Department of Biochemistry

AIIMS BATHINDA

Practical No. 3

Aim : Orientation to Clinical Biochemistry Laboratory : Preanalytical phase-Phlebotomy

and Lab errors

Clinical Biochemistry is a discipline of medicine concerned with the measurement of amounts of

biologically important substances (called analytes) in body fluids.

The methods to measure these substances are carefully designed to provide accurate assessments
of their concentration. The results of clinical chemistry tests are compared to reference intervals
or a medical decision level (MDL) to provide diagnostic and clinical meaning

There are 3 phases of testing process: pre-analytical, analytical, and post-analytical.

1. Pre-analytical phase includes all the activities that take place before testing, such as test
ordering, sample collection, sample transport to the laboratory and initial processing of the
clinical sample.

2. Analytical phase consists of the laboratory activities that actually produce a result, such as
running a sample on an automated analyzer. It also includes maintenance and calibration of
analyzer and running quality control on the analyzer before actual patient sample run.

3. Post-analytical phase comprises transcription and interpretation of results and patient

reporting.

Biological Specimens

Biological samples typically used for clinical chemistry tests:

1.Blood : Whole blood, serum and plasma
2.Urine
3. CSF
4. Fluids : Plural, pericardial, peritoneal, synovial, and amniotic fluid
5. Saliva

Blood is the most commonly used specimen for testing in the clinical laboratory.
Blood consists of two main parts – a fluid portion (called plasma, which contains the dissolved
ions and molecules) and a cellular portion (the red blood cells, white blood cells and platelets).

If a blood sample is collected in a tube containing an additive that prevents the blood from
clotting (called an anticoagulant), the fluid portion of the blood is called plasma.
If the blood is collected in a tube with no anticoagulant, the blood will form a clot. Upon
centrifugation, the clot descends to the bottom of the tube along with the cells. The resultant
liquid above the cells and clot is called serum.

Serum contains all the components of plasma except the clotting proteins, which are consumed
in the cascade of reactions that form the blood clot.

PRE ANALYTICAL PHASE : comprises of following steps

1. Test order by the clinician on the test request form (TRF)
2. Patient identification
3. Patient preparation
4. Sample collection or Phlebotomy (in case of blood collection)
5. Sample transport to the laboratory
6. Sample accessioning and centrifugation

Phlebotomy
Specimen collection is a critical initial step in laboratory diagnosis. Meaningful laboratory
results require careful attention to the specimen source, the method of collection, and the timing,
storage, transport and handling of the collected specimens. In addition,0 a completed request
form with relevant history, if appropriate, is essential for optimal and efficient laboratory workup
of the collected specimens.

Pre-collection check
1. Phlebotomist will ask and confirm patient’s full name, age, address/contact details and
confirm same from the test request form
2. Phlebotomist will check if the patient has followed necessary preparations if applicable.
3. Phlebotomist must arrange basic phlebotomy supplies like Gloves Tourniquet, Needle,
Vacutainers, Adapter/Holder, Alcohol swab, Cotton or Gauze, Bandage or Tape, Needle disposal
unit before blood collection

Steps of Phlebotomy
1. Locate a vein of a good size that is visible, straight and clear.
The median cubital vein lies between muscles and is usually the most easy to puncture. DO NOT
insert the needle where veins are diverting, because this increases the chance of a hematoma.
2. Apply a tourniquet 5-10 centimeters above the intended venipuncture site. It is not
recommended that the tourniquet stays on for longer than 1 minute.
3. Determine the best site for venipuncture.
4. Clean area with an alcohol swab in a circular motion. Allow to dry for 30 seconds. Do not re-
palpate after cleansing
5. Anchor the vein and smoothly insert the needle with bevel up
6. Release the tourniquet as soon as the blood begins to flow
7. Collect the proper amount of blood for tests ordered. Collect blood in tubes in proper order of
draw
8. Ask the patient to keep the pressure on the gauze till the bleeding stops.
9. The needles are disposed in puncture proof container
10. Check site to ascertain if bleeding has completely stopped. Apply sterile Band aid on the
puncture site.
Vacutainers : Blood collection tube with a colored rubber stopper creating a vacuum seal inside
the tube, facilitating the drawing of a predetermined volume of blood.

Order of Draw : It is vacutainer tube sequence a phlebotomist needs to follow while collecting
blood. If the order of draw is not followed, then the additives in one tube can be transferred to
another, causing cross-contamination and inaccurate results.

Order Of Colour Spec. Vol. Tube Mixing Special

Draw Top Content Instructions Instruction
First Adult -10ml Rotate For- Paired
gently to blood culture,
Paed: 5ml mix
Mention the
from which
hand sample is
taken
Second 2.7mL 3.2 % 3- 4 times For coagulation
Sodium determinations.
(Light Blue) Citrate Sample should
be collected
upto mark on
the tube
Third 3.5ml/5.0ml Clot activator 5- 6 times For serum
and gel for
serum determinations
(Gold) separation in chemistry,
serology &
Clot immunoassay.
activator,
Silicone
(Red) 4.0ml coated 5-6 times Red Top to be
(Plastic)
used for Drug
No
Monitoring
anticoagulant

Fourth(Green) 4.0ml Sodium 8-10 times Flow

/Lithium Cytometry,
heparin Cytogenetics,
OFT
Fifth 3.0ml Spray- 8-10 times Routine
coated Hematology
(Lavender) K2EDTA
(plastic)
Sixth 2.0ml Sodium 8-10 times Blood Glucose
fluoride/
(Gray) Na2
EDTA
Instructions for Labeling Samples/Vacutainers

1. The sample labeling is the primary responsibility of the person who is collecting sample.
2. The primary sample is labeled properly allowing identification of the patient which should at
least contain the name, age & unique hospital identification number
Transport of collected sample to the laboratory
1. Transport all samples at ambient temperature (22-26 degree Celsius) within 30 min to one
hour of collection
2. All samples are to be preferably transported in the sample collection bag
Sample receipt at the laboratory
1. Samples are received at the sample receiving counter of the lab
2. The collected samples are matched with details on the test request form
3. The lab label sample with the lab accession number
4. The laboratory can reject the sample according to its rejection criteria

Sample processing/Centrifugation of blood samples

Vacutainer RPM & Time Comments

Yellow Top / Red Top 3000 – 4000 rpm Vacutainer to be kept in upright
for 15 min positions. Allow them to clot for
30 minutes

Samples to be centrifuged within

45-60 minutes of collection

Light Blue Top 3500 – 4000 rpm If sample is to be transported ,

for 15 min separate immediately after
centrifugation

Gray Top 3000 – 3500 rpm Samples to be centrifuged within

for 10 min 45-60 minutes of collection.