboratory worker thus helps the patient to get better by providing accurate test finding to the physician.

Ø The laboratory worker should not offer personal excuses for short-comings in the performance of duty.

Ø Any error should be promptly reported to the superior without fear, so that they can be corrected.

Ø Truth must always be told because a wrong result may lead to a patient’s death.

Ø Proper use of equipments and reagents because equipment and chemicals coast money.

Ø Many patients are not treated until their reports are kept ready. If these reports are delayed, patients cannot be treated early.

Ø It is necessary to keep all reports ready in time (particularly the urgent reports).

Ø In the course of laboratory tastings, the laboratory workers gain a lot of information about patients and their illnesses. The laboratory worker must regard this information as strictly confidential.

Ø Only physician who requested the examinations should receive the patient’s reports.

Ø Every laboratory worker must maintain high moral and professional standards of behavior.

LABORATORY SAFETY

The laboratory worker is surrounded by many dangers such as from-

Ø Handling of infectious material.

Ø Handling of broken glassware.

Ø Accidental spill of corrosive reagents.

Ø Swallowing of corrosive reagents such as conc.H₂SO₄, HCl, NaOH etc.

Ø Swallowing of infectious specimen.

Ø Inhalation of poisonous fumes.

Ø Potential hazards in the form of inflammable chemicals and gas leakages.

IMPORTANT PRACTICAL PRECAUTION

Ø The use of rubber gloves while handling corrosive substances such as strong acid and alkalies and also while handling poisonous chemicals such as potassium cyanide is essential.

Ø The uses of laboratory coats are meant to protect the wearer from chemical splashes and infectious material.

Ø Safety spectacles or goggles should be used while carrying out any procedures where there is risk to the eyes from reagent splashes.

Ø All chemical should be considered as potentially dangerous. Contact with skin and clothing should be avoided.

Ø A gas cylinder and gas taps should be handled with care. Accident can happen through ignorance and incorrect use.

Ø All body fluids such as blood, serum, plasma, urine, CSF, etc. should be handled with great care since they may be potential source of infections.

Ø Laboratory worker must know the meaning of safety signs for the following type of harmful substances.

GLASSWARE

COMPOSITION OF GLASS:-

The laboratory glassware is usually manufactured from borosilicate glass. It is resistant to the action of chemicals with the exception of hydrofluoric acid.

MAIN INGREDIANTS:-

Ingredient	Percent
Silica (SiO₂)	80.6
Boric Oxide (B₂O₂)	12.6
Sodium oxide (Na₂O)	4.15
Alluminium Oxide (Al₂O₃)	2.2

GENERAL GLASSWARE:-

v Beakers:-

These have capacities from 5 to 5000 ml. they are generally in a square form which cylindrical and has a spout. These are used mainly for the preparation of solutions.

v Flasks:-

These have capacities of 25 to 5000 ml. different types of flasks are used such as-

ü Conical flasks:- used for performing titrations.

ü Flat bottomed round flasks:- used for heating liquids.

ü Round bottomed flasks:- these can withstand higher temperature. They can be heated in a naked flame.

ü Volumetric flasks:- used to make final volume of reagent very accurately.

v Measuring cylinders:-

They are available in 10 to 2000 ml capacities. They are used to measure quantity of the liquids.

v Burettes:-

These are used for measuring of variable quantities of liquids. These are used for titration.

v Condensers:-

Used mainly for distillation and for reflux operations.

v Funnels:-

These are available in comprehensive range for variety separations of-

ü Solids from liquids.

ü Liquids from liquids.

ü For pouring liquids, chemicals or solutions into a container.

v Syringes :-

These are mainly used for collection of blood and CSF.

v Pipettes:-

These are used for dispensing controlled quantities of

Liquids.

SOLUTIONS AND REAGENTS

INTRODUCTION:-

Various chemicals are used for preparation of solutions and reagents in a pathological laboratory. A chemical is a substance produced by a chemical process by various manufactures. It is of known compositions examples:- sodium carbonate, potassium hydroxide, hydrochloric acids, glacial acetic acid, etc.

A SOLUTION :-

It is a combination of two substances a solute and a solvent. The dissolved substance is called solute and most of the times the substance present in a relatively greater proportion in the solution is called the solvent.

A REAGENT: -

This term can be applied in particular to any chemical compound or mixture of compounds, usually in solution, employed in chemical analysis or for the detection of biological constituents.

STOCK AND WORKING SOLUTIONS: -

A stock solution is a concentrated solution from which different types of working solutions can be prepared by simple dilutions.

ACIDS AND BASES: -

Acids are proton donors and bases are proton acceptors.

Acid Base + H⁺

ALKALI: - these compounds yield hydroxyl ions on dissociation.

e.g. NaOH Na⁺ + OH^-

The various types of solutions and reagent used in a pathological laboratory are-

· Normal solutions

· Molar solutions

· Percent solutions

· Buffer solutions

· Buffered substances

· Indicators

· Primary standards and

· Other complex reagents.

EQUIPMENTS AND INSTRUMENTS

The various types of equipments and instruments used in clinical biochemistry are as follows-

· Balances

· Hot plate and Magnetic stirrer

· Centrifuges

· Hot air oven

· Incubators

· Water Bathes

· Photometers and Spectrophotometer

· pH Meters

· Distillation plants

· Deionizers

· Cell Counter

· ELISA Strip Reader

· Automatic dispensers and diluters

· Autoanalysers

· Electrophoretic instruments

· Osmometers

· Electrolyte analyser

· Semi-Automatic dry & liquid biochemistry analyser

· Autoclave and microwalles

· Acid –Base Analysers.

TRAINING THE TECHNICIAN

BASIC REQUIREMENT:-

The technician should be very well trained to-

1. Approach the patient pleasantly and confidently.

2. Obtain blood sample properly, quickly and without undue discomfort to the patient.

3. Maintain proper record of collection.

4. Handle the specimen carefully.

5. Analise the specimen accurately.

6. Maintain proper record of reports.

IMPORTANT CHARACTERISTICS OF A GOOD TECHNICIAN

· Pleasant personality.

· Ability to speak well.

· Alertness.

· Enthusiasm.

· Sincerity.

· Working accurately and speed.

· Ability to follow instructions and make corrections.

BASIC STEPS FOR DRAWING A BLOOD SPECIMEN

1. Preparation for blood collection.

a) Each request for a blood specimen must include a number to identify all paper work and specimen associated with each patient. The information given on the blood request form should be recorded on the specimen labels.

Essential items include the following

I. Patient’s complete name and age.

II. Identification no.

III. Date and time if necessary.

IV. Name of physician ordering the tests.

b) The specimen containers should be labeled appropriately before the specimen collection.

2. Ascertaining whether the patient has fasted

Some tests require the patient to fast. Such care is needed to ensure accurate results.

3. Reassuring the patient.

The technician must gain the patient’s confidence

And assure him that, although the venipuncture will be slightly painful, it will be of short duration.

4. Positioning the patient

a) The patient should be made to sit comfortably in a chair and should position his arm on a slanting arm rest, extending the arm straight from the shoulder and it should not bent at the elbow.

b) If the patient wants to lie down, let the patient to lie comfortably on the back. The patient should extend the arm straight from the shoulder. For support, a pillow may be placed under the arm.

Requirement for blood collection

1. Collection tubes, bulbs etc.

2. Sterilized syringes and needles.

3. Sprit or 70% ethanol.

4. Cotton balls or gauze pads.

BLOOD COLLETION:-

1. Checking for paper works and tubes.

The tubes and bulbs should be checked for appropriate kinds and for paper labeling.

2. Selecting vein site.

For most venipuncture procedures on adults, veins located in the arm are used. The median cubital vein is the one used for the patient.

3. Following techniques are useful when encountering a patient with difficult veins.

i. Look for a blood drawing site.

ii. Feel for s vein using the tip of the finger. Think of four things when feeling for a vein, bounce, direction of vein, size of needle, and depth.

iii. Choose the vein that feels fullest.

iv. Try the other arm unless otherwise instructed.

v. Ask the patient to make a fist.

vi. Apply a tourniquet briefly.

vii. Massage the arm from the wrist to elbow.

viii. Cleansing the area to prevent any contamination. Sprit or 70% ethanol is used for cleansing and the area is allowed to dry to prevent possible heamolysis of the blood specimen.

4. Performing the venipuncture

i. The patient’s arm is gripped and thumb of another hand is used to draw skin taut.

ii. The vein is penetrated by positioning the needle at 30° to 40° angle.

iii. After the blood has been drawn the patient should release the fist and the tourniquet is also released.

iv. A cotton ball is held firmly over the venipuncture site as soon as the needle is removed.

v. Collected blood is dispensed in the appropriate tubes or bulbs.

vi. The blood in the anticoagulated bulbs is mixed carefully and blood collected in the tubes or bulbs without anticoagulants is kept on the room temperature.

5. Separation of serum

I. Allow the blood to clot.

II. Loosen the clot slowly and centrifuge the supernatant fluid.

III. By using a Pasteur the pipette, separate the serum from blood cells and store it in a clean and dry test tube.

LAB TESTS

BILLIRUBIN

PRINCIPLE:-

Billirubin is coupled with diazotized sulfanilic acid in the presence of caffeine to give an azo dye. No caffeine is added when “direct billirubin” is determined.

NORMAL VALUES

Total billirubin :- 0.001 mg/dl

Direct billirubin :- 0.00-0.25 mg/dl

Sample material :-

Serum, haparinised plasma or EDTA plasma.

REAGENTS:-

Reagent 1.	Sulfanilic acid solution
Reagent 2.	Nitrite solution
Reagent 3.	Caffeine solution
Reagent 4.	Tartrate solution
Reagent 5.	Sodium chloride solution

Procedure:-

wavelenghth for total billirubin : 578 nm

wavelenghth for direct billirubin : 546 nm

cuvette : 1 cm path length

temperature : 20° C to 30° C

measure against sample blank.

Total billirubin

Pipette into cuvette	Sample blank	Sample
Reagent 1.	0.10ml	0.10ml
Reagent 2.	--------	0.02ml
Reagent 3.	0.50ml	0.50ml
Sample	0.10ml	0.10ml

Mix, let stand at 20°C to 30°C for 5min

Reagent 4

0.50ml

Mix, let stand at 20°C to 30°C for 5min

Read absorbance of sample against sample blank

Direct billirubin:

Pipette into cuvette	Sample blank	Sample
Reagent 1.	0.10ml	0.10ml
Reagent 2.	--------	0.02ml
Reagent 5.	1.00ml	1.00ml
Sample	0.10ml	0.10ml

Mix, let stand at 20°C to 30°C for 5min

Read absorbance of sample against sample blank.

RHEUMATOID FACTOR DETECTION TEST

PRINCIPLE:-

When a serum sample +ve for RF is mixed with latex reagent visible agglutination occurs. In sample negative for rheumatoid factor the latex remains in a smooth suspension form.

PROCEDURE:-

i. Allow all reagents and samples to equiliberate to room temperature before use ensure that the test slide is clean and dry.

ii. Place 50 µl of test serum/ controls on the circle of the test slide.

iii. Add 2 drops of the latex reagent holding the dropper vertically .

iv. Use the mixing stick to mix and spread the reagents over vertically.

v. Gently rotate the slide for 2 minutes, and examine for agglutination immediately under bright light.

RF LATEX REGENT:-

Suspension of polystyrene latex particles of uniform size, coated with human gammaglobulines.

BLOOD GROUP ANALYSIS

INTRODUCTION :-

In 1901 ABO system was discovered by LANDSTEINER . the ABO system consist of four main groups-

1. “A”

2. “B”

3. “AB”

4. “O”

i.e. cell carrying A antigen, B antigen, both A and B antigen and cells that do not carry either A or B antigen respectively.

The blood group antigen react with their corresponding antibodies present in the serum of different blood group.

IMPORTANCE OF ABO BLOOD GROUP

The following conditions required for determination of ABO blood groups-

* In organ transplantation.

* In haemolytic diseases of the new born baby.

* In forensic medicine.

* In malignant diseases.

* In clinical medicine.

PROCEDURE:-

# Take a slide and then mark as A,B,O place an drop of antigen A in the area marked as A & one drop of anti B on the area marked as B & third drop of anti D on the area marked as O.

# Disinfect the first two drops of blood.

# Add three drops of blood, the area marked as A,B and O.

# Mix the antiserum and cell with the stick or toothpicks.

# Observe the haemagglutination, record the result.

HIV TEST

PRINCIPLE:-

HIV antigen are immobilized on a porous immunofiltration membrane. Sample and reagent passes through membrane and are absorbed into the underlying absorbent.

As the patient sample passes through the membrane, HIV antibodies, if present, bind to the immobilized antigen.

Conjugate bind to the Fc portion of the HIV antibodies to give distinct pinkish purple DOT against a white background.

HIV kit contains:

· HIV TRI-DOT test device

· Buffer solution

· Protein A-Conjugate

· -ve & +ve control

· Sample dropper

Test procedure:-

1. Add three drop of buffer solution to the center of the device (HIV TRI-DOT)

2. Hold the dropper vertically and add one drop of patient serum using the sample dropper.

3. Add five drops of buffer solution.

4. Add two drops of liquid conjugate directly from the conjugate vial.

5. Add five drops of buffer solution and read result.

RESULTS:-

1. Non-reactive:- if only one DOT appears (control DOT) specimen is non-reactive for antibodies either HIV1 or HIV2.

2. HIV2:-if two DOTs, one for control and other for HIV2 appear.

3. HIV1:- if two DOTs one for control and other for HIV1 appear.

4. If all three DOTs, one each for control, HIV1, HIV2 appears, the specimen is reactive for antibodies of HIV1 and HIV2.

WIDAL TEST

(STAINED SALMONELLA ANTIGEN, SLIDE TEST)

Clinical significance:-

Rapid-Widal is intended for the detection of Typhoid Ab usually found after two weeks of salmonella infection.

The level of the Ab progressively rise to a maximum by 3-4 weeks if no specific antibiotics are taken. A progressive rise in Ab titer during the period of infection is indicative of salmonella infection.

Principle:-

Rapid widal is based on a reaction between specific species of killed salmonella core(O) Antigens, flagellar(H) Ag, paratyphi”AH”, paratyphi”BH” antigens and the Ab found in patient serum. By staining the bacteria in the Antigens enhance the visibility of the reaction on slide as well as test tube.

Reagents

S.Typhi”O”	5ml
S.Typhi”H”	5ml
S.Paratyphi”AH”	5ml
S.Paratyphi”BH”	5ml
+ve control	0.5ml

Note:- All reagents contain killed bacterial Ag, preservatives and stabilizers

RAPID SLIDE TEST

Clean the glass slide with distilled water and dry. Brings the Antigens to room temperature and mix well before use.

1. Add sample a drop each to “o”,”H”,”AH” and”BH” circles.

2. Add “O” Ag a drop to “O” circle, “H” Ag a drop to “H” circle, “AH” Ag a drop to “AH” circle, “BH” Ag a drop to “BH” circle.mix well using separate disposable plastic sticks.

3. Observe agglutination within 5 min. under a bright light source.

4. Run a –ve control using normal saline as sample.

5. Run a +ve control using the +ve control as sample.

Result:-

· Observe agglutination in comparison with –ve & +ve control.

· A progressive rise in titer is indicative of salmonella infection

BASIC LABORATORY PRINCIPLES AND PROCEDURE