Pre-Analytic Issues in Pre-Analytic or Extra-Terrestrial?

Laboratory Medicine

Daniel J. Fink, MD, MPH

Director, Core Laboratory
New York Presbyterian hospital
Columbia University Medical Center

October 3, 2005

Iatrogenic Green Plasma due to

Pre-Analytic Issues in Isosulfan Blue Dye
Laboratory Medicine

The lab made a mistake!

Pre-Analytic Issues in Phases of Testing

Laboratory Medicine
• Pre-Analytical
– Test Ordering, Specimen Collection, Specimen
Handling
• Analytical
– Test Performance, Quality Control, Result Review
• Post-Analytical
– Result handling, Result Communication, Result
Interpretation

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Why are Pre-Analytical Issues Important? Patient Identification
• Abnormal test results usually are attributed to
disease. This is not always the case. • It is important to identify a patient properly so that
blood is collected from the correct person.
• Important considerations in interpreting laboratory
results include preanalytical and biological variation. • .1% - 1% of specimens are from the wrong patient.
• Two patient identifiers should be used.
• Preanalytical variation is due to factors external to
the patient affecting laboratory specimens prior to • Hospital inpatients should be wearing an identification
testing. Being aware of these factors and following band which should be checked before the blood is
proper procedures will minimize these effects, drawn. Blood should not be drawn from a patient
allowing for more reproducible and accurate results. without a band.

• Biological Variation is due to factors inherent to the • Test forms should be compared to the inpatient's wrist
patient that may or may not be controllable bracelet or verbally confirmed with an outpatient.

JCAHO 2005 Laboratory Services

Biologic Variability National Patient Safety Goals

• Age, sex, race

– Alkaline phosphatase in children Goal: Improve the accuracy of patient identification.
– Neutrophil counts in neonates
– Creatinine, CK in females, blacks
• Diurnal variation Use at least two patient identifiers (neither to be the patient's
– Glucose values obtained during an oral glucose tolerance test tend to be
higher when the test is performed in the afternoon than when the test is location) whenever collecting laboratory samples or
performed in the morning. administering medications or blood products,
• Diet
– High-protein and high-purine diet increase levels of uric acid, urea, and
ammonia in blood compared with vegetarians
Use two identifiers to label sample collection containers in the
• Smoking
– Long-term smoking increases carboxyhemoglobin, hemoglobin, RBC,
presence of the patient.
WBC and MCV values;
– WBC level related to number of packs smoked

Test Ordering Avoiding Specimen Labeling Errors

• Order the relevant test • Match patient identification on the labels to the order and
patient ID using two identifiers
• Know when to order • Draw and label specimens at the bedside, one patient at a time
• Know how often to order • Affix proper specimen labels to the collection tubes
immediately after specimen collection (do not place drawn
• Know how the results will be used tubes in a cup or emesis basin and proceed to another task
before affixing labels)
• Consult with the laboratory director • 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.

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 Specimen Collection Plastic versus Glass Tubes

• Postural Effects • Plastic tubes have replaced glass tubes for

• Collection Tubes and Additives most applications
• Affect of Tourniquet Time • Less breakage, cheaper and lower weight
• Collection from IVs and Catheters • Clot activators needed to be added to serum
• Volume effects tubes
• Avoiding Clots • Give clinically equivalent results for almost
all analytes
• Avoiding Hemolysis

Postural Effects Collection Tube Additives

• Change in posture from supine to erect or sitting causes a

shift in fluid from the intravascular to the interstitial space • Heparin
of about 12%. • EDTA
• An increase of 5% to 15% is seen for most cellular and • Citrate
macromolecular analytes when specimens are collected
• NaF + K Oxalate
sitting as compared to supine.
• Clot Activator
• Conversely, moving from upright to supine can have a
dilutional effect owing to an increase in plasma volume • Serum Separator
• The effect of postural change is accentuated in patients with
a tendency to edema.

Postural Effects Heparin

• Albumin levels are higher among healthy outpatients as

compared to supine healthy hospitalized subjects • Used to collect whole blood or plasma
• Glucose (and other small molecules) move freely between the • Binds to anti-thrombin III to inhibit Xa, IXa, and
interstitial space and the circulation and are least affected by
posture during blood specimen collection. thrombin
• While the free fraction of a metabolite, drug, hormone, or
metal ion is not subject to postural variation, the fraction • Nominal concentration of 12 – 30 U/mL
bound to proteins is affected by posture. Thus, bilirubin
bound to albumin and calcium bound to albumin are affected • Heparin binds calcium so ionized calcium must be
by postural changes. collected using “Calcium Titrated” or “Electrolyte
• A change from upright to supine can reduce (after 5 minutes) Balanced” heparin
cholesterol level by 10% and triglyceride by 12%.

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 EDTA NaF + K Oxalate

• K2EDTA is used to collect whole blood for • NaF + K Oxalate is used to poison glycolytic
hematology studies and plasma for analytes with pathway and to anti-coagulate specimens for glucose
testing
heparin interference
• Glucose decreases by 5 – 7% per hour in specimens
• Acts by binding calcium from adults and by up to 24% per hour in specimens
• Nominal concentration of 1.5 mg/mL from neonates and with very high white counts
• Recent move to K2EDTA from K3EDTA for • Delay in action leads to approximately 9 mg/dL loss
over the first 3 hours after collection
hematology to reduce affect on RBC parameters
• Causes a great deal of hemolysis and not suitable for
other testing

EDTA Effects Clot Activator

• EDTA is hyperosmolar causing cell shrinkage but the • In vitro activation of clotting system to
low pH of EDTA counterbalances this effect by
causing K (and water) to flow into cells.
enhance clot formation
• EDTA may cause platelet clumping and platelet • Tubes contain a silica clot activator attached to
satellitism that may be the result of changes in the the wall with a silicone surfactant
membrane structure occurring when the calcium ion
is removed by the chelating agent, allowing the • Requires inversion of tube for optimal
binding of pre-formed antibodies. function
• Sodium citrate tubes are sometimes collected to • Requires 15 to 30 minutes (instead of 1 hour)
obtain more accurate platelet counts. to complete clot formation.

Citrate Serum Separator Gel

• Citrate is most often used for collection of • Polymer gel with specific gravity between that
coagulation tests of serum (or plasma) and cells
• Acts by binding calcium
• Migrates and forms a barrier during
• Nominal concentration of 3.2% (mol/L)
centrifugation
• Recent move to 3.2% from 3.8% to get more
consistent results for Prothrombin Time, • Certain analytes and therapeutic drugs may
particularly for more sensitive reagents bind to gel over time
• Tubes must be properly filled to within +/- 10
percent of assigned collection volume

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Specialized Additives for Blood Collection Duration of Tourniquet Application
Glycolytic inhibitors
• NaF (2.5 mg/mL of blood with EDTA [1 mg/mL] or potassium oxalate [2 mg/mL]) • Application of the tourniquet for >1 minute can result in
• Lithium iodoacetate (0.5 mg/mL of blood) alone or in combination with lithium heparin (14.3 U/mL of blood)
• Sodium fluoride and lithium iodoacetate require 3 h to fully become effective hemoconcentration, causing an increase in the concentration
• Adding mannose (3 mg/mL of blood) to NaF achieves efficient glycolytic inhibition; Mannose, causes
concentration-dependent interference of large molecules (e.g. serum proteins) that are unable to pass
• Maintaining blood pH at 5.3-5.9 with mixture of citric acid, trisodium citrate, disodium EDTA, and NaF
stabilizes glucose through the capillary wall.
Cell stabilizers
• Acid-citrate dextrose A and B formulations; B formulation has greater amounts of dextrose available to
• Total protein, iron, total lipids and cholesterol increase from
metabolizing cells
• Citrate, theophylline, adenosine, dipyridamole mixture minimizes in vitro platelet activation
5%-7%, bilirubin increases 8% and AST 9%
Proteolytic enzyme inhibitors • Prolonged tourniquet application also promotes anaerobic
• EDTA (1.5 mg/mL of blood) with aprotinin (500-2,000 KIU/mL) stabilizes several polypeptide hormones
• Aprotinin also can be used with lithium heparin (14.3 U/mL) glycolysis resulting in an increase in plasma lactate, a
• A mixture of EDTA, aprotinin, leupeptin, and pepstatin stabilizes parathyroid hormone–related protein
reduction in blood pH, and an increase in blood potassium.
Catecholamine stabilizers
• An antioxidant such as glutathione, sodium metabisulfite, or ascorbic acid at a concentration of 1.5 mg/mL is
used with egtazic acid, EDTA, or heparin
• Repeated fist clenching during phlebotomy can also cause a
• For urine, use 5 mL of a 6 Molar HCl per liter of urine or 250 mg each of EDTA and sodium metabisulfite per
liter of urine
1 – 2 mEq/L increase in potassium.

Collection Tube Additives Collection from IVs and Catheters

• Heparin • Blood should not be collected proximal to an IV site

but preferably from the other arm
• EDTA
• Heparin may contaminate specimens collected from
• Citrate central lines unless flushed out with blood
• NaF + K Oxalate • High glucose and/or low electrolyte values may result
from collecting blood an IV or Central Line
• Clot Activator
• If questionable results are obtained from a sample
• Serum Separator collected through a catheter, the results should be
verified by sending a new sample drawn from a
different site

Order of Draw 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.

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 Example of Contamination with IV Fluid Avoiding Clots

REFERENCE
TEST RESULTS UNITS
INTERVAL • Use a sufficient amount of the correct
Yesterday Today
Sodium 131 108 135-145 mmol/L
anticoagulant
Potassium 4.0 3.0 3.5-5.0 mmol/L • Mix specimen thoroughly after collection
Chloride 87 73 98-110 mmol/L
• Transfer immediately from syringe to tube
Bicarbonate 26 23 22-32 mmol/L
Urea 44.8 38.4 10-20 mg/dL • Do not overfill tubes
Creatinine 1.5 1.3 0.7-1.3 mg/dL
Glucose 108 149 65-110 mg/dL
Protein 7.9 6.1 6.0-8.0 g/dL
Albumin 3.6 2.8 3.5-5.0 g/dL

Syringe Collection Avoiding Hemolysis

• Visual hemolysis was found in 19% of specimens drawn by syringe,

compared to 3% when drawn by an evacuated tube system. • Allow alcohol to dry before collection
• Also, 11% of syringe-collected EDTA samples exhibited clots • Use a larger bore needle
• If a syringe is used, the following can reduce the incidence of hemolysis:
– Pump the plunger 2-3 times prior to collection to loosen the plunger. • Mix gently
– Use a 3-10 mL syringe • Avoid syringe collection if possible
– Ensure that the speed of aspiration does not exceed 1mL of air space
during collection. Excessive aspiration forces cause hemolysis. • Avoid collection from IVs and catheters
– Transfer the blood to the tubes immediately.
– Fill tube by vacuum only. NEVER push down on the plunger; this • Draw slowly when collecting with syringes or
increases the force of the blood flow, creating a high degree of red from catheters
blood cell trauma.
– Use a blood transfer device to transfer syringe-collected blood into a • Transport to lab and centrifuge in a timely fashion
tube. It will enhance safety and improve specimen quality.

Collection Volume Arterial Blood Gases

• Avoid air contamination from a bubble or uncapped specimen

• Overfilled tubes • Delay in analysis can cause high pO2 to fall or low pO2 to rise
• Under filled coagulation tube • Analyze within 30 minutes or place on ice and analyze within
1 hour.
• Under filled hematology tube • Ca2+ binding by heparin can be minimized by using either of
the following:
• Under filling occurs because:
(1) A final concentration of sodium or lithium heparinate of
– Tube was removed too quickly 15 IU/ml blood or less
– Tube slips back from vacutainer needle (2) Calcium titrated heparin with a final concentration of less
than 50 IU/ml blood.
– Air drawn in from butterfly or connector tubing • Heparin Dilution effect can be avoided by use of dry heparin
• Roll specimen to mix heparin and reduce clots

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 Specimen Transport Primary Causes of Hemolysis

• Specimens should be delivered to the • Incomplete drying of skin after cleaning with alcohol
• Vigorous suction with syringe
laboratory promptly after collection • Inappropriate (too small) needle with syringe or vacutainer
• Specimens should not be placed on ice unless • Forcing blood from syringe into tube when it has started to
clot
specified by the laboratory • Shaking of tube instead of gentle agitation or inversion
• Inadequate packing in pneumatic tube container during
• Pneumatic tube transport does not affect transport to the laboratory to prevent shaking
analytical results • 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

Anticoagulants for Blood Collection

• Heparin
• Unfractionated heparin molecular mass 3-30 kd (mean, 14 kd),
as lithium, sodium, or ammonium salts; lithium heparin
preferred for plasma chemistry testing; nominal amount, 14.3
U/mL of blood
Discussion • For ionized calcium, heparin in blood gas syringe can be
calcium-titrated, electrolyte-balanced, or zinc lithium–titrated

• EDTA: salts of EDTA generally used for routine hematology

testing (dipotassium or tripotassium EDTA, disodium EDTA);
nominal amount, EDTA, 1.5 mg/mL of blood

• Citrate: generally used for routine coagulation testing;

trisodium citrate–dihydrate 3.2% (0.109 mol/L)

Essentials of phlebotomy training for

Hematology Physiological Variables
medical students
• Patient identification • A number of physiological variables can be associated with certain patient factors, such
• Completion of requisition form as age. For example, at birth red blood cell count (RBC) and hemoglobin values are
significantly higher than they are in adults, due to the relatively low levels of oxygen in
• Labeling of specimen tubes – content and placement on tube utero. Within the first few months of life they fall substantially and continue to decrease
• Collection of multiple tubes vs. single tube and level out to adult values at about the age of fifteen. In addition, lymphocyte counts
change throughout life; they are highest in children and lowest in the elderly.
• Timing of specimen collection in relation to time of day and drug intake • Smoking, high altitude, patient stance, exercise and pregnancy are some additional
• Posture patient-related variables over which the lab has no control; yet, they may affect patient
• Relation to meals test results.10
• Sites for collection • Additionally, white blood cell (WBC) and platelet counts can be affected by
cryoglobulins. When blood specimens cool from body temperature to room temperature,
• Use of tourniquet the cryoglobulins aggregate and may be falsely identified as platelets and/or WBCs by
• Skin preparation the hematology analyzer. Cold agglutinins have been known to cause spurious reporting
of macrocytosis and decreased RBC counts. Some automated instruments may also
• Collection technique report falsely high WBC counts and platelet counts.3 The blood smear may show
• Appropriate tubes for ordered tests agglutination of RBCs.
• Order of collection of tubes • Platelet satellitism is a phenomenon that only occurs in EDTA anticoagulated blood.
This is due to EDTA-dependent IgG autoantibodies and occurs at room temperature.
• Disposal of syringes, needles When platelet satellitism is present, there may be a false elevation of cell counts.

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 Fibrin Interference Specimen Collection Variables

• 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,
more insidiously, as an invisible microfiber or as strands. Fibrin strands,
though invisible, may directly affect some assays, especially immunoassays.2-
5 Unlike interference from heterophilic antibodies or rheumatoid factor, 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. Important considerations in the preanalytical phase that can have an
effect on fibrin formation are shown in Table 2.

• Table 2. Preanalytical phase considerations that can

• affect fibrin strand formation.
• • Recognition of disease or therapy that may affect clotting time
• • Selection of the appropriate tube type
• • Collection sequence when multiple tubes are collected

Specimen Collection

• Site Preparation: Prior to venipuncture, the site should be cleansed Specimens for therapeutic drug monitoring (TDM) should be obtained after a steady
with alcohol. Before performing the venipuncture, the alcohol should therapeutic concentration of drug or steady state is achieved in the blood. A blood
be allowed to air dry. This will help to ensure that the specimen is not sample obtained just before administering a drug dose after a steady-state level is
contaminated with alcohol, as this can lead to hemolysis. Hemolysis reached will reflect the trough level or the lowest concentration to be obtained with the
established drug dose. If only 1 blood sample is to be collected for TDM, it is preferred
can result in the spurious elevation of such analytes as potassium, that it reflect the trough level. Blood samples obtained when the drug concentration is
lactate dehydrogenase (LD), iron and magnesium in the chemistry lab. at its maximum, will, of course, reflect the peak level. Peak-level specimens need to be
• Proper Venipuncture Technique: During phlebotomy, avoid probing obtained within 1 to 2 hours after intramuscular administration of the drug, 15 to 30
to find the vein and achieve blood flow. Excessive probing and/or minutes after intravenous administration, or 1 to 5 hours after oral administration.
“fishing” to find a vein can result in a poor quality sample, including Timing of specimen collection for TDM, however, depends on the rate of distribution
hemolysis. of the specific drug. If the drug is infused intra-venously, approximately 1 to 2 hours
after the completion of infusion are needed for the distribution phase to be completed,
• Order of Draw: Following the correct order of draw during with exceptions such as digoxin and digitoxin, which need 6 to 8 hours for the
venipuncture will help to ensure accurate test results. The BD and completion of the distribution phase. In general, since the rate of oral drug absorption
CLSI (Clinical and Laboratory Standards Institute, formerly NCCLS) differs from person to person, blood specimens obtained for TDM usually are timed to
reflect trough levels. For the monitoring of certain drugs, such as theophylline,
antibiotics, and antiarrhythmic drugs, it may be necessary and useful to measure peak
levels after intravenous administration.

Specimen Collection Variables Specimen Processing

• Proper Tube Handling and Specimen Processing: Serum and plasma tubes each have their own special handling
requirements.
• Serum Samples
• Serum specimens need to clot completely prior to centrifugation and processing. Blood specimens collected in tubes with a
clot activator should be allowed to clot for 30 minutes to ensure complete clot formation; tubes without an additive need up
to 60 minutes for Blood from patients who are receiving anticoagulant therapy may take longer to clot. Tubes should be
allowed to clot at room temperature, upright in a test tube rack, with the closures on the tubes. Spinning the tube too soon
may result in a gelatinous and/or fibrinous serum sample that will require respinning.
• Plasma Samples do not require clotting prior to centrifugation. This allows the tube of blood to be drawn, mixed and
centrifuged immediately, resulting in a quicker turn-around-time for test results.
• Centrifugation: The next step in sample processing is the centrifugation of the blood collection tubes. Specimens should
be spun for ten minutes at a speed of 1100 to 1300 relative centrifugal force (RCF) in a swinging bucked centrifuge. A
fifteen-minute spin at the same speed is required for spinning tubes in a fixed- angle centrifuge. Serum and plasma tubes
without gel can be spun at a speed of 1000 RCF for ten minutes.
• It is important to spin gel tubes for the recommended time. The gel barrier in the tubes needs time to move and form a solid
barrier between the red cells and the serum or plasma. Also, in PST tubes, the white blood cells and platelets that remain in
the plasma need adequate time to spin out of the plasma. If the PST tubes are spun for less than the recommended 10
minutes, these cells and platelets may remain in the plasma and could cause interference with some chemistry analytes. It is
recommended that SST tubes should not be re-centrifuged after their initial centrifugation. Re-spinning the tubes can result
in elevated potassium values, as excess serum that has been in contact with the red cells will be expressed from underneath
the gel barrier.
• Special Handling of Blood Specimens: Certain chemistry analytes will require the tube of blood to be chilled after
collection in order to maintain the stability of the analyte. A slurry of ice and water is recommended for chilling the tubes
of blood. Examples of specimens that need to be chilled or transported on ice include adrenocorticotropic hormone
(ACTH), angiotensin converting enzyme (ACE), acetone, ammonia, catecholamines, free fatty acids, lactic acid, pyruvate
and renin.
• Other analytes are photo-sensitive, and need to be protected from light in order to remain stable and to ensure that the
laboratory reports an accurate result. This can be done by wrapping the tube of blood in aluminum foil. The most common
example of a light-sensitive analyte is bilirubin. Other chemistry analytes that need to be light-protected include beta-
carotene and erythrocyte protoporphyrin.
• Stability for Whole Blood, Serum and Plasma: A whole blood specimen that is going to be spun down should be
if d d h l df h d bl d ll i hi h f h i O h

8
 Specimen Collection
• Order of Draw: Following the correct order of draw during venipuncture will help
to ensure accurate test results.
• An example of improper order of draw that can lead to an incorrect chemistry
result is drawing an EDTA tube prior to a SST or heparin tube for chemistry
testing. The potential cross contamination of K2 or K3EDTA on the needle from the
lavender top tube to the chemistry tube can lead to an elevated potassium result.
• Proper Tube Mixing: All tubes with additives need to be inverted to mix the
additive evenly with the blood. Plastic serum tubes and SST tubes contain clot
activator and should be inverted 5 times to mix the activator with the blood and
help the specimen clot completely. Other additive tubes, such as heparin, need to
be inverted 8-10 times to mix the anticoagulant with the blood and prevent clotting.
Be sure that tubes are not being shaken vigorously, as this can lead to a hemolysis.
• Correct Specimen Volume: All blood collection tubes need to be filled to the
correct volume. This will ensure the proper blood to additive ratio. For example, if
a 5 mL draw heparin tube is only filled with 3 mL of blood, the heparin
concentration is erroneously high and may potentially interfere with some
chemistry analytes.
• Expiration dates should also be checked on the evacuated tubes. Expired tubes
should not be used, as they may have a decreased vacuum, as well as potential
changes in any additives in the tubes.