Laboratory Investigation of

Hemostasis

LabM 419 Clinical Coagulation

Fall 2009
©Cara Calvo, MS, MT(ASCP), SH(ASCP) – UW, 2009.
Learning Objectives – Upon completion of required
reading, after careful study and following this lecture the student will be able to:
1. Recall the sample collection, handling, and processing protocols necessary to obtain
acceptable specimens for the assays and procedures discussed in this lecture.
2. Correlate sample collection, handling, and processing protocols with potential problems in
coagulation laboratory testing.
3. State the principle, summarize the procedure, interpret test results, and determine the
appropriate clinical utilization for each of the following tests : a) bleeding time, b) platelet
aggregation studies: whole blood and PRP, c) single factor assay, d) bethesda titer, and e)
chromogenic substrate assay.
4. Discuss the clinical utility of the following assays in the lab investigation of hemostasis:
Reptilase time and Euglobulin clot time.
5. Apply knowledge of clot-based plasma procoagulant screens including mixing studies to the
lab investigation of bleeding and thrombotic disorders.
6. Assess the role of point of care testing (POCT) in the laboratory evaluation of hemostasis and
in monitoring anticoagulant therapy.
7. Interpret lab data, correlate lab data with patient information, and synthesize knowledge of
the lab investigation of hemostasis to correctly respond to test questions and solve subject-
matter related case studies.
Foundations of Lab Investigation of
Hemostasis
Coagulation lab provides testing to:
 Aid physicians in determining the cause of patient
hemorrhage or thrombosis
 Monitor therapy of patients who take hemostatic
altering drugs or who undergo procedures that
impact hemostasis
 Screen patient populations in order to establish
population norms and identify inheritance patterns
of bleeding and thrombotic conditions and
diseases.
Review Specimen Requirements

 Test results are only  Types of Samples

as valid as the quality  PLT-poor plasma
of the specimen. (PPP)
 See lecture 7  PLT-rich plasma (PRP)
 Serum
 Lab Exercise 1
 Citrated whole blood
 Review pages 671 –  Whole blood
675 of course text
 Check out UW
On-line Testing Guide

Graphic accessed URL http://pathology.mc.duke.edu/coag/images/16_rgb.jpg, 2009.

Lab Investigation of Primary
Hemostasis
 Platelets
 Numbers
 CBC
 PLT count
 PLT morphology
 Function
 Whole blood aggregation
 Platelet-rich plasma aggregation
 Bleeding Time (BT)

Graphic accessed URL http://www.strokecenter.org/education/ais_pathogenesis/images/platelet_activation.jpg, 2009.

 Platelet Function Tests

 Bleeding Time
 No sample collection!
 Aggregometry
 Sample
 Whole Blood (WB)
 Platelet-Rich Plasma (PRP)

Graphics accessed URL http://www.mclno.org/webresources/pathman/BT_web/bt_paper.jpg, http://www.accumetrics.com/images/img_product_overview.jpg, & http://cmed-tech.com/graphics/platelet2.jpg, 2009.

Bleeding Time
 Time it takes a
standardized skin
incision to stop
bleeding
 Extensive, informative
Issues
patient interview •Response is inversely proportional to PLT
 Reference Range count
•Many patient’s take over-the-counter
 ~2.5 – 9.5 minutes drugs which prolong BT
 Varies with •Relative result correlation across lab
 direction, length, depth where testing is performed

of incision •Results vary with direction, length, depth

of incision methodology
 methodology

Video URL http://www.mclno.org/webresources/pathman/BT_web/Bleeding_time.htm#procedure, 2009.

Sample Quality for PRP PLT
Aggregometry
 Clean venipuncture
 Hemolysis = increased plasma
ADP = prematurely activated
platelets
 Lipemia may obscure OD
during PLT aggregation
 PRP contact with glass =
prematurely activated PLTs
 Keep samples capped to
prevent loss of CO2 – change
sample pH
 Store samples @ room temp –
prevent inhibition of PLT
aggregation
 Perform testing within 3 hours
of sample collection

Graphic accessed URL http://labmed.ucsf.edu/labmanual/mftlng-mtzn/img/BD_plastic_citrate_draw.gif, 2009.

 Whole Blood PLT Aggregometry
 Patient/Test Sample – 3.2%
citrated WB
 Held at room temp (WHY?)
 Tested within 3 hours of
collection
 Mixed 1:1 w/ saline unless PLT
count is < 150 x 109/L
 Principle – electrical impedance
 Reference Range
 COL/EPI < 185 s
 COL/ADP < 120 s
 Issues (prolongation)
 HCT < 35% and/or Figure - anticoagulated whole blood is aspirated from sample
reservoir through capillary and contacts aperture in coated
 PLT < 150,000/L membrane. Platelets adhere, become activated, and finally
 Aspirin, NSAIDS aggregate to form platelet plug. Instrument reports time from start
of test until platelet plug occludes aperture as closure time. ADP,
 Rare PLT aggregation defects Adenosine diphosphate.

Graphic accessed URL http://www.mdconsult.com/das/article/body/148743378-5/jorg=journal&source=&sp=11742823&sid=0/N/206759/f112489002.jpg, 2009

PLT Aggregometry Using PRP
 Patient Sample – 3.2% citrated WB
 Test Sample – PLT-rich Plasma
 Principle – photometry: optical
density of PRP warmed to 37° C is
determined before and after the
addition of various aggregating
agents
 Issues
 Sample quality is critical
 Fibrinogen levels are important
 Agonists must be prepared fresh
daily
 Thrombocytopenia makes result
interpretation difficult
 Complete patient history is essential

Figure 1 - Platelet-rich plasma in an optical aggregometer. Platelet count is

approximately 200 × 109/L, and platelets are maintained in suspension by a
magnetic stir bar turning at 1000 rpm. (Courtesy of Kathy Jacobs, Chronolog, Inc.,
Havertown, PA.)

Figure 2 – Five possible phases of PLT aggregation: 1) baseline, 2) agonist addition and
shape change, 3) primary wave, 4) secretion, and 5) secondary wave.

Graphics accessed URL http://evolvels.elsevier.com/section/default.asp?id=1138_ccalvo7_0001, 2008.

PRP Aggregometry Agonist & Patterns
 ADP (at appropriate concentration)
 Biphasic curve: 1o and 2o waves
(requires intact prostaglandin pathway)
Note: if ADP is added at too low a
concentration or too high a
concentration, will not get biphasic
response
 Epinephrine
 Biphasic curve; requires intact
prostaglandin pathway
 Collagen
 Lag phase followed by 2o wave only
 Ristocetin
 Binds to vWF/GPIb/IX complex and
results in agglutination
 Evaluates adhesion reaction
 Thrombin
 Irreversible aggregation only (does not
require cyclooxygenase)
 Arachidonic acid
 2o wave only; assesses cyclooxygenase
pathway

Graphic adapted from McKenzie, Shirlyn B (2004). Clinical Laboratory Hematology. Pearson Prentice Hall. Figure 39-1, page 787.
Laboratory Investigation of
Secondary Hemostasis
 Coagulation  Fibrinolysis
 Clot-based assays  Immunoassay

 PT, PTT, TT  D-Dimer/FDPs

 Mixing Studies  Chromogenic Assays
 Reptilase Time  Clot-Lysis Assays
 Single Factor assays  Euglobulin Clot Lysis

 Quantitation

 Fibrinogen
 % Activity

 II – XII
 Bethesda Titer
 Calculation

 INR
Clot-based Plasma Procoagulant Screens

 Prothrombin Time (PT)

 Partial Thromboplastin
Time (PTT)
 Thrombin Time (TT)

 Kinetic Fibrinogen

Graphic accessed URL http://peir.path.uab.edu/coagadmin/uploads/coagmech1.gif, 2009.

Reptilase Time

 Atroxin venom has

thrombin-like activity
 Cleaves only
fibrinopeptide A from
fibrinogen
 Test procedure
identical to TT
 Advantage – heparin
insensitive
Single Factor Assays
 Test Sample: PPP
 Principle: A dilution of test plasma is
mixed with equal volumes of factor
deficient plasma and the clotting time
determined. Comparing the degree of
correction of a prolonged clot time
against a standard curve allows the %
activity of the deficient factor to be
determined.
 Reference ranges may be expressed
as either a percentage or in IU/dl.
Many factors, including factors VIII, IX,
X, have reference ranges of 50-150%
alternatively expressed as 0.50-1.50
IU/ml or 50-150 IU/dl
 Issues – same as for PT and PTT

Figure – Single factor assay activity curve is established by

testing (PT or PTT) at least 5 dilutions, including a 1:10
(represents 100% activity) of a normal reference plasma, then
plotting the clot time is seconds versus the % activity for each
dilution on log-log paper.

Graphic adapted from Brown, BA. Hematology Principles and Procedures (1993). Malvern, Mass. Lea & Feiberger. Chapter 5, page 231.
 Concept Clarity
PNP + F8DP Normal clot time
 PNP – Pooled normal
PTFDP + F8DP Prolonged PTT plasma (100% activity
Presume the patient’s plasma is all procoagulants)
deficient in factor VIII.  F8DP – Factor VIII
QUESTION – How deficient?
deficient plasma
 PT FDP – Patient
•Dilute PTFDP with equal volume of factor deficient
F8DP
•Run PTT plasma
•Use STD curve and clot time to find
% activity
Chromogenic Substrate Assays
 Chromogenic substrates
are peptides that react with
proteolytic enzymes under
the formation of color.

EXAMPLE: Factor VIII Assay

 Reagent 1 – IXa, Ca2+, PL,

excess X
 Reagent 2 – Chromogenic
substrate

Graphic accessed URL http://www.diapharma.com/var/diapharma/storage/images-versioned/12386/1-eng-US/fviii_mono11.jpg, 2009.

Popular Chromogenic Substrate
Assays
 Thrombin
 Plasminogen
 Tissue Plasminogen Activator
 Procoagulants
 VIII
 IX
 X
 XII
Bethesda Titer (or How Much Factor Inhibitor Does the Patient
Have? ) *******1 BU = amt of inhibitor that yields 50% factor activity********

 Prepare two-fold serial dilutions of patient plasma in buffered saline.

 To each dilution add an equal volume of reagent normal plasma that
contains 100% factor VIII, and incubate the series 2 hours at 37
degrees centigrade.
 Simultaneously incubate an aliquot of the same reagent normal
plasma.
 Perform a factor VIII activity assay on all incubated dilutions and on the
control plasma.
 The dilution that yields a factor VIII activity that is 50% of the control
activity is the endpoint, and the reciprocal of the dilution is the result in
Bethesda units.
 So if the control result came to 100% factor VIII activity and the 1:16
dilution gave you a result of 50% factor VIII activity, the result would be
16 Bethesda units.

Reference: http://www.fritsmafactor.com/newfritsmafactor/?p=1463#more-1463
Euglobulin Clot Time
 Here are the results for our 12/11/08 quick question, “What assay do you
use to detect abnormalities in fibrinolysis such as DIC?

This question was a little ambiguous in that it didn’t provide a way to answer
multiple assays. Many of us use the D-dimer and fibrinogen assay in parallel
to detect and monitor DIC. Here are the results:
 Fibrin degradation products (FDP): 12 (13.04%)
Euglobulin clot lysis: 1 (1.09%)
Fibrinogen: 10 (10.87%)
D-dimer 69 (75%)
 I suspect more people use fibrinogen with the D-dimer. It is interesting,
however that many of us still use the FDP assay. I suspect this is primarily
used in acute care facilities that do not have an automated D-dimer. It’s also
apparent the old euglobulin clot lysis test has been honorably emeritated (if
that is a word).

Reference: URL http://www.fritsmafactor.com/newfritsmafactor/?p=1521#more-

1521.
Hemostasis POCT
Marcia L. Zucker, PhD
AACC Presentation (12/3/2002): “Point-of-
Care Coagulation Testing “
 Current POCT Test
 Improved clinical outcome  PT
 Reduced LOS – Length of Stay  INR
($$$)
 Improved, timely patient care
 PTT
Cynthia Johns, MSA, MT(ASCP),
 ACT
SH(ASCP)  Fibrinogen
Rodak BF, Fritsma GA, and Doig K. (2007).
Hematology Clinical Principles and
 HiTT (High dose
Applications. St. Louis, Missouri. Thrombin Time)
Saunders Elsevier. Chapter 47

 Improved anticoagulant therapy

monitoring TAT
 References
1. Rodak BF, Fritsma GA, and Doig K. (2007). Hematology Clinical Principles and
Applications. St. Louis, Missouri. Saunders Elsevier. Chapters 45 and 47.
2. Fritsma Factor at URL http://www.fritsmafactor.com/newfritsmafactor/
3. University of Washington, Department of Laboratory Medicine, Reference
Laboratory Services. Handbook of Diagnostic Hemostasis and Thrombosis Test
(2005) at URL
http://depts.washington.edu/labweb/PatientCare/Clinical/Guides/hemostasis.pdf
4. UW Online Testing Guide at URL
http://byblos.labmed.washington.edu/bcard/search.asp
5. Virginia Commonwealth University, Education Resources URL accessible
http://www.pathology.vcu.edu/clinical/coag/education.html
1. Riley, RS et al. Laboratory Evaluation of Hemostasis. 2006.
http://www.pathology.vcu.edu/clinical/coag/Lab%20Hemostasis.pdf, 2009.
6. Tcheung JE, Madan M, Berkowitz SD, et al. Rapid assessment of glycoprotein
IIb/IIIa blockade with the platelet function analyzer (PFA-100) during percutaneous
coronary intervention. American Heart Journal - Volume 141, Issue 2 (February
2001).