Basic coagulation techniques and

Quality control issues

Dr. Shrimati Shetty
Deputy Director
National Institute of Immunohematology ( ICMR
!"M #ospital$ Mum%ai
&inal Diagnosis of a %leeding disorder
'nticoagulant
l
(.)*+ M tri sodium Citrate at a ratio of + parts %lood to )
part anticoagulant is used for all coagulation tests.
,hy not "D-' or #eparin.
l
"D-' irre/ersi%ly chelates Ca ions
l
#eparin acti/ates antithrom%in 0hich is an inhi%itor of
coagulation
'nticoagulant1
l
If the HCT is above 55%

Anticoagulant vol. [x] = 100 heatoc!it x
total vol. of anticoagulate" bloo" !e#ui!e"
$xa%le&
'atient heatoc!it = (0%
100 ) (0 x 5.0 = 0.*+ ,
2reanalytical /aria%les
l
-a%le heol.sis/ li%aeic sa%le
l
I%!o%e! %!o%o!tion of Anticoagulant to bloo"
l
'!olonge" tie inte!val befo!e testing
l
0ifficult %unctu!es
l
1!ee2e tha3ing the sa%les
Coagulation -ests
l
-c!eening coagulation tests
l
Confi!ato!. Tests
,hen a %leeding patient 0al3s in$ 0hat should %e the initial
tests to %e performed.
Screening tests
l
2eripheral smear ( Bernards Soulier syndrome$ macrothrom%ocytopenia$
leu3emia $ throm%ocytopenia
l
Complete Blood Count 4 5--6 7TC'6 ,eu8eias6 th!oboc.to%enia9
l
2-
l
'2--
l
--
l
&4III screening

5nly 2- is a%normal
l
Congenital Causes
1acto! :II "eficienc.
l
Ac#ui!e" Causes
,ive! "isease
;a!fa!in
0IC
Inhibito!s
,A
7alignanc.
2- 6 INR
l
'T easu!es the "eficienc. of all :<
"e%en"ent coagulation facto!s an" also the
integ!it. of ext!insic %ath3a.
l
I=> = ['atient 'T]I-I
[Cont!ol 'T]
5nly '2-- is a%normal
l
Congenital Causes
1acto! :III/ I?/?I/?II
contact facto!s
l
Ac#ui!e" Causes
,ive! "isease
;a!fa!in
0IC
He%a!in
Inhibito!s to facto!s6
,A
2- 7 '2-- prolonged
l
Congenital causes
i.Cobine" "eficienc.
of : @ :III
ii. 1acto! ? "eficienc.
iii. 1acto! :
"eficienc.
iv. 7ulti%le :<
"e%en"ent clotting facto!
"eficienc.
l
Ac#ui!e" causes
,ive! "isease
;a!fa!in
0IC
He%a!in
Inhibito!s to facto!s
vitain < "eficienc.

2-$ '2--$ -- prolonged
l
Congenital causes
i. Afib!inogeneia/
".sfib!inogeneia
ii. 1acto! II "eficienc.
l
Ac#ui!e" causes
0IC
,ive! "isease
Screening for & 4III deficiency
Clot solubilit. test
l
Clot formation 0ith either throm%in or CaCl*
l
Solu%ility of the clot using *8 acetic acid $ )8 mono
chloroacetic acid or urea
l
'fter *9 hours $ the clot is o%ser/ed for solu%ility
l
Different sensiti/ities 0ith different clotting reagents 7
sol/ents
l
Se/ere factor deficiency sometimes gets misdiagnosed as
& 4III deficiency
":IS' test is the sensiti/e assay for detecting & 4III
deficiency
Confirmatory tests6&actor 'ssays
>eagents !e#ui!e"
=o!al %oole" %lasa o! unicalib!ato!
1acto! 0eficient %lasa
A'TT !eagent
CaClA
&actor assays1.
1acto! :III/I?/?I/?II A'TT base"
1acto! II/:II 'T o"e
1acto! :/? can be both 'T/A'TT o"e
&actor ;III <raph
Interpretation of factor results
l
't %irth$ acti/ities of the /itamin ! dependent factors II$ ;II$ I4$ and 4 and
the concentrations of the contact factors 4I and 4II are reduced to a%out
=(8 of normal adult /alues. -he le/els of the factors ;$ ;III$ 4III$ and
fi%rinogen are similar to adult /alues
l
2lasma concentrations of the naturally occurring anticoagulant proteins
(antithrom%in$ protein C$ and protein S are significantly lo0er at %irth
than during the adult years
l
Most %lood coagulation factors and fi%rinogen increase during pregnancy.
&actor (& 4I is the only %lood coagulation factor that decreases.
l
Malignancies
Interpretation
l
-houl" 3e go ahea" 3ith facto! assa.s even
3hen sc!eening tests a!e no!alB
.es6 in case of an. clinical in"ication 3e
shoul" "o the s%ecific facto!s even if 'T/A'TT
is no!al
N22
l
A0)A5 health. "ono!s 6 bloo" g!ou% atche"
l
Co%a!e it 3ith unicalib!ato! 3ith 8no3n
facto! values
l
=eve! use a single in"ivi"ual sa%le as
cont!ol
Deficient 2lasma7 '2-- reagents
l
-houl" have 0% 1:III
l
=egative fo! TT0/inhibito!s
l
0iffe!ent A'TT !eagents have "iffe!ent
sensitivities
&actor 'ssays
l
-eve!e C1% facto!
l
7o"e!ate 1)5%
l
7il" ()D0%
About 5% of the %atients a!e clinicall. il"
"es%ite having C1% facto!
Can 0e diagnose a patient as #' or #B
0ithout doing factor assays.
Mi>ing Studies
5a-ED a"so!be" no!al %lasa6 "eficient in
facto!s :II6 I?6 ? an" %!oth!obin
Age" no!al se!u6 "eficient in facto!s : an"
:III6 %!oth!obin6 an" fib!inogen
Mi>ing Studies1.
>esult Inte!%!etation
=''FA"so!be"
'lasa
Co!!ection 1 :III "eficienc.
=''F Age"
-e!u
=o Co!!ection
=''FA"so!be"
'lasa
=o Co!!ection 1 I? "eficienc.
=''F Age"
-e!u
Co!!ection
Mi>ing Studies1..
7ixtu!e >esult Inte!%!etation
='' F 1 :III 0ef
'lasa
Co!!ection 1 I? "eficienc.
='' F 1 I? 0ef
'lasa
Co!!ection 1 :III "eficienc.
2latelet 'ggregation tests
l
Highl. va!iable !esults
l
0iet6 7e"ication6 %h.sical activit.
l
'latelet !ece%to! stu"ies to confi! "iagnosis
l
Al3a.s confi! "iagnosis b. othe! tests
=eve! give a "iagnosis base" on %latelet
agg!egation aloneG
Disorders diagnosed %y platelet aggregation and
receptor studies
l
:on ;illeb!an" "isease
l
Hlan2anns th!obasthenia
l
5e!na!" -oulie! s.n"!oe
l
-to!age %ool "efect
l
C.cloox.genase "eficienc.
l
othe!s
,hat are the other supporting tests.
l
'latelet !ece%to! stu"ies using antibo"ies
s%ecific fo! %latelet !ece%to!s
H' 1b/I? fo! "iagnosis of 5-- 4C0 DA9
H' IIb/IIIa fo! "iagnosis of HT4 C0D16 C0(19
collagen !ece%to!s 4 C0*(9
Diagnosis of ;,D
l
'latelet agg!egation 3ith >istocetin 4 1.A5 g/l9
absent o! !e"uce"
l
T.%e IIb sho3s inc!ease" agg!egation 3ith 0.5
g/l 3he!eas in no!al cases the!e is no
agg!egation
l
:;1 b. $lect!o%ho!esis sensitive onl. fo! seve!e "ef
l
$,I-A is the test of coni!ation
l
>Cof 6 collagen bin"ing $,I-A an" 7ultie! anal.sis
to subt.%e
Diagnosis of BSS
l
Hiant %latelets in %e!i%he!al sea!
l
=o!al o! !e"uce" %latelet count
l
Absence of agg!egation 3ith 1.A5 g/l
!isticetin
l
Absence of H' 1b/I? !ece%to!s b. flo3
c.toet!.
Diagnosis of <-
l
Absence of agg!egation 3ith (u7 A0'6
Dug/, collagen an" 0.+57 a!ach"onic
aci"
l
Absence of H' IIb/IIIa !ece%to!s b. flo3
c.toet!.
Storage pool defect
l
'!ia!. %hase agg!egation 3ith all agonists
Screening for inhi%itors ( Mi>ing studies
l
='' an" %atient %lasa ixe" an" A'TT
%e!fo!e" at 0 hou!6 1 hou! an" A hou!
l
-houl" exclu"e ,u%us anticoagulants
l
1:III inhibito!s a!e gene!all. %!og!essive 6
1I? inhibito!s/,A ie"iate acting
Screening for inhi%itors
0 h! 1 h! A h!s
=''
'atient
-e%a!atel.
incubate"
Incubate"
7ix
-he Bethesda 'ssay
Speciali?ed In/estigations
l
Th!oboelastog!a%h.
-hrom%oelastography
-hrom%inoscope
-hrom%inoscope
2&' )((
Quality control e>ercises
l
IIC
l
$IC
Some e>amples
Case 1
'e!i%he!al sea!) giant %latelets seen
'latelet count 1*0?10*/u,
'T C1D -ecs/ ' 15 secs J
A'TT CAK secs/' *A secsJ
TT C 1( secs/' 15 secs
1 ?III ) =
>
I
'
A

>
is
t
o
c
e
ti
n
5
%
A
0
'
6
c
ol
la
g
e
n
6
A
A

K
0
)
1
0
0
%
H
'
1
b
/I
?
!
e
c
e
%
t
o
!
s

hi
g
hl
.
!
e
"
u
c
e
"
0
ia
g
n
o
si
s
&
5
-
-
Some e>amples 1..
l
Case A
'- no!al
'latelets A(0? 10*/ul
'T C1D secs/ ' *A secs
A'TT C *0 secs ' 5L secs
TT C 15 secs/ ' 1( secs
1 ?III ) =
l
1
?


K
(
%
J

1

:

1
5
%
1

:
II
I
L
%

0
i
a
g
n
o
s
i
s
&
C
o

b
i
n
e
"
"
e
fi
c
i
e
n
c
.
o
f
1

:

a
n
"
:
II
I

-han3 you