Sample collection and processing

1. Sample Collection & Processing
Dr. Rajju Tiwari
Final Year PG Biochemistry
PGIMS,Rohtak

2. Types of Biological Specimens
• Blood - serum ; plasma
• Urine
• Saliva
• Amniotic fluid
• Cerebrospinal fluid
• Synovial fluid
• Peritoneal fluid
• Pleural fluid
• Pericardial fluid

3. Standard precautions for infection
control
Personal protective equipments(PPE)
1. lab coat
2. Gloves
3. mask
4. Eye protection
Proper hand hygiene
alcohol-based antiseptic hand cleaners/senitiser are used
Handle all blood specimens as potentially infectious
material
Never recap needles , use needle cutter to destroy
needle

4. Artery
Capillary
BLOOD
Phlebotomy is the process of making an incision in
a vein with a needle to collect blood
Person authorized to perform the procedure are-
Laboratory technicians/trained nurses and intern
MBBS students

5. Sample collection procedure
 Determine the patient identity before collecting sample by checking
details on requisition form
 Ask the patient about: fasting status in case of sample to be collected in
empty stomach and time of any medication taken
 Check the requisition form for requested test, patient information, and
any special requirements
 Select a suitable site for venipuncture and tourniquet should be applied
2-3 inches above elbow joint

6. Site of collection
The vein sites should be examined and
considered in the following order:
 Median Cubital
 Cephalic
 Basilic (avoid if possible because it is closest
to the brachial artery and median nerve)
 Wrist Area (top and side only)
 Back of the Hand

7. If a vein is not readily apparent, a number of techniques may be used to make
palpation easier:
 Massage arm or apply warm cloth to dilate vein
 Bend arm very slightly, relaxing the muscles of the antecubital
 Lower arm below the heart to allow veins to fill to capacity
 Close patient’s hand to dilate vein
 Pumping of fist should be avoided
 it falsely elevates potassium, phosphate, lactate ,lowers pH
and hence increases ionized calcium conc

8. Precautions when using a tourniquet
 Do not apply tourniquet above a central venous catheter or the side of a
mastectomy
 The tourniquet should not be left on the site for longer than one minute
during the collection due to the possible occurrence of
hemoconcentration and localised stasis which may result in high value
for protein based analytes and PCV
 Clean venipuncture site with 70% alcohol/ isopropyl using circular
motion from centre to periphery
 Allow the area to dry before venipuncture
 Anchor the vein and smoothly insert the needle with bevel up
 Release the tourniquet as soon as blood begins to flow

9.  Identify the correct vaccutainer
 Collect the proper amount of blood in proper order according to
National Committee For Clinical Laboratory (NCCLS) guidelines-

11. Syringe method-
 If using a syringe, be careful not to pull too hard on the plunger as this
may damage red cells
 Do not force blood from the syringe into the tubes, which may also
cause damage to red cells
 Using the syringe method of collection, the order of tube fill is
different-
 Blood culture bottle are filled first
 Tubes for coagulation testing ( blue vaccutainer)
 Tubes with anticoagulant
 Tube without anticoagulant must be filled last
 To avoid hemolysis- mix tubes with anticoagulant additives gently
5-10 times

12. Additive carry-over
 This can occur when blood in a additive tube touches the needle during
venipuncture or during transfer from a syringe
 Most problem are seen with EDTA
 Least with HEPARIN, as it occurs in blood naturally
 Carry-over least likely to occur if tubes fill from the bottom up which
keeps the tube contents away from the needle
 Order of draw reduces the risk of specimen contamination and additive
carry-over

13. If an incomplete collection or no blood is obtained
 Change the position of needle
 Adjust the angle, the bevel may be against the vein wall
 Loosen the tourniquet
 Try another tube, it may be due to loss of vaccum in vaccutainer
this may lead to short draw and incorrect ratio of blood and anticoagulant
• Plastic (polyethylene terphthalate) tubes are preferred over glass tubes as
they decrease likelihood of breakage and hence exposure to infectious
material.

14. Precautions
 If swelling is noticed during venipuncture, immediately release
tourniquet, remove needle and apply pressure using gauze pad
 If first attempt fails, pull needle back slowly, relocate vein, pull skin
taut (anchor vein), and redirect needle
 Do not attempt to stick patient more than two times contact another
phlebotomist
 If patient feels a shooting, electric-like pain, tingling, or numbness,
remove the needle, notify nurse or physician, and document the
incident

15. Apply direct pressure to puncture site for 30-60 seconds or until
bleeding stops
- For patients with a known bleeding disorder immediately after
removing butterfly needle apply direct pressure to puncture site until
bleeding stops for up to 10 minutes, even if blood was not obtained

16. Order of Draw from Catheter Lines
1. Draw 3–5 ml of blood in a syringe and discard
2. Blood for blood culture
3. Blood for anticoagulated tubes (lavender, green light blue, etc.)
4. Blood for clot tubes (red)

17. Skin puncture
 method of choice in paediatric patients
 Accessible veins in sick infants must be reserved exclusively for
parenteral therapy
 Skin puncture is useful in adults with (1) extreme obesity, (2)
severe burns, and (3) thrombotic tendencies.
 Skin puncture is often preferred in geriatric patients because
skin is thinner and less elastic; thus a hematoma is more likely to
occur from a venipuncture

18. Sites for skin puncture
Adults : finger tip
earlobe
Infants: heel
big toe (not in <1 year)

19.  Best locations for a finger
stick is the 3rd and 4th
fingers of the non-
dominant hand
 Perform the stick to side of
the center of the finger.
 NEVER use the tip or
center of the finger
Finger stick procedure

20. • Cleanse fingertip
• Avoid fingers that are cold,
cyanotic, swollen, scarred or
covered with a rash
 Massage to warm the finger
NOTE: Do NOT squeeze or apply
strong repetitive pressure to the
site, this may result in hemolysis or
increase tissue fluid in the blood
causing incorrect glucose results

21. Heel stick
 hold the heel with the forefinger at the
arch and the thumb proximal to the
puncture site at the ankle.
 After blood collection is complete, elevate
the heel place a piece of clean dry cotton e
until bleeding has stopped

22.  Record the date and time of sample collection
 Check for sample label
 Check if sample transported in appropriate boxes with biohazard signs

23. transport of Sample
 A requisition form must accompany each sample submitted to
laboratory
 Requisition form must contain proper information:
initials of Patient name, CR No./ OPD no. age and sex, diagnosis,
doctor’s signature
 Date and time of sample collection should be mentioned Maintain
identification
 Transport on ice - BGA
lactate
 protect from light – bilurubin

24.  Sample rejection criteria-
- Unlabelled specimen
- Incorrect container
- Insufficient specimen
- Unsuitable specimen

25. Sample processing
Pre
centrifugation
Centrifugation
Post centrifugation

26. Pre centrifugation Phase
Ideally, all measurements should be performed within 45 minutes to 1
hour after collection
Blood should be stoppered in the original container until ready for
separation
To be kept at room temp till plasma /serum is separated

27. Centrifugation Phase
 Plasma
Centrifuge the anticoagulated blood for at least 15 minutes at 2000
to 3000 g to obtain cell-free plasma
 Serum
When coagulation is complete, centrifuge the sample for at least 10
minutes at a minimum speed of 1500 g
 When separating serum or plasma, the temperature should not
drop below 15 °C or exceed 24 °C

28. Precautions during centrifugation
 Temperature maintenance
 Keep tubes stoppered
to decrease evaporation
prevent aerosolization
prevent CO2 loss and hence
pH changes

29. The Lipaemic Sample
This can be due to food intake, after intestinal absorption,
triglycerides are present in plasma as chylomicrons and their
metabolites (remnants) for 6 to 12 h

30. To avoid lipaemia
 patient should fast at least 12 hours before blood samples are
taken
 In patients receiving parenteral infusion of lipids a period of 8
hours of interruption of the treatment is necessary
 Centrifugation
for s.lipids – at 12000g for 10 mins
 The method of choice for removal of turbidity from
serum and plasma is a 10 min centrifugation in a
centrifuge with 10000 g

31. Prevention of bilirubin interference
 The high prevalence of hyperbilirubinaemia in patients from
intensive care, gastroenterological surgical or paediatric departments
--Blanking procedures are useful to eliminate spectral bilirubin
interferences
 K4 [Fe(CN)6] effectively eliminates bilirubin interference in H2O2-
forming enzymatic methods

32. Sample storage period
 OPD Lab- 24 hrs at 2-8oC
 Departmental lab - 24 hrs at 2-8oC
 Emergency lab – 6hrs at 2-8oC
Turnaround time
 OPD lab – 7 hrs after sample collection
 Departmental lab – 7 hrs after sample receiving
 Emergency lab- 3 hrs after sample receiving

33. URINE
 Random –for spot glucose, ketone bodies
 first-morning-voided urine – used for microscopic examination,
and HCG measurement
 24-hour total volume collection- for protein, creatinine
- Begin the urine collection in the morning after emptying the
bladder for first time, note the exact time and collect urine till next
morning sample
- Store the bottle at room temperature or in refrigerator
- Any urine passed with bowel movement should also be collected

34. Thank you!