Pre-Analytic Issues in

Laboratory Medicine

Mr.Sreekanth P G
Medical Biochemist

How do you put a giraffe in a

refrigerator?
How do you put an elephant in a
refrigerator?
When the lion called a meeting of the
animal kingdom, who didn't
participate ?

And the correct answers are...

To put a giraffe in a refrigerator, you open

the door, put the giraffe in, and close the

door.
To put an elephant in a refrigerator, you
open the door, take the giraffe out, then put
the elephant in.
When the lion called the meeting of the
animal kingdom, the elephant couldn't come
because he was in the refrigerator

 Logic is not always so logical.

Common sense is not so commonly found
Laboratory errors won't go away just
because it seems logical that there should
be fewer errors with more expensive
equipment, more computerization, and more
automation.

Pre-Analytic Issues in
Laboratory Medicine

Mr.Sreekanth P G

Laboratory Testing
Impacts Nearly Everyone

Infant

Child

Teen

Adult

Senior

Accurate, reliable lab testing is essential

to all aspects of health care

ERRORS
May occur
Can not be eliminated but can be
minimised

QUALITY ASSUARANCE
The practice which encompasses all
eneavors, procedures, formats and
activities directed towards ensuring that
aspecified quality product achieved and
maintained

Quality System

Quality Assurance

Quality Control

Phases of Testing
Pre-Analytical
Test Ordering, Specimen Collection, Specimen
Handling
Analytical
Test Performance, Quality Control, Result
Review
Post-Analytical
Result handling, Result Communication, Result
Interpretation

LABORATORY TESTING PROCESSES AND THEIR

POTENTIAL ERRORS
PRE ANALYTICAL ERRORS
PROCESS
POTENTIAL ERRORS
Test ordering inappropriate test
Handwriting not legible
Wrong patients ID
Special requirements not specified
Specimen acquisition
Incorrect tube or container
Incorrect patient ID
Inadequate volume
Invalid specimen
(hemolysed or diluted)
Collected at wrong time
Improper transport conditions

ANALYTICAL ERRORS
Analytical
Instrument not calibrated Measurement
correctly
Specimens mix up
Incorrect volume of specimen
Interfering substances present
Instrument precision problem
Test reporting

Wrong patient ID
Report not legible
Report delayed
Transcription error

POST ANALYTICAL ERRORS

Test interpretation Interfering substance not
recognized
Specificity of the test not understood
Precision limitation not recognized
Analytical sensitivity not appropriate
Previous values not available for
comparison

Why are Pre-Analytical Issues

Important?
Abnormal
test results usually are attributed to
disease.
Important considerations in interpreting laboratory
results include preanalytical and biological
variation.
Preanalytical variation is due to factors external to
the patient affecting laboratory specimens prior to
testing. Controllable
Biological Variation is due to factors inherent to
the patient that may or may not be controllable

Patient Identification
It is important to identify a patient properly so

that
blood is collected from the correct person.
.1% - 1% of specimens are from the wrong
patient.
Two patient identifiers should be used.
Hospital inpatients should be wearing an
identification band
Test forms should be compared to the inpatient's
wrist bracelet or verbally confirmed with an
outpatient.

Avoiding Specimen Labeling Errors

Match patient identification on the labels to the order

and
patient ID using two identifiers
Draw and label specimens at the bedside, one patient
at a time
Affix proper specimen labels to the collection tubes
immediately after specimen collection Do not draw extra
unlabeled tubes
The person who collects the specimen should label the
specimen
Avoid secondary labeling where the specimen is
labeled by hand and then printed labels are attached
later.

PHLEBOTOMY
In the 20th century

phlebotomy was introduced

as a diagnostic tool

Prior to that it was considered

to be curative

Specimen Collection
Postural Effects
Collection Tubes and Additives
Affect of Tourniquet Time
Collection from IVs and Catheters
Volume effects
Avoiding Clots
Avoiding Hemolysis

Postural Effects
Change in posture from supine to erect or

sitting causes a shift in fluid from the

intravascular to the interstitial space
An increase of 5% to 15% is seen for most
cellular and
macromolecular analytes when specimens are
collected
sitting as compared to supine.
Conversely, moving from upright to supine can
have a
dilutional effect owing to an increase in plasma
volume
The effect of postural change is accentuated in
patients with a tendency to edema.

Postural Effects
Albumin levels are higher among healthy outpatients as
compared to supine healthy hospitalized subjects
Glucose (and other small molecules) move freely
between the
interstitial space and the circulation and are least affected
by posture during blood specimen collection.
While the free fraction of a metabolite, drug, hormone,
or metal ion is not subject to postural variation, the
fraction bound to proteins is affected by posture.
Thus, bilirubin bound to albumin and calcium bound to
albumin are affected by postural changes.
A change from upright to supine can reduce (after 5
minutes) cholesterol level by 10% and triglyceride by
12%.

Site Preparation:
Before performing the venipuncture, the alcohol should
be allowed to air dry. This will help to ensure that the specimen is not
contaminated with alcohol, as this can lead to hemolysis. Hemolysis
can result in the spurious elevation of such analytes as potassium,
lactate dehydrogenase (LD), iron and magnesium in the chemistry
lab.
Proper Venipuncture Technique:
During phlebotomy, avoid probing
to find the vein and achieve blood flow. Excessive probing and/or
fishing to find a vein can result in a poor quality sample, including
hemolysis.
Order of Draw: Following the correct order of draw during
venipuncture will help to ensure accurate test results.)

 Plastic versus Glass Tubes

Plastic tubes have replaced glass tubes for
most applications
Less breakage, cheaper and lower weight
Clot activators needed to be added to serum
tubes
Give clinically equivalent results for almost
all analytes

Collection Tube Additives

Heparin
EDTA
Citrate
NaF + K Oxalate
Clot Activator
Serum Separator

First tests known

Diabetes
Patient urinates on the floor. If
the urine contains sugar, ants
will crawl to lick the urine. This
test was used up to 20 years ago

 Heparin
Used to collect whole blood or plasma
Binds to anti-thrombin III to inhibit Xa,
IXa, and thrombin
Nominal concentration of 12 30 U/mL
Heparin binds calcium so ionized
calcium must be collected using Calcium
Titrated or Electrolyte Balanced heparin

EDTA
K2EDTA is used to collect whole blood
for hematology studies and plasma for
analytes with heparin interference
Acts by binding calcium
Nominal concentration of 1.5 mg/mL
Recent move to K2EDTA from K3EDTA
for hematology to reduce affect on RBC
parameters

EDTA Effects
EDTA is hyperosmolar causing cell
shrinkage but the
low pH of EDTA counterbalances this effect
by causing K to flow into cells.
EDTA may cause platelet clumping and
platelet satellitism
Sodium citrate tubes are sometimes
collected to obtain more accurate platelet
counts.

 When doing a platelet count (using an ETDA anticoagulant blood

sample) a low platelet count is obtained with some samples. This due
to platelet clumps showing many platelets as ONE or gating these
platelets out because their clumped size is close to that of a micro
RBC.
Platelet satelitism is another suspect.
When these patients samples are repeated using a CITRATE sample
instead,
TWO different categories emerge: a- A normal platelet count is
obtained with a so-called "EDTA artefact patients".
The other blood indices are inaccurate - ONLY the platelet count is
correct. The CBC should be reported with the CITRATE platelets and
EDTA WBC & RBC data.
b- A low platelet count is obtained (almost identical to EDTA
samples). This is a genuine LOW platelet count that can be
confirmed with both slide viewing and manual counting of platelets.
So, before reporting that platelet count as LOW: Check the sample
for micro clots. Make a blood film and check if they are really low.
Check it with a CITRATE sample (to get a count if it is normal). DO
NOT REPORT CBC OF A CITRATED SAMPLE - only the Platelet
count!

Citrate
Citrate is most often used for collection of
coagulation tests
Acts by binding calcium
Nominal concentration of 3.2% (mol/L)
Recent move to 3.2% from 3.8% to get
more consistent results for Prothrombin
Time, particularly for more sensitive reagents
Tubes must be properly filled to within +/10 percent of assigned collection volume

NaF + K Oxalate
NaF + K Oxalate is used to poison glycolytic

pathway and to anti-coagulate specimens for glucose

testing
Glucose decreases by 5 7% per hour in
specimens from adults and by up to 24% per hour in
specimens from neonates and with very high white
counts
Delay in action leads to approximately 9 mg/dL
loss over the first 3 hours after collection
Causes a great deal of hemolysis and not suitable
for other testing

Clot Activator
In vitro activation of clotting system to
enhance clot formation
Tubes contain a silica clot activator attached to
the wall with a silicone surfactant
Requires inversion of tube for optimal
function
Requires 15 to 30 minutes (instead of 1 hour)
to complete clot formation.

Serum Separator Gel

Polymer gel with specific gravity between
that of serum (or plasma) and cells
Migrates and forms a barrier during
centrifugation
Certain analytes and therapeutic drugs
may bind to gel over time

Duration of Tourniquet
Application
Application of the tourniquet for >1 minute can result in

hemoconcentration, causing an increase in the

concentration of large molecules (e.g. serum proteins)
that are unable to pass through the capillary wall.

Total protein, iron, total lipids and cholesterol increase

from 5%-7%, bilirubin increases 8% and AST 9%

Prolonged tourniquet application also promotes

anaerobic glycolysis resulting in an increase in plasma

lactate, a reduction in blood pH, and an increase in blood
potassium.

Repeated fist clenching during phlebotomy can also

cause a 1 2 mEq/L increase in potassium.

Collection from IVs and

Catheters
Blood should not be collected proximal to an IV site
but preferably from the other arm
Heparin may contaminate specimens collected from
central lines unless flushed out with blood
High glucose and/or low electrolyte values may result
from collecting blood an IV or Central Line
If questionable results are obtained from a sample
collected through a catheter, the results should be
verified by sending a new sample drawn from a
different site

Collection from IVs and

Catheters
If a syringe is used, a small volume (<=10 mL) syringe
is recommended so that clotting in the syringe during
phlebotomy is avoided.

If samples must be obtained from a catheter, heparin

contamination and dilution must be avoided. The line
should be flushed with 5 mL of saline and the first 5 mL of
blood or six dead space volumes of the catheter discarded.

Order of Draw

Syringe Collection
Visual hemolysis was found in 19% of specimens drawn by

syringe,
compared to 3% when drawn by an evacuated tube system.
Also, 11% of syringe-collected EDTA samples exhibited clots
Following can reduce the incidence of hemolysis:
Pump the plunger 2-3 times prior to collection to loosen the
plunger.
Use a 3-10 mL syringe
Ensure that the speed of aspiration does not exceed 1mL of
air space during collection. Excessive aspiration forces cause
hemolysis.
Transfer the blood to the tubes immediately.
Fill tube by vacuum only. NEVER push down on the plunger;
this increases the force of the blood flow, creating a high
degree of red blood cell trauma.
Use a blood transfer device to transfer syringe-collected blood
into a tube. It will enhance safety and improve specimen quality.

Collection Volume
Overfilled tubes
Under filled coagulation tube
Under filled hematology tube
Under filling occurs because:
Tube was removed too quickly
Tube slips back from vacutainer needle
Air drawn in from butterfly or connector
tubing

Avoiding Clots
Use a sufficient amount of the correct
anticoagulant
Mix specimen thoroughly after
collection
Transfer immediately from syringe to
tube
Do not overfill tubes

Avoiding Hemolysis

Allow alcohol to dry before collection

Use a larger bore needle
Mix gently
Avoid syringe collection if possible
Avoid collection from IVs and catheters
Draw slowly when collecting with syringes or
from catheters
Transport to lab and centrifuge in a timely
fashion

Arterial Blood Gases

Avoid air contamination from a bubble or uncapped

specimen
Delay in analysis can cause high pO2 to fall or low pO2 to
rise
Analyze within 30 minutes or place on ice and analyze within
1 hour.
Ca2+ binding by heparin can be minimized by using either of
the following:
(1) A final concentration of sodium or lithium heparinate of
15 IU/ml blood or less
(2) Calcium titrated heparin with a final concentration of less
than 50 IU/ml blood.
Heparin Dilution effect can be avoided by use of dry heparin
Roll specimen to mix heparin and reduce clots

Specimen Transport
Specimens should be delivered to the
laboratory promptly after collection
Specimens should not be placed on ice
unless specified by the laboratory
Pneumatic tube transport does not affect
analytical results

Primary Causes of Hemolysis

Incomplete drying of skin after cleaning with alcohol
Vigorous suction with syringe
Inappropriate (too small) needle with syringe or

vacutainer
Forcing blood from syringe into tube when it has
started to
clot
Shaking of tube instead of gentle agitation or inversion
Inadequate packing in pneumatic tube container during
transport to the laboratory to prevent shaking
Prolonged contact of plasma or serum with cells
Chilling (or freezing) of whole blood specimens
For skin puncture specimens, squeezing tissues too
hard
during collection

Fibrin Interference
Residual fibrin, long recognized as a possible interferent in
the clinical laboratory, may be present as a result of
improper specimen handling during and after collection.
It can be present in primary tube samples either as a visible
clot, which may physically occlude the instrument
sample probe or,as an invisible microfiber or as strands.
Fibrin strands,though invisible, may directly affect some
assays, especially immunoassays.
Fibrin interference is usually not reproducible and
disappears with time as the fibrin settles out of the
sample.
Care taken during the preanalytical phase can help to
reduce the presence of fibrin strands in the processed
specimen.

 Stability for Whole Blood, Serum and

Plasma: A whole blood specimen that is
going to be spun down should be centrifuged
and the serum or plasma removed from the
red blood cells within two hours after the
venipuncture.
The sample will be stable at room
temperature for eight hours, and up to 48
hours at 2-4 degrees C.
After 48 hours, the serum specimen should
be frozen at 20 degrees C in an aliquot tube

Assembled venipuncture set;

Various tube holders used in
venipuncture

Blood Tubes

Figure 2-3
Venipuncture
1

Blood collection tube with gel

separator before (left) and after
(right) centrifugation

Bloo
d

Gel

Seru
m
Gel
Clotte
d
blood

Acceptable sites for skin

puncture to collect blood from
an infants foot.

NO
!

Yes

Figure 2-6
Microcollection tubes

Table 2-3
Differences in Composition between Plasma and Serum
Value Ratio

Analyte

Plasma value greater than

serum value

Calcium
Chloride
Lactate dehydrogenase
Total protein

No difference between
serum and plasma values

Bilirubin
Cholesterol
Creatinine

Plasma value less than

serum value

Albumin
Alkaline phosphatase
Aspartate aminotransferase
Bicarbonate
Creatin kinase
Glucose
Phosphate
Potassium
Sodium
Urea
Uric acid

Percent (%)
0.9
0.2
2.7
4.0

1.3
1.6
0.9
1.8
2.1
5.1
7.0
8.4
0.1
0.6
2.0

Table 2-4
Difference
in
composition
between
Value Ratios
Analyte
Percent (%)
capillary
andthan
venous
serum
Capillary
value greater
Glucose
1.4
venous value

Potassium

No difference between
capillary and venous values

Phosphate
Urea

Capillary value less than

venous value

Bilirubin
Calcium
Chloride
Sodium
Total protein

0.9

5.0
4.6
1.8
2.3
3.3

Common sense in
Laboratory Practice
Mr.Sreekanth P G
Lecturer In Medical Biochemistry
University of Calicut

How do you put a giraffe in a

refrigerator?
How do you put an elephant in a
refrigerator?
When the lion called a meeting of the
animal kingdom, who didn't
participate ?

And the correct answers are...

To put a giraffe in a refrigerator, you open

the door, put the giraffe in, and close the

door.
To put an elephant in a refrigerator, you
open the door, take the giraffe out, then put
the elephant in.
When the lion called the meeting of the
animal kingdom, the elephant couldn't come
because he was in the refrigerator

 Logic is not always so logical.

Common sense is not so commonly found
Laboratory errors won't go away just
because it seems logical that there should
be fewer errors with more expensive
equipment, more computerization, and more
automation.

Laboratories should focus their

efforts on pre and post analytic
errors rather than analytic errors.
WRONG!
All errors are critical.
Laboratories need to expand their
monitoring to cover pre and post analytic
errors, but must continue to monitor
analytic errors.

Glucose
320 mg/dl vs 640 mg/dl
Tendancy to normalise
Standards /calibrators
Possible upper value?

Linearity
instrument
reagent
Dilutions solve the issue
undiluted
dilution
1/10 dilution

500
500
250
300
50 60

Reducing the time

End point assays
Assumes the reaction is complete
Not acceptable for high conc.
Kinetic assays- Never
Reaction rate /min
Delta A * factor

Calcium
Tourniquet
Contaminated glassware
Use acid washed tubes /disposable plastic
tubes

Urine M/E
Pus cells 6 10/HPF

15-20/HPF
Volume
Centrifugation

HDL Direct Vs Precipitation

Low values with direct method
Speed and time Centrifugation
Creatininine
Bilirubin
Antibiotics
urea: Creat ratio

 Bilirubin and Urine BS BP

Na +, K+
CKMB > CPK
Checking Reagent OD

 Blood Glucose
1100 mg/dl
CPK
6000 U/L
RA Factor
2000 IU/ml
Bilirubin
32 mg/dl
Creatinine
25 mg/dl
ASO
2000 IU/ml
K

 Lab should operate at high standards of

proffessional and technical competance in
the interest of patients , society and
government

Quality Assurance in
Healthcare
The Right result, at the
Right time,
on the
Right specimen, from the
Right patient, with result interpretation
based on
Correct Reference data, and at the
Right price

 Quality assurance in healthcare is a

modern MYTH
- a Mighty Yearning (by the public),
Testimony (by healthcare providers), and
Hope (by all of us) that things will work
okay if and when we need medical care.

WHO IS RESPONSIBLE FOR

QUALITY?

To be responsible for Quality in

YOUR LABORATORY

 How do you get to the other side of a river

infested with crocadiles?

 Answer - you jump into the river and swim

to the other side. This is the right method
because all crocodiles are at the meeting
called by the lion. Don't you remember?