HEMOSTASIS SPECIMEN COLLECTION AND HANDLING

GENERAL OVERVIEW
 Hemostasis laboratory procedures are performed on
 Sample: Venous blood and Plasma
 Anticoagulant: EDTA (for cell counting) and 3.2% Sodium Citrate (specifically for coagulation tests; it
chelates *in reversible manner* Ca2+ that is needed for coagulation, it keeps blood anticoagulated).
 Specimens used in testing
 Whole blood (venous blood; EDTA): For Platelet Aggregometry – a test that measures the aggregation of
the platelets and Platelet Count.
 Platelet-rich plasma (PRP) (plasma spx): For Platelet Aggregometry and Platelet Function Tests
 Platelet-poor plasma (PPP) (plasma spx): For Coagulation Testing/Assays
PATIENT MANAGEMENT DURING HEMOSTASIS SPECIMEN COLLECTION
 No fasting needed but they should avoid vigorous activities (because it can cause erroneous results) and should
rest quietly for 30 minutes prior to collection for hemostasis testing, patients also need to avoid caffeine
intake for 2 hours (soft drinks also have caffeine).
 DRUGS THAT AFFECTS TESTING: (should assess 2 weeks history); patient should be 1 week free of any drugs.
 Aspirin: Inhibits plts aggregation specifically the action of cyclooxygenase.
 Coumadin (warfarin): Inhibits prothrombin group (FV,VII,FIX, FX) and regulators Protein C,S and Z;
coumadin gets the K needed for g-carboxylation; If coumadin is really need for the medication of the px,
ask the doctor before proceeding with the test.
 Observe/measured through prothrombin time/ PT test – measures coag factors found in extrinsic
pathway; prothrombin group is found in extrinsic and common pathway except FIX.
 Heparin: Inhibits most of the clotting factors
 Measured through activated partial thromboplastin time/ aPPT test
* Coumadin and Heparin are both blood-thinners*
 Penicillin: It can alter blood vessels walls that can cause hemolysis and hemolysis will facilitate in the early
activation of the coag factors.
* In antibiotics, it is only limited with penicillin.
 COLLECTION TUBES:
 Most hemostasis specimens are collected in plastic blue-stopper (blue-top, blue-closure) sterile
evacuated blood collection tubes containing a measured volume of 0.105 to 0.109 M (3.2%)
buffered sodium citrate anticoagulant; still needs to be a plastic coated despite of its anti-
coagulant property as it’s reaction with Ca 2+ is reversible and glass materials pre-activates FXII.
 Tubes of uncoated soda-lime glass (common in EDTA, ESR, SPS) are unsuitable because of their
negative surface charge activates platelets and plasma procoagulants.
 Siliconized (plastic-coated) glass tubes are available, but their use is waning because of concern
for potential breakage, with consequent risk of exposure to blood borne pathogens; it is
siliconized to prevent the blood to have in contact with glass tubes because it can pre-activate the
coagulation (pre-activates FXII) as glass materials have a –ve charge surface.
* There are citrate tubes that are made of plastic.
NOTE: Syringes used for evaluation of hemostasis: Plastic, Polystyrene, Silicone.
* The syringes back then are made up of glass; the first ever syringe developed was made up of glass.
HEMOSTASIS SPECIMEN COLLECTION PROTOCOL
 PREFFERED METHOD OF COLLECTION
 Evacuated blood collection tube system to lessen the exposure to ambient air.
 However, some may require syringe collection and initial “discard tubes” in special circumstances.
 If the hemostasis specimen is part of a series of tubes to be filled from a single venipuncture site, it
must be collected first or immediately after a non-additive tube.
ORDER OF DRAW FOR HEMOSTASIS TEST IN ETS:
a) Non-additive tube – discard tube/ serological tube; serves as cleaning tube to remove TF/FIII from
tissue juices.
b) Light blue top tube

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 c) EDTA
d) Plastic syringe
NOTE: If non-additive tubes are unavailable, use and discard a preliminary blue-topped tube.
 Blood to Anticoagulant Ratio
 A short draw—that is, a specimen with a smaller volume than the minimum specified by the
manufacturer generates erroneously prolonged clot-based coagulation test results because the excess
anticoagulant relative to blood volume neutralizes test reagent calcium.
 Excess anticoagulant: Prolonged PT and aPPT; increased time/value.
 Clotted specimens
 Useless for hemostasis testing even if the clot is small. A few seconds after collection, the phlebotomist
must gently invert the specimen at least five times to mix the blood with the anticoagulant and prevent
clot formation.
 Clotted specimen: Prolonged PT and aPPT as coag factors and plts are all consumed.
* QNS and clotted blood spx are rejected in coag tests; request for re-extraction or if the personnel who extracted
don’t want to replace the spx, make a letter indicating you received a clotted or QNS sample and have the
signature of personnel who extracted or note on the remarks that it is clotted or underfilled *
 Excessive specimen agitation
 Phlebotomist must never shake the tube, test results from visibly hemolyzed specimens are unreliable
for coagulation assay, and the specimen must be recollected; RBC have phospholipids which have the
property the same tissue thromboplastin/TF, it has tissue thromboplastin like substances and will
combine with ADP (also from hemolyzed RBC), when plts and and coag factor met those two, it’ll pre-
activated.
 Causes hemolysis leading to red cell being rupture, procoagulant activation, and platelet activation.
 Excessive agitation: Shortened PT and aPPT
* If received spx is hemolyzed and is still subjected for testing, note on the remarks that is it hemolyzed sample*
 Excessive needle manipulation; blind shot/fishing
 Promotes the release of procoagulant substances (result from leakages of blood/injury) from the skin
and connective tissue, which contaminate the specimen and cause clotting factor activation.
 Excessive manipulation: Shortened PT and aPPT as the coag factors and plts are already pre-activated
from in-vivo coagulation.
NOTE: Traumatic venipuncture releases tissue thromboplastin.
 Tourniquet application
 Phlebotomist must remove the tourniquet within 1 minute of its application to avoid blood stasis.
 Stasis is a condition in which venous flow is slowed resulting to local accumulation of FVIII and von
Willebrand factor which may cause in false shortening (because of pre-activation) of clot-based coagulation
test results.
 Tourniquet: Shortened PT and aPPT

common in EDTA, subject for re-extraction

*Plt storage are room temperature: 16 or 18–24⁰C.

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SELECTION OF NEEDLES FOR HEMOSTASIS SPECIMENS

 Whether evacuated collection tubes or syringes are used, the

bore of the needle should be sufficient to prevent hemolysis
(happens if the size of the bore isn’t appropriate with the
pressure of the blood) and activation of platelets and plasma
procoagulants. If the overall specimen is < 25 mL, a 20- or 21-
gauge thin-walled needle is used.
 Larger specimen (≥ 25 mL) 19-gauge needle is required.
 23-gauge needle is acceptable for pediatric patients or patients
whose veins are small, but the negative collection pressure must
be reduced.
*Gauge of the needles should agree with the volume of blood to be
extracted; the higher the volume, the higher the gauge*
ANTICOAGULANTS USED FOR HEMOSTASIS TESTING
 3.2% Buffered sodium citrate (0.105-0.109M)
 Blood to anticoagulant ratio:
 Anticoagulant used for hemostasis testing; binds calcium ions to prevent coagulation, and the
buffer stabilizes specimen pH if the tube stopper remains in place
 IMPORANT NOTE:
 Adjustment of sodium citrate volume for elevated hematocrit
a) 9:1 blood-to-anticoagulant ratio is effective, provided the patient’s hematocrit is 55% or less
b) In PCV, the decrease in plasma volume relative to whole blood unacceptably raises the
anticoagulant-to-plasma ratio, which causes falsely prolonged results for clot-based
coagulation
o FORMULA: 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐴𝐶 = (1.85𝑥103)(100 − 𝐻𝑐𝑡)
(100−𝐻𝑐𝑡)
o FORMULA: 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐴𝐶 =
(595)(𝑏𝑙𝑜𝑜𝑑 𝑣𝑜𝑙.)
OTHER ANTICOAGULANTS USED FOR HEMOSTASIS SPECIMENS
 EDTA
 Anticoagulant used in collecting specimens for complete blood counts, including platelet counts
 Not used for coagulation testing because calcium ion chelation by EDTA is irreversible, interfering
with coagulation assays and inhibits thrombin-fibrinogen reaction
 Required for specimens used for molecular diagnostic testing, such as testing for factor V
Leiden mutation or the prothrombin G20210A mutation
 Heparin
 Used for platelet retention test/glass bead retention test (test for platelet adhesion)
 Never been validated for use in plasma coagulation testing but may be necessary in cases of
platelet satellitosis as a substitute for specimens collected in EDTA or sodium citrate
 Citrate theophylline adenosine dipyridamole
 Used to halt in vitro platelet or coagulation activation for specialty assays such as those for the
platelet activation markers platelet factor 4 (PF4) and platelet surface membrane P-selectin
(measured by flow cytometry) or the coagulation activation markers prothrombin fragment 1+2 and
thrombin-antithrombin complex
 NOTE: Oxalates shortens clotting time because it forms insoluble precipitates
HEMOSTASIS SPECIMEN STORAGE TEMPERATURE
 Sodium citrate-anticoagulated whole blood specimens are placed in a rack and allowed to stand in a
vertical position with the stopper intact and uppermost
 pH remains constant if the specimen is sealed
 Specimens are maintained at 18° C to 24° C never at refrigerator temperatures

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  IMPORTANT NOTE:
 Storage at 1° C to 6° C activates FVII and FXI; it destroys platelet activity through uncontrolled
activation, and causes the cryoprecipitation of large VWF multimers
 Never be stored at temperatures >24° C because heat causes deterioration of FV and FVIII
HEMOSTASIS SPECIMEN STORAGE TIME
 Specimens collected for PT testing may be held at 18° C to 24° C and tested within 24 hours of the time of
collection; specimens collected for partial thromboplastin time (PTT) testing also may be held at 18° C to 24°
C, but they must be tested within 4 hours of the time of collection, provided that the specimen does not
contain unfractionated heparin anticoagulant
 If a patient is getting unfractionated heparin therapy, specimens for PTT testing must be centrifuged within 1
hour of the time of collection, and the plasma, which should be PPP, must be tested within 4 hours of the time
of collection.

PREPARATION HEMOSTASIS SPECIMENS FOR ASSAY

 Whole-blood specimens used for platelet aggregometry
 Blood for whole-blood platelet aggregometry or lumiaggregometry must be collected with 3.2%
sodium citrate and held at 18° C to 24° C until testing
 Chilling destroys platelet activity; aggregometry should be started immediately and must be
completed within 4 hours of specimen collection

 Platelet-rich plasma (PRP) specimens used for platelet aggregometry

 Platelet count:
 Citrated blood is first checked visually for clots and then centrifuged at 50xg for 30 minutes with
the stopper in place to maintain the pH; the supernatant PRP is transferred by a plastic pipette to a
clean plastic tube, and the tube is sealed and stored at 18° C to 24° C until the test is begun

 Platelet-poor plasma (PPP) used for coagulation studies

 Platelet count:
 Clot-based plasma coagulation tests require PPP-plasma
 Citrated blood is centrifuged at 1500xg for 15 minutes in a swinging bucket centrifuge; alternatively, the
angle-head StatSpin Express 2 generates 4400xg and can produce PPP within 3 minutes

IMPORTANT NOTES:
 The presence of >10,000 platelets/mL in plasma affects clot-based test results

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 Platelets are likely to become activated in vitro and releases phosphatidylserine which triggers
plasma coagulation and neutralizes LA if present interfering with LA testing
 Platelets also secrete fibrinogen, FV and FVIII, and vWF that may desensitize PT and PTT assays and
interfere with clot-based coagulation assays
 Platelets release platelet factor 4 (PF4), a protein that binds and neutralizes therapeutic heparin in
vitro, falsely shortening the APTT and interfering with heparin management
 Laboratory practitioners inspect hemostasis plasmas for hemolysis (red), lipemia (cloudy, milky), and
icterus (golden yellow from bilirubin). Visible hemolysis implies platelet or coagulation pathway
activation.
 Lipemia and icterus may affect the end-point results of optical coagulation instruments