PRESENTED BY,

N. SWETHA
M.SC MEDICAL BIOCHEMISTRY
 BLOOD COLLECTION
Most of the investigations in a biochemistry
laboratory are from blood specimens
Sites : veins, arteries or capillaries
Specimen of choice : venous blood
Method : venipuncture
VENIPUNCTURE
PRELIMINARY STAGE :

• Confirm the identity of the patient

• Verify the tests requested
• Make sure that the patient is fasting, if required for
the test
• Estimate the volume of blood to be drawn
• Select the appropriate type of tube for blood, plasma
or serum required
SITES FOR VENIPUNCTURE :
Common site : veins in the antecubital fossa
Veins :
Median cubital vein
Cephalic vein
Basilic vein
Other sites : veins in the wrist or ankle
PROCEDURE :
Clean the skin with 70% isopropanol and allow to dry
Tourniquet is fastened tight enough to the patient's arm
to restrict the venous flow
Ask the patient to make fist to make the veins palpable
and prominent
A sterile disposable needle with syringe should be
inserted into the vein (150 angle to the arm )
Once blood appears, the tourniquet is released
When desired amount of blood has been drawn, a small
cotton pad soaked with spirit is placed on the puncture
site and the needle is withdrawn
Transfer the blood to an appropriate container
EVACUATED TUBE SYSTEM
• Preferred method
• Made of borosilicate glass or plastic tubes
• Evacuated blood tubes are :
More convenient and easier to handle
Minimizes the risk of specimen contamination
• Vacuum in the evacuated tubes are lost over time
BASIC COMPONENTS :

• Multisample needle : Allows collection of multiple

tubes during venipuncture
• Tube holder : Plastic cylinder with a small opening
for needle at one end and tube at the
other end
• Evacuated tubes : Contain premeasured vacuum that
automatically draws volume of blood
needed
COLLECTION WITH EVACUATED BLOOD TUBES

Skin is cleaned
Needle is screwed to the collection tube holder and
the tube is gently inserted into the holder
Needle should be guided gently into the patient's vein
Once the needle is in place, the tube should be
pressed forward into the holder to puncture the
stopper and release the vacuum
When blood begins to flow into the tube, the
tourniquet is released
Contd…

Tube is filled until the vacuum is exhausted

Once the tube is completely filled, it is withdrawn

from the holder and replaced by another tube, if
necessary

When blood collection is complete, the needle is

withdrawn and small cotton pad soaked with spirit is
placed on the puncture site
SKIN PUNCTURE

An open collection technique in which the skin is

punctured by a lancet and a small volume of blood
collected into a micro device

Skin puncture is the method of choice in :

Pediatric patients
Where sample volume is limited
Patients with extreme obesity and severe burns
SITES FOR SKIN PUNCTURE :

• Infants : Lateral or medial plantar surface of

heals
• Older children : Plantar surface of big toe
Ear lobe
• Adults : Palmar surface of the last digit of
2nd, 3rd or 4th finger
 PROCEDURE :
The skin is cleaned as for a venipuncture
When the skin is dry it is punctured by a sharp sterile
lancet
Depth of incision : < 2.5 mm
The finger is held in such a way that gravity assist the
collection of blood on the finger tip
Massage of the finger to stimulate blood flow should be
avoided
The first drop of blood is wiped off
Subsequent drops of blood are transferred to the
appropriate collection tube held at slight angle to the
skin
ARTERIAL PUNCTURE
Frequently used for blood gas analysis
Method of choice in :
Critically ill patients of respiratory diseases
Cardiovascular disease
Patients undergone major surgery

SITES FOR ARTERIAL PUNCTURE :

• Radial artery
• Brachial artery
• Femoral artery
PROCEDURE :
Insert the needle into the artery
Heparinized syringe should be used to collect the
blood
Expel any air bubble from the syringe if any and the
syringe should be sealed quickly
Blood and anticoagulant should be mixed well by
gentle inversion of the syringe
Syringe containing blood should be kept on ice, to
slow down red cell metabolism and to reduce
glycolysis
 ANTICOAGULANTS
STOPPER COLOUR ANTICOAGULANT ACTION

GREEN HEPARIN PREVENTS THE

FORMATION OF FIBRIN

LAVENDER EDTA CALCIUM CHELATING

AGENT

BLUE SODIUM CITRATE CALCIUM CHELATING

AGENT

GREY OXALATE CALCIUM CHELATING

AGENT

GREY SODIUM FLUORIDE PREVENTS GLYCOLYSIS

RED NO ADDITIVE
 URINE COLLECTION
The type of urine specimen to be collected depends on the
test to be performed
Three methods of collection of urine :
Random collection – Urine collected at any time of the day
Early morning specimen is preferred
Timed collection – Obtained at specific time of the day
During GTT for assessment of
glucose excretion
24 hr collection – Required only when it is necessary
to know the entire day's volume of
Urine output
PRESERVATION OF URINE SPECIMENS :

 Preservation – Essential to maintain integrity

Inhibit bacterial growth
 Best method of preservation – Immediate refrigeration
 Preservatives :
Toluene
Thymol
Chloroform
Formalin
Hcl
Acetic acid
CEREBROSPINAL FLUID
CSF is present within the subarachnoid space
surrounding the brain and spinal cord
Most frequently tested body cavity fluid
Obtained by lumbar puncture
( L3 – L4 inter lumbar space )
Main function – protects the brain and spinal cord
from injury
 CLINICAL APPLICATION OF CSF EXAMINATION :

 In diagnosis of :

Neurological disorders
Cerebrovascular accident
Meningitis
Encephalitis
Lymphoma
Subarachnoid hemorrhage
INTRODUCTION
Three phases of laboratory testing :

Pre-analytical – Specimen collection, transport and

processing
Analytical – Testing
Post-analytical – Result transmission, interpretation
and follow-up
PREANALYTICAL ERRORS
DEFINITION :

Factors that affect the quality of a specimen before

the tests are performed

Occur outside the laboratory

PATIENT IDENTIFICATION
ERRORS
IMPROPER PATIENT ID :

According to CLSI
Patient identification by asking patients to state their
full name, date of birth and unique ID number
Patient identification from identification bracelet
ID bracelet attached to the patient
Test requisition or computer generated labels bought
to bed side
 IMPROPER SPECIMEN LABELLING :

Handwriting not legible

Collection tubes unlabeled
Incorrectly labeled

Labels should bear the following :

Patient's name
ID number
Date and time of collection
 FACTORS AFFECTING TEST RESULTS
DURING BLOOD COLLECTION :

Wrong venipuncture technique

Hemolysis of RBC' s
Use of wrong containers
Instability of substances in blood
PHLEBOTOMY TECHNIQUE ERRORS
TOURNIQUET APPLICATION :

Prolonged tourniquet application can result in

hemoconcentration
If the tourniquet is tied too high, veins may not be
prominent
Tourniquet tied too close to the venipuncture site can
cause hematoma
 SITE SELECTION :

Specimen collection personnel should avoid the

following sites :

Sites with IV infusion

Scarred, burned or tattooed areas
Damaged veins
Edematous areas
Hematoma
Mastectomy
 HEMOLYSIS :

Rupture of RBC s can lead to unreliable results

Hemolysis causes a false increase in the
concentration of the substances being tested

To avoid hemolysis :

Use clean, sterile and dry syringe
Withdraw blood slowly and steadily
Allow sufficient time for the blood to clot
Avoid vigorous shaking of tubes after collection
 TRANSPORT ERRORS
Specimen must be transported immediately after
collection
Centrifugation within 2 hrs of collection
Transport in leak – proof plastic bags
Filled collection tubes should be transported in an
upright position
Temperature
Protection from light
Tubes should remain close until the time of testing
DEFINITION
Automation is the mechanization of conventional
manual methods
Improves the standard of the laboratory
Handle large number of samples
Less involvement of the analyst
Use of auto analyzers provide quicker and accurate
results
CLASSIFICATION OF AUTOANALYZERS
SEMI AUTOANALYZERS :

 Initial stages are performed manually

 Setting of the unit is automatic with regard to
wavelength, time, temperature and interpretation
of result
 Only one analysis can be done at a time
CONTINUOUS FLOW AUTO ANALYZERS :

Samples flow in a sequence

Each sample in the batch is subjected to the same
analyte

Two types :
Single channel continuous flow analyzer
Multi-channel continuous flow analyzer
 DISCRETE ANALYZER

BATCH ANALYZER RANDOM ACCESS

ANALYZER
One reagent is stored in More than one reagent
the machine at a time. can be stored.
One batch of particular The computer is
biochemical tests can be programmed to carry out
performed automatically any number of selected
with all the samples tests on individual samples
STEPS INVOLVED IN AUTOMATION
SAMPLE IDENTIFICATION :

• The tube containing each of the sample is labeled at

the time of collection of blood or other fluids for
analysis.
• On reaching the lab where it is to be tested, the
sample is recorded by computerized procedure after
which the samples are processed.
 BAR CODING :

• A bar coded label is placed onto the sample container

and is read subsequently by a bar code reader.

• Bar coding is a sample recognition system, which

allows electronic identification of the samples and
analysis needed and relays this information to the
automated analyzers.
 SAMPLE PREPARATION :

• Adequate quantity of blood is collected in vacutainer

tubes .
• After centrifugation, the tubes are placed in the sample
carousel.
• Since the specimen probe is equipped with a level sensor,
the required amount of serum can be pipetted
automatically.
SAMPLE HANDLING, TRANSPORT AND DELIVERY :

• The tubes holding the samples are kept covered till

the time of analysis to avoid evaporation or spillage.

• For analysis, the sample is loaded on loading zone of

the analyzer.
 SAMPLE PROCESSING:

• Automation of the analysis of analytes requires the

capability of removing the interfering substances from
blood for the analyte to be tested

REAGENT HANDLING AND STORAGE :

• Reagents are stored in plastic or glass containers

• They can be stored at 4°C if the assay is delayed and
before analysis, it is brought to room temperature.
REAGENT IDENTIFICATION AND DELIVERY :
Labels on reagent containers include the following
information :
Reagent identification
Volume of the contents
Expiry date
Reagent containers carry bar codes that contain most of
the above information .
Liquid reagents are taken up and delivered to mixing
chambers.
The reagent probes are washed and flushed , if more than
one reagent is used to prevent reagent carry over.
 MIXING AND INCUBATION :

The mixing of specimen and reagent takes place in

thermo - cuvettes
Mixing can be achieved by :
Forceful dispensing
Magnetic stirring
Rotating paddle
The reaction mixture is incubated at 370 C
 MEASUREMENT DEVICES :
The cuvette is passed through photo-electronic
instruments
Measurement devices include :
Spectrophotometers
Flurometers
Flame photometers or
Radioactive counters
After taking the absorbance, the results are prepared
by software attached
 SIGNAL PROCESSING AND CONTROL OF
MICROPROCESSORS :

The computer processes the digital data into useful

output

Output signals are automatically processed and the

results are made available in form of readings/ graphs
 CRITERIA FOR SELECTION
OF AUTOANALYZER

Sample load
Cost of the instrument
Number of parameters
Sensitivity and accuracy of test results
Time taken for reporting
 ADVANTAGES
The analyzers are computer based with self
monitoring features
Require only microlitre quantity of samples and
reagents
Easy to operate
Results are accurate and reliable
Difficulties in manual estimations are eliminated
Workload can be finished within short span of time
 DISADVANTAGES
Automated systems are expensive
Difficult to maintain
Requires trained service personnel
Impractical for small number of samples
Back-up procedure must be available in case of
instrument failure