Automation in biochemistry, Micro biology and Hematology of 21st century

INTERNSHIP REPORT
SUBMITTED
TO
Department of Medical Laboratory Technology
Loyola College (Autonomous), Chennai-600061
Submitted By
ATTULURI VAMSI KUMAR
(17-PML-014)
Guided By
DR. SHERAFIN JANCY VINCY

CERTIFICATE
This is to certify that the INTERNSHIP TRAINING PROGRAMME
work has been carried out by ATTULURI VAMSI KUMAR (17-
PML-014) of Ist
M.Sc. Medical Laboratory Technology, Loyola College
(Autonomous), Chennai, in partial fulfillment of the requirements for the degree
of MASTER OF SCIENCE in MEDICAL LABORATORY
TECHNOLOGY and it has not formed the basis for the award of any other
degree or diploma.
DR. SHERAFIN JANCY VINCY
(Guider and Supervisor, Coordinator of medical lab technology)

ACKNOWLEDGEMENT
This report documents the work done during the internship training program at
southern railway headquarters hospital, Perambur Chennai-23, India.
I wish to express my sincere gratitude to Dr. NIRMALA DEVI Managerial
director (M.D) of southern railway hospital, Perambur for giving me the
opportunity to do my internship at her highly esteemed laboratory. Also I would
like to thank DR. Jaishri. Head of lab department, who took special interests
and gave full support to learn the subject and skills.
I am grateful to DR. SHERAFIN JANCY VINCY (Coordinator-MLT) for
her valuable guidance, advice, suggestion and encouragement rendered to me at
every stage.
The guidance and support received from all the members who contributed to
this study was vital for the completion of this study. I am grateful to all of them
for their constant support. I have tried my best to keep this report simple yet
technically correct. I hope I succeed in my attempt.
ATTULURI VAMSI KUMAR

SOUTHERN RAILWAY HEADQUARTERS HOSPITAL
Also known as the Perambur railway hospital is a 500- bed hospital
of the Southern Railway located in Ayanavaram, Chennai. It is spread
across a land measuring 15 acres and was established during the
British rule. The hospital has specialized in 3 disciplines; the National
Board of Examination (NBE) has accredited the hospital for
recognition in postgraduate training. The hospital has also been
accredited by international institutions such as Royal college of
surgeons for imparting training in PG courses. It is also an approved
institution for training nurses. The hospital was established chiefly to
serve the employees of the railways. The hospital, however, caters to
general public as well at a low cost. The hospital is a national referral
centre for cardiology and cardiovascular surgery for the Indian
Railways
SOUTHERN RAILWAY HEADQUARTERS HOSPITAL

CONTENTS
1. PHLEBOTOMY / SAMPLE COLLECTION
2. HEMATOLOGY
3. BIOCHEMISTRY
4. MICROBIOLOGY
5. BLOOD BANK & SEROLOGY

PHLEBOTOMY
Introduction
The drawing of blood by venipuncture for transfusion, apheresis,
diagnostic testing, or experimental procedures.
A person who perform phlebotomy is called a “phlebotomist”.
Steps involved
o

 Assemble the needle, the barrel, and the first tube you wish to use as
in the figures above. The needle should not be uncovered until ready
to perform venipuncture. Place any additional tubes to be used in a
convenient location, keeping some spares handy. The gauze, alcohol
pads, and bandages should be ready. (Note: some phlebotomists may
elect to do this step before applying the tourniquet; this is preferable.)
BLOOD COLLECTION: ROUTINE VENIPUNCTURE AND SPECIMEN
HANDLING
Venipuncture procedure:

The venipuncture procedure requires both knowledge and skill to
perform. Each phlebotomist generally establishes a routine that is
comfortable for her or him. Several essential steps are required for
every successful collection procedure:
1. Identify the patient.
2. Assess the patient's physical disposition (i.e. diet, exercise, stress,
basal state).
3. Check the requisition form for requested tests, patient
information, and any special requirements.
4. Select a suitable site for venipuncture.
5. Prepare the equipment, the patient and the puncture site.
6. Perform the venipuncture.
7. Collect the sample in the appropriate container.
8. Recognize complications associated with the phlebotomy
procedure.
9. Assess the need for sample recollection and/or rejection.
10. Label the collection tubes at the bedside or drawing area.
11. Promptly send the specimens with the requisition to the
laboratory.
Equipment
The following are suggested supplies for blood specimen collection.
• Non sterile exam gloves
• Puncture resistant sharps container
• Alcohol wipes
• Tourniquet
• Appropriate specimen collection lab tubes
• 2 x 2 gauze
• Tape
• Evacuated tube holder
• Multi-sample blood collection needle
Order form / requisition:
A requisition form must accompany each sample submitted to the
laboratory. This

Requisition form must contain the proper information in order to
process the specimen.
The essential elements of the requisition form are:
• Patient's surname, first name, and middle initial.
• Patient's ID number.
• Patient's date of birth and sex.
• Requesting physician's complete name.
• Source of specimen. This information must be given when
requesting microbiology, cytology, fluid analysis, or other testing
where analysis and reporting is site specific.
• Date and time of collection.
• Initials of phlebotomist.
• Indicating the test(s) requested
LABELING THE SAMPLE:
• A properly labeled sample is essential so that the results of the test
match the patient. The key elements in labeling are:
o Patient's surname, first and middle.
o Patient's ID number.
o NOTE: Both of the above MUST match the same on the requisition
form.
o Date, time and initials of the phlebotomist must be on the label of
each tube.
What is a vacutainer:
A Vacutainer blood collection tube is a sterile glass or plastic test tube
with a colored rubber stopper creating a vacuum seal inside of the tube,
facilitating the drawing of a predetermined volume of liquid.
Order of draw
Blood collection tubes must be drawn in a specific order to avoid cross-
contamination of
Additives between tubes. The standard order of draw is:

 First - blood culture tube (yellow-black stopper)
 Second - non-additive tube (red stopper or SST)
 Third - coagulation tube (light blue stopper). If just a routine
coagulation assay is the only test ordered, then a single light blue
stopper tube may be drawn. If there is a concern regarding
contamination by tissue fluids or thromboplastins, then one may
draw a non-additive tube first, and then the light blue stopper tube.
 Last draw - additive tubes in this order: SST (red-gray, or gold,
stopper). Contains a gel separator and clot activator.
 Sodium heparin (dark green stopper)
 PST (light green stopper). Contains lithium heparin anticoagulant
and a gel separator.
 EDTA (lavender stopper)
 ACDA or ACDB (pale yellow stopper). Contains acid citrate dextrose.
 Oxalate/fluoride (light gray stopper)
NOTE: Tubes with additives must be thoroughly mixed. Erroneous test
results may be obtained when the blood is not thoroughly mixed with the
additive.
VENIPUNCTURE SITE SELECTION:
Although the larger and fuller median cubital and cephalic veins of the arm
are used most frequently, wrist and hand veins are also acceptable for
venipuncture.
Certain areas are to be avoided when choosing a site,
• Extensive scars from burns and surgery - it is difficult to puncture the
scar tissue and obtain a specimen.
• The upper extremity on the side of a previous mastectomy - test results
may be affected because of lymphedema.
• Hematoma - may cause erroneous test results. If another site is not
available, collect the specimen distal to the hematoma.
• Intravenous therapy (IV) / blood transfusions - fluid may dilute the
specimen, so collect from the opposite arm if possible. Otherwise,
satisfactory samples may be drawn below the IV by following these
procedures:
1. Turn off the IV for at least 2 minutes before venipuncture.

2. Apply the tourniquet below the IV site. Select a vein other than the one
with the IV.
3. Perform the venipuncture. Draw 5 ml of blood and discard before
drawing the specimen tubes for testing.
• Cannula/fistula/heparin lock - hospitals have special policies regarding
these devices. In general, blood should not be drawn from an arm with a
fistula or cannula without consulting the attending physician.
• Edematous extremities - tissue fluid accumulation alters test results.
PERFORMANCE OF A VENIPUNCTURE:
 Approach the patient in a friendly, calm manner. Provide for their
comfort as much as possible, and gain the patient's cooperation.
 Identify the patient correctly.
 Properly fill out appropriate requisition forms, indicating the test(s)
ordered.
 Verify the patient's condition. Fasting, dietary restrictions,
medications, timing, and medical treatment are all of concern and
should be noted on the lab requisition.
 Position the patient. The patient should sit in a chair, lie down, or sit
up in bed. Hyperextend the patient's arm.
 Apply the tourniquet 3-4 inches above the selected puncture site. Do
not place too tightly or leave on more than 2 minutes.
 The patient should make a fist without pumping the hand.
 Select the venipuncture site.
 Prepare the patient's arm using alcohol prep. Cleanse in a circular
fashion, beginning at the site and working outward. Allow to air dry.
 Grasp the patient's arm firmly using your thumb to draw the skin
taut and anchor the vein. The needle should form a 15 to 30 degree
angle with the surface of the arm. Swiftly insert the needle through
the skin and into the lumen of the vein. Avoid trauma and excessive
probing.

 When the last tube to be drawn is filling, remove the tourniquet.
 Remove the needle from the patient's arm using a swift
backward motion.
 Press down on the gauze once the needle is out of the arm,
applying adequate pressure to avoid formation of a hematoma.
 Dispose of contaminated materials/supplies in designated
containers.
 Mix and label all appropriate tubes at the patient bedside.
 Deliver specimens promptly to the laboratory.
TROUBLESHOOTING GUIDELINES
if an incomplete collection or no blood is obtained
 Change the position of the needle. Move it forward (it may not be
in the lumen)
 Or move it backward (it may have penetrated too far).

 Adjust the angle (the bevel may be against the vein wall).
 Loosen the tourniquet. It may be obstructing blood flow.
 Try another tube. There may be no vacuum in the one being used.
 Re-anchor the vein. Veins sometimes roll away from the point of the
needle and puncture site.
PERFORMANCE OF A FINGERSTICK
 Follow the procedure as outlined above for greeting and identifying
the patient. As always, properly fill out appropriate requisition forms,
indicating the test(s) ordered.
 Verify the patient's condition. Fasting, dietary restrictions,
medications, timing, and medical treatment are all of concern and
should be noted on the lab requisition.
 Position the patient. The patient should sit in a chair, lie down or sit
up in bed.
 Hyperextend the patient's arm.

 The best locations for finger sticks are the 3rd and 4th fingers of the
non-dominant hand. Do not use the tip of the finger or the center of
the finger. Avoid the side of the finger where there is less soft tissue,
where vessels and nerves are located, and where the bone is closer to
the surface. The 2nd (index) finger tends to have thicker, callused
skin. The fifth finger tends to have less soft tissue overlying the bone.
 Avoid puncturing a finger that is cold or cyanotic, swollen, scarred,
or covered with a rash.
 Using a sterile lancet, make a skin puncture just off the center of the
finger pad.
 The puncture should be made perpendicular to the ridges of the
fingerprint so that the drop of blood does not run down the ridges.
 Wipe away the first drop of blood, which tends to contain excess
tissue fluid.
 Collect drops of blood into the collection device by gently massaging
the finger.
 Avoid excessive pressure that may squeeze tissue fluid into the drop
of blood.
 Cap, rotate and invert the collection device to mix the blood collected.
 Have the patient hold a small gauze pad over the puncture site for a
couple of minutes to stop the bleeding.
 Dispose of contaminated materials/supplies in designated
containers.
 Label all appropriate tubes at the patient bedside.
 Deliver specimens promptly to the laboratory.

COMPLICATIONS FROM PHLEBOTOMYPOSSIBLE
COMPLICATIONS FROM PHL
Problems obtaining a specimen
 Blood Sample That Cannot Be Obtained
Probing is not recommended. Probing is painful to the patient. In most
cases another puncture in a site below the first site, or use of another vein
on the other arm, is advisable.
It is advisable not to attempt a venipuncture more than twice. Notify the
patient’s
Registered Nurse
Another person should attempt to draw the specimen
If another person is asked to draw a patient, the new person must re--
identify the patient.
 If an incomplete collection or no blood is obtained
 Change the position of the needle. Move it forward (it may not be in
the lumen) or move it backward (it may have penetrated too far).
 Adjust the angle (the bevel may be against the vein wall).
 Re-anchor the vein. Veins sometimes roll away from the point of the
needle and
puncture site.
If blood stops flowing into the syringe/tube
 The vein may have collapsed; resecure the tourniquet to increase
venous filling. If this is not successful, remove the needle, take care of
the puncture site, and redraw.
 The needle may have pulled out of the vein when switching tubes.
Hold equipment firmly and place fingers against patient's arm, using
the flange for leverage when withdrawing and inserting tubes.

PATIENT COMPLICATION
 Problems other than an incomplete collection
 Hematoma
A hematoma forms under the skin adjacent to the puncture site - release
the tourniquet immediately and withdraw the needle. Apply firm pressure.
To prevent a hematoma
• Puncture only the uppermost wall of the vein (just under the skin)
• Remove the tourniquet before removing the needle
• Use the major superficial veins (the large veins just under the skin)
• Make sure the needle fully penetrates the uppermost wall of the vein.
(partial
Puncture may allow blood to leak into the tissues just under the skin)
• Apply pressure to puncture site
 Petechiae
Little red spots, ranging in size from pinpoint to several millimeters in
diameter.
Petechiae consist of extravasated blood. This complication may be a result
of a
coagulation abnormality, such as a platelet defect and should be brought to
the attention
of the patient’s healthcare provider.
 Syncopy (fainting)
Patients may become dizzy and fain at the thought or sight blood, this is the
most
common complication phlebotomy. It is caused because of rapid fall in
blood pressure.
An automatic nervous system reaction, (psychosomatic trigger), usually
based on fear.
Treatment and safe handling of an unconscious patient is a necessity of any
qualified
Phlebotomist.
• Abort draw: Remove tourniquet, needle and bend arm
• Call for assistance

• Using good body mechanics, slide patient to floor, keeping hand firmly
behind the
cervical spine area. Protect head and neck from injury!
• Elevate feet above heart and monitor blood pressure, breathing, etc.
• Use ammonia only if patient is not responsive within 5 minutes and blood
pressure remains low.
• Assist to upright position in stages (monitor B/P with each change in
position)
This is a gradual process. If patient stands up to quickly, he will most likely
to
faint again due to drop in B/P.
 Scarred Vein
Areas that have been burned or scarred should be avoided during
phlebotomy. Burned
area is very sensitive and susceptible to infection, whereas veins under
scarred area are difficult to palpate.
: Samples & Collection
TYPES OF BLOOD SAMPLES
1. Whole Blood
A blood sample that is drawn and mixed immediately with an anticoagulant
to maintain the integrity of the blood cells and prevent clotting, allowing
whole blood analysis to be accurate. The blood remains in liquid state.
2. Serum
The liquid portion of whole blood that has been allowed to clot. The clotting
factors are bound in the clot. (Blood collected in a tube with no additive will
clot within 15-45 minutes. One 10 ml tube of whole blood will yield about
3-4 ml of serum. This is the only tube that should not be inverted).
3. Plasma
The liquid portion of blood that has not been allowed to clot. Usually,
formed when

freshly drawn blood is mixed with anticoagulants. The clotting factors are
present in the plasma. This sample is mixed 6-8 times and immediately
centrifuged and plasma removed.
COLLECTION TUBES
SST- Serum Separator Tube (two types): Gold/Red-Gray Marble
Draw a sufficient amount of whole blood into a plain, red-top tube or serum separator
(SST®) tube. If using an SST® tube, gently invert the tube several times to activate
clotting. Allow blood to clot at ambient temperature for 20-30 minutes. Centrifuge for
10 minutes to separate serum from clot and transfer the serum to a screw-capped,
plastic vial if required; this should be completed within 1 hour of obtaining the
specimen.
Additive : Polymer gel and powdered glass clot activator
Stopper Type : HemogardTM Gold top/Conventional Red-Gray Marble
Tube Type/Size : Plastic tube 13 x 100/16 x 100
Specimen Type : Serum
Draw Amount: 5.0 ml/8.5 ml
Inversions : 5
Laboratory Use : Sterile SST® brand tube for serum clot activator determinations
that require serum in chemistry or infectious disease testing. Gel separates
serum from cells. Tube inversion ensures mixing of clot activator with
blood and clotting within 30 minutes.
Mint Green Stopper- PST, Plasma Separator Tube: HOSPITAL PATIENTS ONLY
NOTE: After the tube has been filled with blood, immediately invert the tube several times
in order to prevent coagulation.
Green Stopper Tube (two types): Dark Green/Clear Green
Lavender Stopper Tube (two types): Dark Lavender/Clear Lavender
Additive : Lithium Heparin, Polymer gel plasma separator
Stopper Type : HemogardTM
Tube Type/Size : Plastic tube 13 x 75
Specimen Type : Whole Blood, Plasma
Draw Amount: 3.0 ml
Inversions : 8
Additive : Sodium Heparin

Draw Amount: 4.0 ml/2.0 ml (pediatric tube)
Inversions : 8
Laboratory Use : For plasma determinations in chemistry. Tube inversion prevents
clotting.
Dark green 4.0 ml Sodium Heparin used for chromosome studies.
Additive : EDTA-K2
Draw Amount: 4.0 ml/2.0 ml (pediatric tube)
Inversions : 8
Laboratory Use : Dark Lavender 4.0 ml for whole hematology, ammonia, lead, HIV,
RNA quantization determinations and for blood bank testing. Tube inversion
prevents clotting.
Light Blue Stopper Tube (two types): Solid Light Blue/Clear Light Blue
NOTE: It is imperative that the tube be completely filled. The ratio of blood to
anticoagulant is critical for valid results. Immediately after draw, invert the tube 6 to 10
times in order to activate the anticoagulant.
Gray Stopper Tube
Red Stopper Tube
Additive : Sodium Citrate (3.2%, 0.109M)
Tube Type/Size : Glass 13 x 75
Draw Amount: 2.7 ml/1.8 ml (pediatric tube only)
Inversions : 4 (gently)
Laboratory Use : For coagulation determinations of plasma specimens. Tube
inversion prevents clotting.
Note: Certain tests require chilled specimens. Follow recommended procedures
for collection and transporting of coagulation specimen.
Additive : Sodium fluoride/Potassium oxalate
Tube Type/Size : Plastic 13 x 75
Draw Amount: 4.0 ml

Inversions : 8
Laboratory Use : For glucose, toxicology determinations. Antiglycolytic additives
stabilize glucose values for up to 24 hours at room temperature. Tube
inversion ensures proper mixing of additive and blood.
Additive : Clot Activator (powdered glass)
Tube Type/Size : Plastic 13 x 75
Specimen Type : Serum
Draw Amount: 6.0 ml
Inversions : 5
Laboratory Use : For serum determinations in chemistry, serology and blood bank
testing. Can be used as sterile transport tube.
Royal Blue Stopper Tube : (two types)
No additive/EDTA
There are 2 types of royal blue top Monoject® tubes - one with EDTA anticoagulant and
the other plain. These are used in the collection of whole blood or serum for trace
metals analysis.
Yellow Stopper Tube: (two types)
Additive : None/EDTA
Draw Amount: 7.0 ml
Inversions : None
Laboratory Use : For trace element, toxicology and nutrition determinations.
Special stopper formulation offers the lowest verified levels of trace elements available.
Refer to specific test for proper tube.
Additive : ACD, solution A
Stopper Type : Conventional
Specimen Type : Whole blood
Draw Amount: 8.5 ml
Inversions : 8
Laboratory Use : Tissue typing and some Red Cross testing. Refer to specific test
for proper tube.
Additive : ACD, solution B
Stopper Type : Conventional
Specimen Type : Whole blood

Draw Amount: 6.0 ml
Inversions : 8
Laboratory Use : Tissue typing. Refer to specific test for proper tube.
BLOOD CULTURE
 purpose
The detection of septicemia
 preparation of the patient
1. Explain that the physician has ordered a series of test and you will have
to
perform several sticks.
2. Clean the skin first using alcohol (using concentric rings from the inside
out)
3. Follow this with an iodine swab using same technique
4. Allow iodine to dry before performing the venipuncture. Once the iodine
is dry,
do no palpate the vein again unless you have “sterilized” your own gloved
finger
as you did for site puncture.
 Procedure
Each laboratory uses its own particular blood culture system. The protocol
for the
collection of cultures also varies from hospital to hospital. The following
are certain
procedural steps that are common to all blood culture methods.
1. Paint the septum of the blood culture bottle(s) with iodine.
2. For the first culture, if possible, collect a specimen from each arm. The
amount of
blood to be drawn depends on the culture system used. (usually 5-10 ml)
Draw
the blood in sterile syringes only.
3. After completion of the draw, replace the needle used to make the
venipuncture
with a new sterile one. Inject the sample into the blood culture bottle and
quickly,

but gently mix to avoid clotting.
4. In subsequent cultures, one venipuncture will be enough; however, each
one
should be obtained from alternate arms.
5. After returning to the laboratory, you may be required to “vent” one of
the culture bottles if a two bottle system is used. Check with the lab
regarding the proper procedure to follow.
 VALUES: Normal blood cultures should be sterile. The growth of
microorganisms in the blood is a life threatening situation.
ORAL GLUCOSE TOLERANCE TEST (GTT)
 purpose
To confirm diabetes mellitus; to aid in diagnosis of malabsorption
syndrome and hypoglycemia.
 preparation of patient
1. The patient should not eat, drink coffee or alcohol, smoke or exercise
vigorously
for at least 10 hours prior to or during the testing.
2. If this testing is to be done on an outpatient basis, inform the patient of the
time
involved.
 procedure
1. Patient’s height and weight if obtained to determine amount of glucose
solution to give. Several calculators and methods of calculation are
available for this purpose.
2. Draw a fasting sample in a gray top tube. Also collect a fasting urine
specimen.
3. Give the patient the predetermined amount of glucose solution to drink.
Make
sure solution is chilled. NOTE THE TIME. (Patient must drink all the
solution in
a 5 minute time limit.)
4. Draw a specimen at 30 minutes, 1 hour, 2 hours, and 3 hours. Also collect
urine
samples at each blood collection. NOTE TIMES.

Special Considerations:
If the patient becomes nauseated or faint, note for vomiting and should this
occur within the first 30 minutes of test, discontinue and notify the
physician. Encourage patient to drink more water during the test to
promote adequate urine excretion.
This test is considered a timed test and therefore the physician can make
the most
accurate diagnosis if the testing is followed as closely as possible. If it is
impossible to collect any specimen at the appointment time, notify the lab.
Measure Bleeding Time
Supplies needed:
• Disposable exam gloves
• Alcohol or povidone-iodine sponges
• Surgicutt*, template, spring-loaded blade, or similar device.
• Tourniquet
• Blood pressure cuff
• Watch with second hand
• Filter paper
• 2 x 2 gauze pads
• Puncture-resistant sharps container
• Plastic bags for used supplies
• Steri-strips, butterfly bandage, or other bandage
Procedure Guidelines:
1. Perform your beginning procedure actions. Double-check the requisition
slip.
2. Support the patient’s arm on the bed or other surface, palm up. Make
sure the
patient is comfortable and can maintain this position for the duration of
the
procedure.
3. Apply the blood pressure cuff to the upper arm. Do not inflate it.
4. Apply gloves.

5. You will perform the test approximately 4 inches below the antecubital
space.
Cleanse the site with alcohol or povidone-iodine. Wipe in a circular
motion.
Begin in the center of the puncture site and extend the circle 3 inches in
diameter.
6. Allow the alcohol or other skin prep to dry.
7. Remove the Surgicutt*, template, or other product from the package.
Twist off the tab on the site, Taking care not to touch the blade or activate
the trigger.
8. Inflate the blood pressure cuff until the gauge reads 40mmHg. You must
stat the
test within 60 seconds of inflating the cuff.
9. Apply the Surgicutt or other device to the prepared skin, approximately 4
inches
below the antecubital space. Position the device so the blade is parallel to
the
bend in the elbow.
10. Depress the trigger while monitoring the second hand on your watch.
Remove the blade from the skin with one second of depressing the
trigger. Record the time.
Discard the device containing the blade in the puncture-resistant container.
11. Absorb the blood with the edge of the filter paper. Position the paper
near the
incision, without touching the wound directly. Placing the paper directly
on the
incision will interfere with the results of the test.
12. With the filter paper, blot the bleeding every 30 seconds. When he
blood no
longer stains the paper, stop timing. Discard the filter paper in the
plastic bag.
Record the time the test ended.
13. Deflate the blood pressure cuff.
14. Wipe remaining blood from the skin.
15. Apply a Steri-strip, butterfly bandage, or dressing to the incision.
16. Remove the gloves and discard in the plastic bag.
17. Remove the blood pressure cuff.

18. Perform your procedure completion actions.
Collecting a Blood Sample Using a Butterfly Needle Syringe
Supplies needed:
• 2 pairs of disposable exam gloves
• Face shield or goggles
• Surgical mask
• Fluid-resistant gown or lab coat
• Tourniquet
• 10 ml syringe
• Sterile 23 gauge butterfly needle
• Sterile 20 gauge needle
• Bandage tape to secure the butterfly in place
• Blood collection tubes
• Labels for collection tubes
• Permanent black pen for labeling tubes
• Laboratory requisition forms
• Puncture- resistant needle disposal container
• Plastic bag for used supplies
• Bandage
1. Perform your beginning procedure actions.
2. Check the requisition slip to determine what specimen to collect. Select
the proper tubes.
3. Assemble the needle and syringe. Uncoil the butterfly tubing. Move the
plunger back and forth to break the seal.
4. Apply a tourniquet and locate a vein. Select the largest, most stable vein
in the area. When palpated, the site should feel firm and rebound slightly.
5. Cleanse the site with alcohol. Wipe in a circular motion. Begin in the
centre if the
venipuncture site and extend the circle out 2 inches in diameter.

6. Allow the alcohol or other skin prep dry thoroughly.
7. Remove the needle cover, holding the needle by the wings, with the bevel
facing up, in your dominant hand.
8. Stabilize the vein by holding it with your non-dominant thumb,
approximately 1 inch below the puncture site.
9. Insert the needle into the patient’s vein. You will feel a change of
pressure when the needle enters the vein. Advance the entire length of the
needle.
10. Rest your dominant hand on the patient’s arm. Make sure that the
needle does not move. Blood should begin to flow into the attached tubing.
11. Gently tape the butterfly wings against the skin to hold the needle in
place.
12. Holding the syringe with the dominant hand, slowly pull back on the
plunger, filling the syringe with blood.
13. Release the tourniquet when the last drop of blood is obtained. Pull the
upper end of the tourniquet downward. Avoid pulling upward, as this may
cause the needle to come out of patient’s arm.
14. Place a 2x2 gauze pad 1 inch above the insertion site. Avoid using
cotton balls.
Cotton balls tend to stick to the insertion site and when removed, remove
the platelet plug, causing bleeding.
15. Quickly withdraw the needle. Immediately bring the gauze pad down
over the site, and apply pressure. Maintain pressure for 3 to 4 minutes, or
until bleeding stops.
16. Cover the puncture site with a bandage.
17. Remove the butterfly tubing from the syringe. Carefully discard them in
the puncture resistant container.
18. Open the package for the 20 gauge needle and attach the needle to the
syringe.
20. After leaving the room, apply full personal protective equipment.
21. Transfer the blood to the vacuum tubes in a rack by inserting the needle
through the rubber stopper, allowing the tube to fill. Allow the rack to
support the tube when the needle is inserted. Avoid holding it with your
hand.
22. Fill the tubes in order of the draw.
23. Gently invert the tubes several times to mix the samples. Avoid shaking.

24. Discard the needle and syringe in the puncture- resistant container.
25. Label the tubes according to the facility policy.
26. Transport the blood to the lab, following facility policy.
Drawing Blood Using a Lancet for Micro draw or Infant
Heel Stick
Supplies needed:
• Disposable exam gloves
• Lancets
• Microvette collection devices
• Labels for collection
• Permanent black pen for labeling tubes
• Laboratory requisition forms
• Puncture- resistant needle disposal container
• Plastic bag for used supplies
• Bandage or spot adhesive bandages.
1. Perform your beginning procedure actions. Check the requisition slip to
determine what specimen to collect. Select the proper tubes.
2. Identify the specimen collection site.
3. Cleanse the site with alcohol. Wipe in a circular motion. Begin in the
center of the puncture site and extend the circle out 2 inches in diameter.
4. Allow the alcohol or other skin prep to dry.
5. Hold the plastic end of the lancet in your dominant hand. With your non-
dominant hand, break the plastic cover off the end to expose the needle.
6. Hold the lancet at 45 degree angle. With the sharp of the lancet, pierce
the skin. For an adult fingerstick, make the stick perpendicular to the lines
in the fingerprints.
Follow the directions for the type of lancet you are using. If the lancet has a
plunger, depress it to pierce the skin while holding pressure on the site.
7. Remove the lancet. Discard it in the puncture-resistant sharps container.

8. Wipe the first drop of blood away with a sterile 2 x 2 sponge. You will
need the rest to fill the containers.
9. Hold the collection tube near the collection site. Position the tube almost
horizontally, with the end slightly down. Squeeze the skin slightly, allowing
blood to flow into the tube. Do not squeeze hard, as this forces tissue fluid
into the sample, diluting it. If blood does not flow freely, create suction by
placing your gloved finger over the end of the capillary tube, or by
squeezing the small bulb. Fill the tube approximately 2/3 to ¾ full. Usually,
two or three tubes are filled.
10. Apply a gentle pressure with the 2 x 2 gauze to the skin to prevent
painful bleeding inside the tissues. The patient can hold the gauze sponge
in place until the bleeding stops. Avoid using cotton balls. Cotton balls tend
to stick to the puncture site, and when removed, remove the platelet plug,
causing bleeding.
11. Wipe any remaining blood from the skin, and cover with an adhesive
bandage.
12. Label the sample while in the patient’s room.
HEMATOLOGY
INTRODUCTION
Hematology is the branch of medicine concerned with the study of the
cause, prognosis, treatment, and prevention of diseases related to blood. It
involves treating diseases that affect the production of blood and its
components, such as blood cells, hemoglobin, blood proteins, bone marrow,
platelets, blood vessels, spleen, and the mechanism of coagulation. Such
diseases might include hemophilia, blood clots, other bleeding disorders
and blood cancers such as leukemia, multiple myeloma, and lymphoma.
The laboratory work that goes into the study of blood is frequently
performed by a medical technologist or medical laboratory scientist. Many
hematologists work as hematologist-oncologists, also providing medical
treatment for all types of cancer.

SCOPE:
 Blood
 Venous blood
 Venipuncture
 Hematopoiesis
 Blood tests
 Cord blood
 Red blood cells
 Erythropoiesis
 Erythropoietin
 Iron metabolism
 Hemoglobin
 Glycolysis
 Pentose phosphate pathway
 White blood cells
 Platelets
 Reticuloendothelial system
 Bone marrow
 Spleen
 Liver
 Lymphatic system
 Blood transfusion
 Blood plasma
 Blood bank
 Blood donors
 Blood groups

 Hemostasis
 Coagulation
 Vitamin K
 Complement system
 Immunoglobulins
(Abnormality of the hemoglobin molecule or of the rate of hemoglobin synthesis)
 Anemias (lack of red blood cells or hemoglobin)
 Hematological malignancies
 Coagulopathies (disorders of bleeding and coagulation)
 Sickle Cell anemia
 Thalassemia
Laboratory assessments of blood formation and blood disorders :
 Full blood count - A count of the total number of red blood cells,
white blood cells and platelets present in blood.
 Blood film - Blood is smeared over a glass slide that is stained with
specific dyes and viewed under a microscope. The number, shape and
size of blood cells and the presence of any abnormal cells or
immature cells are noted. The stain used for reticulocytes or
immature red blood cells is Heilmeyer's reticulocyte stain. Staining
may flag up abnormally shaped red blood cells such as sickle cells or
spherocytes.
 Staining may also detect blood parasites such as malaria,
toxoplasmosis, and microfiliariasis.
 Assessment and staining of immature platelets or megakaryocytes
may also be performed.
 Assessment of granulocytosis.
 The erythrocyte sedimentation rate (ESR) may be tested.
 The bone marrow may be examined.
 Iron status and anemias are assessed using tests such as serum
ferritin, vitamin B12 and folate levels.

 The Coombs' test or antiglobulin test may be used for blood typing
and blood matching prior to blood transfusion, for example.
 Platelet function in bleeding and coagulation may be checked using a
test called prothrombin time.
 Diascopy is a technique used to determine whether a lesion is
vascular, nonvascular or hemorrhagic.
 D-dimer assessment may be performed to check for thrombotic
disorders.
 Electrophoresis may be used to examine proteins in the blood such as
hemoglobin and to check for hemoglobinopathies such as
thalassemia or sickle cell anemia.
 The enzyme G6PD may be assessed in sickle cell disease.
 Fine-needle aspiration of lymph nodes and tumors.
 Examination of spleen biopsy.
 Immunocytochemical techniques for detecting antigens both inside
and on the surface of cells.
 Assessment of storage diseases such as Gaucher's disease, Niemann-
Pick disease and glycogen storage disease.
 Assessment of chronic myeloproliferative disorders using bone
marrow and blood examination.
 Assessment of hemophagocytic syndromes.
 Karyotyping to look for chromosomal disorders and abnormalities
List of Hematology tests:
 Complete Blood cell count
 Total Erythrocyte count
 Total Leukocyte count
 Total Eosinophil count
 Total platelet count
 Differential Leukocyte count
 Total Reticulocyte count
 Erythrocyte Sedimentation Rate
 Hematocrit(Packed cell volume)
 Hemoglobin
 Red cell indices

 Sickle Cell anemia
COMPLETE BLOOD CELL COUNT
 WBC (white blood cell count) :
 These help to fight infections. If you have high WBC levels, it tells your
doctor you have inflammation or infection somewhere in your body. If
it’s low, you could be at risk for infection.
 The normal range is 4,500 to 10,000 cells per microliter (cells/mcL). (A
microliter is a very tiny amount – one millionth of a liter).
 RBC (red blood cell count) :
 This is the number of red blood cells you have. These are important
because they carry oxygen through your body. They also help
filter carbon dioxide. If your RBC count is too low, you may
have anemia or another condition. (If you have anemia, your blood has
fewer red blood cells than normal.) The normal range for men is 5
million to 6 million cells/mcL; for women it’s 4 million to 5 million
cells/mcL.
 HB or HBg(Hemoglobin) :
 This is the protein in your blood that holds the oxygen. The normal
range for men is 14 to 17 grams per deciliter (gm/dL); for women it’s
12 to 15 gm/dL.
 HEMATOCRIT
 How much of your blood is red blood cells. A low score on the range
scale may be a sign that you have too little iron, the mineral that helps
produce red blood cells. A high score could mean you’re dehydrated or
have another condition. The normal range for men is between 41% and
50%. For women the range is between 36% and 44.
 MCV(Mean corpuscular volume):

 This is the average size of your red blood cells. If they’re bigger than
normal, your MCV score goes up. That could indicate low vitamin
B12 or folate levels. If your red blood cells are smaller, you could have a
type of anemia. A normal-range MCV score is 80 to 95.
 PLATELETS :
 These play a role in clotting. This test measures the number of platelets
in your blood. The normal range is 140,000 to 450,000 cells/mcL
ERYTHROCYTE SEDIMENTATION RATE
An erythrocyte sedimentation rate (ESR) test is sometimes called a
sedimentation rate test or sed rate test. This test doesn’t diagnose one
specific condition. Instead, it helps your doctor determine whether you’re
experiencing inflammation.
The doctor will look at ESR results along with other information or test
results to help figure out a diagnosis. The tests ordered will depend on your
symptoms. This test can also monitor inflammatory diseases.
In this test, a tall, thin tube holds a sample of your blood. The speed at
which the red blood cells fall to the bottom of the tube is measured.
Inflammation can cause abnormal proteins to appear in your blood. These
proteins cause your red blood cells to clump together. This makes them fall
more quickly.
NORMAL RANGE:
ESR test results are measured in mm/hr, or millimeters per hour.
The following are considered normal ESR test results:
 Women under age 50 should have an ESR under 20 mm/hr.
 Men under age 50 should have an ESR under 15 mm/hr.
 Women over age 50 should have an ESR under 30 mm/hr.
 Men over age 50 should have an ESR under 20 mm/hr.

 Newborns should have an ESR under 2 mm/hr.
RED CELL INDICES
Estimation of red cell indices has great concern in interpreting clinical
abnormalities like classification of anemia. These have been the derived
parameters of Mean cell volume (MCV), Mean cell hemoglobin (MCH).
Mean cell hemoglobin concentration (MCHC). More recently red cell
distribution with (RDW) has also been included. These are the
quantitative measurement of the average size by volume (V),
hemoglobin content divided by weight (W) and hemoglobin
concentration (w/v) of the RBC’s.

SEROLOGY
INTRODUCTION:
 Serology is the scientific study of serum and other bodily fluids. In
practice, the term usually refers to the diagnostic identification
of antibodies in the serum. Such antibodies are typically formed in
response to an infection (against a given microorganism), against
other foreign proteins (in response, for example, to
a mismatched blood transfusion), or to one's own proteins (in
instances of autoimmune disease).
 Serological tests may be performed for diagnostic purposes when
an infection is suspected, in rheumatic illnesses, and in many other
situations, such as checking an individual's. Serology blood tests help
to diagnose patients with certain immune deficiencies associated
with the lack of antibodies, . In such cases, tests for antibodies will be
consistently negative.
 Serological methods are diagnostic methods that are used to identify
antibodies and antigens in patients sample which is serum and
plasma. There are some classical serological methods like
Agglutination and Precipitation that are used to identify infectious
diseases and human blood grouping typing.
 There are several serology techniques that can be used depending on
the antibodies being studied. These
include: ELISA, agglutination, precipitation, complement-fixation,
and fluorescent antibodies and more recently chemiluminescence.

 Some serological tests are not limited to blood serum, but can also be
performed on other bodily fluids such as semen and saliva, and
cerebrospinal fluid (CSF) which may contain antibodies.
 Serological tests may also be used in forensic serology, specifically for
a piece of evidence (e.g., linking a rapist to semen sample).
Why serological test needed?
To understand serologic tests and why they’re useful, it’s helpful to know a
little about the immune system and why we get sick.
Antigens are substances that provoke a response from the immune system.
They are most often too small to see with the naked eye. They can enter the
human body through the mouth, through broken skin, or through the nasal
passages. Antigens that commonly affect people include the following:
 bacteria
 fungi
 viruses
 parasites
The immune system defends against antigens by producing antibodies.
These antibodies are particles that attach to the antigens and deactivate
them. When your doctor tests your blood, they can identify the type of
antibodies and antigens that are in your blood sample and identify the type
of infection you have.
Sometimes the body mistakes its own healthy tissue for outside invaders
and produces unnecessary antibodies. This is known as an autoimmune
disorder. Serologic testing can detect these antibodies to help your doctor
diagnose an autoimmune disorder.

Types of Serologic Tests
Because antibodies are so diverse, various tests are useful for detecting the
presence of different types:
 An agglutination assay shows whether antibodies exposed to certain
antigens will cause particle clumping.
 A precipitation test shows whether the antigens are similar by
measuring for the presence of antibody in body fluids.
 The Western blot test identifies the presence of antimicrobial antibodies
in your blood by their reaction with target antigens.
What Do the Results Mean?
Normal Test Results
Your body produces antibodies in response to antigens. If testing shows no
antibodies, it indicates you don’t have a current or past infection. Results
that show there are no antibodies in the blood sample are normal.
Abnormal Test Results
Antibodies in the blood sample often mean you’ve had an immune system
response to a specific antigen from either a current or a past exposure to a
disease or foreign protein.
The testing may also diagnose an autoimmune disorder. In that case,
antibodies to normal or non-foreign proteins or antigens would be present
in the blood.
The presence of certain types of antibodies can also mean that you’re
immune to one or more antigen. This means that future exposure to the
antigen or antigens won’t result in illness.
Serologic testing can diagnose multiple illnesses, including:

 brucellosis, which is caused by bacteria
 amebiasis, which is caused by a parasite
 measles, which is caused by a virus
 rubella, which is caused by a virus
 HIV
 syphilis
 fungal infections
What Happens After Serologic Testing?
The care and treatment provided after serologic testing can vary. It often
depends on whether antibodies were found. It may also depend on the
nature of your immune response and its severity.
An antibiotic or another type of medication may help your body fight the
infection. Even if your results were normal, your doctor might order an
additional test if they still think you might have a specific type of infection.
The bacteria, virus, parasite, or fungus in your body will multiply over time.
In response, your immune system will produce more antibodies. This
makes them easier to detect as time goes on and the infection gets worse.
The tests results may also show the presence of antibodies related to
chronic conditions, such autoimmune disorders.

BIOCHEMISTRY
1. VITROS® 5600 INTEGRATED ® IMMUNODIAGNOSTIC AUTOMATIC SYSTEM.
VITROS® 5600 is an integrated system that brings together Centralize
sample loading and prioritize up to 50 samples to optimize workflow, with
a system that processes both chemistry and immunoassay samples
simultaneously and eliminate the need to manually move samples between
modules or to create aliquots. It is designed in such a way to remove the
obstacles and reducing turnaround time and increasing our ability to
provide meaningful results.
FEATURES
 Increase capacity with 150 reagent positions that allow over 100
assays to be on-board at once.
 Provide greater efficiency, lower maintenance and minimal
calibration by integrating our proven Micro Slide, Micro Tip, Micro
Well, Intel I check and Micro Sensor technologies
 Optimize turnaround time and productivity through innovative
Sample Centered processing
 Provide high reportable result efficiency that improves labor
effectiveness and cuts costs
 Verify system processing and result reporting while reducing errors
Assays
The VITROS®
5600 System broad menu of quality assays covers 90% of the
menu needs and 99% of the testing volume for typical labs,1
with over 120
state-of-the-art assays across a broad range of disease areas, including
1. Bio-Chemistry
 Sugar
 GTT
 LFT
 HbA!c
 Urea
 Creatinine
 Electrolytes
 Cholesterol
 Lipid profile

VITROS®
5600 System
2.COBAS e 411 ANALYZER
Cobas e 411 offers rapid STAT and turnaround time, an on-board
capacity of 18 tests and throughput of up to 88 tests per hour. Sample
carrier options include disc or Roche/Hitachi five-position rack For
immunological analysis of serum or plasma.
Principle
It works on ECL Electrochemiluminescence principle, “Electro” refers to
electrical stimulation. + “Chem” indicates a chemical reaction. +
“Luminescence”means “produces light.” = Electrochemiluminescence (ECL)
Cobas e 411 uses ECL which is a unique and highly sensitive luminescence
(light) detection system that amplifies the signal we want and reduces any
signals we don’t want to deliver unmatched low-end sensitivity and broad
dynamic measuring ranges.
The measuring cell uses an amplified signal to detect ultra-low
concentrations of analyte. By precisely controlling electricallyinitiated
reactions, ECL technology virtually eliminates unnecessary repeats and
reruns, providing accurate results the first time.
Features

 Easy to operate The customized keyboard and easy-to-learn software
make training and operation simple and keep user involvement to a
minimum.
 Unique programming-by-loading concept Barcode-based data entry
is carried out automatically by loading reagents, controls and
calibrators onto the system – a rapid, robust and safe procedure.
 STAT facilities for urgent samples cobas e 411 disk system features
two STAT sample positions that can be accessed at any time,
delivering results rapidly in response to clinicians‘ requests .
 The cobas e 411 rack system features a STAT port for immediate
access emergency testing.
 Innovative technology Novel Electrochemiluminescence (ECL)
technology provides superior analytical performance. Increased
sensitivity means that extremely low levels of antigen, as well as
subtle changes in levels, can be detected.
 The very wide measuring range facilitates cost and time efficient
testing by reducing the need to dilute and repeat samples
Assays

3. AU 480 Beckman Coulter
The AU480 is an automated chemistry analyzer that measures analytes in
samples, in combination with appropriate reagents, calibrators, quality
control (QC) material, and other accessories. The system is for in vitro
diagnostic use only. The AU480 performs automated analysis of serum,
urine, and other fluids.
Features
1.Improved convenience
 New and intuitive graphical user interface
 Embedded maintenance videos
 Universal applications
 Plug and play calibration via 2-D barcode
2. Efficiency
 Reagent reduction
 Low consumable usage
 Microsampling ideal for pediatrics
3.Continuous loading sample feeder
 Positive sample identification
 Beckman Coulter flexible racks to handle all commonly used tube
sizes in parallel
 Barcoded racks to define different sample materials
 80 samples on board at any time
 Automatic repeat and reflex capability
4. Flexible STAT rotor
 Dedicated STAT rotor with 22 positions and integrated barcode
reader
 Full STAT capabilities during routine operation
 Refrigerated area for calibrators and controls
 Combined routine and emergency work area
5. Large reagent compartment
 76 refrigerated reagent compartments
 Ready to use liquid stable reagents

 Automatic bottle switch capabilities
 Advanced calibration
 Liquid detection and remaining test number calculation
6. Cuvette wheel with low maintenance
 Non disposable quartz cuvettes
 Software driven carry over elimination
 Dry incubation bath
 Environmental
friendly
3. AU 480 Beckman Coulter
MICROBIOLOGY
Introduction
Microbiology section of the laboratory provides state-of-art services for
diagnosis of infectious diseases – bacterial, mycobacterium, and fungal,
parasitic and viral infections. Serological investigations to determine the
antibody/antigens of HIV, HBV, HCV, Dengue, Leptospira, and Cryptococcus
etc. are performed besides routine tests like Widal, RA, and ASO etc.
Processes conducted in laboratory.
1. Sample collection
2. Barcoded with patient details
3. Patient details along with type of sample are recorded in computer system.
4. Transported to allocated department
5. Confirmation/identification of sample with patient details in the computer

6. Analysis is done :- inoculation
-incubation
- Isolation
- Inspection
- Identification
7. Results obtained and recorded
HOW THE MICROBIOLOGY LAB DIAGNOSE THE INFECTIONS
Steps involved:-
(1): collection of the appropriate specimens.
For instance if an infection of the bladder is a possibility, the physician may order a
urinalysis that looks at the cell types present in the urine, such as red and white blood
cells, as well as a urine specimen for a culture to grow the organism that is causing the
infection.
Specimen may be collected from a number of different sources including blood, urine,
throat swabs. A good quality specimen means the best chance of diagnosing disease.
(2) Test methods conducted
Based on the physician requisition report the laboratory decides what technique is best
to identify the infectious disease and the organism likely causing the problem. All of the
techniques look for some product of an organism. The time it takes to identify the
organism is dependent on the technique used as well as how complicated the testing
process may be.
The knowledge and skills that are learnt in this particular department is many. But I
would like to reflect on the following things,
• Culture Media
• Culture Methods
• Identification of Bacteria
CULTURE MEDIA
Bacteria have to be grown (cultured) for them to be identified, as only rarely can they be
recognised by their morphology alone. The study of bacteria involves the study of
bacterial populations rather than of single bacterial cells. Bacteria occur as mixed
populations. By appropriate procedures they have to be grown separately (isolated) on
culture media and obtained as pure cultures for study.

Types of Culture Media:
Media are of different types on consistency and chemical composition.
A. On Consistency:
1. Solid Media. Advantages of solid media:
 Bacteria may be identified by studying the colony character;
 Mixed bacteria can be separated. Solid media is used for the isolation of bacteria
as pure culture. 'Agar' is most commonly used to prepare solid media. Agar is
polysaccharide extract obtained from seaweed. Agar is an ideal solidifying agent
as it is : (a) Bacteriologically inert, i.e. no influence on bacterial growth,
 It remains solid at 37°C
 It is transparent.
2. Liquid Media. It is used for profuse growth, e.g. blood culture in liquid media. Mixed
organisms cannot be separated.
B. On Chemical Composition:
1. Routine Laboratory Media
2. Synthetic Media. These are chemically defined media prepared from pure chemical
substances. It is used in research work.
Routine Laboratory Media:
These are classified into six types:
(1) Basal media,
(2) Enriched media,
(3) Selective media,
(4) Indicator media,
(5) Transport media,
(6) Storage media.
1. Basal Media.

Basal media are those that may be used for growth (culture) of bacteria that do not need
enrichment of the media. Examples: Nutrient broth, nutrient agar and peptone water.
Staphylococcus and Enterobacteriaceae grow in these media.
2. Enriched Media.
The media are enriched usually by adding blood, serum or egg. Examples: Enriched
media are blood agar and Lowenstein-Jensen media. Streptococci grow in blood agar
media.
3. Selective media.
These media favour the growth of a particular bacterium by inhibiting the growth of
undesired bacteria and allowing growth of desirable bacteria. Examples: MacConkey
agar, Lowenstein-Jensen media, tellurite media (Tellurite inhibits the growth of most of
the throat organisms except diphtheria bacilli). Antibiotic may be added to a medium
for inhibition.
4.Indicator (Differential) Media.
An indicator is included in the medium. A particular organism causes change in the
indicator, e.g. blood, neutral red, tellurite. Examples: Blood agar and MacConkey agar
are indicator media.
5.Transport Media.
These media are used when specimen cannot be cultured soon after collection.
Examples: Cary-Blair medium, Amies medium, Stuart medium.
6.Storage Media.
Media used for storing the bacteria for a long period of time. Examples: Egg saline
medium, chalk cooked meat broth.
Common Media In Routine Use:
Nutrient Broth.
500 g meat, e.g. ox heart is minced and mixed with 1 litre water. 10 g peptone and 5 g
sodium chloride are added, pH is adjusted to 7.3. Uses: (1) As a basal media for the
preparation of other media, (2) To study soluble products of bacteria.
Nutrient Agar.
It is solid at 37°C. 2.5% agar is added in nutrient broth. It is heated at 100°C to melt the
agar and then cooled.

Peptone Water.
Peptone 1% and sodium chloride 0.5%. It is used as base for sugar media and to test
indole formation.
Blood Agar.
Most commonly used medium. 5-10% defibrinated sheep or horse blood is added to
melted agar at 45-50°C. Blood acts as an enrichment material and also as an indicator.
Certain bacteria when grown in blood agar produce haemolysis around their colonies.
Certain bacteria produce no haemolysis.
Types of changes :
(a) beta (p) haemolysis. The colony is surrounded by a clear zone of complete
haemolysis, e.g. Streptococcus pyogenes is a beta haemolytic streptococci,
(b) Alpha (a) haemolysis. The colony is surrounded by a zone of greenish
discolouration due to formation of biliverdin, e.g. Viridans streptococci,
(c) Gamma (y) haemolysis, or, No haemolysis. There is no change in the medium
surrounding the colony.

Chocolate Agar or Heated Blood agar. Prepared by heating blood agar. It is used for
culture of pneumococcus, gonococcus, meningococcus and Haemophilus. Heating the
blood inactivates inhibitor of growths.
MacConkey Agar. Most commonly used for enterobacteriaceae. It contains agar,
peptone, sodium chloride, bile salt, lactose and neutral red. It is a selective and indicator
medium :
(1) Selective as bile salt does not inhibit the growth of enterobactericeae but inhibits
growth of many other bacteria.
(2) Indicator medium as the colonies of bacteria that ferment lactose take a pink colour
due to production of acid. Acid turns the indicator neutral red to pink. These bacteria

are called 'lactose fermenter', e.g. Escherichia coll. Colourless colony indicates that
lactose is not fermented, i.e. the bacterium is non-lactose fermenter, e.g. Salmonella.
Shigella, Vibrio.
Mueller Hinton Agar. Disc diffusion sensitivity tests for antimicrobial drugs should be
carried out on this media as per WHO recommendation to promote reproducibility and
comparability of results
EMB (Eosin-methylene blue) Agar. A selective and differential medium for enteric
Gram-negative rods. Lactose-fermenting colonies are coloured and nonlactose-
fermenting colonies are nonpigmented. Selects against gram positive bacteria

XLD (Xylose Lysine Deoxychoiate). It is used to isolate Salmonella and Shigella species
from stool specimens. This is a selective media.
SS (Salmonella-Shigella) Agar. It is a selective medium used to isolate Salmonella and
Shigella species. SS Agar with additional bile salt is used if Yersinia enterocolitica is
suspected

CULTURE METHODS
The culture methods used depend on the purpose for which they are intended. In the
clinical laboratory, the indications for culture are mainly to:
 isolate bacteria in pure culture;
 demonstrate their properties;
 obtain sufficient growth for preparation of antigens
 and for other tests;
 type isolates by methods such as bacteriophage and
 bacteriocin susceptibility;
 determine sensitivity to antibiotics;
 estimate viable counts; and
 maintain stock cultures.
The methods of culture ordinarily used in the laboratory are the
 streak,
 lawn, stroke,
 stab,
 pour plate
 liquid cultures.

Special methods are employed for culturing anaerobic bacteria.
IDENTIFICATION OF BACTERIA
Once a bacterium is obtained in pure culture, it must be identified. The following
characteristics are studied in the process.
Morphology:
The morphology of the bacterium depends on a number of factors such as
 the strain studied,
 nature of the culture medium,
 temperature
 time of incubation,
 age of the culture
 The number of subcultures it has undergone.
The characteristics noted are
 shape,

 size,
 arrangement,
 motility,
 flagella,
 spores
 capsules.
All these cannot be made out in a single medium.
Staining reactions:
The age of the culture is important. In older cultures, staining characteristics either vary
or are not brought out well. Simple stains bring out the morphology best. Differential
and special stains are necessary to make apparent characteristics like flagella, capsules,
spores and metachromatic granules. The Gram stain divides bacteria into the Gram
positive and the Gram negative;
The Ziehl-Nelsen stain divides bacteria into acid fast and non-acid fast. The fluorescent
antibody technique enables identification by surface antigens.
The study of morphology and staining characteristics helps in the preliminary
identification of the isolate.
Cultural characteristics:
These provide additional information for the identification of the bacterium. The
characteristics revealed in different types of media are noted. While studying colonies
on solid media, the following features are noted: (shape, size, elevation, margins, surface,
edges, colour, structure, consistency, emulsifiability, degree of growth, nature)
Metabolism:
The requirements of oxygen, need for carbon dioxide, capacity to form pigments and the
production of haemolysis help in classification.
Fermentation and other biochemical properties:
The more important and widely used tests are described below:
Sugar fermentation: This is tested in sugar media. Add production is shown by change in
the colour of the medium to pink or red, and the gas produced collects in Durham s tube.
Indole production: This is tested in a peptone water culture after 48 or 96 hours of
incubation at 37 C. This test demonstrates the production of indole from tryptophan.
Add 0.5 ml Kovac’s reagent and shake gently. Red colour indicates a positive reaction.
Kovac’s reagent consists of:
• Paradimethylaminobenzaldehyde 10 g

• Amyl or isoamyl alcohol 150 ml
• Concentrated HCL 50 ml
This is prepared in small quantities and stored in the refrigerator.
Methyl red test (MR): This test is used to detect the production of acid during the
fermentation of glucose and the maintenance of pH below 4.5 in an old culture. Five
drops of 0.04% solution of methyl red are added to the culture in glucose phosphate
medium which had been incubated at 30°C for five days, mixed well and read at once.
Red is positive while yellow signifies a negative test.
Voges-Proskauer test (VP): This test depends on the production of acetyl methyl
carbinol from pyruvic acid as an intermediate stage in its conversion to 2:3 butylene
glycol. In the presence of alkali and atmospheric oxygen, the small amount of acetyl
methyl carbinol present in the medium is oxidised to diacetyl
which reacts with the peptone of the broth to give a red colour.
The test is performed by adding 0.6 ml of a 5% solution of a-naphthol in ethanol and 0.2
ml of 40% KOH to 1 ml of a glucose phosphate medium culture of the organism
incubated at 30°C for five days or 37°C for 48 hours. In a positive reaction, a pink colour
appears in 2-5 minutes, deepening to magenta or crimson in half an hour. In a negative
reaction, it remains colourless for half an hour. Traces of pink colouration should be
ignored.

Citrate utilisation:
Koser s citrate medium uses citrate as the sole source of carbon. The ability to use this
substance is indicated by the production of turbidity in the medium.
Indole, MR, VP and citrate tests are very useful in the identification and
classification of enteric Gram negative bacteria. These tests are commonly
referred to by the sigla IMViC tests.

Nitrate reduction:
This is tested after growing the bacterium for five days at 37°C in a broth containing 1
per cent KNO3. The test reagent consists of a mixture of equal volumes of solutions of
sulphanilic acid and a-naphthylamine in 5N acetic acid mixed just before use. 0.1 ml of
the test reagent is added to the culture. A red colour developing within a few minutes
signifies a positive reaction, while absence of colour indicates a negative reaction. This
is a test for the presence of the enzyme nitrate reductase which reduces nitrate to
nitrite.
Urease test:
This test is done in Christensen s urease medium. Inoculate the slope heavily and
incubate at 37°C. Examine after four hours and after overnight incubation. The test
should not be considered negative till after four days of incubation. Urease positive
cultures produce a purple-pink colour. Urease producing bacteria reduce urea to
ammonia which is responsible for the colour.
Catalase production:

Place a loopful of H202 on colonies on nutrient agar. Prompt effervescence indicates
catalase production. Culture media containing blood are unsuitable for the test as blood
contains catalase.
Oxidase reaction:
This reaction is due to cytochrome oxidase which catalyses oxidation of reduced
cytochrome by oxygen. A 1.0-1.5% solution of tetramethyl p-phenylene diamine
hydrochloride is poured over the colonies. Oxidase-positive colonies become maroon,
purple and black in 30 sec to 1 minute. The test can also be done by Kovac’s method. A
strip of filter paper soaked in the oxidase placed in a petri dish and the colony to be
tested is smeared on the paper in a line about 5 mm long. In a reaction, the smeared
area turns dark in 10 seconds. The solution should be freshly prepared.
AUTOMATED MACHINES.
(1) Microbial detection system (microbial analyser)
-Blood culture
Blood kept in the microbial analyser for 7 days. If a positive micro-
organism are there it shows green. If negative it shows red.
To assign and place a vial to a station.

a) Take the new vial to be placed in the instrument and open the
instrument doors.
b) With the instruments barcode reader, scan the menu option vial
entry .be sure you hear a beep sound.
c) Scan the vials barcode label again listen for the beep sound
d) Find the station with green, red leds illuminated
e) Carefully insert the vial into the illuminated station.
f) Repeat the same process for each of the new vials.
To remove negative vials (batch)
a. Open the instrument doors
b. Scan the menu option remove negative
c. Locate all the stations with the flashing green leds
d. Remove these vials
e. Do not scan their vial labels
To remove positive vials
I. Open the instrument doors
II. Scan the menu option remove positive
III. Find the station with the flashing green, flashing red leds
IV. Remove the vials and scan the vials sequence number barcode
V. Be sure you hear a beep sound
VI. Repeat the same process to remove all positive vials
VII. The acknowledged alarm sound is heard after all positive vials are
removed.
After removal the blood is going for further test for detection of the present
microbe.
DISPOSAL OF BIOMEDICAL WASTE
All the bio-hazardous and non-bio hazardous materials and processed
sample are segregated and disposed in the respective colour coded bags as
per treatment of biomedical waste procedure in the department manual.
(1) White
Needles
Syringes with fixed needles
Scalpels

Blades
Slide
Any metal sharp
(2) Red
Vacutainers
Syringes without needls
Gloves
Tubings
Catheters
Urine bags
Microwells, agsay cups
Micro tips
Disposable sample containers
(3) Yellow
Human anatomical waste/pathological waste
Human tissue
Organs
Body parts
Fetus <7 months
Items contaminated with blood
Body fluids
Cotton swabs
Bags containing residual
Discarded blood
Blood components
Discarded linen
Mattresses
Beddings (contained with blood, body fluids)
Microbiology lab waste like culture plates
(4) Black
Mon-contaminated materials.

BLOOD BANK
INTRODUCTION
A place where blood is collected from donors, typed, separated into
components, stored, and prepared for transfusion to recipients. A blood
bank may be a separate free-standing facility or part of a larger laboratory
in a hospital.
Separation of blood:
Typically, each donated unit of blood (whole blood) is separated into
multiple components, such as red blood cells, plasma and platelets. Each
component is generally transfused to a different individual, each with
different needs.
An increasingly common blood bank procedure is apheresis, or the process
of removing a specific component of the blood, such as platelets, and
returning the remaining components, such as red blood cells and plasma, to
the donor. This process allows more of one particular part of the blood to
be collected than could be separated from a unit of whole blood. Apheresis
is also performed to collect plasma (the liquid part of the blood) and
granulocytes (white blood cells).
Who receives blood:
Accident victims, people undergoing surgery and patients receiving
treatment for leukemia, cancer or other diseases, such as sickle cell disease
and thalassemia, all utilize blood. Over 20 million units of blood
components are transfused every year in the US.
Giving blood to yourself:
Patients scheduled for surgery may be eligible to donate blood for
themselves, a process known as autologous blood donation. In the weeks
before non-emergency surgery, an autologous donor may be able to donate
blood that will be stored until the surgical procedure.
Typing and testing blood:
After blood is drawn, it is tested for the ABO blood group type and the Rh
type (positive or negative), as well as for any unexpected red blood cell

antibodies that may cause problems in the recipient. Screening tests are
also performed for evidence of donor infection with hepatitis viruses B and
C, human immunodeficiency viruses (HIV) 1 and 2, human T-lymphotropic
viruses (HTLV) I and II and syphilis.
Storage of blood:
Each unit of whole blood is normally separated into several components.
Red blood cells may be stored under refrigeration for a maximum of 42
days, or they may be frozen for up to 10 years. Red cells carry oxygen and
are used to treat anemia. Platelets are important in the control of bleeding
and are generally used in patients with leukemia and other forms of cancer.
Platelets are stored at room temperature and may be kept for a maximum
of five days.
Fresh frozen plasma, used to control bleeding due to low levels of some
clotting factors, is usually kept in the frozen state for up to one year.
Cryoprecipitated AHF, which contains only a few specific clotting factors, is
made from fresh frozen plasma and may be stored frozen for up to one year.
Granulocytes are sometimes used to fight infections, although their efficacy
is not well-established. They must be transfused within 24 hours of
donation.
Other blood products: Other products derived from blood include albumin,
immune globulin, specific immune globulins and clotting factor
concentrates. These blood products are commonly made by commercial
manufacturers.
American Association of Blood Banks (AABB): The AABB is a key
international association of blood banks, including hospital and community
blood centres, transfusion and transplantation services and individuals
involved in transfusion and transplantation medicine. The AABB
establishes the standards of care for patients and donors in all aspects of
blood banking; transfusion medicine; hematopoietic, cellular and gene
therapies; and tissue transplantation. More than 2000 community and
hospital blood banks, hospital transfusion services and laboratories and
over 8000 individuals from the US and 80 countries outside the US make up
the AABB

DONOR SELECTION
Purpose
To assess the suitability of blood donor
SCOPE AND APPLICATION:
This SOP describes the donor selection criteria for blood donation, one of
the most important steps in protecting the safety of the blood supply. The
process is intended to identify elements of the medical history and
behaviour or events that put a person at risk for transmissible disease or
personal medical risk, either temporarily or permanently.
RESPONSIBILITY:
A qualified Medical Officer must determine the eligibility of donors. Donor
selection is based on a medical history questionnaire and a limited physical
examination done on the day of donation including the results of pre-
donation screening tests.
MATERIAL REQUIRED:
Donor Questionnaire
Donor Card

PROCEDURE:
CRITERIA FOR SELECTION OF BLOOD DONORS
Accept only voluntary / replacement non-remunerated blood donors if
following criteria are fulfilled:
The interval between blood donations should not be less than three months.
The donor shall be in good health, mentally and physically fit and shall not
be a person having multiple sex partners or a drug-addict or a jail inmate.
The donors shall fulfil the following requirements, namely:-
Age: donor should be in age group of 18 to 60 years;
Weight: should not be less than 45 kg / 100 lbs.;
Temperature: below 99.5 0 F;
Pulse: 60 – 100 / min;
Blood pressure: Systolic pr. – 100 to180 mm Hg & Diastolic pr. – 60 to 100
mm Hg without medication.
Hemoglobin: should not be less than 75% (or in case of Male 12.5 g/dl, in
case of female 11.5g /dl)
Donor should be free from acute respiratory distress;
Donor should be free from any skin disease specially at the site of
phlebotomy;
Donor should be free from transfusion-transmitted diseases as far as
possible determined by history and examinations mentioned above.
There should not be any puncture site or scar mark on arms or forearms
indicative of professional donors or intravenous drug abusers.
Deferral of the donor for the period mentioned below:

CONDITIONS PERIOD OF DEFERMENT
(a) Abortion 6 months
(b) History of blood
transfusion
12 months
(c) Surgery 12 months
(d) Typhoid 12 months after recovery
(e) History of Malaria duly
treated
3 months endemic area
3 years non endemic area
(f) Tattoo 6 months
(g) Immunization (Cholera,
Typhoid,
Diphtheria, Tetanus)
15 days
(h Rabies vaccination 1 year after vaccination
i) Hepatitis in family or close
contact
12 months
(j) Immunoglobulin 12 months
(k) Tooth extraction 14 days
(l) Eczema After recovery
(m) After child birth 6 months
(n) Local infection After recovery
Deferral of the donor permanently suffering from the following diseases:
Cancer

Heart disease
Abnormal bleeding tendencies
Unexplained weight loss
Diabetes – controlled on insulin
Hepatitis B infection
Chronic nephritis
HIV/ AIDS
Liver disease
Tuberculosis
Polycythemia Vera
Asthma
Epilepsy
Leprosy
Schizophrenia
Endocrine disorders
Rheumatic fever
Hepatitis C infection
Private interview:
Detailed sexual history should be taken. Positive findings should be
recorded on confidential notebook.
Informed Consent:
Information should be given about following things:
Need for blood

Need for voluntary donation
Regarding transfusion-transmitted infections
Need for questionnaire & honest answers
Safety of blood donations
Processing & use of donated blood
Screening tests for donated blood
DOCUMENTATION
All details should be included in the donor questionnaire form.
BLOOD COLLECTION
Purpose
To assess the blood donor with help of blood donor selection criteria
SCOPE & APPLICATION:
This describes a procedure for blood collection from the donor, using an
aseptic method. Blood is collected in a sterile closed system bag with a single
venepuncture. A correct performance of venepuncture is essential for the
quality and safety of the blood donation. Successful venepuncture results not
only in safe collection of a full unit of blood suitable for separation of
components with good quality yields, but also contributes to the comfort and
satisfaction of the donors the donors thus encouraging re-attendance.
RESPONSIBILITY:
The Medical Technologist (Lab) or Nurse under supervision of trained
registered doctor is responsible for blood collection from the donor after

verifying the donor screening details, checking the unit number labels and
preparing the phlebotomy site.
Materials :
 Cotton/ Gauze swabs.
 Artery Forceps.
 Pilot Tubes:
 1 test tube with anticoagulant for serology ii.
 1 test tube without anticoagulant for TTI
 Tourniquet.
 Oxygen Cylinder with accessories.
 Rubber Gloves.
 First Aid Tray.
 Tubing Stripper.
 Electronic Tube Sealer.
 Needle Destroyer.
 Blood collecting Bags.
 Discard Jar with 10% Sodium Hypochlorite.
 Artery Forceps, Scissors.
 Tapes
 Blood Bag Mixer (Bio mixer)
 Comfortable donor couch or chair
 70% alcohol swab.
PROCEDURE:
Make the donor lie down with a pillow under the head or recline in a
comfortable donor chair. Loosen tight garments.
Identify the donor by name. Enter the bag and segment numbers on the donor
card/form.
Ask the donor if he/she is in a comfortable position. Give the donor a hand
roller/squeezer to hold.
Select appropriate blood bag for blood collection.

Clean the venepuncture site with 70% alcohol swab. Disinfect the skin a of
venepuncture site about 5 cm diameter from the centre to periphery in a
circular manner. Scrub the area for at least 30 sec or till froth forms. Do not
touch the area after cleaning is done. Repeat the whole process if the puncture
site is touched. Dispose the used swab unit into the waste basket. Dry the skin
area with unit the puncture is made.
Set the bio mixer for the required volume of blood to be collected and place
the bag on it.
Apply the tourniquet on donor arm.
Clamp the bleed line of the blood bag using plastic forceps to ensure that no
air enters the tubing or bag once the needle cover is removed.
Keep the bevel of the needle facing upward and the shaft at an angle of 150 to
the arm.
Once the needle is beneath the skin, release the clamp.
Insert the blood bag needle into the vein for about 1 to 1.5 cm by a bold single
prick to ensure smooth flow of blood and secure on the arm with adhesive
strips.
Advise the donor to gently squeeze the hand roller to improve blood flow.
If the venipuncture is unsuccessful do not make further attempt in the same
arm. Take the donor’s permission for a second attempt. Use a new bag.
Once blood enters the bag tubing, press the bio mixer ‘start’ switch to allow
the blood to flow into the bag. After the programmed volume of blood is
collected, the bio mixer automatically clamps the tubing.
Clamp the bloodline at 2 sites and cut in the middle. Collect blood in the pilot
tubes from the tubing so that blood flows directly into the tubes from the
donor arm.
Release the tourniquet and remove the needle gently from the donor’s vein
pressing the phlebotomy site. Fasten a cuff around the donor’s arm in a flexed
position. Seal the blood bag tubing with the tube sealer.

Burn the needle of the bag in the needle incinerator. Discard the tubing with
the burnt needle in a container of sodium hypochlorite solution.
Observe the donor is donor for 30 minutes after the blood collection is over.

POST DONATION CARE
Purpose
To provide utmost care to blood donor after donation
The donor needs to be observed after blood collection, in order to attend to
any adverse reactions in the immediate post-donation period. Time of
observation should be minimum 30 minutes.
RESPONSIBILITY:
Post donation care should be taken by nurse. In case of adverse reaction, it will
be managed by doctor.
MATERIAL REQUIRED:
Sterile Cotton.
Adhesive tape.
Leaflet for post donation instructions.
PROCEDURE:
To prevent adverse reactions like giddiness ask the donor not to get up from
the chair/cot for 5 minutes even if he feels perfectly all right.
Observe for another 10 minutes in the refreshment area whilst having light
refreshments.
Inspect the venipuncture site before the donor leaves the donor room. Apply
an adhesive tape only after oozing stops. If there is persistent oozing at the site

of venipuncture, apply pressure to stop oozing. Then apply adhesive at the site
of venipuncture. If oozing still purchase apply pressure with ice. If there is
hematoma apply ice gently over the area after 5 minutes. Inform the donor
about the expected change in skin colour. If still bleeds consult with doctor.
Instruct the donor to drink adequate fluid in the day and avoid strenuous
activities.
No smoking for thirty minutes.
No driving on that day.
DOCUMENTATION:
Give a leaflet of post donation instructions to the donor.
Record donor profile in the register.
Note any adverse reaction on the donor record.
If any problem occurs after returning home, inform the Blood Transfusion
Center. Give thanks to the donor.
Observe the donor is donor for 15 minutes
Drink more fluids than usual in the next four hours.
Remove bandage after few hours.
PREPARATION OF RED CELL SUSPENSION
Purpose
To prepare cell suspension for blood ABO blood grouping

SCOPE AND APPLICATION
This procedure applies to all testing that requires red cell suspension
preparation.
RESPONSIBILITY
It is the responsibility of Medical Technologist (Lab) in the red cell serology
laboratory performing a given test to prepare the appropriate red cell
suspension. Every morning, the technologist must prepare A, B& O red cell
suspension for the routine use. .
MATERIALS REQUIRED:
Calibrated Centrifuge
0.9% saline
Anti-coagulated blood sample of donor
Donor unit segment
Test tubes
Pasteur pipette
Disposal box
2 plastic beakers
Racks to hold sample tubes
Principle:
The ratio of serum to red cells may affect the sensitivity of agglutination tests.
A 5% red cell suspension is a common reagent in many serological procedures.

The suspension need not be exactly 5%; an approximation achieves the
appropriate serum-to-cell ratio for most test procedures.
Pooled Cell Suspension:
Label tubes with A, B & O groups.
Place 1 drop of red cells each from 3 of A group sample tubes into the A
labelled tube.
Place 1 drop of red cells each from 3 of B group sample tubes into the B
labelled tube.
Place 1 drop of red cells each from 3 of O group sample tubes into the O
labelled tube.
Fill the tube ¾ full with 0.9% saline to re-suspend the cells.
Centrifuge the tubes for at least 1 minute at 1000 rpm.
To make 5% red cell suspension, add 1 drop of RBC to 19 drops of saline.
Test the pooled cells prepared by adding the antisera (anti-A, B, AB & D) in use.
Limitations:
Haemolysis of red cells from improper washing may result in false results.
DOCUMENTATION:
Enter the results of donor unit numbers from which pooled cells are produced
in the donor register.
Record the results of testing with the anti-sera in use.
Enter the manufacturer’s name and batch number of the anti-sera.

ABO BLOOD GROUPING
Purpose
To determine the determine the correct ABO group of an individual and ensure
the reliability of the result
RESPONSIBILITY
It is the responsibility of Medical Technologist (Lab) in the serology area to
perform the ABO grouping of donors and patients. One Medical Technologist
(Lab) performs red cell testing and the other serum testing. If a discrepancy is
encountered in cell and serum grouping, all tests should be repeated by the
same Medical Technologist (Lab) using by new reagent. If the discrepancy
persists, the sample should be repeated collection for correct result. It is the
responsibility of all staff performing the ABO grouping to ensure that quality
controlled reagents and proper cell concentrations are used. It is the
responsibility of Medical Officer to sign the report; in case of emergency the
Medical Technologist (Lab) can sign the report.
MATERIALS REQUIRED:
Refrigerator to store samples and reagents at 2 – 8 0 C
General purpose centrifuge
Microscope
Blood samples of donor/ patient.
Anti-A, anti-B, anti-AB anti-sera
20% A, B & O pooled cell suspension
0.9 % saline

(* All reagents must be used in accordance with the manufacturer’s
instructions.)
Tiles
Plastic pipette
Disposal bucket with Na hypochlorite
Plastic beakers - 2
Principle:
ABO system is the only system in which there is a reciprocal relationship
between the antigens on the red cell and the naturally occurring antibodies in
the serum. Routine grouping of donor and patient must therefore include both
cell and serum tests, each serving as check on the other. The procedure is
based on the principle of agglutination of antigen positive red cells in the
presence of antibody directed towards the antigen.
Cell testing:
Separate the cell & serum from blood sample
Label the tiles with patient & donor number and test identification
Prepare 20 % cell suspension for red cells being tested
Add 2 drops of anti-A, anti-B and anti-AB reagent in the labelled areas
Add 1 drop of 20% cell suspension of the red cells to be tested
Gently mix the contents
Examine for agglutination within 2-4minutes
Interpret & record test results
Serum testing:

Add 2 drops of test serum in all labelled areas
Add 1 drop of 20% pooled A reagent cells to the area labelled Ac
Add 1 drop of 20% pooled B reagent cells to the area labelled Bc
Add 1 drop of 20% pooled O reagent cells to the area labelled Oc
Mix the contents gently and examine for agglutination within 2-4minutes
Interpret & record test results.
Results:
Depending on presence (+) or absence (-) of agglutination, the test is described
as positive or negative.
Observe anti AB as positive control and O cell as negative control.
Confirm the cell grouping results with those obtained in serum grouping and
vice versa.
Interpretation:
Agglutination of tested red cells and serum constitute positive test results.
Positive reactions characteristically show 3+ to 4+ agglutination.
Absence of agglutination represents negative test result.
The interpretation of serum and cell tests for ABO group is as follows:
REACTION FOR ABO GROUP SYSTEM (Forward and Reveres Group).
FORWARD GROUPING
(Cell Grouping)
REVERSE GROUPING (Serum
Grouping)
Interpret
at ion
(ABO

Groupin
g)
Anti-A
+
Test Cells
Anti-B
+
Test Cells
Anti-AB
+
Test Cells
*(P.C)
Test
Serum
+
A-Cells
Test
Serum
+
B-Cells
Test Serum
+
O-Cells
*(N.C)
+++ --- ++++ --- +++ ---- A
--- +++ ++++ +++ --- ---- B
--- --- --- +++ +++ ---- O
+++ +++ ++++ --- --- ---- AB
Note:- P.C= Positive Control.
N.C= Negative Control.
Any discrepancy between cell and serum typing tests should be resolved
before an interpretation is recorded for the donor/ patient.
DOCUMENTATION:
Enter the results of donor /patient grouping in the grouping register and also
record the following details-
Date on which the test is run.
Name of the reagents used.
Lot number of the reagents
Initials of the technologist who performed the test.
Initials of the Supervisor who verified the result

Rh D TYPING
Purpose
To determine the correct RhD typing of an individual and ensure the
reliability of the result
This Standard Operating Procedure provides the method to be followed to
determine the Rh D type of an individual and ensure the reliability of the result.
This procedure describes the method for detection of Rh D antigen on the red
cells.
RESPONSIBILITY:
It is the responsibility of Medical Technologist (Lab) in the serology area to
perform the Rh D
typing of an individual using one monoclonal & one blended reagent. If a
discrepancy is encountered between the two batches of anti-D, the test should
be repeated and confirmed by Indirect Coombs’ Test. It is the responsibility of
all staff performing the Rh D typing to ensure that quality controlled reagents
and proper cell concentration are used.
MATERIAL REQUIRED:
Refrigerator to store samples and reagents at 2- 80 C
Microscope
Blood samples of donors/ patients
Anti-D Blended

Anti-D Monoclonal
Rh control reagents (positive & negative)
0.9 % saline
Test tubes
Plastic pipette
Glass slides
Disposal box
Glass beakers - 2
Racks to hold test
Principle:
Testing with anti-D is necessary to determine if red blood cells possess or lack
Rh D blood group antigen. Presence of agglutination is a positive test result,
which indicates the presence of the D antigen on the red blood cells.
D Typing:
Label the tiles with patient & donor number and test identification
Prepare 20% cell suspension for cells being tested.
Add 2 drops of anti-D reagents (Monoclonal & Blended) in D1, D2 marked.
Add 2 drops of positive & negative control reagents in the properly labelled
areas
Then add 1 drop of 20% patient cell suspension to reagent & control areas
Mix gently & wait for 2-4 minutes.
Read, interpret and record the test and control results immediately.

Results
Depending on presence (+) or absence (-) of agglutination, the test is described
as a positive or negative.
Interpretation:
Agglutination in D1 & D2 indicates that the red cell under investigation is D
positive.
Absence of agglutination in D1 & D2 indicates D negative at this point. All
negative results must be verified under microscope.
In case of any discrepancy, the result is to be confirmed by ICT method.
The interpretation of Rh D type is as follows:
Rh-D tying Result Remar
k
s
D1 + Pt`s
Cells
D2 + Pt`s
Cells
Positive
Control
Negative
Control
+++ +++ ++++ ---- D Positive
-- --- ++++ ---- D Negative
+++ --- ++++ --- Confirm by ICT
DOCUMENTATION:
Enter the results of donor /patient grouping in the grouping register and also
record the following details-

Date on which the test is run
Name of the reagents used
Lot number of the reagents
Initials of the technologist who performed the test.
Initials of the Supervisor who verified the result
ANTIBODY SCREENING
This procedure applies to all testing that requires antibody screening, including
donor units and prenatal specimens.
MATERIALS REQUIRED:
Equipment:
Refrigerator to store samples & reagents at 2-8 0 C.
Deep freezer to store enzyme, albumin & AHG reagent.
Table top centrifuge.
Microscope.
Water bath.
Reagents:
Group O pooled cells
Enzyme

22% Bovine albumin
Antihuman globulin reagent
IgG sensitized control cells
0.9% saline
Distilled water
Clotted blood sample of donor
Test tubes
Pasteur pipette
Glass slide
Disposal box.
2 plastic beakers
Racks to hold sample tubes.
PROCEDURE:
Principle:
The antibody screen test is used to detect unexpected antibodies. In this test,
pooled O cells are combined with serum under investigation. The addition
enhancer medium enzyme/ albumin helps to promote the interaction of red
cells and antibodies allowing antigen-antibody reactions to occur.
Antibody screen:
Label tubes with donor/ patient and test identification. Test tube (number)
Add 2 drops of serum to each tube.
Add 1 drop of enzyme to the tube labelled ‘enzyme’.

To each of the tube labelled ‘saline’, ‘enzyme’ and ‘ICT’, add 1 drop of 5%
pooled O red cell suspension.
Incubate the tube labelled ‘saline’ for 1hr. at room temperature.
Place the tubes labelled ‘enzyme’, ‘albumin’ and ‘ICT’ into the incubator at 370
C for 1hr.
After 1 hr, examine the tube labelled ‘saline’ for agglutination.
Remove the tubes labelled ‘enzyme’, ‘albumin’ and ‘ICT’ from the incubator
and add 1 drop of albumin to the tube labelled ‘albumin’.
Again place tubes labelled ‘albumin’ and ‘ICT’ to the incubator for ½ hr. more.
After ½ hr. incubation, tubes are from the incubator and tube labelled
‘albumin’ are examined for agglutination.
Positive (IGg serum) control and Negative (AB donor serum) control
Add pooled cell
Results:
Gently re-suspend the red cell button and examine for agglutination.
Examine all negative results microscopically.
Proceed to perform indirect anti-globulin test on tube labelled ‘ICT’.
Wash the cells 3 times with saline. Decant completely after last wash.
Add 2 drops of AHG reagent to the cell button.
After 6-8 minutes, read and record the results.
Add 1 drop of IgG sensitized cells to all negative results. This shows a positive
result.

Interpretation:
Haemolysis or agglutination in any test may indicate the presence of an
unexpected antibody.
The absence of agglutination in all tests is a negative test
After addition of IgG sensitized cells to a negative test, the presence of
agglutination indicates that the AHG added was capable of reacting and that
the negative result is valid.
DETECTION OF INCOMPATIBILTY BETWEEN PATIENT AND DONOR
Purpose
To detect the ABO incompatibility before transfusion of blood
This procedure is applied for compatibility testing of all patients requiring
transfusion.
MATERIAL REQUIRED:
Equipment:
Refrigerator to store samples at 2-80 C
Microscope
Clotted blood sample of donors/patients
Blood sample with anticoagulant of donor/patients

0.9% saline
Tiles
Plastic pipette
Glass slides
Disposal bucket with Na hypochlorite
2 plastic beakers
Racks to hold donor and patient sample tubes
Principle:
The cross-match is used to detect unexpected blood group antibodies in
patient’s serum against antigens on donor cells and to detect antibodies in
donor serum against antigens on patient’s cells which are complete antibody
active at room temperature and cause direct agglutination. Positive reaction in
any test indicates incompatibility.
Cross-match:
Label areas with donor/ patient and test identification
Prepare 20% cell suspension from donor and patient red cells
Add 2 drops of patient serum to area of major cross matching
Add 2 drops of donor serum to area of minor cross matching
Add 1 drop of 20% donor red cell suspension in area of major cross matching
Add 1 drop of 20% patient’s red cell suspension in area of minor cross
matching
Lightly mix the contents and examine for agglutination.
Read, interpret and record the results

Interpretation:
Hemolysis or agglutination in any test indicates incompatibility.
Absence of hemolysis/ agglutination in all tests indicates compatibility.
Examine all visually negative reactions under microscope.
ANTIGLOBULIN CROSS-MATCH
Purpose
To detect incompatibility between donor and patient at 370C
This procedure applies to compatibility testing of all Multi-transfused patients
and transfusion recipients who currently demonstrative or have a history of
clinically significant antibodies.
RESPONSIBILITY:
It is the responsibility of the Medical Technologist (Lab) in the cross match
facility of the red cell serology laboratory to perform the anti-globulin cross
match using quality controlled reagents and proper dell concentrations. One
MT performs the tests and another checks it. If any unexpected blood group
antibody is detected, inform the staff of Advanced Red Cell Serology to carry
out further investigations.

MATERIAL REQUIRED:
Equipment:
Refrigerator to store samples & reagents at 2-60C.
Table top centrifuge.
Microscope.
Clotted blood sample of patient.
Segment from donor unit.
Donor red cells suspended in saline.
Antihuman globulin reagent (anti-lgG+anti-C3d).
lgG sensitised control cells.
0.9% Saline.
Distilled water.
Serum tubes.
Pasteur pipettes.
Glass slides.
Rubber teats.
Disposal box.
2 plastic beakers.
Aluminium racks to hold test tubes.

PROCEDURE:
Principle:
The cross match through the anti-globulin phase permits detection of clinically
significant incompatibilities caused by incomplete antibodies that sensitized
cells at 370C, but do not directly cause agglutination.
Cross-match:
Label tube with patient/ unit and test identification.
Add two drops of patient serum to each tube.
Prepare a 5% cell suspension in saline from each donor unit segment.
Add 1 drop of donor’s 5% red cell suspension to the tube.
Incubate tubes at 370C for minimum 15 minutes. (Follow manufacturer’s
directions when using commercial reagents).
Wash the cells a minimum of 3 times with saline. Decant completely after last
wash (washing can be done manually or in automated cell washer).
Add two drops of antihuman globulin reagent to the dry cell button. viii. Mix
well and centrifuge at 1000 rpm for 1 minute.
Re-suspend and read for agglutination. Grade and record test results
immediately.
To all negative anti-globulin tests add 1 drop of lgG-sensitized control cells.
Centrifuge, resuspend and read for agglutination. Grade and record test results.
After the addition
of lgG-sensitised control cells to a negative test, the presence of agglutination
indicates that the AHG serum added was capable of reacting and that the
negative anti-globulin test is valid.

Interpretation:
Hemolysis or agglutination indicates the presence of a serologically
incompatible cross-match. This result is interpreted as incompatible.
Absence of agglutination and hemolysis is a negative test result and indicates a
serologically compatible cross-match. This result is interpreted as Compatible.
DOCUMENTATION:
Enter all results on the transfusion record card and OT/Ward transfusion
register. Enter only the results of compatible units in the blood compatibility
form. The Medical Technologist (Lab) who performed the test and the one who
checked the results sign all records.

RED CELL CONCENTRATE PREPARATION
Purpose
To prepare blood component from whole blood through blood donation
SCOPE AND APPLICATION
For judicious use of blood it is necessary to use the components as per the
need than use of whole blood. From the whole blood Red cell concentrate
(RCC), FFP and Platelet Concentrate (PC) are separated.
RESPONSIBILITY
It is the responsibility of Medical Technologist (Lab) to separate component
from the whole blood collected in multiple bags. The Medical Technologist (Lab)
will work under the direct supervision of blood transfusion expert.
EQUIPMENT AND MATERIALS REQUIRED:
Freshly collected whole blood
Refrigerated centrifuge machine
Metal clips and tube sealer
Plasma extractor
Double pan weighing balance
Double bags / triple bags
Manuals of all equipment for reference regarding use and maintenance of each
equipment.

PROCEDURE:
Principle:
Red Cell Concentrate (RCC) is obtained by removal of supernatant plasma from
centrifuged whole blood. The volume of plasma removed determines the
hematocrit of the component. A hematocrit of 80% or lowers ensures the
presence of adequate glucose for red cell metabolism for specified days of
storage.
Preparation of packed red cells:
Process the blood collected within 6 hours.
Keep the bags vertical for 10–15 minutes.
Note the weight of the primary bags and record in the register.
Balance the bags in the buckets using dry rubber or unused bags.
Keep equally balanced bucket diagonally opposite each other in refrigerated
centrifuge.
Position the bags in buckets parallel to the direction of the spin.
Centrifuge the bags at 1500 rpm for 10 minutes at 1 to 60 C unless also
preparing platelets. If platelet concentrate is to be prepared then set the
temperature of refrigerated centrifuge machine at 20 to 24 0C (Standardize the
speed of the centrifuge according to the machine used).
Break the seal of the tubing connecting to the satellite bag and express the
Platelet Rich Plasma (PRP) into the satellite bag leaving 50-60 mL plasma along
with red cells.
Mix the contents thoroughly and seal the tubing and detach the bags.
Keep the primary bag containing packed cells in quarantine storage in blood
bank refrigerator kept in the component room.

Automation in biochemistry, Micro biology and Hematology of 21st century

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