thanks

1. 1
Sample collection and processing
Part 1
Dr. Urender singh
2nd yr PG Biochemistry
Faculty of Medicine & Health Scinces
SGT Medical College
Gurgoan

2. • Accuracy -To ensure that analytical results
obtained are representative of actual analyte
conc. in patient and, thus of his/her
physiological/pathological state.
• Safety
• Minimize patient discomfort and
complications
• Avoid recollection
2
Aim of sample collection

3. • Blood- venous/ arterial/ capillary- whole blood ; serum ; plasma
• Urine
• Faeces
• Saliva
• Solid tissues
• Amniotic fluid
• Cerebrospinal fluid
• Synovial fluid
• Peritoneal fluid
• Pleural fluid
• Pericardial fluid
Example of biological specimen
3

4. Venipuncture
Skin puncture
Arterial puncture
Blood collection
4

5.  Personal protective equipments(PPE)
1. lab coat
2. Gloves
3. mask
4. Eye protection
 Proper hand hygiene
now a days alcohol-based antiseptic hand cleaners are used instead of hand
washing if the hands are not visibly soiled
if not available and clean visibly soiled hands are there,detergent should be used
followed by alcohol based cleaner
 isolation this separate certain pts from others and limit their contact with
hospital personnel and visitor
Handle all blood specimens as potentially infectious material. Never recap needles ,dispose in puncture
proof sharps container
Standard precautions for infection
control
5

6. 1. Phlebotomy Room- private area such as an isolated room
• counter or work table for all of the blood handling equipment and
supplies
• phlebotomy chair should be available
2. Blood Processing Room-
• refrigerated centrifuge
• a small refrigerator
• sufficient counter space for the processing of blood specimens.
• A -70°C freezer should be in or near the room
• sink and running water .
• Use one counter area for processing the blood samples and
another counter area for completing paperwork.
Facilities required
6

7.  Disposable latex gloves (use non-latex, e.g. nitrile or
vinyl, if employee and/or patient has a latex allergy).
 Isopropyl alcohol
 Gauze or lint free tissues
 Needle with safety feature
 Venous blood collection system (e.g,Vacutainer)
 Syringe (if vacuum blood collection system
unavailable)
 Safety transfer device (if using syringe)
 Tourniquet (preferably latex free) or blood pressure
cuff
 cont……
Equipment Preparation
7

8. 8
Puncture proof container marked"Bio
hazardous".
Red and yellow biomedical waste disposal bags
Band Aids (optional)
Blood specimen collection tubes appropriate for tests
ordered
Disinfectant (10% household bleach) for bench tops
Other personal protective equipment, e.g., lab coat or
face shield, if appropriate

9. 1.Patient Preparation
1. Identification— at least 3 items of identification should be used
• Name
• Age
• Sex
• Medical record / C R No.
• Room no. ,bed no.
• Address for outpatient
For alcohol levels testing and medicolegal cases –chain of custody must be
established . Additional identification like photograph may be required.
2. Enquire for allergy to latex , alcohol , band aid any
Venipuncture
9

10. Timed specimens –
a. to monitor changes in a patient's condition—eg. monitor a
cardiac marker to determine if it is rising or decreasing
b. to measure how well a substance is metabolized –eg. Two
hour postprandial specimen and/or a glucose tolerance test
4. position of patient- patient should be comfortably seated or
supine ,and should have been in this position for at least 20 mins
before the specimen is taken
10
3. Timing of blood collection
Specific instructions must be followed
a.Circadian variation
b.Travel
c.Menstrual calendar

11. 5. Verify –
If a fasting specimen is required, confirm that the fasting order has
been followed-
To be fasting the patient should not have consumed even a lifesaver,
stick of gum or other sugar-containing food items during the fasting
period, or has “accidentally” ingested a sip of juice, black coffee, tea,
single bite of other food items, or any amount of alcohol.
h/o of any long distance travel
h/o of exercise/ stenous physical activity within 8 hours
h/o of smoking in past one hour
h/o of alcohol ingestion
h/o any intake of medicines ,herbal preparations
h/o of any bleeding disorder
h/o of fainting on blood collection
such factors should be mentioned on the requisition slip of the
patient
11

12. 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
• Lower Extremity/Foot/Ankle avoid
performing on diabetic patients or those
with obvious circulatory problems)
• Scalp (performed only by highly
experienced phlebotomists)
Site of venipuncture
12

13. 13
Preparation of Venipuncture Site -
Using sterile alcohol prep. pad (70% isopropyl
alcohol), cleanse site using concentric outward
moving circles. Allow site to air dry. Do not blow on
site.
Using index finger, palpate (feel) for vein, its direction
and location

14. Apply the Tourniquet
 3-4 inches above venipuncture site
 60mmHg pressure if blood pressure cuff is used
Precautions when using a tourniquet:
Do not apply tourniquet above a central venous catheter or
midline, on arm with a kidney access device (Cannula, Fistula,
Shunt).
The tourniquet should not be left on the site for longer than one
minute during the actual collection due to the possible occurrence
of hemoconcentration and infiltration of blood into tissue.
If the patient is sensitive to the tourniquet, apply over sleeve or
towel.
14

15. • Changes in serum composition when venous occlusion is prolonged from 1
to 3 mins.
• Increased-total protein,Fe,total lipid, cholesterol,billirubin
• Decreased-potassium.
• Composition of blood drawn first, is most representative of Composition of
circulating blood.
• Hence first drawn tubes should be for analytes like calcium ,that are
importent to critical medical decisions.
• Conc. Of protein bound analytes is also increased due to stasis.
Effects of prolonged venoocclusion
15

16. Never leave the tourniquet on longer than one minute during the
examination. If more time is needed, release the tourniquet
and then re-apply.
If a vein is not readily apparent, the following 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. Avoid having the patient
"pump" the hand
It falsely elevates potassium, phosphate,
lactate ,lowers pH and hence increases ionized calcium conc.
16

17. Perform the Venipuncture –
• vacutainers
• Syringes
• butterfly needle
Choose appropriate
Needle/ butterfly size-
Adults -19 to 22
Children- 21 to 23
17

18. Hold dry gauze over puncture site with arm raised for 30-
60 seconds or until bleeding stops .
If puncture site continues to bleed slightly, apply a
pressure bandage to site using clean folded gauze, band-
aid or paper tape. Tell patient to leave the bandage on
site for at least 15 minutes.
For patients with a known bleeding disorder (Haemophilia,
von Willebrand's Disease ), 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.
Label the Specimen Containers
Proper disposal of gauze,needle and holder, gloves etc.
18

19. • If swelling is noticed during venipuncture, immediately release tourniquet, remove
needle and apply pressure using gauze pad
• On patients with a bleeding disorder (Haemophilia or von Willebrand's Disease),
use a 21 No. gauge butterfly needle or smaller. Do not probe if blood is not
obtained immediately. Remove needle.
• If your first attempt fails, pull needle back slowly, relocate vein, pull skin taut
(anchor vein), and redirect needle. Lateral needle relocation should never be
attempted to access the basilic vein, since nerves and the brachial artery are in
close proximity.
• 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.
Precautions
19

20. Inappropriate sites for venipuncture
 Burn, Scar, Tattoos
 Damaged vein
 Edema
 Hematoma
 Arm with cannulas, fistulas or vascular grafts
20

21. Procedure related
• Hematoma formation
• Iatrogenic anemia
• Inadvertent arterial puncture
• Infection of the site
• Nerve injury
Patient related
‣ Fainting(syncope)
‣ Excessive bleeding
‣ Allergies
‣ Obesity
‣ seizures
Complications
21

22. • method of choice in paediatric patients, especially infants
• Venipuncture of deep veins in paediatric patients may also rarely
cause
- cardiac arrest
- Haemorrhage
- thrombosis
- venous constriction followed by gangrene
- damage to organs or tissues accidentally punctured
- infection
• Accessible veins in sick infants must be reserved exclusively for
parenteral therapy.
Skin puncture
22

23. 23
Skin puncture is useful in adults with
1) Extreme obesity
2) Severe burns or bandages
3) Extensive vein damage
4)Thrombotic tendencies
5)Sample applied directly to point of care testing or
filter paper
Skin puncture is often preferred in old age patients because skin is
thinner and less elastic; thus a hematoma is more likely to occur from
a venipuncture.

24. Adults : Finger tip
Earlobe
Children: Heel
Big toe (not in <1 year)
Finger stick:
• It may be necessary to warm the skin puncture site to increase
blood flow to the site. A warm, moist towel (or other warming
devices) at a temperature no higher than 42oC may be used to
cover the site for three to five minutes. This technique
increases blood flow to the site up to 7 fold, does not burn the
skin, and does not result in significant changes for routinely
tested analytes.
• Thoroughly cleanse the chosen site with 70% isopropanol.
Allow the skin to air-dry.
Sites-
24

25. 25
Obtain the sample from the third or fourth (middle or ring) finger
Choose a site that is on the side of the fingertip, midway between
the edge and midpoint of the fingertip. The puncture should be
made perpendicular to the fingerprint ridges
Use a lancet to make a deep puncture (1.5 mm) at the chosen site
Using a dry gauze, wipe away the first drop of blood, making certain
the area is completely dry. The first drop is most likely to contain
excess tissue fluid.

26. 26
Collect the blood sample in a collection device appropriate for
the test to be performed.
Do not massage or milk to avoid contamination with tissue
debris
Apply a piece of gauze, (or cotton ball), to the puncture site, using
slight pressure until the bleeding has stopped. For older children
and adults, offer a band-aid

27. • hold the heel with the forefinger at the arch
and the thumb proximal to the puncture
site at the ankle. If using a lancet the blade
should be not longer than 2.4 mm to avoid
injury to the calcaneus (heel bone)
• Collect the specimen in microcollection
capillary blood tubes or
• On filter paper for neonatal screening or
molecular genetics testing
• After blood collection is complete, elevate
the infant’s foot above the body and apply
pressure using sterile gauze until bleeding
has stopped .Do not apply adhesive
bandages.
Heel stick
27

28. Skin puncture blood is a mixture of undetermined proportions
of blood from arterioles ,venules , capillaries, interstitial and
intracellular fluids.
Venous vs capillary serum
28
Capillary>
venous
% Capillary =
venous
Capillary <
venous
%
Glucose 1.4 phosphate bilurubin 5.0
potassium 0.9 urea calcium 4.6
chloride 1.8
sodium 2.3
Total protien 3.3

29. Indications-
• assessment of PO2 when pulse oximetry is abnormal
• there may be changes to acid/base status as in
respiratory,metabolic,kidney diseases
• Symptoms of oxygen,carbon dioxide or pH imbalance
like difficulty breathing,nausea or vomitting
• assessment of ventilator efficacy
• Head or neck trauma,injuries that affect breathing
• Prolonged anesthesia as in cardiac bypass or brain
surgery
• Rapid Hb
Arterial puncture
29

30. 30
Site-
Adult -In order of preference, the radial, brachial, and femoral arteries
can be selected. Unacceptable sites are those that are irritated,
edematous, near a wound, or in an area of an arteriovenous (AV)
shunt or fistula
Neonate- indwelling umbilical artery catheter
Children- capillary puncture to obtain arterialized capillary
blood

31. • 1. Have the patient make a fist and
occlude both the ulnar (opposite the
thumb side) and radial arteries (closest to
the thumb) by compressing with two
fingers over each artery
• 2. Have the patient open their fist and
observe if the patient's palm has become
bleached of blood
• 3. Release the pressure on the ulnar
artery (farthest from the thumb) only and
note if blood return is present. The palm
should become perfused with blood.
Adequate perfusion is a positive test
indicating that arterial blood may be
drawn from the radial artery. Blood
should not be taken if the test is negative.
Serious consequences may result if this
procedure is not followed and may result
in the loss of the hand or its function
Modified Allen Test
31

32. • The amount of anticoagulant
should 0.05 mL liquid heparin
(1000 IU/mL) for each milliliter of
blood.
• Using too much heparin is
probably the most common pre-
analytic error in blood gas
measurement
• the blood should fill the syringe
fairly quickly.
• The colour of the blood should be
bright red, not dark red.
Procedure of art. puncture
32

33. • There should be no air space in the syringe at the
completion of collection.
• Roll syringe between your hands 15-20 times to
mix the anticoagulant in the syringe.
• Label specimen.
• Transport specimen on crushed ice. The
specimen should be cool not frozen, (maintain a
temperature of 1–5°C) to minimize leukocyte
consumption of oxygen
Important Points to remember
33

34. Complications are unusual but can include:
 Local pain
 Haematomas
 A rare vasovagal episode
 Extremely rarely, an expanding
aneurysm of the radial artery has been reported
after frequent punctures
34

35. • Serum- The watery portion of blood that remains after
coagulation has occured; it is obtained after centrifugation.
Historically, serum was the preferred assay
material for determining extracellular concentrations of
constituents in blood.
• Plasma- the noncelloular component of anticoagulated
whole blood, plasma contains clotting factors.
Today, plasma is preferred for many, but not
all, laboratory investigations because the constituents in
plasma are better reflecting the pathological situation of a
patient than in serum and because of better turn around
time(TAT)
Serum or Plasma?
35

36. Plasma
Advantage of plasma
 Time saving: Plasma
samples can be centrifuged
directly after sample
collection, unlike serum, in
which coagulation is
completed after 30 minutes
 Higher yield: 15 to 20 %
more in volume of plasma
than of serum can be
isolated from the same
volume of blood
Disadvantage of plasma.
 Contamination with cations:
NH4 +, Li+, Na+, K+.
 Assay interference caused by
metals complexing with EDTA
and citrate.
 inhibition of alkaline phosphatase
activity by zinc binding,
 inhibition of metal dependent cell
activation in function tests,
 binding of calcium (ionized) to
heparin . 36

37. Plasma cont…
• Advantage of plasma
 Prevention of coagulation-
induced interferences:
Coagulation in primary and
secondary tubes that were
already centrifuged, may
block suction needles of the
analyzers when serum
tubes are used; this is
prevented by using
anticoagulants.
 Disadvantage of plasma
 Interference by fibrinogen in
total protein measurement and
protein electrophoresis.
 Inhibition of metabolic or
catalytic reactions by heparin .
 Interference in the distribution
of ions between the intracellular
and extracellular space by EDTA,
citrate.
 Reduced storage stability for
certain analytes
37

38. Serum
Advantages of serum
 Nearly cell-free
 Good storage stability for
most analytes
 Wide range of assays
available
 Disadvantages of serum
 Longer TAT
 Instrument or test interference
from fibrin, especially with
anticoagulation therapy
• Increase in the concentrations
of platelet components in
serum as compared to plasma
(e.g. potassium, phosphate,
magnesium, aspartate
aminotransferase, lactate
dehydrogenase, serotonin,
neurone-specific enolase, zinc).
38

39. Plasma >serum % Plasma=
serum
% Plasma<
serum
%
Calcium 0.9 Bilrubin Albumin 1.3
Chloride 0.2 Cholesterol ALP 1.6
LDH 2.7 creatinine AST 0.9
Total protein 4.0 Bicarbonate 1.8
CK 2.1
Glucose 5.1
Phosphorus 7.0
Potassium 8.4
Sodium 0.1
Urea 0.6
Uric acid 0.2 39

40. 40
Sample collection and processing
2nd part
by Dr. Urender singh
2nd yr PG Biochemistry
Faculty of Medicine & Health Sciences
SGT Medical College
Gurgoan

41. • Heparin ( mucoitin polysulfuric acid)-effective anticoagulant
in small quantities without significant effect on many
determinations.
• Heparin is available as lithium heparin (LiHep) and sodium
heparin (NaHep) in green top tubes
• Heparin accelerates the action of antithrombin III, neutralizing
thrombin and preventing the formation of fibrin. 2 mg of
calcium/sodium/lithium/ammonium salt used /10 ml blood
(1000 units /ml)
• lithium heparin may be used for most chemistry tests except
for lithium and folic acid levels; for lithium, a royal blue
sodium heparin, Na2EDTA, can be used instead. Sodium
heparin cannot be used for assays measuring sodium levels
but it is recommended for trace elements, leads and
toxicology.
Anticoagulants and additives
…41

42. Disadvantages of heparin are high cost, temporary action,
blue background formation in smears stained with wright’s
stain, inhibition of acid phosphatase and polymerase activity
Liquid heparin causes dilution error in various analysts. so,
dried (lyophilized) heparin is recommended for critical care
testing
Calcium-titrated heparin at a concentration of 40 to 60
IU/ml blood (dry heparinisation) and 8 to 12 IU/ml blood
(liquid heparinisation) is recommended for the determination
of ionized Calcium.
42

43. Ethylenediamine tetra-acetic acid (EDTA)
anticoagulant of choice for haematology, isolation of genomic DNA
and qualitative and quantitative virus determination by molecular
techniques as it preserves cellular components of blood.
It’s available in lavender top tubes as a liquid or spray-dried in the
disodium, di- or tripotassium salt form (K2EDTA in plastic, spray-dried,
and K3EDTA in liquid form in glass tubes)
 K3EDTA is a liquid and will dilute the sample ∼1-2%. K2EDTA is spray-
dried on the walls of the tube and will not dilute the sample
43

44. • Converts calcium to non ionized form
• A conc. of 34-38 g/l is used in ratio 1 part to 9 parts of
blood
• For coagulation testing, a light blue top tube containing
3.2% sodium citrate is commonly used because it
preserves the labile coagulation factors, and its effect is
easily reversible with addition of calcium
• Black top tubes also contain buffered sodium citrate and
generally used for Westergren sedimentation rates.
They differ from the light blue top tubes in that the
ratio of blood. Anticoagulant is 9:1 in the light blue top
tubes and 4:1 in the black top tubes.
Citrate

45. • sodium/ potassium/ lithium/ ammonium oxalates
form insoluble complexes with calcium ions
• 1 to 2g/l mostly used
• >3g/l causes haemolysis
• Cause shrinkage of cells by drawing water except
combined ammonium (3parts) +potassium(2parts)
oxalates
• Inhibits ALP, ACP, LDH , AMYLASE
oxalates
45

46. • Used as inhibitor of glycolysis (inhibits glycolysis for
24 hrs at 25degree C) in combination with
EDTA/oxalate.
• As preservative, effective at concentration of 2g/l of
blood. 3 to 5 times higher conc. required when used
alone for anticoagulation.
• Prevents glycolysis by forming an ionic complex with
Mg++, thereby inhibiting the Mg++ dependent enzyme,
enolase
Sodium fluoride
46

47. • Antiglycolytic agent (inhibits glycolysis for 24 hrs) at
conc. Of 2g/l
• No effect on urease ( so it is preferred when a single
sample we want to do the urea and sugar)
• Inhibits CK
Sodium iodoacetate
47

48. Stopper color Anticoagulant/additive Specimen type/use Mechanism of action
Red (glass) None Serum/chemistry and
serology
N/A
Red (plastic/Hemogard) Clot activator Serum/chemistry and
serology
Silica clot activator
Lavender (glass) K3EDTA in liquid form Plasma/hematology Chelates (binds) calcium
Lavender (plastic) K2EDTA/spray-dried Plasma/hematology Chelates (binds) calcium
Pink Spray-dried K2EDTA Plasma/blood bank Chelates (binds) calcium
White EDTA and gel Plasma/molecular
diagnostics
Chelates (binds) calcium
Light blue Sodium citrate Plasma/coagulation Chelates (binds) calcium
Light blue Thrombin and soybean
trypsin inhibitor
Plasma/coagulation Good for fibrin
degradation products
Black Sodium citrate Plasma/sed rates –
hematology
Chelates (binds) calcium
48

49. Light green/black Lithium heparin and gel Plasma/chemistry Inhibits thrombin formation
Green Sodium heparin, lithium heparin Plasma/chemistry Inhibits thrombin formation
Royal blue Sodium heparin, Na2EDTA Plasma/chemistry/
toxicology
Heparin inhibits thrombin
formation Na2EDTA binds
calcium
Gray Sodium fluoride and lithium
iodoacetate
Plasma/glucose testing Inhibits glycolysis
Yellow Sterile containing sodium
polyanetholesulfonate (SPS)
Serum/microbiology culture Aids in bacterial recovery by
inhibiting complement,
phagocytes and certain
antibiotics
Yellow Acid citrate dextrose (ACD) Serum/blood bank, HLA
phenotyping and paternity
testing
RBC preservative
Tan (glass) Sodium heparin Plasma/lead testing Inhibits thrombin formation
Tan (plastic) K2EDTA Plasma/lead testing Chelates (binds) calcium
Yellow/gray and orange Thrombin Serum/chemistry Clot activator
Red/gray and gold Clot activator separation gel Serum/chemistry Silica clot activator
49

50. •During centrifugation, blood is forced into a
thixotropic inert, polymer gel (specific gravity
1.04) material located at the base of the tube.
• The gel undergoes a temporary change in
viscosity during centrifugation and lodges
between the packed cells and the top serum
layer .
Serum separator gel
50

51. Special considerations
• Stoppers may contain zinc ,interfering with its estimation
• Stoppers may contain TEBP ( tris [2 butoxyethyl ] phosphate ) a
constituent of rubber which may interfere with measurement of
certain drugs.
• With time the vacuum in evacuated tubes is lost , this may lead
to short draw and incorrect ratio of blood and anticoagulant
• The silicone coating also decays with age
• Soda lime glass tubes may release trace elements like calcium
and magnesium
•Plastic (polyethylene terphthalate) tubes are preferred over glass
tubes as they decrease likelihood of breakage and hence exposure
to infectious material.
51

52. • 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.
Additives-
Compounds added to biological specimen to prevent them
from clotting or to prevent the constituents of a specimen.
52

53. This is a special sequence of tube collection
that reduces the risk of specimen
contamination and additive carry-over.
EXCEPTION: Venous pH and ionized calcium are
to be drawn before any tubes, including
routine or fungal/AFB blood cultures
Order of draw
53

54. • Designed to deliver blood first for those tests most
affected by micro clot formation.
• This method assumes that the blood entering the
syringe last is the freshest and that this blood will be
the first blood out of the syringe during the transfer
process.
• Because the clotting process is activated the
minute the blood starts to fill the syringe, it is
important to transfer the blood quickly and to fill
anticoagulant tubes before the serum tubes.
The order of draw for the syringe system
54

55. order of draw is as follows:
1st. Sterile specimens (blood culture tubes or bottles)
2nd. Light blue stopper for coagulation studies.
3rd. Lavender stopper - EDTA
4th. Green stopper - heparin
5th. Gray stopper - oxalate/fluoride
6th. Red stopper or red/gray stopper (gold Heamogard) - non
additive and gel separator, respectively.
7th. Orange stopper - thrombin
55
The order of draw for the syringe system

56. The haemolytic sample
Hemolysis mean, the libration of hemoglobin after RBC have
ruptured.
It can occur in-vivo or in vitro. Plasma or serum assume pink to red
colour.
Cause of Hemolysis:-
1. Using too small a needle
2. Forcing the blood through needle
3. Shaking the tube or bulb too vigorously after blood collection
4. Presence of excess of anticoagulant
5. Centrifuging blood sample at high speed before completion of
clotting
6. Freezing or thawing of blood
7. Presence of water in the container

57. Chemical Test Affected By Haemolysis
1. S.potassium
2. S. inorganic Phosphorus
3. SGOT
4. SLDH
5. S. Acid Phosphatase

58. 1. Visual detection- possible above 300 mg/L
Samples are always intensely red coloured.
2. Spectrophotometric detection
For slightly haemolyzed sample, however, appropriate
sample blank and biochromatic reading can be used
to eliminate effect of hemoglobin interference.
Detection and measurement of haemoglobin in
serum or plasma
58

59. 3. Analytical measurement
In vivo vs in vitro hemolysis
• haptoglobin is reduced during intra-vessel haemolytic
process
• After in-vitro haemolysis all constituents of erythrocytes,
including potassium concentration, lactate dehydrogenase
and aspartate aminotransferase activities, increase in
addition to the concentration of free haemoglobin in
plasma or serum.
• In contrast, haptoglobin concentration in plasma/serum
of haemolytic samples remains unchanged.
59

60. • Rise of intracellular constituents in the extra-cellular space
Cell constituents with an intracellular concentration 10 times
higher than the extra-cellular concentration will increase in
plasma/serum during haemolysis (e.g. potassium,
magnesium, phosphate, lactate dehydrogenase, aspartate
aminotransferase, aldolase etc )
• Interference with analytical procedure
Adenylate kinase released from erythrocytes causes an
increase of creatine kinase and CK-MB activity especially
when inhibitors of adenylate kinase in the assay mixture are
inadequate
‣ Optical interference by haemoglobin
the colour of haemoglobin increases the absorption at a
respective wavelength or changes the blank value.
Mechanisms of interference by haemolysis
60

61. The icteric sample
• Icteric plasma contains high levels of bilirubin. Normal
levels of bilirubin are about 0.5 mg/dL. In cases of
hyperbilirubinemia, levels will exceed 1.5 mg/dL and
plasma will become affected.
• Icteric plasma samples have a high prevalence in samples
from patients in the ICU, as well as gastroenterology,
surgical and pediatric patients. Conc. of bilirubin greater
than 2.5 mg/dL can lead to clinically relevant changes of
anti-thrombin. Higher conc. can interfere with other
coagulation tests.

62. • Spectral interference
1. Bilirubin has a high absorbance between 340 nm and
500 nm wavelengths
2. The reduction of absorption as a result of oxidation
bilirubin in alkaline solution is the main cause for
bilirubin interference in modifications of the Jaffé
method without deproteinisation
3. In a strongly acid solution the absorption of
conjugated bilirubin shifts to the UV wavelengths.
Therefore bilirubin interferes in the determination of
phosphate using the phosphomolybdate method through
its reducing effect
Mechanisms of bilirubin interference
62

63. • Chemical interference –
1. Bilirubin interferes in oxidase/peroxidase based
test systems. Bilirubin reacts with H2O2 formed in the
test system which causes systematically lower results
in enzymatic procedures that are used for the
measurement of glucose, cholesterol, triglycerides,
urea and creatinine
2. Bilirubin competitively interferes with dyes binding
to albumin
63

64. • The visual inspection of plasma or serum samples for the
detection of hyperbilirubinaemia is often not sensitive
enough. This is particularly true when samples are
simultaneously stained by other pigments (e.g.
haemoglobin and its derivatives).
• Hyperbilirubinaemia is directly detected in diluted
samples that are measured at 450 and 575 nm . The
direct procedure of bilirubin measurement is only
applied for the determination of hyperbilirubinaemia in
newborns
Detection and documentation of increased bilirubin
concentrations in clinical samples
64

65. • With the nutritional supply of carotines or
carotinoids, bilirubin concentration by direct
measurement is overestimated
65

66. • Method selection -The high prevalence of
hyperbilirubinaemia in patients from intensive care,
gastroenterological surgical or paediatric
departments makes it pertinent to select analytical
methods that are less susceptible towards bilirubin
interference.
• Blanking procedures are useful to eliminate spectral
bilirubin interferences
Prevention of bilirubin interference
66

67. • A mixture of non-ionic tensides may reduce
bilirubin interference like in the
spectrophotometric determination of inorganic
phosphate using phosphomolybdate
• Immunological procedures for the measurement of
serum albumin can be used to replace dye binding
methods that are susceptible to bilirubin
interference
67

68. Lipaemia is a turbidity of serum or plasma
which is caused by elevated lipoprotein
concentrations and which is visible by the
eye.
• Post-centrifugal coagulation of serum
samples of heparinized patients can also be
the cause of turbidity.
The Lipaemic Sample
68

69. • Increased triglyceride concentration in plasma/serum. This
can be due to-
I. Food intake ( after intestinal absorption triglycerides are
present in plasma for 6 to 12 h.)
II. Altered lipid metabolism
III. Metabolic disorders causing hypertriglyceridemia
IV. lipid infusions
69
Causes of lipaemia (turbidity)

70. Identification and quantification of lipaemia
Optical and photometric methods for serum and
plasma samples
If triglyceride concentrations above 300 mg/dL
Lipaemia in plasma or serum is visually observed
70

71. • 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 chylomicrons – at 1000g
for s.lipids – at 12000g for 10 mins
• Ultra-centrifugation must be employed for the
separation of low density lipoproteins and high
density lipoproteins.
Means to avoid lipaemia and interferences caused by
turbidity
71

72. • Optical clearing systems- Commercial test kits may
contain detergents such as triton X-100, cholic and
desoxycholic acid, lipase or cholesterol esterase to
remove turbidity in plasma or serum samples.
The method of choice for removal of turbidity from
serum and plasma is a 10 min centrifugation in a
micro-centrifuge with 10 000 g.
72

73. 73
Sample collection and processing
3rd part
Dr. Urender singh
2nd yr PG Biochemistry
F MHS
SGT Medical College
Gurgaon

74. Handling of specimen for Analysis
1. Identification
2. Preservation
3. Separation and storage
4. Transport

75. 1.Identification
 Min. information-
Name, location, Identifying number,
Date and time of collection.
 No specific labeling” handling with special
care”
 All specimens should be treated as special
and potentially infectious.

76. 2.Preservation
• For these tests, must be 4 °C from start to
until the specimens are analyzed, e.g ammonia,
blood gas determinations, blood pH , acid phosphatase,
lactate, pyruvate, and certain hormone tests gastrin and
renin.
• Transfer of these specimens to the laboratory
must be done by placing the specimen
container in ice water.
• A notable decrease in pyruvate and increase
in lactate con. within a few minutes at
ambient temperature

77. • Specimens for bilirubin or carotene and for some
drugs, such as methotrexate, must be protected from
both daylight and fluorescent light to prevent
photodegradation.
• For molecular diagnostic laboratories, challenge to
recover RNA from transported specimens.
• Specimens from liver, spleen, or heart have large
amounts of RNA, but specimens from skin, muscle,
and bone have lower RNA content.
• Tissue samples should be frozen immediately.
Alternatively, a blood specimen should never be frozen
before separation of the cellular elements because of
hemolysis and released heme that may interfere.

78. • 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
of lab should not drop below 15 °C or exceed 24 °C.
3.Separation and storage
78

79.  Plasma/ serum should be separated within 2 hours.
 Premature separation of serum may permit formation
of fibrin, which can clog sampling devices in testing
equipment.
 If it is impossible to centrifuge a within 2 hours, the
specimen should be held at room temperature rather
than at 4 °C to decrease hemolysis.
 Plasma should be removed from the primary tube
promptly after centrifugation and held at −20 °C .
 Frost-free freezers should be avoided because they
have a wide temperature swing during the freeze-thaw
cycle.
 4 °C or −20 °C is not the optimum storage temperature
for all tests; some lactate dehydrogenase isoenzymes
are more stable at room temperature than at 4 °C.

80.  Specimen tubes should be centrifuged with stoppers for
reduce evaporation maintains anaerobic conditions (
carbon dioxide and ionized calcium ) and prevent
aerosolization of infectious particles.
 Removal of the stopper before centrifugation allows loss of
CO2 and increase pH (acid phosphatase )
Cryopreservation- one method to store and maintain samples
for extended periods of time of white blood cells and DNA .
Whole blood specimens can be centrifuged, and white cells
removed and cryopreserved at −20 °C until these cells are
required for DNA extraction.

81.  For longer periods of storage, isolated DNA
can be stored at-70°C.
 After these extracted DNA samples have
completely thawed, it is important to fully mix
the sample to ensure a homogeneous
specimen.
 The extracted DNA should not be exposed to
repetitive cycles of freezing and thawing
because this can lead to shearing of the DNA

82. Blood Alcohol
• Alcohol distributes into the aqueous compartments of
blood; because the water content of serum is greater than
that of whole blood, higher alcohol concentrations are
obtained with serum as compared with whole blood e.g
1:18
• So serum is the best choice of specimen.
• Specimens should be kept capped to avoid evaporative
loss.
• Blood may be stored, when properly sealed, for 14 days at
room temperature or at 4 °C without preservative.
• For longer storage or for nonsterile postmortem
specimens, sodium fluoride should be used as a
preservative.

83. Estimation of Alcohol consumed
• Dr. Erik M.P.Widmark, a Swedish physician,
N =W• ρ •[Ct + β • t]/(d •Z)
N = number of drinks
W = body weight (kg)
ρ (rho) = volume of distribution (L/kg) (0.68 for males, 0.55 for
females)
Ct = blood alcohol concentration (kg/L)
β = rate of ethanol elimination (0.15 g/L/h)
t = time since first drink (h)
d = specific gravity of alcohol (0.8)
Z = amount of ethanol alcohol per drink (L) (15 mL of ethanol in
a standard drink)
• Frequently the time since the first drink is unknown; the
formula can be modified.
N =W• ρ •[Ct ]/(d •Z)

84. 4.Transport
• We assumed that transport time is as 72 hours.
• The specimen tube should be good constructed.
• Reduced pressure of 0.50 atmosphere (50 kPa) may be
encountered during air transport.
• Polypropylene and polyethylene containers are usually
suitable for specimen transport. Glass should be
avoided.
• Not use Polystyrene tube because it may crack when
frozen.

85. • Sample delivery for molecular analysis ( usually DNA
only) will be delayed due to extracted nucleic acid,
can be transported in a buffer solution or water, or
it can be dried down and shipped as a loose
powder.
• DNA should be transported at ambient
temperatures and should not be exposed to
extremely high temperatures because it will begin
to degrade.

86. • Corrugated, fiberboard, or Styrofoam boxes
designed to fit around a single specimen tube are
commonly used. A padded shipping envelope
provides adequate protection for shipping single
specimens.
• When specimens are shipped as drops of blood on
filter paper (e.g., for neonatal screening), the paper
should be enclosed in a paper envelope to ensure
that the sample remains dry.

87. • For transport of frozen or refrigerated specimens,
a Styrofoam container should be used. The
container walls should be 1 inch (2.5 cm) thick to
provide effective insulation.
• The container should be vented to prevent
buildup of carbon dioxide under pressure and a
possible explosion. Solid carbon dioxide (dry ice)
is the most convenient refrigerant material for
keeping specimens frozen, and temperatures as
low as −70 °C can be achieved.

88. 88
Other biological samples

89. • Random –for spot glucose, ketone bodies ,molecular
testing for infectious agents such as Chlamydia(STD)
• First-morning sample- most conc. and has a lower pH due
to decreased respiration during sleep – used for
microscopic examination , for proteins and chorionic
Gonodotropin measurement.
• Midstream specimen-best for investigating bladder
disorders
Urine
89

90. 90
Timed specimen- obtained at:-
1. specific times of day (urobilinogen 2-4 PM )
2. during certain phases of micturition e.g. first 10 ml
for urethritis , midstream for bladder diseases
3.In some metabolic disorders, urine must be collected during or
immediately after symptoms of the disease appear- Acute Neurovisceral
Attack in Porphyria is measurement of urinary PBG( porphobilinogen)
and Lead exposure increases urinary ALA ( alfa linolenic acid).
24-hour total volume collection-
1 .The preferred container should be 4-L , plastic, and clean with the
correct preservative added.
2. Instruct the patient to start with an empty bladder and to end with an
empty bladder

91.  Creatinine excretion is based on muscle mass, and
since a patient's muscle mass is relatively constant,
creatinine excretion is also reasonably constant.
Therefore, one can measure creatinine on several 24-
hour collections to assess the completeness of the
specimen and keep this as part of the patient's record.
Catheter specimen – urine examination for critically ill
and urinary tract obstruction.
91

92. 92
IMPORTENT
Urine should not be collected at the same time for two or
more tests requiring different preservatives.
Aliquots for an analysis such as a microscopic examination
should not be removed while a 24-hour collection is in
process.
Removal of aliquots is not permissible even when the
volume removed is measured and corrected, because the
specific gravity, volume, and composition of the urine varies
throughout the day, and test results will be affected.

93. Collection of Urine from Children
• For Infant scrotal or perineal area is cleaned and
dried first, and skin oils are removed. A plastic U-Bag
is placed around the infant’s genital area.
• To obtain a sterile urine specimen for culture from an
infant, a suprapubic tap is performed.

94. Urine Ethanol
•Urine has been used as an alternative, less invasive specimen
for the determination of alcohol use.
•During the postabsorptive phase following alcohol ingestion,
the concentration of alcohol in urine is roughly 1.3 times that
in blood.
•However, the use of urine alcohol measurements to estimate
blood concentrations is discouraged because the ratio of 1.3
is highly variable.
•The detection of alcohol in urine represents ingestion of
alcohol within the previous 8 to 12 hours.

95. Result Reason
Change in color Breakdown or alteration of chromogen or other
urine constituent (e.g. hemoglobin, melanin,
homogentisic acid, porphyrins)
Changes in odor Bacterial growth, decomposition
Increased turbidity Increased bacteria, crystal formation,
precipitation of amorphous material
Falsely low pH Glucose converted to acids and alcohols by
bacteria
Falsely elevated pH Breakdown of urea by bacteria forming
ammonia
False negative glucose Utilization by bacteria (glycolysis)
95

96. False negative ketone Volatilization of acetone. Breakdown of
acetoacetate by bacteria
False negative bilirubin Destroyed by light. Oxidation to biliverdin
False negative urobilinogen Destroyed by light
False positive nitrite Nitrite produced by bacteria after specimen is
voided
False negative nitrite Nitrite converts to nitrogen and evaporates
Increased bacteriuria Bacteria multiply in specimen before analysis
Disintegration of cells/casts Unstable environment, especially in alkaline
urine, hypotonic urine or both
96

97. Preservative for 24 hr sample Tests
None (refrigerate) Amino acids, amylase, chloride, copper, creatinine,
glucose, heavy metals (arsenic, lead, mercury),
histamine, immunoelectrophoresis, lysozyme,
methylmalonic acid, microalbumin,
mucopolysaccharides, porphobilinogen, porphyrins,
potassium, protein, protein electrophoresis, sodium,
urea, uric acid, xylose tolerance
10 g boric acid Aldosterone, cortisol
10 mL 6N HCL Calcium, catecholamines, citrate, cystine,
homovanilic acid, hydroxyproline, magnesium,
metanephrines, oxalate, phosphorus,
0.5 g sodium fluoride Glucose
Equal amounts of 50% alcohol,
Saccomanno's fixative, SurePath or Preserve
CT
Cytologic examination
97

98. • For occult blood- “the presence of a bleeding ulcer or malignant
disease in the gastrointestinal tract.
• In the newborn, the first specimen from the bowel (meconium)
may be used for detection of maternal drug use during the
pregnancy .
• Screening for tryptic activity (cystic fibrosis in children)
• For analysis of Polio-melliteus virus.
• Fecal nitrogen and fat in 72 hour specimen in malabsorption.
• Differential and quantitative analysis of stool DNA integrity
proposed as biomarker for detection of colorectal cancer
• No preservatives required.
• Transport specimen in cold ice box.
Faeces
98

99.  Obtained usually by lumbar puncture.
USE
 Diagnosis of meningitis, CVA, metastasis
 In molecular diagnosis for rapid identification of infectious
agent and T and B cell gene rearrangement in
hematological malignancies.
PROCESSING
 Rapid processing is a clinical requirement. So
antiglycolytic agents usually not added.
99
CEREBROSPINAL FLUID

100. • Prenatal diagnosis of congenital disorders- fetal
maturity, Rh isoimmunization, intrauterine
infection, cystic fibrosis, sickle cell anemia, Tay-
Sachs disease, and thalassemia.
• 10 ml aspirated in syringe
• Sterile containers, such as polypropylene test
tubes or urine cups, are used to transport the fluid
to the laboratory.
Amniotic fluid
100

101. Synovial Fluid
 Synovial fluid is a clear thixotropic fluid that
serves as a lubricant in a joint, tendon sheath, or
bursa.
 Sterile plain tubes- culture ,glucose and protein
measurements.
 EDTA tube- total leukocyte, differential, and
erythrocyte counts.
 Microscopic slides are prepared for staining with
Gram’s or other stains indicated, and for visual
inspection.

102. Pleural, Pericardial, and Ascitic Fluids
• The fluid may be removed to determine whether it
is an effusion or an exudate—a distinction made
possible by protein or enzyme analysis.
• The fluid may also be examined for cellular
elements.
• In the molecular diagnostic laboratory identify the
infectious agent.
• Possibly for the detection of cancer cells.

103. 1. Measurement of blood group substance
2. Determination of secretor status
3. Saliva can be used as a source of DNA or RNA.
4. Ethanol measurements and for the detection of drugs of abuse,
but it is not a frequently used sample for ethanol
determinations
103
Saliva

104. Buccal Cells
• Collection of buccal cells has been identified
as providing an excellent source of genomic
DNA
• useful after blood transfusions and bone
marrow transplantation.
• collect buccal cells: rinsing with mouthwash
and using swabs or cytobrushes.

105. Malignant tissue for estrogen ,
progesterone receptors 0.5 – 1 gm
 Frozen quickly, within 20 minutes –
liquid nitrogen/ dry ice +alcohol
105
Solid tissue

106. Breath Ethanol
• The fundamental principle for use of breath
analysis is that alcohol in capillary alveolar blood
rapidly equilibrates with alveolar air in a ratio of
approximately 2100 : 1 (blood : breath).
• Before breath alcohol analysis, a deprivation
period of 15 minutes is required to allow for
clearance of any residual alcohol that may have
been present in the mouth e.g. very recent
drinking, use of alcohol-containing mouthwash,
vomiting of alcohol-rich gastric fluid.

107. Thank you!
107