• SCENE 1 :- You have to collect blood for glucose
estimation from a hemiplegic patient with the
right healthy arm running an i.v dextrose drip.
Which site will you choose ?
• SCENE 2 :- You have to transport a 24 hr urine
sample for estimation of Uric acid .Which
preservative will you use ?
• SCENE 3 :- You have to send samples for study of
cell morphology. Which container will you use ?
The Quality Assurance Cycle
•Data and Lab
TYPES OF BIOLOGICAL SAMPLES
– WHOLE BLOOD
– OTHER BODY FLUIDS LIKE saliva, spinal, synovial,
amniotic, pleural, pericardial and ascitic fluid
The process of collecting a blood sample is
called as phlebotomy.
Use : Most common sample collected is venous
1) PRELIMINARY STEPS:-
– IDENTIFY :- Name ,Age, Sex, Medical Record Number,
Date of Birth, Address( MOST IMPORTANT PART OF
SAMPLE COLLECTION )
– VERIFY TESTS REQUIRED
– ESTIMATE AMOUNT OF BLOOD TO BE DRAWN
– SELECT APPROPRIATE BULBS or Tubes(VACUTAINERS)
• LOCATION –
• VEIN OF CHOICE-median cubital vein in the
antecubital fossa as the vein is large and very close
• Veins on back of the hand or ankle (AVOID in
diabetics and patients with poor circulation)
• In IPD it is advisable to collect blood during insertion of
• If it is absolutely necessary to collect blood from an
arm running fluids - SHUT OFF fluids for 3 minutes and
discard first 5ml of blood
• Use a site below infusion site as retrograde blood flow
through veins doesn’t occur.
• PATIENT POSITION:-
– patient should be comfortable :seated or supine .
– Never perform on a standing patient.
– Arm to be extended straight from wrist to shoulder.
• WEAR PROTECTIVE EQUIPMENT e.g. gloves, gown , masks
(preventing aerosol transmission) to prevent transmission
of infection from patient
Note - do enquire about latex allergy as gloves, tourniquets
are commonly made of latex
• 19-22 gauze commonly used .(larger the gauze-
smaller the bore ).
• For children use 22-23 .
• If collecting a larger volume say 20 – 30 ml use
18or wider bore needle.
• TO AVOID VENEPUNCTURE ON A PARTICULAR
– i.v fluid running on that side
– Hemiplegia or other sensory loss ( RISK OF AN
INFECTED ARM AND GANGRENE LATER –PATIENT WILL
NOT FEEL PAIN)
– infections, cellulitis
– arterio-venous fistula
– AVOID repeated aspiration from a single arm
– sclerosed veins : repeated transfusions ,
chemotherapy –LEAVE IT TO THE SPECIALIST ONLY
TRY OTHER ARM, ANKLE VEINS, FEMORAL –TAP
• 2) SITE PREPARATION: - Disinfect with
a) prepackaged alcohol
b) Gauze pad saturated with 70% isopropanol
c) benzalkonium chloride solution 1:750
• Cleaning of puncture site must be done in a
circular motion and from the site outward.
• Skin should be allowed to dry as alcohol can
cause hemolysis and interfere with results.
• 3) TIMING – The time at which a specimen is obtained
is important for constituents that have diurnal
variation e.g. cortisol and samples of therapeutic
drug monitoring .
• Timing also very important for measurement of
glucose and alcohol.( medicolegal importance-
concentration may change later)
• Types of timed samples:-
– FASTING SAMPLE is best for biochemical investigations (12-
14 hrs or overnight fast)
– POST PRANDIAL SAMPLE is taken 2 hours after a meal
– RANDOM SAMPLE can be taken anytime.
4) VENOUS OCLUSION –
• It is done to distend the veins.
• Place a tourniquet 4-6inches above an intended
• A BP cuff can be used ; the pressure kept not more
than 60 mm Hg.
• It should not be kept for more than one minute
– Decreased pH
– increased lactate
– increased proteins
– increased ionized calcium
– increased potassium
• Pumping and taping of hand to be avoided.
• 5) ASPIRATION: -
RULE :- ASPIRATE SLOWLY
– Use an evacuated blood tube (VACUTAINER)
• Direct fill
• Advantages of evacuated blood tube – more
convenient, easier to use
• Drawback- costlier
• If using a syringe maintain the 15 degree
contact angle .
• 6) SEAL PUNCTURE SITE USING A STERILE SWAB
• 7) LABELLING: - Label after every collection . No
batch labelling ( Put the sticker or write neatly
on tube after collecting each sample only)
• 8) DESTROY AND DISCARD NEEDLE: - USE A NEEDLE
• 9) AUTOCLAVE all disposables
• ARTERIAL BLOOD:-
Use :- Blood gas analysis
• Required specially training and performed by
doctors or specially trained technicians only.
• SITES: - RADIAL ARTERY, BRACHIAL ARTERY,
• TRANSPORT TO POINT OF TESTING :-
– USE HEPARINIZED SYRINGE
– !!HURRY !!
– !!!EVERY SEC VALUES CHANGE !!!
• CAPILLARY BLOOD:-
Skin is punctured with a lancet and a small volume of
blood is collected .
a) Limited sample volume e.g. paediatric ward.
b) Repeated venepunctures have caused severe vein
c) Burns or bandaged areas- veins are not available for
d) Blood collected on special filter papers for neonatal
screening and molecular genetics testing
– tip of a finger
– An ear-lobe
– Heel or big toe of infants
• Clean site
• Puncture with a sharp stab but not deeper than
2.5cm to avoid contact with bone
• Use different site for each puncture to prevent
• Don’t press or massage as it causes tissue debris
and tissue fluid comes out – it is not same as
• First drop of blood is wiped off and the
subsequent drops are collected in appropriate
• Fill rapidly as site will clot
Patient preparation –
• Preparation of Patient for OGTT:-
• The Patient is instructed to have good
• carbohydrate diet for 3 days prior to the test.
• Diet containing about 30-50gm of carbohydrate
should be taken on the evening prior to the test.
• Patient should avoid oral hypoglycemics for at
least 2 days prior.
• No Strenuous exercise on previous day.
• Patient should be on 12 hr fast 8PM to 8 AM next
morning. NO TEA –COFFEE – BISCUITS etc .
• No smoking during the test
• Preparation of Patient for LIPID PROFILE :-
• The Patient is instructed to have normal diet for 3
days prior to the test. No extra butter/ghee or other
oils in food. Avoid meat and eggs day before testing.
• Patient should avoid oral cholesterol lowering
medicines (eg.STATINS)for at least 2 days prior.
• No Strenuous exercise on previous day.
• Patient should be on 12 hr fast 8PM to 8 AM next
( NO TEA – COFFEE IN MORINING)
• No smoking during the test .
Containers and preservatives for blood
• PLAIN BULB – No anticoagulants .Used for all investigations on
serum eg.enzymes, bilirubin, uric acid etc
• FLUORIDE BULB – Potassium oxalate and sodium fluoride ( 3 :1)
• Sodium fluoride inhibits Glycolytic enzyme ENOLASE. Potassium
oxalate forms insoluble complexes with Ca+2 and prevents
• Use: Blood sugar estimation
• CHEMISTRY BULB – Potassium oxalate - 20 mg/ 10 ml blood .It
forms insoluble complexes with calcium ions and prevents
• Use: Urea estimation
• CITRATE BULB – Sodium citrate - 60 mg/10ml blood. Citrate
converts Ca+2 to a soluble non-ionized form
• Use: Prothrombin time, Blood grouping, ESR .Should not be used to
• WINTROBE BULB – Ammonium oxalate and Potassium oxalate -
forms insoluble complexes with Ca+2 and prevents coagulation.
Use: Routine haematological investigations, cell morphology of
body fluids. It should not be used to measure urea by urease
method as it contains ammonium oxalate.
• PARAFFIN COATED – Inner surface coated with paraffin to make
surface smooth and prevents cell lysis.
Use: to study cell morphology of body fluids.
• EDTA BULB- EDTA - 20mg/10ml.It chelates divalent cations like
Use: Platelet count, bone marrow examination, isolation of genomic
• HEPARIN BULB- Heparin – inhibits factors IX and XI and also binds
anti -Thrombin III and increases its activity. (2mg/10 ml)
Use: Arterial blood gas analysis and osmotic fragility test. It can be
used for a wide range of analytes but it is costly. It inhibits enzyme
ACID PHOSPHATASE and unsuitable for its assay.
• Note – While mixing anti-coagulant with
sample it is important not to shake vigorously
as this causes hemolysis.
• TYPES OF SPECIMENS:-
• RANDOM – obtained at anytime of the day
• EARLY MORNING SAMPLE – A clean, early morning fasting
sample is the most preferred sample for detection of abnormal
constituents like proteins, or compounds like HCG.
• DOUBLE VOIDED SPECIMEN is used in GTT. It is the urine excreted
during a timed period after a complete emptying of bladder
• MID-STREAM SAMPLE- For bacteriological examinations, the first
10 ml of voided urine is discarded then urine is collected.External
genitalia must be cleaned properly for such collection.
• CATHETERISATION of the bladder through the urethra for urine
collection is carried out only in special circumstances, i.e., in a
comatose or confused patient. This procedure risks introducing
infection and traumatizing the urethra and bladder, thus producing
iatrogenic infection or hematuria.
• Suprapubic aspiration of urine from bladder:-
– Urethral injury( eg Ruptured Urethra ) – take
sample from suprapubic catheter
– Urethral obstruction (Tumors, Strictures etc
causing complete obstruction)
– Prolonged catheterization especially for Urine
culture and sensitivity .
• 24-HOUR SAMPLE - Obtained by collecting urine
excreted in 24 hours. E.g. 8am of one day to 8am of
next day discarding early morning sample of first day.
(Volume - 1200 to 1500ml.)
• Substances added to urine to reduce bacterial action or
chemical decomposition or to solubilize constituents
that might precipitate out of solution.
Commonly used are :-
• HCl -6mol/L -30ml per 24 hour collection (pH below 3)
• Na2CO3 ,NaOH is used to preserve specimens for Uric
acid, urobilinogen and porphyrins. (pH – 8- 9). Uric acid
will form clump like precipitate with HCl.
Toluene and light petroleum are also good
preservatives but as they are lighter than
urine they float on urine and contaminate
pipettes during processing.
Others; - Thymol, Acetic acid, Na2CO3 ,HNO3
, Boric acid.
• OTHER BODY FLUIDS
• Uses Meningitis, CVA, demyelinating diseases like
multiple sclerosis, meningeal involvement in
• Procedure – Lumber Puncture
• Contraindications of lumbar puncture – A very
high intra-cranial pressure in infants and children
can cause instant death if LP is carried out as CSF
gushes out too fast and brain stem herniation
occurs- check for PAPLLOEDEMA
Uses – Ascites of any cause – cirrhosis, peritonitis etc
Procedure -Abdominal tap at most dependent portion at
Pleural fluid and Pericardial fluid – Pleural and
Uses – prenatal diagnosis of congenital disorders,
assessment of fetal maturity & Rh iso immunization
• ANALYSED FOR THE FOLLOWING –
1) FAECAL FAT – obstructive jaundice and malabsorption
2) PARASITES – microscopy
3) HEME AND OCCULT BLOOD – for GI bleeding
4) FAECAL NITROGEN- malabsorption
• Care to be taken to prevent contamination with urine during
• Usually fresh specimens to be used to avoid need for preservation.