CLINICAL LABORATORY TRAINING

REPORT
Submitted to Bharathiar University for
the award of the degree of

MASTER OF SCIENCE IN
APPLIED MICROBIOLOGY

By

MARIATHERESA
21MAMY008

Guided by
MR.B.VENKATRAJAH. M.Sc., M.Phil., PGDBI.,MBA., M.A
MICROBIOLOGY

DEPARTMENT OF MICROBIOLOGY
SCHOOL OF BIOLOGICAL SCIENCES
CMS COLLEGE OF SCIENCE AND COMMERCE
COIMBATORE
AUGUST 2022
DECLARATION
 DECLARATION

I, AMALA MARIA GRACE JOSE, hereby declare that the training report entitled

“CLINICAL LABORATORY TRAINING REPORT” submitted to the Bharathiar

University, in partial fulfillment of the requirements for the award of the Degree of Master of

Science in Applied Microbology is a record of original and independent training underwent by me

during August 2022 under the guidance of MR.B.VENKATRAJAH, Assistant Professor, Department

of Microbiology, School of Biological Sciences, CMS College of Science and Commerce, Coimbatore,

and it has not formed the basis for the award of any Degree / Diploma / Associate ship / Fellowship or

other similar title to any candidate in any University.

AMALA MARIA GRACE JOSE

CERTIFICATE
 CERTIFICATE
This is to certify that the training report entitled “CLINICAL LABORATORY

TRAINING REPORT” submitted to the Bharathiar University, in partial fulfillment of

the requirements for the award of the Degree of Master of Science in Biotechnology is a

report of original training underwent by MARIA THERESA, (21MAMY008) during

August, 2022 of her study in the Department of Microbiology, School of Biological

Sciences,, CMS College of Science and Commerce, Coimbatore, under my supervision and

guidance and this training report has not formed the basis for the award of any Degree /

Diploma / Associate ship / Fellowship or other similar title to any candidate in any

University.

Counter Signed with seal

GUIDE COURSE CO-ORDINATOR DIRECTOR

ACKNOWLEDGEMENT
 First and foremost, I thank God almighty who leads me in each step in the progress
towards the successful completion of our dissertation and for his immense blessing on us
throughout the period of our studies.

I would like to express my sincere thanks to Dr. H. Balakrishnan, Principal, CMS

College of Science and Commerce, Coimbatore, for providing all facilities in the department and
college.

I extend my sincere thanks to Dr. T. Vinoth Kumar, Director, School of Biological

Sciences, CMS College of Science and Commerce, Coimbatore, for this encouragement and
suggestions to complete the work successfully.

I wish to thank my guide MR. B. Venkatrajah for his guidance and support and I
would also like to express my gratitude to All the Staff Members of School of Biological
Science, CMS College of Science and Commerce, Coimbatore.

With deep since of gratitude I extend my heartfelt thanks to CISTRON

LABORATORIES for providing me with all the necessary facilities for the research.
CONTENTS
 CONTENTS

S. NO TITLE PAGE NO
1 INTRODUCTION 1
2 EXPERIMENTS AND RESULTS 5
3 SUMMARY AND CONCLUSION 38
4 BIBLIOGRAPHY 40
5 APPENDIX 41
INTRODUCTION
 1. INTRODUCTION

Body fluids

Bodily fluids are liquids originating from inside the bodies of living things.
They include fluids that are secreted or excreted from the body. Approximately 60-
65% of body water is contained within the cells (in intracellular fluid) with the other
35-40% 0f body water contained outside the cells (in extracellular). This fluid
component outside thecells includes the fluid between the cells (intestinal fluid),
lymph and blood. There are approximately 6 to10 liters of lymph in the body,
compared to 3.5 to 5 liters of blood.
Importance of body fluids

The importance of body fluids is as follows,

• Body temperature regulation.

• Bitterer blood flow that provides efficient transport of nutrients and

substances to all organs of the body.

• Aid in the excretion of toxins and waste products in the body.

• Keeps the skins moist.

• Aid in the digestive process.

What are diseases?

A disease is an abnormal condition affecting a living organism. Diseases are
generally understood to be medical conditions that involve a pathological process
associated with a specific set of symptoms. Localized diseases affect specific parts
of the body; disseminated diseases spread to other parts of the body; and systemic
diseases affect the entire body.

Categories of diseases include autoimmune, bacterial, blood, cancer, digestive,

heart, nerve (or neurodegenerative), sexually transmitted or thyroid. Diseases may be
communicable or non-communicable. External sources that can cause disease
include acquired viruses or bacteria, and internal causes of disease include
autoimmune or genetic dysfunction. Some diseases are chronic, meaning that they
are continually present and may present symptomatically during a long duration.
Laboratory test
Many infectious diseases have similar signs and symptoms. Samples of your
body fluids can sometimes reveal evidence of the particular microbe that's causing
your illness. Blood tests:-A technician obtains a sample of your blood by inserting a
needle into a vein, usually in your arm.
Urine tests:-This painless test requires you to urinate into a container. To avoid
potential contamination of the sample, you may be instructed to cleanse your genital
area with an antiseptic pad and to collect the urine midstream.
Throat swabs:-Samples from your throat, or other moist areas of your body, may be
obtained with a sterile swab.
Stool sample:-It is instructed to collect a stool sample so a lab can check the sample
for parasites and other organisms.

Clinical biochemistry
Clinical biochemistry refers to the analysis of the blood plasma (or serum) for
a wide varietyof substances-substrates, enzymes, hormones, and their use in
diagnosis and monitoring of disease. Analysis of other body fluids (e.g., urine, ascitic
fluids, CSF) is also included. Mostcurrent laboratories are now highly automated to
accommodate the high workload typical of a hospital laboratory. Tests performed are
closely monitored and quality controlled.

Clinical laboratory
Clinical laboratory is a place where tests are usually done on clinical
specimens in orderto obtain information about the health of a patient as pertaining to
the diagnosis, treatment, and prevention of disease. Clinical laboratories are thus
focused on applied science mainly on a production-like basis, as opposed to research
laboratories that focus on basic science on an academic basis.

Types of laboratory
Clinical
biochemistry
Chemistry performs a wide variety of tests using the most current technology.
It is defined as the scientific study of matter and the various compounds of the
elements as
it relates to the human body. Common tests analyzed in the chemistry laboratory are
glucose, cholesterol, creatinine, potassium, liver and heart enzymes, thyroid tests and
hormone tests.
Haematology

Haematology is the study of blood, blood morphology and blood diseases.

Haematologists count and classify blood cells into different categories. Coagulation is
the study of the clotting activity of blood.

Microbiology
Microbiology is the study of microorganisms including algae, bacteria, fungi,
protozoa and viruses. Any bodily fluid or tissue can be cultured for infectious disease.
Once bacteria grow in culture, it can be tested against many different antibiotics to find
the most effective for fighting the infection while limiting opportunities for antibiotic
resistance.

Immunology
Immunology is the study of immune products such as antibodies produced by the
body in response to foreign material.

Pathology:
Pathology is the branch of medicine, which treats the essential nature of disease,
especially the structural and functional changes in tissues and organs of the body, which
cause or are caused by disease.
 2. MATERIALS AND METHODS

The training been underwent at SOWRAM CLINICAL LABORATORY during

the period the analysis been taken in the human pathological sample. The techniques
which were been trained during, been described as follows.

Clinical techniques of general Instructions

• Approach the patient pleasantly, confidently and in a friendly manner.

• Inform the patient that blood is going to be drawn from him/her and that it
willnothurt and get his/her confidence and co-operation.

• Collect the requisition form for individual‟s patients from the front office
andverifyif the form contains all the necessary details. (Name, age, sex,
tests requested).
EXPERIMENTS AND RESULTS
HAEMATOLOGY
 3. EXPERIMENTS AND RESULTS

HAEMATOLOGY
BLOOD GROUPING AND RH
PRINCIPLE
This test is based on the principle of direct agglutination. The erythrocytes of a person
contain antigens on the surface on the membrane. When these antigens are allowed to react
with the corresponding antibodies, antigens-antibody reactions are produced. Normal
erythrocytes will clump or agglutinate when mixed with Anti A, Anti B, Anti A & B, if they
possess A, B, AB antigens respectively.

SAMPLE
Use whole blood as specimen.

REAGENTS
• Anti A, Blood grouping reagent (commercially available)

• Anti B, Blood grouping reagent (commercially available)

• Anti AB, Blood grouping reagent (commercially available)

• Anti D, Blood grouping reagent (commercially available)

• Anti A1, Lectin (commercially available )

• Glass slides

• Applicator sticks

• 0.9% isotonic saline

Slide agglutination method
Take a clean grease free microscope slide. Draw a line in the middle of the slide using
a wax glass marking and label the left portion Anti A, and add a drop of Anti A, and the
right as Anti B. Add 1 drop of well mixed 3-5% cell suspension to each side. With a tooth
pick, separate for each side mix the cells and Anti-sera well. Gently rotate the slide for
mixing. Place the slide against a white background. After 2 minutes, examine both
macroscopically and microscopically.

RESULTS AND DISCUSSION

• If agglutination is observed with anti A blood group reagent, then the patient’s
blood group is A.

• If agglutination is observed with anti-A1 Lectin reagent, then the patient’s

blood group is A1.

• If agglutination is observed with anti B blood group reagent, then the patient’s
blood group is B.

• If agglutination is observed with anti A and anti B blood group reagent, then
the patient‟s blood group is AB.

• If agglutination is not observed with both anti A and anti B blood group
reagent then the patient‟s blood group is O.

• If agglutination is observed with anti D blood group reagent, then the patient’s
Rhtype is positive. All Rh negative is reconfirmed with du test.

Fig.1 Blood grouping

Table.1 Blood grouping

Monoclonal Monoclonal Monoclonal Result

S.
Reaction Antibodies antibodies Antibodies blood
No.
A B D group
A
1 Agglutination + - +
Positive
A
2 Agglutination + - -
Negative
B
3 Agglutination - + +
Positive
B
4 Agglutination - + -
Negative
AB
5 Agglutination + + +
Positive
AB
6 Agglutination + + -
Negative
O
7 Agglutination - - +
Positive
O
8 Agglutination - - -
Negative

ESR-ERYTHROCYTE SEDIMENTATION RATE

PRINCIPLE
The “sedimentation rate” is a non-specific indicator of disease and is common
performed ESR refers to the rate cells fall in mm/ time. this test is primarily used to monitor
with inflammatory disease particularly rheumatoid arthritis.
SAMPLE
Venous blood -2ml

SPECIMEN
EDTA anticoagulant whole blood that is less then 4 hours old; the EDTA tube must
be at least half full.

MATERIALS
 • 3.8% sodium citrate – 0.4ml
• Auto zero westergerns tubes
• Sediplast rack
• Plastic pipettes
PROCEDURE
Sodium citrate -0.4 ml Blood sample-1.6 ml
• Fill in the Westergerns tube exactly to the zero mark.
• Place it in stand vertically.
•Reading may be made at 5 minutes interval over a period of half and hour or at the end of
half an hour.
NORMAL REFERENCE VALUE
• Male : 0-10 mm/hr
• Female : 0-20 mm/hr

INTERPRETATION
Increased sedimentation rates are found in all collagen disease (SLE) infections,
pneumonia, syphills, inflammatory disease, toxaemia, anaemia, nephritis, nephrosis.
Normal sedimentation rate (no increase) Polycythemia vera, hyper fibrinogenemia, sickle cell
anaemia, congestive heart failure, hereditary sperocytosis, pyruvate kinase deficiency.

COMPLETE BLOOD COUNT-AUTOTOMATED HEMATOLOGY ANALYSER

PRINCIPLE

White blood cells, red blood cells and platelets are counted by the principle of impedance
variation this principle takes advantage of the fact that blood cells are less conductive to
electrical current than the cell diluting fluid. WBCs are counted after lysing the RBCs by a
detergent (ionic or non-ionic) which does not lyse the WBCs themselves. After dilution (1:
50 for WBC count and Hb 1: 50,000 for RBC count Indices and platelet count) the diluted
sample is directed to two sides of the instrument.

SAMPLE
Use whole blood as sample collected in K3 EDTA.

INSTRUMENT

• Advia 60 OT Automated Haematology Cell Counter.

 • Haemo mixer.

Fig.2 Advia 60 OT Automated Haematology Cell Counter.

PROCEDURE
• Switch on the instrument by pressing the ON/OFF Switch located in the rear panel
of the instrument.

• Wait for Approximately 3 min for the initialization process to be completed.

• Change of Front Panel LED from Red to Green indicates that the initialization
process is completed. Process QC / patient samples when the background limits are
within the acceptable range.

• Run any one of the Blood samples to prime the instrument.

• Mix the sample thoroughly but gently using the hemomixer just prior to
processingthe sample.

• Enter the patient ID or run using the <ID> keys after verifying the ID on the
sample matches with that on the Requisition Form.

• Present the sample after gentle and through mixing under the sampling probe.

• Press the < START> key on the front panel or the sampling bar located behind the
sampling needle. The Green - Red LED Starts Blinking.

• Remove the sample tube when the LED Stops blinking. Result will be displayed
on the LCD screen. Enter the results in the Daily Results Register Haematology.
 REFERENCE RANGE
Potential Sources of variability: The following factors will affect cell counting
in anautomated haematology analyzer.
Visible clots Fibrin micro clots or cryoprecipitate in the specimen.

Table .2 Blood cell count

Parameter Reference
Range
WBC Adults 4000-10,000 cells/cu.mm
count
RBC count Male : 4.5 to 6.0 x 106cells/cu.mm; Female: 4.0 to 4.5 x
106 cells/cu.m
Haemoglob in Men 14 – 18g/100ml Women 12-16 g/ 100ml

Platelet 250,000 – 500,000/CU.MM

count
Haematocrit Male: 42 – 52 Female 36 – 48%

MCV 70-90 microns

MCH 14-18 gm/dl
MCHC 45-55%

URINE COMPLETE ANALYSIS PRINCIPLE

Specific Gravity: The test detects the ion concentration of urine. In the presence of two
indicators methyl red & bromothymol blue which give colours changes ranging from blue- green
to orange with increasing low ionic concentration.

pH: This test is based on the double indicator principle that gives a broad range of colours
covering the entire urinary pH range. The test strip contains a combination of methyl-red (pH
range 5.0-6.0.) and bromothymol- blue (pH range 8.0-9.6) as indicators. The colour gradations
extend form orange via green to blue.

 Protein: The test is based on the colour change of the indicator, tetrabromo thymol blue,
in the presence of protein. A positive reaction is indicated by a colour change from
yellow which is negative to a green –gold for a trace reaction through green & then to
greenish-blue for elevated levels.

 Glucose: The test area is impregnated with glucose-oxidase / peroxidase together with
potassium iodide and a blue background dye. The oxygen liberated in the final reaction
binds with the dye to produce a series of colour changes 30 seconds after wetting the
strip with urine.
 Ketone bodies: The test is based on the reaction of ketone with nitro prusside. The
resulting colour ranges from tan when no reaction takes place to buffy pink through pink to
purple for a positive reaction.

 Urobilinogen: A stable diazonium salt 4-methoxybenzen diazonium reacts

immediately with urobilinogen in the strong acid environment of the test to effect a
change from tan to pink

 Bilirubin: The test for bilirubin is based on the coupling of bilirubin with a stable
diazonium salt (2-4 dichloro aniline diazonium) in the acid environment of the test area
of the strip. The colour ranges from tan to orangish when no bilirubin is present through
various shades of tan to reddish brown with increasing levels of billirubin.

SAMPLE
Instruct patients to collect a minimum 15 ml of a random sample of urine in a clean
dry container (preferably collected from the laboratory) Instruct patients to void directly in
to the container and while collecting allow the first portion of urine to escape. Use fresh
well mixed uncentrifuged urine as specimen for the test, process the samples within 1 hour
of sample received.

INSTRUMENT

Fig.3 Urisys 1100

TEST PROCEDURE
• Ensure the urine sample is at room temperature and mix the urine sample thoroughly
before testing. Take a test strip from the pack and close it again immediately.

• Briefly, no longer than 1 sec, dip the test strip into the urine making sure that all the
test areas are moistened. If the strip is dipped for a longer time reagents from the
testareas of the strip will dissolve in the urine.

• When removing the strip, wipe the edge of the strip against the rim of the specimen
container to remove excess urine or dab the side edge of the strip on a clean
 Absorbent surface. If reading visually compares the test results carefully with the
color chart on the label after the appropriate time period (60 to 120 seconds) or
place the strip with the pads facing upward on the test strip tray and press okThe
report will be printed one by one.

REFERENCE RANGE
• pH : 5-9

• Specific Gravity: SG of random urine ranges from 1.010- 1.030.

• Nitrite: Normally no detectable amount of nitrite is present in urine.

• Protein: Normally no detectable amount of protein is present in urine

• Ketone: Normally, no ketones are present in urine.

• Glucose : Normally no detectable amount of glucose present in urine

• Bilirubin: Normally, no bilirubin is detectable in urine
 Urobilinogen: Present in normal amounts

• Leucocytes : Normal urine yields negative results

Pathological leucocyte concentration > 20 leucocytes/µl

Blood Normally no blood is present in urine. Blood may be in the urine of menstruating
females.

Table.3 Urine analysis

Determinat Normal Pathologic
Abnormal
ion Finding definition
Diabetes insipidus
Volume of >500ml
1 50-200ml and polyuria
urine
<20ml Oliguria, Anuria
Hepatic and post
Pale yellow Dark yellow
hepatic condition
Colour of
2 Reddish Chyluria, Hematuria
Urine
White Black urine Alkaptonuria
Dark yellow Biliverdin present
Appearance Presence of abnormal
3 Usually clear Turbid
of leukocytes
 Urine Milky Chyle
Usually pH less than
acidic PH 4.8 More, Fever, Ketosis
4.88 to 7.5 acidic urine
4 Reaction pH More
than 7.9
Severe vomiting
Alkaline
Urine
Odour of Fruity Acidosis, Ketosis
5 Aromatic
urine Ammonical Cystitis
Specific Varies from
Low specific Chronic nephritis &
6 Gravity of 1.003 to
gravity diabetes incipidus
urine 1.060

BLOOD BLEEDING TIME

PRINCIPLE

Bleeding time measures the ability of these platelets to arrest bleeding and therefore
measures platelets number and function.
SAMPLE

Blood sample.

MATERIALS
• Syringe

• Glass tubes -12 * 75 mm

TEST PROCEDURE

Blood - 1 ml

Evaluate tubes at 1 hour for retraction of the clot from the sides of the tubes. Although
rarelyperformed, the clot retraction test measures the ability of platelets contractile proteins
to reduce clot size.

RANGE
Normal value for bleeding time 2.5 minutes.
BIOCHEMISTRY
 GLUCOSE

PRINCIPLE
Glucose is determined after enzymatic oxidation in the presence of glucose oxidase.
The h final colour is directly proportional to the glucose concentration and is measured at
505 nm.

SAMPLE
Use plasma or urine as specimen.2 ml of venous blood from a peripheral vein in a
sodium fluoride K3 EDTA tube. Separate the plasma within 2 hours of collection. Separate
the plasma by centrifugation at 2500- 3000 rpm for 3 -5 minutes. Process the sample on the
sameday. For Urine, use 24 hour urine as specimen using thymol as preservative. Do not use
hemolysed, contaminated samples for testing.
MATERIALS
RX Imola and RX Daytona Clinical Chemistry Analyzer.

Fig.4 RX Imola and RX Daytona Clinical Chemistry Analyzer.

REAGENTS
• MOPS Buffer: 50 m mol/l, pH 7.06.2.3 Phenol: 11 m mol/l
• Phosphate buffer: 50 m mol/l, pH 7.0

• 4-aminophenazone: 0.77 m mol/1

• Glucose oxidase: ≥1.5kU/l

• Peroxidase : ≥1.5kU/l

PROCEDURE
• Before Switching on, check the pre-start inspection. Once pre-start inspections are
done, Switch “ON” the Computer, Printer, and the analyzer. Wait for initialization
process to be completed.
 • After initialization is completed, ensure that the screen displays a standby mode.
• Check water tank, wash solution, cuvette, and waste tank.
• Clean outside of sample/reagent probe, stains on the internal surface, and RCV
tray. Log on the system, check system overview, and prepare the system before
startroutine operation.

• Daily before processing the patient samples load the Internal Quality Control as per
Quality Control procedure. (BL/CB/L).

• Remove controls from the sample disk or rack once QC is validated. Start routine
sample for analysis. Place the barcode samples on the sample disk or enter the
patient ID and cross verify SID code on the sample matches with that on the
requisition form.

• Press the <START> key on monitor display, and begins to process patient
samples. Once the process is completed, result will be displayed on the monitor
screen, andautomatically will be transferred to the LIS through interface.

• Enter the results in the Daily Results Register Biochemistry.

CALCULATION OF RESULTS

The system performs all calculations internally to produce the final reported result.

The system does not calculate the final result for sample dilutions made by the

operator.
In these cases the result produced by the operator must be multiplied by the operator. If the
dilution factors are entered in to the system during the sample programming by the
operatorthe system automatically.

REFERENCE VALUE
• Fasting: Adult: 74-99
• Children: 60-100
• Random: 80-120
• Post prandial: 90-140

INTERPRETATION OF RESULTS
• Elevated levels are found in pancreatitis, pituitary and thyroid dysfunction,
renalfailure and liver diseases.
 • Low glucose levels are found in insulinoma, hypopituitarism, neoplasms, insulin
induced hypoglycaemia.

CHOLESTROL

PRINCIPLE
Cholesterol Esterase hydrolyses cholesterol esters into free cholesterol and fatty acids.
In the second reaction cholesterol oxidase converts cholesterol to cholest-4-en-3-one and
hydrogenperoxide. In presence of peroxidase, hydrogen peroxide oxidatively couples with
4- aminoantipyrine and phenol to produce red quinoneimine dye which has absorbance
maximum at 510 nm (500-530). The intensity of the red colour is proportional to the amount
of total cholesterol in the specimen.

SAMPLE
Use only serum / plasma as specimen for the test.Collect 2 ml of venous blood in a
plain red 1topped vacutainer tube or yellow capped gel tubeAllow the tube to stand for 30
minutes atroom temperature and separate the serum by centrifugation at 2500 - 3000 rpm for
5 - 10 minutes. Ensure complete clot formation has occurred and there are no fibrin threads.
Process all samples on the same day within 2 hours of collection. If processing is done
after8 hours of collection, separate the serum and aliquot it storage vials at 2- 80C.
If assay is likely to be done after 48 hours of collection, store the serum at - 20 0CDo not use
hemolysed, contaminated samples for testing.

Type of container and additive

Use Red topped Plain vacutainer tubes / green topped heparinised vacutainer tubes,
yellow capped gel tubes for collecting samples. Do not add any additives / preservatives.

MATERIALS

RX Imola and RX Daytona Clinical Chemistry Analyzer.

REAGENTS
• Pipes buffer: 80 m mol/l, pH 6.7

• 4-aminoantipyrine: 0.25 m mol/l

• Phenol: 6 m mol/l

• Peroxidase: ≥ 0.5 U/ml

• Cholesterol esterase: ≥ 0.15 U/ml

 • Cholesterol oxidase : ≥ 0.10 U/ml

PROCEDURE
• Before Switching on, check the pre-start inspection. Once pre-start inspections are
done, Switch “ON” the Computer, Printer, and the analyzer. Wait for initialization
process to be completed.

• After initialization is completed, ensure that the screen displays a standby mode.

• Check water tank, wash solution, cuvette, and waste tank.

• Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray.

• Log on the system, check system overview, and prepare the system before
startroutine operation.

• Daily before processing the patient samples load the Internal Quality Control as
perQuality Control procedure. (BL/CB/L).

• Remove controls from the sample disk or rack once QC is validated.

• Start routine sample for analysis. Place the barcode samples on the sample disk or
enter the patient ID and cross verify SID code on the sample matches with that on
the requisition form. Press the <START> key on monitor display, and begins to
process patient samples.

• Once the process is completed, result will be displayed on the monitor screen, and
automatically will be transferred to the LIS through interface.

• Enter the results in the Daily Results Register Biochemistry.

REFERENCE RANGE
• < 200 mg/dl -Desirable blood cholesterol.

• 200-239 mg/dl- Borderline-high blood cholesterol.

• ≥ 240 mg/dl - High blood cholesterol.

INTERPRETATION OF RESULTS
• Increased Serum levels are seen in the following conditions:
 • Familiahypercholesterolem.
• Nephrotic syndrome.

• Biliary obstruction.

• Hypothyroidism.

• Pregnancy.

• Decreased serum levels are seen in the following conditions:

• Hyperthyroidism.

• Malnutrition.

• Chronic anemia.

• Thyroiditis.

• Severe liver insufficiency.

HDL-CHOLESTEROL
PRINCIPLE
The assay has 2 distinct reaction steps as given below.
Reaction 1: Elimination of chylomicron, VLDL-Cholesterol and LDL-Cholesterol by
cholesterolesterase, cholesterol oxidase and subsequently catalase.
Reaction 2: Specific measurement of HDL-Cholesterol after release of HDL-Cholesterol by
detergents.
SAMPLE
• Use only fasting serum as specimen for the test.

• Collect 2 ml of venous blood in a plain red topped vacutainer tube / yellow capped
gel tube. Allow the tube to stand for 30 minutes and separate the serum by
centrifugation at 2500- 3000 rpm for 5 – 10 minutes.

• Do not use lysed serum for testing as it may give very high resµlts. o Do not use
contaminated / turbid samples for testing.

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

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

MATERIALS
RX Imola and RX Daytona Clinical Chemistry Analyzer.

REAGENTS
Enzyme Reagent 1:
• Cholesterol Esterase

• Oxidase

• Catalase

Enzyme Reagent 2:
• Peroxidase

• Sodium hydrazine

PROCEDURE
• Before Switching on, check the pre-start inspection. Once pre-start inspections are
done, Switch “ON” the Computer, Printer, and the analyzer. Wait for initialization
process to be completed.

• After initialization is completed, ensure that the screen displays a standby mode.

• Check water tank, wash solution, cuvette, and waste tank.

• Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray

• Log on the system, check system overview, and prepare the system before start
routine operation. Daily before processing the patient samples load the Internal
Quality Control as per Quality. Control procedure. (BL/CB/L).

• Remove controls from the sample disk or rack once QC is validated. Start routine
sample for analysis. Place the barcode samples on the sample disk or enter the
patient ID and cross verify SID code on the sample matches with that on the
requisition form. Press the <START> key on monitor display, and begins to process
patient samples. Once the process is completed, result will be displayed on the
monitor screen, and automatically will be transferred to the LIS through interface.
 • Enter the results in the Daily Results Register Biochemistry.

REFERENCE RANGE
• Low: < 40.0 mg/d L

• High: ≥ 60.0 mg/d L

INTERPRETATION OF RESULTS
• Values >60 mg/d L are considered a negative risk factor for CHD and are considered
protective.

• Values > or = 80-100 mg/d L may indicate metabolic response to certain medications
such as hormone replacement therapy, chronic liver disease, or some form of chronic
intoxication, such as with alcohol, heavy metals, industrial chemicals including
pesticides.

• Values <40 mg/d L correlate with increased risk for CHD.

• HDL values < or = 5 mg/d L occur in Tangier disease, in association with cholestatic
liver disease, and in association with diminished hepatocyte function.

LDL-CHOLESTROL
PRINCIPLE
The assay has 2 distinct reaction steps as given below
Reaction 1: Elimination of chylomicron, VLDL-Cholesterol and HDL-Cholesterol by
cholesterol esterase, cholesterol oxidase and subsequently catalase.
Reaction 2: Specific measurement of LDL-Cholesterol after release of LDL-Cholesterol by
detergents in Reagent 2.
SAMPLE
• Use only fasting serum as specimen for the test.

• Collect 2 ml of venous blood in a plain red topped vacutainer tube / yellow capped
gel tube. Allow the tube to stand for 30 minutes and separate the serum by
centrifugation at 2500- 3000 rpm for 5 – 10 min .

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

• Do not use contaminated / turbid samples for testing.

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

• If analysis is not done on the same day /within 3 hours of collection, separate the
serum and store it at 2-8 °C for up to 7 days.
 • If assay is likely to be done after 48 hours of collection, store the serum at - 200C.

MATERIALS

RX Imola and RX Daytona Clinical Chemistry Analyzer.

REAGENTS

Enzyme Reagent 1:

• Cholesterol Esteras E
• Cholesterol Oxidase

• Catalase Enzyme Reagent 2:

• Peroxidase

• Sodium Azide

PROCEDURE
• Before Switching on, check the pre-start inspection. Once pre-start inspections are
done, Switch “ON” the Computer, Printer, and the analyzer. Wait for initialization
process to be completed.

• After initialization is completed, ensure that the screen displays a standby mode.

• Check water tank, wash solution, cuvette, and waste tank.

• Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray

• Log on the system, check system overview, and prepare the system before start
routine operation. Daily before processing the patient samples load the Internal
Quality Control as per Quality Control procedure. (BL/CB/L).

• Remove controls from the sample disk or rack once QC is validated. Start routine
sample for analysis. Place the barcode samples on the sample disk or enter the
patient ID and cross verify SID code on the sample matches with that on the
requisition form.

• Press the <START> key on monitor display, and begins to process patient samples.

Once the process is completed, result will be displayed on the monitor screen, and
automatically will be transferred to the LIS through interface.
 • Enter the results in the Daily Results Register Biochemistry.

REFERENCE RANGE
• Optimal: < 100 mg/dl

• Near or above optimal: 100 - 129 mg/dl

• Borderline High: 130-159 mg/dl

• High: 160- 189 mg/dl

• Very high: >190 mg/dl

• Very high: >190 mg/dl

INTERPRETATION OF RESULTS
Evaluation of cardiovascular risk is based on the following range of values established by
the National Cholesterol Education Program (NCEP):
• Desirable: <100 mg/dl

• Low risk: 100mg/dl to 129 mg/dl

• Borderline high: 130 mg/dl to 159 mg/dl

• High:160 mg/dl to 189 mg/dl

• Very high: > or =190 mg/dl

Decreased values may indicate hypobetalipoproteinemia.

Non-detectable low density lipoprotein cholesterol indicates abetalipoproteinemia.
Relatedpolyneuropathy may exist in affected individuals.

ALBUMIN
PRINCIPLE
Albumin combines with Bromocresol green (BCG) Reagent to form a coloured
product. Theintensity of the colour formed is directly proportional to the albumin present in
the specimenand is measured at 578 nm.
PRIMARY SAMPLING
• Use only serum as specimen for the test.
 • Collect 2 ml of venous blood from a peripheral vein in a plain red topped
vacutainer tube or yellow capped gel tube, separate the serum within 2 hours of
collection.
• Allow the tube to stand at room temperature till complete clot formation occurs (for
30 minutes) and then separate the serum by centrifugation at 2500- 3000 rpm for 3
–5minutes. Ensure complete clot formation has occurred and there are no fibrin
threads.

• Process all samples on the same day within 2 hours of collection. If processing is
done after 8 hours of collection, separate the serum and aliquot it storage vials at 2-
80C.

• If assay is likely to be done after 48 hours of collection, store the serum at - 200C.
• Do not use haemolysed, contaminated samples for testing.

MATERIALS

RX Imola and RX Daytona Clinical Chemistry Analyzer.

REAGENTS

• Bromocresol Green

• Succinate buffer: 75 m mol/l; pH 4.2.

• Bromocresol green: 0.15 m mol/l.

• Brij 35.

• Preservative.

PROCEDURE
• Preservative + BCG Albumin-BCG complex

• Before Switching on, check the pre-start inspection. Once pre-start inspections are
done, Switch “ON” the Computer, Printer, and the analyzer. Wait for initialization
process to be completed.

• After initialization is completed, ensure that the screen displays a standby mode.

• Check water tank, wash solution, cuvette, and waste tank.

 • Clean outside of sample/reagent probe, stains on the internal surface, and RCV tray

• Log on the system, check system overview, and prepare the system before start
routine operation. Daily before processing the patient samples load the Internal
Quality Control as per Quality Control procedure. (BL/CB/L).
• Remove controls from the sample disk or rack once QC is validated. Start routine
sample for analysis. Place the barcode samples on the sample disk or enter the
patient ID and cross verify SID code on the sample matches with that on the
requisition form. Press the <START> key on monitor display, and begins to process
patient samples. Once the process is completed, result will be displayed on the
monitor screen, and automatically will be transferred to the LIS through interface.

• Enter the results in the Daily Results Register Biochemistry.

REFERENCE RANGE
• Adults: 3.50 – 5.20 g/dl

• Neonates 3.8 - 4.2 g/dl

INTERPRETATION OF RESULTS
Albumin level in blood increases due to dehydration, reduced plasma water content,
stasis during vein puncture which can cause fluid to escape into the extra cellular
compartment.
Albumin levels in blood is found to be decreased in conditions like excessive
protein loss from kidney, skin, or intestine, decreased synthesis due to dietary, hepatic
disease or mal absorption, increased catabolism in fever, untreated diabetes mellitus and
hypertension.
SEROLOGY
 SEROLOGY
WIDAL TEST
PRINCIPLE
The main principle of widal test is that if homologous antibody is present in patients
serum, it will react with respective antigen in the reagent and gives visible clumping on the
test card and agglutination in the tube. The antigens used in the test are “H” and “O” antigens
of Salmonella Typhi and “H” antigen of S. Paratyphi. The paratyphoid “O” antigen are not
employed as they cross react with typhoid “O” antigen due to the sharing of factor 12. “O”
antigen is a somatic antigen and “H” antigen is flagellar antigen. Bacterial suspension which
carry antigen will agglutinate on exposure to antibodies to Salmonella organisms. Patients‟
suffering from enteric fever would possess antibodies in their sera which can react and
agglutinate serial doubling dilutions of killed, coloured Salmonella antigens in a
agglutination test.

PROCEDURE
SLIDE TEST
 Place one drop of positive control on one reaction circles of the slide.
 Pipette one drop of Isotonic saline on the next reaction cirlcle. (-ve Control).
 Pipette one drop of the patient serum tobe tested onto the remaining four
reactioncircles.
 Add one drop of Widal TEST antigen suspension „H‟ to the first two reaction
circles. (PC & NC).
 Add one drop each of „O‟, „H‟, „AH‟ and „BH‟ antigens to the remaining
four reaction circles.
 Mix contents of each circle uniformly over the entire circle with separate
mixingsticks.
 Rock the slide, gently back and forth and observe for agglutination
macroscopicallywithin one minute.

SEMI-QUANTITATIVE METHOD
 Pipette one drop of isotonic saline into the first reaction circle and then place 5,
10,20, 40, 80 ul of the test sample on the remaining circles.
 Add to each reaction circle, a drop of the antigen which showed agglutination
withthe test sample in the screening method.
  Using separate mixing sticks, mix the contents of each circle uniformly over the
reaction circles.
 Rock the slide gently back and forth, observe for agglutination
macroscopicallywithin one minute.

STANDARD TUBE TEST

 Take 4 sets of 8 Kahn tubes/test tubes and label them 1 to 8 for O, H, AH and
BH antibody detection.
 Pipette into the tube No.1 of all sets 1.9 ml of isotonic saline.
 To each of the remaining tubes (2 to 8) add 1.0 ml of isotonic saline.
 To the tube No.1 tube in each row add 0.1 ml of the serum sample to be tested
andmix well.
 Transfer 1.0 ml of the diluted serum from tube no.1 to tube no.2 and mix well.
 Transfer 1.0 ml of the diluted sample from tube no.2 to tube no.3 and mix
well.Continue this serial dilution till tube no.7 in each set.
 Discard 1.0 ml of the diluted serum from tube No.7 of each set.
 Tube No.8 in all the sets, serves as a saline control. Now the dilution of the
serumsample achieved in each set is as follows: Tube No. : 1 2 3 4 5 6 7 8
(control) Dilutions 1:20 1:40 1:80 1:160 1:320 1:640 1:1280.
 To all the tubes (1 to 8) of each set add one drop of the respective
WIDALTEST antigen suspension (O, H, AH and BH) from the reagent vials
and mix well. Cover the tubes and incubate at 37° C overnight (approximately
18 hours).
 Dislodge the sedimented button gently and observe for agglutination.

INTERPRETATION OF RESULT
SLIDE TEST
Agglutination is a positive test result and if the positive reaction is observed with 20
µl of test sample, it indicates presence of clinically significant levels of the corresponding
antibody in the patient serum.
No agglutination is a negative test result and indicates absence of clinically
significant levels of the corresponding antibody in the patient serum.
 Fig.5 widal card test
TUBE TEST
 The titre of the patient serum using Widal test antigen suspensions is the
highestdilution of the serum sample that gives a visible agglutination.
 The sample which shows the titre of 1:100 or more for O agglutinations and 1:200
ormore for H agglutination should be considered as clinically significant (active
infection). Example: In the figure, titre is 160.
 Demonstration of 4-fold rise between the two is diagnostic.
 H agglutination is more reliable than O agglutinin.
 Agglutinin starts appearing in serum by the end of 1st week with sharp rise in 2nd
and3rd week and the titre remains steady till 4th week after which it declines.

Fig.6 widal tube test

CRP TEST
PRINCIPLE
The C-Reactive Protein test is based on the principle of the latex agglutination. When
latex particles complexed human anti-CRP are mixed with a patient‟s serum containing C
reactive proteins, an visible agglutination reaction will take place within 2 minutes. C-
Reactive Protein (CRP), also known as Pentraxin 1, is a non-glycosylated protein in the
Pentraxin family that also includes Pentraxin 2/SAP and Pentraxin 3/TSG-14. CRP is an
acute phase reactant, a protein made by the liver and released into the blood within a few
hours after tissue injury, the start of an infection, or other cause of inflammation.

A high level of CRP in the blood is a sign that there may be an inflammatory process
occurring in the body. Inflammation itself isn‟t typically a problem, but it can indicate a
host of other health concerns, including infection, arthritis, kidney failure, and pancreatitis.
High CRP levels may put patients at increased risk for coronary artery disease, which can
cause a heart attack.

PROCEDURE
QUALITATIVE TEST
 Bring all reagents and serum sample to Room Temperature and mix latex
reagent gently prior to use. Do not dilute the controls and serum.
 Place 1 drop of Serum, Positive control and Negative control on separate
reaction circle on glass slide.
 Then add 1 drop of CRP latex reagent to each of the circles.
 Mix with separate mixing sticks and spread the fluid over the entire area of the cell.
 Tilt the slide back and forth slowly for 2 minutes observing preferably under
artificial light.
 Observe for visible agglutination.

SEMI-QUANTITATIVE TEST
 Prepare dilution of the specimen with physiological saline 0.9% as indicated in
the table
Table.4

 Then proceed for each dilution as qualitative test.

INTERPRETATION OF RESULT

Fig.7 CRP test

Positive: Agglutination of latex particles, indicating the presence of C – reactive protein at a

significant and detectable level.

Negative: No agglutination for Semi-Quantitative Test Results, the last dilution of serum
with visible agglutination is the CRP titre of the serum.

ANTI STREPTOLYSIN

O TEST PRINCIPLE

The ASO titer tends to rise a week following infection and peaks at 3 to 5 weeks,
begins to fall at 8 weeks, and returns to pre-infection levels at around 8 months. Stronger
ASO responses tend to occur with throat infection than skin infection, possibly because
free cholesterol present in skin binds to streptolysin O, thereby decreasing its
immunogenicity. Qualitative and semi-quantitative determination of anti-streptolysin-O
antibodies (ASO) in serum can be performed using a rapid latex agglutination test. ASO
latex reagent is a stabilized buffered suspension of polystyrene latex particles that
have been coated with Streptolysin O. When the latex reagent is mixed with a serum
containing ASO, agglutination occurs.
PROCEDURE

 Bring all test reagents and samples to room temperature.

 Use a disposable pipette to draw up and place one free-falling drop of each
undilutedsample into its identified circle of the slide. Retain each pipette for mixing
in step 5.
 Deliver one free-falling drop of positive and negative control into its identified
circle.
 Mix the ASO latex reagent by gently shaking. Add one free-falling drop of a reagent
to each control and sample.
 Using the flattened end of the appropriate plastic pipette as a stirrer (step 2),
thoroughly mix each sample with reagent within the full area of the circle.
 Discard the disposable pipette.
 Slowly rock the slide for exactly two (2) minutes and observe for agglutination
undera high-intensity light.
 Record results.
 Re-wash glass slide for future use.

INTERPRETATION OF RESULT

A test sample is considered to contain ASO antibodies in excess of 200 IU/ml when
agglutination (clumping) is observed when compared to the result of the negative control.

Fig.8 ASO Test

ASO titer may be interpreted either by comparison of acute and convalescent-phase

titers or against reference upper limit of normal values. Rise in titer (≥ twofold) from the
acute phase to the convalescent phase is the best evidence of antecedent infection with group
A streptococci. In the area, where rheumatic fever or poststreptococcal glomerulonephritis is
endemic, a single initial titer more than (>) upper limit of normal value is diagnostic.

VDRL TEST

PRINCIPLE

VDRL stands for Venereal Disease Research Laboratory test. It is used for serological
diagnosis of syphilis and it is an example of Slide flocculation test. In this test cardiolipin
antigen is used as reagent to detect auto-antibody in serum of patients.

Cadiolipin antigen is an alcoholic extract of bovine heart muscle to which lecithin and
cholesterol are added. Cardiolipin reacts non-specifically to cardiolipin auto-antibodies (IgM
and IgG) to form flocculation (floccular agglutination).

Autoantibody in response to cardiolipin is produced within 2-3 weeks of treponemal

infection due to tissue damage. The stimulating antigen for auto-antibody production is
mitochondrial membrane. These autoantibodies can react with cardiolipin.

The autoantibodies are not only produced in case of syphilis but also in other
treponemal infection, occasionally during pregnancy, in acute malaria, leprosy, tuberculosis,
viral pneumonia, leptospirosis, cancer, infectious mononucleosis, and sometimes after certain
vaccination. So this VDRL test is non –specific test. For the test, at first a drop of antigen is
placed on a slide and then a drop of serum is added to it. The slide is rotated to mix the
content. In case of positive test, flocculation occurs.

PROCEDURE

 Take patient’s serum, heat is for 30 minutes at 56°C and then allow to cool to room
temperature
 Take a measured volume of diluted antigen suspension (a colloidal suspension of
tissue cardiolipid or chemically synthesized cardiolipin) and add to a measured
volume of serum in a glass slide.
 Rotate the slide for about 4 minutes,
 Examine the slide microscopically for clumping of Ag-Ab complex using 10X
objective and eye piece.
INTERPRETATION OF RESULT

Fig.9 VDRL Test

 Reactive (Positive test): If Ag-Ab clump of large or medium size then it

is reported as reactive (Positive VDRL test)
 Weakly reactive: If Ag-Ab complex is small sized, it is weakly reactive
 Non-reactive: If no Ag-Ab clump is formed, it is non-reactive
 All reactive and weakly reactive serum require serial dilution to
estimate antibody titer.
SUMMARY AND CONCLUSION
 3. SUMMARY AND CONCLUSION
Executive Summary Diagnostic tests are an essential component of the health care
system, providing vital information that impacts provider decisions regarding the
prevention, diagnosis, treatment, and management of disease. With advances in technology
over the last several decades, diagnostics have become even more integral to the practice of
medicine today, enabling, for example, more personalised and more preventive treatments.

While testing capabilities continue to evolve rapidly through new development of

new technologies, there are certain concept of measurement used to evaluate their
effectiveness that are fundamental and unchanging. Viewed narrowly, diagnostic tests are
those that aid in identifying illness in a person who present with symptoms by confirming or
ruling out the presence of a specific disease or infection. As our knowledge of human body
has grown, so too has the definition of “diagnostic tests”. This term is regularly used as a
reference to tests that contribute to molecular testing have opened up a new area of diagnostic
that enable providers to evaluate the likelihood that someone will develop an inheritable
disease potentially many years in the future. Understanding the foundation of Testing of all
Diagnostic Tests, regardless of what role they play in care or what technology is used to
perform them, require some minimum level of accuracy and precision, and sensitivity in
order to demonstrate they are effective.
BIBLIOGRAPHY
 4. BIBLIOGRAPHY

Adlard, K.(2008) Examining the push-pull method of blood sampling from centralvenous
access devices. Journal of pediatric Oncology Nursing.

Ahmed, S.V., Jayawarna, C. & Jude, E.(2006)Post lumber puncture headache:diagnosis

and management. Post graduate Medical Journal.

Chown B; Lewis M; Kaita K.(1957)..al, “a new kell blood – group phenotype”. Nature
180(4588):711.

Ellen Jo Baron et.al, Bailey and Scott, (1994). Diagnostic Microbiology, 9th Edition, Mosby.
Nickel RG; Willadsen SA; Freidhoff LR; et al.(August 1999). “Determination of Duffy
genotypes in three populations of African descent using PCR and sequence specific
oligonucleotides”. Human Immunology. 60(8):738 – 42.

Reynolds J, Moyes R.B. Breakwell DP, 2009. Differential staining of bacteria: acid fast stain.
Current protocols in Microbiology.
Appendix