1-22-10 Tests of Coagulation (mainly on screening testing) 1. Discuss the use of laboratory testing in the evaluation of bleeding risk.

Primary hemostasis involves platelets, vWF, an fi!rinogen, "hich together form the #primary platelet plug$% &t occurs mostly in the epithelium, mucosa, an en othelium% 'ysfunctional primary hemostasis results in ecchymoses, "et purpura in the !uccal an rectal mucosa, an petechiae (thin( )*+&,&-.)% ,creening tests for primary hemostasis inclu e/ History of patient an family/ valua!le tool for assessing !lee ing ris(% &nclu es as(ing a!out previous surgeries, menstrual o!stetric history, an family history of !lee ing% PB smear/ can pic( up a!normal loo(ing platelets Bleeding time/ a normal !lee ing time is less than 9 minutes. )lee ing time tests for everything involve in platelet plug formation/ platelet num!er, normal su!en othelial collagen, normal vWF, effective interaction of su!en othelial "WF an platelets, an normal platelet factors% PFA-1 / involves the passing of whole blood though a narro" tu!e coate "ith collagen01'P or 2P&% &t is reporte as closure time (CT)% ,econ ary hemostasis involves the coagulation casca e/ aPTT is the &-T*&-,&C path"ay (3&&, 3&, &3, 4&&&) PT is the 23T*&-,&C path"ay (!"")% 4&& !in s Tissue Factor% The common casca e proteins are 3, &&, 4, an &% 1ll #can !e fol e in half5$% The ultimate result is prothrom!inthrom!in, "hich converts fi!rinogenfi!rin% The coagulation casca e is evelope in vitro, so it6s not e7actly reflective of "hat goes on in the !o y% 'ysfunctional secon ary hemostasis results in muscle an joint !lee s (thin( )&. )822',)% ,creening tests for secon ary hemostasis inclu e/ History of patient an family P# (prothrom!in time)/ reflects extrinsic path"ay% &nvolves the com!ination of brain tissue thromboplastin an citrated plasma (negatively-charge citrate !in s Ca29 an pulls it out of the system, there!y halting the coagulation cascas e)% Calcium is then a e , an clotting time is measure % -ormal is 1$-1%s "&' is .(-1.$s aP## (activate partial throm!oplastin time)/ involves citrated plasma, phospholipids, an contact activator% Then a in Ca29 an measure the time to clot% -ormal is $%-))s #*# (throm!in time)/ involves thrombin an citrated plasma -ormal is 1 -1%s Fir!rinogen+1 mg,d- prolongs TCT5 Note that PB smear is NOT done for secondary hemostasis% :ou cannot see the coagulation casca e in a smear5

1 itional note/ the lecture mentions specific tests for platelet function% They inclu e .latelet aggregometry (test platelet response to specific agonists), v/F factor antigen (;+1-T&T1T&42 measure of vWF/1g), an 'istocetin *ofactor Assay (tests F+-CT&<-18 a!ilities of vWF)% :ou perform these tests as after esta!lishing a primary hemostasis pro!lem using the screening tests !lee ing time or PF1-100 (see case of heavy menses in lecture)% They are iscusse in greater etail in the ne7t <6Connell lecture% &f a patient has a quantitative eficiency in vWF, treat "ith DDA!P, "hich enhances the release of vWF% $. Describe ho0 the screening tests for evaluation of .rimary and secondary hemostasis are .erformed. ,creening tests for primary hemostasis/ Bleeding time/ in vivo measure of time it ta(es for a s(in incision to clot% 1s mentione a!ove, it in irectly assesses all steps of platelet plug formation, inclu ing platelet num!er, interaction !et"een platelets an su!en othelial vWF (involving "b"1), an platelet-platelet interactions (involving ""b"""a an fi!rinogen)% -ormal a ult has !lee ing time => min &nherite isor ers that prolong !lee ing time/ 2lan3mann4s #hrombasthenia/ efective ""b"""a Bernard-5oulier/ efective "b"1 4on Wille!ran 6s 'isease/ pro!lems "ith vWF Platelet granule efects 1c?uire isor ers that prolong !lee ing time +remia/ i%e% uring (i ney failure Paraproteinemia/ e7cess gammaglo!ulin ,evere anemia 8iver isease 1spirin an -,1&'s PFA-1 (platelet function tests)/ passes anticoagulate "hole !loo through a narro" tu!e "ith a mem!rane coate "ith collagen91'P, or 2P&% PF1-100 is superior to !lee ing time !ecause it effectively screens for platelet ysfunction, mo erate-to-severe vW', an accounts for spirin effects% Closure time (CT)/ time it ta(es to occlu e (clot) the aperture ,creening tests for secon ary hemostasis/ aP## "ill get a prolonge aPTT "ith eficiencies in @AWB, pre(alli(rein, 3&&, 3&, &3, 4&&&, 3, 4, &&, an Fi!rinogen% 1 normal aPTT is $%-))s% aPTT "ill only !egin prolonging if there6s a !"#$"% deficiency in any of the aforementione factors% &n other "or s, you only nee C0D of the factors to have a normal aPTT5 27ceptions inclu e 4&&& an 3& eficiencies, "hich !egin prolonging aPTT "ith a E0-F0D eficiency% The curve for &3 aPTT is also very flat, "hich means that it prolongs later !ut oes not get very high% :ou coul have no &3 an only see a slight elevation in aPTT (!ut "ill start seeing severe !lee ing)%

1 eficiency in @AWB, pre(alli(rein, or 3&& "ill prolong aPTT !ut "ill have no physiological effect &i'e' no secondary hemostasis()% 1 s.urious .rolongation of aP## can occur in three "ays/ :ou have an elevate citrate6.lasma ratio (i%e% you have too much citrate relative to plasma in the test tu!e) :ou acci entally have he.arin in the test tu!e% @eparin is an anticoagulant5 :ou have a clotted sam.le% 1 clot "ill pull factors out of the sample% &f you get a prolonge aPTT, the ne7t step is to "or( it up !y performing a % 6% mi7ing study, "here E0D patient6s plasma is mi7e "ith E0D control plasma (again, you o -<T o P) !loo smears for secon ary hemostasis)% The i ea !ehin this test is that you only nee C0-F0D of normal for each coagulation casca e factor to maintain a normal aPTT% ,uch lo" levels means that a ing E0D normal plasma shoul correct the pro!lem if it6s a eficiency (even if you have Gero factor5)% ThusH &f the aPTT is normal "ith a E0/E0, the patient has a deficiency in the intrinsic or common path"ay% With this result, assay factors XII) XI) IX) and VIII% &f the aPTT remains prolonge ( oesn6t correct) "ith a E0/E0, the patient has an inhibitor in the intrinsic or common path"ay (the inhi!itor "ill mo ify the normal patient6s plasma in the E0/E0 mi7)% With this result, test for Lupus Anticoagulant or Anticardiolipin Antibodies% &f these are negative,t est for inhi!itors specific to coagulation factors (i%e% FVIII inhibitor)% @emophilia a/ ecrease F4&&& activity @emophilia !/ ecrease F&3 activity P#/ as mentione !efore, PT is performe "ith the com!ination of citrate plasma an !rain tissue throm!oplastin% Calcium is then a e , an the clotting time is measure % 1 normal PT is 12-1Es, "hile a normal &-* is 0%I-1%2s% PT starts prolonging "ith a % -8 9 eficiency in 4&&, 3, 4, &&, or fi!rinogen+1 % The most common cause for a prolonged PT is a heterozygous deficiency in FVII or a gene polymorphism' @omoGygote eficiency of 4&& is incompati!le "ith life% &f you get a prolonge PT, the ne7t step is to "or( it up !y performing a % 6% mi7ing study% +nli(e aPTT, you nee F0-E0D of normal F4&& for PT to not !e prolonge % ,till, the results can !e interprete in the same "ay/ &f the PT is correcte , it6s a eficiency% &f the PT remains prolonge , it6s an inhi!itor% With this result, test for anti-Factor && in Lupus Anticoagulant% -ote that 8upus anticoagulant "ill result in a prolonge PT 1-' aPTT% #*# (or TT)/ throm!in time com!ines !ovine0human throm!in "ith citrate plasma% Throm!in activates fi!rinogen, so throm!in time tells us if there6s a defect in fibrinogen%

Aore specifically, TCT measures the *1T2 <F C<-42*,&<- <F F&)*&-<.2-F&)*&-% Things that affect the rate of fi!rinogen conversion/ a fibrinogen level of =100mg0 8 prolongs TCT ( on6t have enough fi!rinogen to have a long throm!in time5)% This is seen in afribinogenemia, he.arin test for presence of heparin using the 'e.tilase test, "hich clots fi!rinogen in the presence of heparin% lo" quality of fibrinogen cause !y hereditary dysfibrinogenemia, cirrhosis, he.atocellular carcinoma, an ne0born infants% Para.roteinemia an renal failure also prolong the TCT% 1 normal TCT is 10-1Es 1 prolonge TCT "ill !e accompanie !y a prolongation of ** T+, OT+,- T+-,, screening tests. increased bleeding time) increased aPTT) and increased PT% That6s !ecause fi!rinogenfi!rin is involve in !oth e7trinsic an intrinsic coagulation casca es, an fi!rinogen is also an essential component of the platelet plug% &f ou get a prolonge TCT, the ne7t thing to or er "oul !e serum and urine .rotein electro.horesis 0ith immunofi7ation% This "oul specifically test for paraproteinemia% ). "dentify the strengths and 0eaknesses of different screening tests of coagulation. Bleeding time/ results are highly performer- epen ent it is not a relia!le pre ictor of proce ure-associate hemorrhage in patients "ithout a !lee ing history% &t is !etter to loo( for a history of bleeding% 1 normal !lee ing time does not rule out operative hemorrhage% :ou can6t operate on someone Just !ecause they have a normal !lee ing time% )lee ing time is not relia!ly correlate "ith aspirin or -,1&',% PFA-1 / ,trengths/ superior to !lee ing time as a screen for platelet ysfunction, mo erate-tosevere vWF, an 1spirin effect 1 fe" a itional notes on clot !rea( o"n FibrinolysisKclot !rea( o"n% The (ey me iator of the process is .lasmin. :uglobulin -ysis #ime (a(a dilute 0hole blood clot lysis time)/ this test measures the time to brea/ down a clot in either a sample of plasma &ELT0 or whole blood &DWB LT0 ,hortene "ith cirrhosis; al.ha$-anti.lasmin deficiency, .lasminogen activator inhibitor-1 <PA"-1= deficiency, an systemic fibrosis Prolonge "ith renal disease, "hich increases P1&-1 levels (rare)