Focus Article

Garbage In - Garbage Out: Talking Trash About PreAnalytical Variables (Part 1)

Presented by: Marlies Ledford-Kraemer, MBA, MT(ASCP)SH
Introduction
Variation, whether biological or methodological (pre-analytical, analytical, or postanalytical phases of testing), can have a significant impact on patient outcomes such as
diagnosis, treatment, and therapeutic monitoring. Understanding how these variations
affect coagulation laboratory testing will lead to providing more reliable test results and
lay a foundation for quality improvement.
For

coagulation

testing,

more

so

than

other

laboratory disciplines, the old adage garbage in garbage out lies at the heart of the pre-analytical
variable story. This Focus Article is one in a series
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to Lab

of four to address variation in the coagulation

laboratory. The series will address: 1) pre-analytical
variables affecting routine coagulation assays, 2)
pre-analytical issues that impact esoteric assays, 3)
analytical variation in test performance, and 4) postanalytical concerns.

A natural conclusion to this

series will be an assessment of how processes in

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to Dr

Pre-Analytical Variables (Part 1)

the three methodological phases can be evaluated and stabilized thereby leading to a
cycle of continuous quality improvement. Look for these companion articles on the
CLOT-ED web site at www.CLOT-ED.com.
The pre-analytical phase of testing, according to various authors, may or may not
include the patient as a variable.

Since biological variation affects the patients

hemostatic function in vivo as well as in vitro, this article will not include patient
variables. Herein the term pre-analytical encompasses everything that happens to a
patient specimen up to the point of actual testing (analytical phase). For the routine
coagulation laboratory, variables associated with the pre-analytical phase include:
Test ordering/requisition
Patient identification
Phlebotomy
Specimen transport
Specimen examination
Centrifugation & aliquots
Time to testing & storage
Test Ordering
Laboratory testing begins with a need. That is, a patient 1) presents with a potential
hemostatic/thrombotic problem, 2) requires clearance for a surgical procedure, or 3)
uses a therapeutic agent that necessitates monitoring. The physician acts upon that
need (history and physical presentation) by requesting certain coagulation tests. The
knowledge of the physician in which test(s) to order and
translating that information to the chart is the initial driver for a
good outcome. The subsequent transcription of that physician
order to a requisition (computerized or not) by a nurse or ward
clerk is equally critical. Potential errors in transcription can also
occur in the laboratory by incorrect ordering of tests in the
laboratory information system (LIS).

A College of American

Test
Desired

Test
Ordered

INR

PT

Anti-Xa

FX

FV Leiden

FV

PT 20210

PT or FII

FVIII

FVII

FXI

FIX

Pathologists Q-Probe study performed in 660 institutions showed 4.8% of outpatient

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Pre-Analytical Variables (Part 1)

requisitions contained at least one type of order entry error (one or more tests were not
entered into the LIS or a test was ordered that was not on the requisition). In other
words, irrespective of how good your laboratory performed a FVIII, it is irrelevant when
a FXIII was requested!
According to NCCLS guidelines H3-A5 (2003) a requisition form should contain the
following:
Patient name & age from identification plate
An identification number
Date & time to obtain specimen
Accessioning number
Physicians name
Department or location
Critical to a coagulation laboratory is the suggested other information, that is,
knowledge

of

anticoagulant

(warfarin,

heparin,

anti-thrombins)

or

antiplatelet

medications.
Patient Identification
For a positive outcome, correct identification of the patient is the critical link between
test result and patient. It is imperative that the phlebotomist ensures that the patient
denoted on the requisition form is the individual from whom the blood is to be drawn.
For the patient who is conscious, the phlebotomist must require the patient to provide
full name and/or some other unique identifier. NCCLS document H3-A5 also provides
guidelines for identification of patients who are minors, unconscious, mentally
incompetent, or are unidentified emergency cases. Subsequent to the venipuncture,
the phlebotomist must, immediately and in the patients presence, label the drawn
tubes. Each specimen tube must be identified with the patients full name, accession
number (hospital number), date & time of collection, and phlebotomist initials.

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Pre-Analytical Variables (Part 1)

Phlebotomy
Phlebotomy (derived from the Greek phlebos [vein]) is defined as the act of puncturing a
vein for the purpose of withdrawing blood.

For such an apparently simple act,

numerous articles, book chapters, and web sites are devoted to the topic. Upon closer
inspection one realizes that this vital step releases the in vivo world to the in vitro test
tube. The very act of the venipuncture initiates the hemostatic response. It is this
singular reason that explains the fragility of specimens for coagulation testing. Table
1 on page 5 summarizes the seventeen steps entailed in the venipuncture procedure
(as outlined in NCCLS document H3-A5) and highlights specific issues that can impact
testing.
Various blood collection systems may be used for obtaining a blood specimen. These
include: 1) one or two syringe(s)/needle, 2) winged (butterfly) cannula/syringe, 3)
indwelling cannula (vascular access device)/syringe, or 4) an evacuated tube/needle.
Use of a syringe/needle may increase the risk for obtaining a hemolyzed sample.
Moreover the larger the syringe, the greater the chance that clotting may occur,
therefore volumes of < 20 ml are recommended.

Winged collection sets may be

necessary in pediatric or geriatric settings or for a patient with difficult venous access. It
is critical that a discard tube is used to fill the dead space (tubing from butterfly to
syringe) before obtaining a specimen for coagulation testing. Should this not be done,
the blood to anticoagulant ratio will be adversely impacted.

Obtaining a specimen

through a venous access device should be avoided. If no alternative exits, one must
flush the line with 5ml of saline and discard the first 5ml of blood in order to minimize
heparin contamination.

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Pre-Analytical Variables (Part 1)

Table 1: Venipuncture Procedure and Impact on Testing

Hematocrit varies according to posture and increases from

lying to sitting to standing
Water moves from intravascular compartment to interstitium
resulting in ~12% decrease in plasma volume and by that
increase in macromolecules (shorten PT & APTT)
20-30 minutes are required for hematocrit to adjust to change
in body position

Small needle bore size causes mechanical disruption of platelets & hemolyzes cells
Appropriate needle gauge to be used is based
on amount of blood to be drawn, age of patient, & size of veins
19-21 gauge for adults suggested

Procedural Steps

Tourniquet should be
applied for no longer
tha n
o ne
m inu te
(remove as soon as
blood begins to flow) to
minimize stasis and local
hemoconcentration
Patients hand should be
closed (better exposure
of vein) but not pumping
(changes concentration
of certain analytes)
Preferred veins are the
median cubital or cephalic veins
Patients arm should be
in a downward position
to prevent backflow
Angle of insertion should
be as shown

Accession request to identify needed paperwork & supplies for

obtaining blood specimen

Approach & identify the patient

Verify patient's dietary restrictions and latex sensitivity; select

appropriate gloves & tourniquet

Assemble necessary supplies & appropriate collection tubes

according to test requests

Position the patient

Apply tourniquet, close patient's hand, select vein

Put on gloves

Clean venipuncture site

Perform venipuncture; as blood begins to flow, ask patient to

open hand

10 Use correct order of draw for collection tubes

11 Release & remove tourniquet
12 Place gauze pad over puncture site

Prefe rable to use

evacuated blood collection tube with full
draw
Siliconization of glass
tubes reduces contact
activation
Plastic tubes
PET outer tube resists breakage &
retains vacuum
Polypropylene
used
for inner tube is a
non-activa ting surface
Smaller diameter tubes
minimize headspace
and by that platelet
activation
Tube stopper is critical
in maintaining vacuum,
p re ven ting
di valen t
cation leaching into
citrate, and preventing loss of CO2
(increases pH)

13 Remove needle & activate manufacturer's safety features

Draw should not be prolonged or traumatic

(probing with needle
introduces tissue thromboplastin) as this accentuates coagulation activation (PT &/or APTT
shortened)
Blood
flow should be
uninterrupted
Upon tube filling, immediately invert, gently, 3-4
times
Numerous inversions or
vigorous shaking can
a ct i va te
p l a te l e ts
(release PF4 that neutralizes heparin)

14

Apply pressure to the site, ensure that bleeding has stopped, &
bandage the area

15 Label tubes & record time of collection

16 Chill specimens (if required)
17 Distribute collection tubes to laboratory

Adapted from NCCLS Document H3-A5 (Vol 23 No 32), 2003

Blood culture tube

GLASS Non-Additive SERU M tube
Coagulation tube
PLASTIC SERUM tube (with or without clot activator,
with or without gel)
Heparin tube
EDTA tube
Glycolytic inhibitor tube

Anticoagulant should
be 3.2% of dihydrate
form of trisodium citrate
FV is more stable in
citrate than any other
anticoagulant
Ma y be buffered or
non-buffered
Plasma is not buffered since hemoglobin is absent
At pH above 8.0, FV
& FVIII deteriorate
Use
of citric acid
maintains pH at 5.8

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Pre-Analytical Variables (Part 1)

The collection device of choice for coagulation testing is an evacuated blood collection
tube.

The tube, however, is not completely evacuated and it is this residual gas,
following the ideal gas law, which allows for the filling of the tube. As blood
starts to fill the tube, the residual gas is restricted to an ever decreasing
volume thereby causing the pressure of the gas to increase.

When this

pressure reaches ambient pressure, the draw is complete. True to the ideal
gas law, temperature and altitude can significantly impact the interior gas
pressure. NCCLS guidelines state that the draw volume must not be more
than 10% below that stated at the time of manufacture.
Materials used for making tubes include glass and plastic.

Safety concerns have

increased the use of plastic tubes since these tubes, made of polyethylene
terephthalate (PET), are less susceptible to breakage. The disadvantage is that PET
tubes have lower water-vapor barrier properties. Since water can diffuse out of these
tubes over time, the dilution provided by the remaining liquid in the tube (sodium citrate
anticoagulant) will be reduced and affect blood to anticoagulant ratios. It is therefore
imperative that manufacturers expiration dates are adhered to closely.

Another

consideration with the use of plastic tubes is the re-evaluation of a laboratorys

reference and therapeutic intervals. In a comparison between glass and plastic tubes,
Flanders and colleagues noted only a disparity in Thrombin Time values. Caveats to
their study include use of only normal samples, tubes from a single manufacturer, and
no notation of PT and APTT results. Contrastingly, van den Besselaar, et al, and BironAndreani, et al, concurred in their studies that PT values from patients receiving oral
anticoagulants were significantly different (higher) when collected in plastic versus
siliconized glass tubes from various manufacturers. Accordingly tube surface properties
can significantly influence ISI determinations.
A historical haunting in coagulation sampling has been the discard tube. Studies by
Yawn (1996), Gottfried (1997), Adcock (1997), and Brigden (1997) with their respective
colleagues, showed that no statistical differences occurred for PT, INR, and/or APTT
between a first and second draw tube. To that end, NCCLS guideline H21-A4 indicates
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Pre-Analytical Variables (Part 1)

that it is acceptable practice to use the first draw tube if only PT, INR, or APTT are
requested. However, for other coagulation testing this practice has not been sanctioned
since there are no current published data to suggest that this practice [use of discard
tube] is unnecessary.

Should a patient require testing in addition to coagulation

testing, then the order of draw as noted in Table 1 should be used. As discussed
above, when using winged blood collection sets or obtaining blood from venous access
devices, a discard tube (volume) is mandatory.
Coagulation can not occur without calcium ions. As such agents that bind calcium
(sodium citrate) permit blood fluidity in the test tube.

This process is reversed in

coagulation testing such as the PT and APTT wherein plasma samples are recalcified
and the coagulation process becomes engaged. Since plasma is tested and citrate is
concentrated in the plasma compartment, it stands to reason that as more citrate is
present in the test tube, the more it will complex to calcium introduced into the assay
and by that make available less calcium to promote clotting (clotting process is slowed).
Values for PT and APTT are longer in 3.8% (129mmol/L) than 3.2% (109mmol/L)
sodium citrate tubes.

This effect on the PT was elegantly shown by Duncan and

colleagues, who demonstrated that the International Sensitivity Index (ISI) was ~10%
lower when using 3.8% sodium citrate. Their findings were corroborated by Adcock, et
al who noted a significant difference in INR results from patients on oral anticoagulant
therapy when using both citrate concentrations and more pronounced with a lower ISI
reagent. Moreover, manufacturers determine reagent ISI values from plasmas collected
in 3.2% sodium citrate. Collectively, there came a realization that the INR could better
be standardized by using one citrate concentration. In the Fourth Edition Approved
Guideline H21-A4, NCCLS states that only concentrations between 105 and 109
mmol/L, (3.13% to 3.2%), respectively, should be used.

This decision aligns with

recommendations from the International Society for Thrombosis & Haemostasis and the
European Committee for Clinical Laboratory Standards.

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Pre-Analytical Variables (Part 1)

Variances such as under filling of collection

tubes or an elevation in patient hematocrit
also impact the citrate-calcium axis. Though

X = (100-Hct)/(595-Hct)
then take value for X and
(X)(volume of tube) = ml anticoagulant

the use of 3.2% citrate anticoagulant reduces

these variations, they are not eliminated.
Evacuated

tubes,

when

filled

properly,

maintain a ratio of one part anticoagulant to 9

parts blood.

This ratio is disrupted by

hematocrit values above 0.55 L/L (55%) and

must be adjusted as noted in the box on the

Example:
Hct = 65% & Blue tube volume = 2.7ml
X = (100-65)/(595-65) = 35/530 = 0.066
0.066 x 2.7ml = 0.178ml Na citrate in place
of standard volume of 0.27ml
See Appendix, NCCLS document H21-A4 (2003)
for convenient to use table

right. Both hematocrit and fill variances will result in excess citrate in plasma that,
similar in action to higher citrate concentrations, prolong the PT and APTT.
Under filling of evacuated tubes begs the question, how low is too low? With minimal
use of 3.8% citrated tubes, the answer has become easier since the higher citrate
concentration demanded more stringent adherence to the blood/anticoagulant ratio. A
1998 study performed by Adcock and colleagues demonstrated that fill volumes of only
60% for PT and 70% for APTT were sufficient for providing accurate test results. A
caveat to their findings is that it may be reflective of the reagent/instrument system
used. Hence NCCLS suggests that manufacturers recommendations be followed for
proper fill volumes.
Specimen Transport
How a specimen tube is handled from patient to laboratory is laden with pitfalls of which
most are out of the control of a laboratory. One must consider not only transport within
a hospital but also intra- and inter-city movement of samples via courier services. The
means of transport, exposure to heat (reference laboratory collection boxes), vibration
(pneumatic tube systems), position of tubes (upright preferable), and overall time to
delivery can dramatically affect test results.

For example, elevated temperatures

enhance degradation of factors V & VIII whereas prolonged exposure to cold (>7 hours)
may activate Factor VII. It is for the latter reason that whole blood or plasma samples
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Pre-Analytical Variables (Part 1)

are no longer required to be transported on ice. Traumatic handling such as shaking,

vibration, and agitation can lead to hemolysis and platelet activation causing falsely
shortened clotting times.
Specimen Examination
Specimen tubes must undergo physical inspection at two stages: prior to and following
centrifugation. Initial examination should note that the specimen is indeed in a blue
top tube, the collection volume is appropriate for tube fill volume, and no clots or fibrin
strands are observed.

Historically the latter was achieved by inserting wooden

applicator sticks into the tube.

Today this practice is not recommended for safety

reasons (aerosol contamination). Subsequent to centrifugation, the plasma should be

checked for hemolysis, icterus, and lipemia.
Centrifugation and Aliquots
Platelet poor plasma (PPP) used for coagulation testing is obtained by centrifugation at
a speed and time that results in a residual platelet count of less than 10 x 109/L
(10,000/L). A simple method for checking the residual platelet count of PPP is to use
an automated cell counter. This number can be reached by centrifuging specimens at
1,500 gravity (g) forces (rcf) for no less than 15 minutes (either room or refrigerated
temperature is acceptable). Relative centrifugal force (rcf) is calculated by knowing the
rotating radius of the centrifuge and rotational speed (revolutions per minute [rpm]). A
rcf nomogram is provided in NCCLS document H18-A2. In order to minimize remixing
of plasma and red cells, a swing-out bucket (angle) rotor should be used and the brake
not applied at the end of centrifugation.
Testing Interval & Storage
All tubes, whether specimens (whole blood) or samples (separated plasma), must be
stoppered in order to minimize loss of CO2 (causes pH to increase leading to prolong
PT and/or APTT). Specimens should be tested within the time intervals outlined in
Table 2. If testing can not be performed within these time restrictions, plasma should
be separated from cells using a plastic, disposable pipette. Care must be exercised to
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Pre-Analytical Variables (Part 1)

ensure that the platelet layer is not disturbed. According to Woodhams and colleagues,
stability of coagulation proteins is best when sample volumes of ~1ml are stored in

Table 2: Testing Intervals & Storage Temperatures

Storage Temperature

Test

1 PT
2

APTT
Non Heparinized

Room
o
18 - 24 C
o
[64 - 75 F]

No Centrifugation Whole Blood

No

Yes

Centrifugation

No

Yes

Yes

Yes

Yes

Yes

Yes

Yes

4 hours

Yes

Yes

4 hours

Specimen

Plasma on Cells

No Centrifugation Whole Blood

Centrifugation
Centrifugation
3 APTT Heparinized (within 1 hour of
collection)
4 Other Assays

Maximum Time Interval

Refrigerated
o
2-4 C
o
[36 - 39 F]

Action

Centrifugation

Plasma on Cells
Plasma on cells or
plasma removed
within 1 hour
Plasma on cells or
plasma

Specimen Acquisition
to Analysis

24 hours
4 hours

NCCLS Document H21-A4 (Vol 23 No 35), 2003

microtubes having a minimum of dead space. The number of aliquots to be prepared

depends on number of tests ordered, ability to batch tests, and specific requirements of
a reference laboratory if testing is to be performed off-site. Plasma aliquots may be
stored for 2 weeks at 20 oC however, it is imperative that a frost-free freezer not be
used. The cycling of temperature, which eliminates frost build-up, causes repeated
thawing and freezing of samples leading to factor deterioration. The current NCCLS
recommendations for long-term storage indicate that samples may be kept for up to 6
months at 70 oC. This recommendation may be somewhat optimistic as shown by
Plumhoff and colleagues who demonstrated decreases of approximately 20% (after 3
days) and 10% (after 7 days) for factors VIII and V, respectively, when stored at 70 oC.
A study conducted by Woodhams, et al, showed stability (defined as less than 10% loss
of activity) of coagulation factors up to 18 months at this temperature. However, they
also noted that factors VIII and XI showed deterioration above this level at 6 months.
For testing, frozen plasmas should be thawed rapidly at 37 oC (prevents denaturation of
fibrinogen) and tested immediately (acceptable to hold at 4 oC for a maximum of 2
hours).
Previous NCCLS recommendations for testing intervals and subsequent storage were
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Pre-Analytical Variables (Part 1)

quite stringent (samples to be refrigerated or frozen if testing not completed in 2 or 4

hours, respectively). While this imposed considerable hardship relating to compliance,
the current recommendations raise concerns regarding specimen integrity should addon testing be considered. The laboratory must now be able to clearly delineate from
where specimens come (warfarin anticoagulant clinic, heparinized patient) and
recognize that sample acceptability for one type of assay may not be appropriate for
another (factor assays, for example). McGlasson has recommended that all samples
be handled as if further testing was to be performed so that the specimen is not
compromised. This would not burden the patient with an additional venipuncture or the
healthcare system with unnecessary follow-up testing caused by poor sample integrity.
Conclusion
A review of the literature by Bonini and colleagues revealed that 32-68% of all
laboratory errors occur in the pre-analytical phase. The frequency of overall errors
ranged from 0.05 to 0.60% of patients and/or tests. These numbers reflect only those
errors that produced an observable detrimental effect. Utilizing the mean of their noted
frequencies (0.33%) and applying that to the approximately 300 million coagulation tests
performed annually in the United States, one arrives at a substantial error rate (990,000
per year). It will require education, training, and interdepartmental cooperation to bring
about a reduction in pre-analytical variables. Approaching that goal will increase time
savings, reduce costs, and most importantly improve patient outcomes.
References
1.
2.

Koepke JA, Rodgers JL, Ollivier MJ. Pre-instrumental Variables in Coagulation Testing. Am J Clin Pathol 1975;64:591-6.
Walker, ID. Blood Collection and Sample Preparation: Pre-analytical Variation in Laboratory Techniques in Thrombosis A
Manual, 2nd Edition. Jespersen J, Editor. Kluwer Academic Publishers, 2000.

3.

Narayanan S. Preanalytical Aspects of Coagulation Testing. Haemtologica 1995;80(Sup 2):1-6.

4.

Lawrence JB. Preanalytical Variables in the Coagulation Laboratory. Lab Med 2003;34:49-57.

5.

Bush V, Cohen R. The Evolution of Evacuated Blood Collection Tubes. Lab Med 2003;34:304-10.

6.

Flanders MM, Crist R, Rodgers GM. A Comparison of Blood Collection in Glass Versus Plastic Vacutainers on Results of
Esoteric Coagulation Assays. Lab Med 2003;34:732-35.

7.

Van den Besselaar AMHP, Meeuwisse-Braun J, Witteveen E, Van Meegen E. Effect of Evacuated Blood Collection Tubes on
Thromboplastin Calibration. Thromb Haemost 1998;79:1062-3.

8.

Biron-Andreani C, Mallol C, Seguret F, Schved J-F. Plastic versus Siliconized Glass Tubes: Evaluation in Current Laboratory
Practice. Thromb Haemost 2000;83;800-1.
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Pre-Analytical Variables (Part 1)

9.

Yawn BP, Loge C, Dale J. Prothrombin Time: One Tube or Two. Am J Clin Pathol 1996;105:794-97.

10. Gottfried EL, Adachi MM. Prothrombin Time and Activated Partial Thromboplastin Time Can be Performed on the First Tube.
Am J Clin Pathol 1997;107:681-3.
11. Adcock DM, Kressin DC, Marlar RA. Are Discard Tubes Necessary in Coagulation Studies? Lab Med 1997;28:530-33.
12. Brigden ML, Graydon C, McLeod B, Lesperance M. Prothrombin Time Determination: The Lack of Need for a Discard Tube
and 24-Hour Stability. Am J Clin Pathol 1997;108:422-26.
13. Duncan EM, Casey CR, Duncan BM, Lloyd JV. Effect of Concentration of Trisodium Citrate Anticoagulant on Calculation of the
International Normalised Ratio and the International Sensitivity Index of Thromboplastin. Thromb Haemost 1994;72:84-8.
14. Adcock DM, Kressin DC, Marlar RA. Effect of 3.2% vs 3.8% Sodium Citrate Concentration on Routine Coagulation Testing.
Am J Clin Pathol 1997;107:105-10.
15. Adcock DM, Kressin DC, Marlar RA. Minimum Specimen Volume Requirements for Routine Coagulation Testing: Dependence
on Citrate Concentration. Am J Clin Pathol 1998;109:595-99.
16. Wenk RE. Disposables as Sources of Preanalytical Contamination and Misdiagnosis. Am J Clin Pathol 1997;107:395-7.
17. Adcock DM, Kressin DC, Marlar RA. The Effects of Time and Temperature Variables on Routine Coagulation Tests. Blood
Coag Fibrinolysis 1998;9:463-70.
18. Woodhams B, Girardot O, Blanco M-J, Colesse G, Gourmelin Y. Stability of Coagulation Proteins in Frozen Plasma. Blood
Coag Fibrinolysis 2001;12:229-36.
19. Plumhoff EA, Thompson CK, Fisher PK, Bowie EJW, Nichols WL. Effects of Specimen Storage and Handling on Coagulation
Testing. Thromb Haemost 1993;69:866 (Abst).
20. McGlasson DL. Laboratory Variables That May Affect Test Results in Prothrombin Times (PT)/International Normalized Ratios
(INR). Lab Med 2003;34:124-8.
21. Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in Laboratory Medicine. Clin Chem 2002;48:691-98.
22. NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard-Fifth Edition.
NCCLS Document 2003;H3-A5:Vol 23 No 32.
23. NCCLS. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays; Approved
Guideline-Fourth Edition. NCCLS Document 2003;H21-A4:Vol 23 No 35.
24. NCCLS. Procedures for the Handling and Processing of Blood Specimens; Approved Guideline-Second Edition. NCCLS
Document 1999;H18-A2:Vol 19 No 21.

Address for Correspondence

Marlies Ledford-Kraemer, MBA, BS, MT(ASCP)SH
CLOT-ED, Inc
130 N Rolling Hill RD
Islamorada, FL 33070
T: (305) 393-1920
F: (305) 853-5611
e-mail: marlies@CLOT-ED.com
Web Site: www.CLOT-ED.com

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