Laboratory #2

Protime
Laboratory #2
Protime /Prothrombin(PT)
Skills= 15 Points
Objectives
The student will be able to:
1. Perform the manual prothrombin time on two (2) controls( one normal and one
abnormal) and one (1) patient within + 2SD of known values.
2. Accuratel report results with !"# accurac.
$. Properl %raph &ualit control results with 1''# accurac.
(. State the reference ran%e for prothrombin times.
". )nterpret accuratel the results of routine P* testin%.
+. ,-plain the clinical si%nificance of the Prothrombin time.
.. ,-plain the mechanism of action of /oumadin dru%s.
0. 1eco%ni2e the optimal ran%e for patients on therapeutic anticoa%ulants.
!. ,valuate patient P* results and su%%est possible factor deficiencies.
!aterials
1. 12 - ." test tubes
2. 3-ford pipettes 2'' and 1'' 4l
3. Pipette tips
4. 5ater bath $./ or 6eat block
5. *est tube rack
6. 7imwipes or %au2e
7. Stop watch
8. /alcium chloride8tissue thromboplastin (P* rea%ent)
. /ontrols9 :evel ) and ))) (normal and abnormal)
!. Patient citrated plasma specimens
"e#erences
Powers! Lawren"e #.! $ia%nosti" &ematolo%y! '. 482
Linne! (.(.! ) *in%srud! +.,. -asi" Te"hni.ues /or the ,edi"al Laboratory!
se"ond edition
-rown! -.0.! &ematolo%y: Prin"i'les and Pro"edures! 5
th
edition! 1ha'ter 5

Princi$le
Pa%e 1 o/ %
!L&' 122()Lab #2
Laboratory #2
Protime
*he P* measures functional activit of the e-trinsic and common pathwas. *he P* evaluates
patients suspected of havin% an inherited or ac&uired deficienc in these pathwas. *he procedure
uses a tissue thromboplastin rea%ent with /a/l(/alcium /hloride) to provide a one step procedure
for evaluatin% plasma clottin%.
*his test was devised on the assumption that when an optimal amount of calcium and an e-cess of
thromboplastin are added to decalcified plasma9 the rate of coa%ulation depends on the
concentration of prothrombin in the plasma. *he prothrombin time is therefore the time re&uired for
the plasma to clot after an e-cess of thromboplastin and an optimal concentration of calcium have
been added. /lot formation can be detected b optical or electromechanical methods9 usin%
manual9 semiautomated or automated devices.
*he normal values for the prothrombin time ran%e from 1'.' to 1$.' seconds. An elevated
prothrombin time ma indicate the presence of vitamin 7 deficienc9 D)/9 liver disease9 presence of
;SP<s or a deficienc in one or more of the followin% factors=
;actor ) (;ibrino%en)
;actor )) (Prothrombin)
;actor > (Proaccelerin9 :abile ;actor)
;actor >)) (Proconvertin9 Stable ;actor)
;actor ? (Stuart@Prower ;actor)
;actor ?))) (;ibrin Stabili2in% ;actor)
)n addition9 inhibitors can cause prolon%ed P*<s.
*he prothrombin time is also used to follow the pro%ress of patients treated with Dicumarol (a
therapeutic coumarin anticoa%ulant dru% used to inhibit clottin%9 especiall for preventin% post@
operative thrombosis and pulmonar embolism)
Antithrombotic dru%s are bein% used increasin%l and the present some ver real ha2ards to the
patient. )f the de%ree of anticoa%ulation is insufficient9 rethrombosis or embolism can occur. )f there
is too much anticoa%ulation9 fatal hemorrha%e can take place. *he laborator is responsible for
advisin% the phsician about the level of anticoa%ulation achieved. )n %eneral9 the P* or P** results
of patients on therapeutic anticoa%ulants should be 1A to 2A times normal. 1emember that man
labs are also reportin% P* results as an )B1 value for patients on anticoa%ulant therap. 5e will be
usin% the )B1 in a later lab. *wo cate%ories of antithrombotic dru%s are the coumarins9 which act as
vitamin 7 anta%onists9 and heparin. /oumarin dru%s9 such as Dicumarol9 are monitored b use of
Pa%e 2 o/ %
!L&' 122()Lab #2
Laboratory #2
Protime
the one@sta%e prothrombin time test. /oumadin dru%s inhibit the recclin% of vitamin 7 which is
needed b the Prothrombin %roup for snthesis.
*he rea%ents necessar for the prothrombin time test are primaril calcium chloride and tissue
thromboplastin. *hromboplastin converts prothrombin to thrombin ans supplies a source of
phospholipid and tissue factor. *hromboplastin rea%ents are prepared from tissue factor suspended
in phospholipid. *hese rea%ents must be prepared in specific concentrations and purchased
commerciall. /alcium chloride serves as a cofactor in the coa%ulation cascade.
A commercial control is tested with each batch of prothrombins. ,ach prothrombin control must be
prepared before use accordin% to the manufacturersC directions. /ontrol values and limits will var
with the brand and control used. Proper use of the control can detect deterioration of the
thromboplastin9 use of a calcium solution of the wron% concentration9 or use of the wron% incubation
temperature. ;or the purposes of our lab= :evel ) will be the normal control9 whereas :evel 2 or $ will
be the abnormal control.
Procedure:
1. 1econstitute tissue thromboplastin accordin% to instructions. :abel the thromboplastin
with the time9 date and initials. *he thromboplastin rea%ent is stabile for . das after
reconstitution. Allow to sit 1'@1" minutes9 then invert %entl several times. Mix well
prior to pipetting any of this reagent at any step in this procedure.
2. Pipet 1@2 mls 9 usin% a plastic pipet9 of the tissue thromboplastin@calcium chloride
rea%ent ( P* rea%ent) into a 12 - ." mm test tube and place in a $. incubator. *he
level of the thromboplastin should not e-ceed the hei%ht of the heat block.
3. Pipet 100 L or .1 mL of normal control(:evel 1) into each of $ test tubes.
4. Allow at least one (1) minute for the control to reach $./.
5. Pipet 200 L or .2 mLof P* rea%ent into the tube containin% the control. Start the
stop watch simultaneously.
6. Di- the tube and leave in the heat block for a minimum of .@0 seconds. *hen remove9
wipe the e-terior9 tilt back and forth %entl until a visible clot is formed. As the clot
forms9 the mi-ture will %elatini2e and ma turn cloud.
7. Stop the stop watch immediatel when the clot be%ins to form and record the time in
seconds. /arr out 1 si%nificant fi%ure passed the decimal point. ;or e-ample9 if our
result is 12.2$ seconds9 report as 12.2 seconds.
8. 1epeat the procedure for the second run of normal control. 1ecord the time.
. )f the results from run 1 and run 2 are within + 1 second from each other9 avera%e the
two results and report with appropriate units. ;or manual P*9 results should match
within 1.' second (if result is less than 2' seconds). 1esults over 2' seconds should
match within 2.' seconds.
Pa%e * o/ %
!L&' 122()Lab #2
Laboratory #2
Protime
12. )f results are not within re&uired limits9 a third run should be performed and
avera%e the two that match within acceptable limits. e sure and cross out any
!alues you are not using for the final calculation. "nclude measurement unit of
seconds on report sheet.
11. /hart E/ results on %raphs posted in the lab.
12. 1epeat steps $@1' for the abnormal control( :evel $)9 as well as the patient
sample.
#uality $ontrol:
1. /ontrols= :evel 1 ( normal) and :evel $(abnormal)
2. Adherence to %ood laborator practice and careful followin% of the recommended
procedures will result in clinicall accurate and reproducible results.
3. 3nce rea%ents and controls are reconstituted9 put a time9 date and initial on the bottle.
4. /ontrols should be ran once per shift or with each chan%e of rea%ent.
5. /ontrols should be lo%%ed into either an :)S ( :aborator )nformation sstem) or on
%raph paper to detect shifts and trends in &ualit control values.
6. Stabilit of controls once reconstituted is 0 hours.
Sources of %rror&'rou(leshooting:
1. Associated with specimen
a. )nappropropriate ratio of anticoa%ulant to blood
b. ;ailure to correct citrate volume if hematocrit F ""#
c. /lotted9 hemol2ed or lipemic samples
d. :ack of PPP
e. Dela in testin% or processin%
f. )nappropriate stora%e
2. Associated with stora%e
a. )ncorrect preparation of rea%ents
b. ;ailure to properl store rea%ents
c. Gse of rea%ents beond reconstituted stabilit time or e-piration date
d. /ontaminated rea%ents
$. Associated with procedure
a. )ncorrect temperature
b. )ncorrect incubation times
c. )ncorrect volumes of sample9 rea%ents or both
Pa%e + o/ %
!L&' 122()Lab #2
Laboratory #2
Protime
Prothrombin Time "es,lts
-ame.....................
/ate......................
.
Points= 12
001hart 2,ality 1ontrol res,lts on 3ra$h
00 4ncl,5e meas,rement ,nits
PT -ame an5 4/# ",n 1 ",n 2 ",n * 6inal
"es,lt
7ithin
"e#erence
"an3e8
-ormal
control
&bnorm
al
control
Patient
Pa%e 5 o/ %
!L&' 122()Lab #2
Laboratory #2
Protime
Pa%e 9 o/ %
!L&' 122()Lab #2
Laboratory #2
Protime
-ame...................
/ate....................
Lab # 2: Protime
3tudy 4uestions
Points5 15
1. #hat "oa%ulation /a"tor de6"ien"ies "an be dete"ted usin% the
'rothrombin time as a
s"reenin% test7 83 'ts9
2. #hat is the re/eren"e ran%e /or Protimes7 81 't9
3. #hat thera'euti" anti"oa%ulant is monitored usin% the PT7 :;'lain how it
wor<s. 82 'ts9
4. =/ a 'atient is on thera'euti" anti"oa%ulant! how many times normal do we
want the PT
to be7 81 't9.
5. #hat rea%ent8s9 are utili>ed in 'er/ormin% the PT7 :;'lain the 'ur'ose o/
ea"h rea%ent used. 82 'ts9
6. $oes the PT more a""urately monitor de6"ien"ies in the intrinsi" or the
e;trinsi" 'athway7 #hat initiates this 'athway7 81 'ts9
Pa%e ( o/ %
!L&' 122()Lab #2
Laboratory #2
Protime
7. ?ame the /a"tor whi"h! i/ de6"ient will "ause a 'rolon%ed PT but will not
a@e"t the PTT. 81 't9.
8. Aor a normal 'rothrombin time! all these /a"tors are re.uired in ade.uate
"on"entrations :;1:PT 8"ir"le one 9 8 1 't9
a. ;actor )
b. ;actor ))
c. ;actor >
d. ;actor >))
e. ;actor >)))
. ?ame three 'otential sour"es o/ error that "an o""ur in the PT test. 83
'oints9
Pa%e % o/ %
!L&' 122()Lab #2