Lab diagnosis of bleeding disorders Dr chithra p

malabar dental college and research centre

1. LABORATORY DIAGNOSIS
OF BLEEDING DISORDERS
PRESENTED BY
Dr. CHITHRA P
Senior Lecturer
Dept of Oral Medicine & Radiology
Malabar Dental College & Research Centre, Manoor Chekannoor
Road, Mudur PO, 679578,

2. INTRODUCTION
Bleeding disorders constitute an important group of
disorder in haematology.
Bleeding disorders result when the blood’s ability to
form a clot at the site of blood vessel injury is
impaired.
Bleeding disorders are classified as disorders of
primary hemostasis (platelet or vascular disorders) or
disorders of secondary hemostasis (coagulation
protein disorders).
Differentiation is an essential step in the diagnostic
workup

3. HEMOSTASIS
Hemostasisisdefinedasaphysiologicalresponsetoblood
vesselinjuryandbleeding,whichentailsa co-ordinated
processinvolvingthebloodvessel,platelets,andbloodclotting
proteins(i.e.coagulationfactors).
Guyton, Hall. Textbook of Medical Physiology
Hemostasis can be divided into primary and secondary
hemostasis

4. BLEEDING DISORDERS

5. BLEEDING DISORDERS

6. DIAGNOSIS OF BLEEDING
DISORDERS

7. Indiagnosisofhemostaticdisorders,itisimportantto
haverelevantclinicalinformationbeforeinterpreting
theresults.
Fromthehistory,itisimportanttodifferentiate
hereditarydisordersfromacquired.
Hereditarydisordersgenerallystartearlyinage,or
recurrentinnatureandmayhaveapositivefamily
history.
Ontheotherhand,acquireddisordersoccuratanyage
andhaveanunderlyingpredisposingcause.
DIAGNOSIS OF BLEEDING DISORDERS

8. History of medication should include replacement
therapy, use of anticoagulants like heparin besides
intake of analgesics, antibiotics etc.
Depending on the type of bleed, it is important to
differentiate between platelet and coagulation factor
bleeds.
In general, platelet bleeds present as peticheal
hemorrhage, mucosal bleeds, whereas coagulation
factor disorder presents as deep abdominal bleeds,
muscle bleeds, hemostasis, ecchymotic patches.
DIAGNOSIS OF BLEEDING DISORDERS

9. Itisimportanttogoinastepwiseapproachsothat
minimumtestsareundertakentomakeadefinitive
diagnosisandtoavoidunnecessarytestsandlaboratory
load.
Inaddition,suchaninvestigativeapproachbecomes
costeffectiveandpatientfriendly.
Ashortscreeningcoagulationshouldbethestarting
point.
Dependingontheabnormalitiesobservedintheshort
screening,extendedscreeningtestscanbeperformed
followedbyspecializeddiagnostictests.
LABORATORY TESTS

11. COLLECTION OF BLOOD
Venous blood should be taken by means of a clean venepuncture,
with minimum stasis(remove tourniquet as soon as the needle is in
the vein) using a 21 gauge number needle(length not 3.5cms) and a
plastic syringe and transferred rapidly in a plastic(polypropylene) or
siliconized glasstube (13x100mm), containing 3.2% trisodium citrate
solution.

12. The blood should be allowed to run down by the side of the tube, it
should never be squirted into the test tube, the tube should be screw
capped and quickly but gently mixed by inverting at least 5-7 times
without producing frothing.
The blood specimen should be transported to the laboratory as
quickly as possible. Centrifuge within 30 minutes after
venepuncture and carry out actual test within 4 hrs.
Centrifuge the sample at 3500 to 4000 rpm for 15 minutes at 4°C in
a refrigerated centrifuge
Keep the samples in a box. Containing small saturated with water to
eliminate air spaces, until tested or during testing
COLLECTION OF BLOOD

13. SCREENING TESTS

14. Quantitativeplateletdisordersaredetectedviaa
plateletcount.
Thisshouldbeperformedinallpatientswitha
suspectedbleedingdisorder.
Samplesarecollectedandanalyzedeithermanually
(byhemocytometer)orwithanautomatedcell
counter.
PLATELET COUNT

15. Examinationofabloodsmearallowsforrapidestimationof
plateletnumbers.Thisisessentialintheemergencysettingwhere
automatedcountsmaynotbeavailable.
Smearexaminationalsoservestoverifyfindingsofautomated
countersbecauseplateletclumpingcanresultinartifactuallylow
counts,adecreasedplateletcountshouldalwaysbeverifiedvia
smearevaluationand,ifnecessary,thecountrepeatedonafreshly
drawnbloodsample.
Attentionshouldbepaidtoplateletmorphology,asthepresenceof
largeplatelets(macroplatelets)isgenerallyindicativeof
megakaryocytichyperplasiaandaregenerativeresponse.
PLATELET COUNT

16. BLEEDINGTIME
Principle: The bleeding time test measures the time that elapses
after infliction of a standard wound and the arrest of bleeding. It
depends largely on the rate of formation of platelet plug and is
mostly independent of fibrin forming coagulation mechanisms.
TEMPLATE METHOD
The patient is seated comfortably at a warm ambient temperature
with one forearm resting horizontally on a table with the flexor
surface upper most.
A scalpel blade is so arranged on the holder (with the help of
gauge) that the tip protrudes 1mm through the template.
A sphygnomano-meter cuff is placed round the patient’s upper arm
and inflated to 40mm Hg.

17. Skin of the forearm is cleaned with spirit and allowed to dry.
Template is pressed firmly on the forearm with the slit placed in
cephalo-caudal direction and about 5 cm distal to the crease of
the elbow, taking care of superficial veins.
With the help of the scalpel blade an incision 9 mm in length and
1 mm in depth is made with a quick movement.
BLEEDINGTIME

18. Immediatelyastopwatchstarted.Thesidesoftheincisionare
gentlyblottedwithafilterpaperevery30secondstoremovethe
bloodoozingout,untiltheincisionisdry(avoidtouchingthe
wounditselfwiththefilterpaper).
Stopthestopwatchandnotethereading.Cleanthewoundwith
spirit.Opposewoundedgesandputadhesiveplasterforatleast
48hr.
NormalRange:2-5minutes
Interpretation:Bleedingtimeisameasureofcapillaryfunction,
plateletnumberandplateletfunction.Itisprolongedinconditions
suchasThrombocytopeniaandPlateletfunctiondefects,Von
Willebrand’sDisease,Capillarydefect
BLEEDINGTIME

19. PROTHROMBIN TIME
Principle:The prothrombintime test belongs to a group of blood
tests that assess the clotting ability of blood.
The test is also known as the pro time or PT test. The blood is
collected in a tube that contains sodium citrate to prevent the
clotting process from starting before the test.
The blood cells are separated from the liquid part of blood (plasma).
The PT test is performed by adding the patient’s plasma to a protein
in the blood (thromboplastin) that converts prothrombinto
thrombin.
The mixture is then kept in a warm water bath at 37°C for one to
two minutes. Calcium chloride is added to the mixture in order to
counteract the Sodium citrate and allow clotting to proceed.
The test is timed from the addition of the calcium chloride until the
plasma clots. This time is called as the prothrombintime.

20. Normal value : The normal prothrombin time is 11-15
seconds. A prothrombin time within this range indicates
that the patient has normal amounts of clotting factors VII
and X. A prolonged PT time is considered abnormal.
Abnormal value:The prothrombin time will be prolonged
if the concentration of any of the tested factors is 10% or
more below normal plasma values.
PROTHROMBIN TIME

21. Aprolongedprothrombintimeindicatesadeficiencyin
anyoffactorsVII,X,V,prothrombin,orfibrinogen.It
maymeanthatthepatienthasavitaminKdeficiency,a
liverdisease,ordisseminatedintravascularcoagulation
(DIC).
Theprothrombintimeofpatientsreceivingwarfarin
therapywillalsobeprolonged-usuallyintherangeofone
andonehalftotwotimesthenormalPTtime.PTtime
thatexceedsapproximatelytwoandahalftimesthe
controlvalue(usually30secondsorlonger)isgroundfor
concern,asabnormalbleedingmayoccur.
PROTHROMBIN TIME

22. Limitations:Spuriousresultsmayoccurifthebloodto
anticoagulantratioisnot9:1.ThePTclottingtimesmay
beprolongedbysubstancesincludingcorticosteroids,
EDTA,oralcontraceptives,asparaginase,clofibrate,
erythromycin,ethanol,Tetracyclineandanticoagulants
suchasheparinandcoumarin.ThePTmaybeshortened
bysubstancesincludingantihistamines,butabarbital,
caffeine,oralcontraceptives,phenobarbitalandvitamin
K.
PROTHROMBIN TIME

23. ACTIVATEDPARTIALTHROMBOPLASTIN
TIME(APTT)
Principle : APTT is the time required for plasma to
be clotted when maximal surface contact activation
and optimal phospholipid and calcium concentration
are provided.
Results are expressed as time seconds or as ratio.
Normal range: Depends on the reagents used in the
laboratory. Usually ranges from 29-35 seconds.

24. Interpretation :APTT is sensitive to the deficiencies or
abnormalities of both intrinsic and common coagulation factors i.e.
Factors I, II, V, X, VIII, IX, XI, XII, Fletcher factor and Fitzgerald
factor.
The test is more sensitive to an abnormality occurring in the early
stage of coagulation mechanism i.e. factors leading upto the
generation of Factor Xa and less sensitive to later stage i.e. Factor II
fibrinogen.
A deficiency levels < 20-50% of Factor XII, XI, IX, XIII, X or V
would give prolonged APTT. Where as a deficiency upto 10% of
Prothrombin and 0.5-1g/litre of fibrinogen or less would give an
abnormal APTT.
ACTIVATEDPARTIALTHROMBOPLASTINTIME(APTT)

25. APTT is also prolonged when there is an inhibitor present in
patient’s plasma. Therefore whenever a prolonged APTT is
detected, screening test for inhibitor must be performed. It is also
an important test for the control of heparin therapy.
Therapeutic range-60-100 seconds.
ACTIVATEDPARTIALTHROMBOPLASTINTIME(APTT)

26. APTT MIXINGSTUDYWITHNORMALSERUM/ ADSORBED
PLASMA
Principle : Plasma samples found to have a prolonged APTT are
further investigated to define the abnormality by performing mixing
or correction tests.
Correction of the abnormality by the additive indicates that the
reagent must contain the substance deficient in the test sample.
An abnormal APTT is repeated on 50:50 mixtures of a known
congenitally deficient plasma and the test plasma, or on 50:50
mixtures of aged/adsorbed plasma and test plasma until correction is
obtained and the missing factor identified.

28. CLOTSTABILITYTEST
Principle : Clot formed in the presence of factor XIII and
Ca2+ are stable for at least 1 hr in 1% monochloracetic
acid solution and in 5 mol/litre urea, whereas clots
formed in the absence of factor XIII dissolve rapidly.
Normal –Clot stable
If clot dissolves : Factor XIII deficiency

29. THROMBINTIME(TT)
Principle:Thrombinisaddedtotheplasmaandclotting
timemeasured.Theclottingofcitratedplasmaisaffected
bytheconcentrationandreactionoffibrinogen,andalso
bythepresenceofinhibitorysubstances.
Thenormalrangeis15–17seconds.
Interpretation:Thrombintimeisprolongedinthe
presenceof heparin,hypofibrinogenemia,
dysfibrinogenemiasandfibrindegradationproducts.

31. FIBRINSPLITPRODUCTS(FSPS) / FIBRIN
DEGRADATIONPRODUCTS(FDPS)
FSPs are the end-products of fibrinolysis, and are generated
when fibrinogen, soluble fibrin, or cross-linked fibrin is lysed.
Commercial latex agglutination kits (Thrombo-Wellcotest,
Wellcome Reagents, Beckenham, England) constitute a rapid,
semi-quantitative method for FSP determination.
The test utilizes latex particles coated with anti-human FSP
antibodies, which also cross-react with the FSPs of other
species.

32. Thistestalsohelpstodetectfibrinogen.Specialtubesareused
whichcontainthrombin,toclotthesampleandremovethe
fibrinogenfromsolution,aswellasaninhibitoroffibrinolysisto
preventfurtherfibrinbreakdown.
ElevatedconcentrationscommonlyoccurinDICbutarenot
universallypresent,norspecific,forthesyndrome.Increased
concentrationsmayalsooccurinhepaticdisease,duetoenhanced
fibrinolysisandreducedclearance.Falseelevationsmayoccurin
patientsonheparintherapyorthosewithdysfibrinogenemia.
FIBRINSPLITPRODUCTS(FSPS) / FIBRIN
DEGRADATIONPRODUCTS(FDPS)

33. D-DIMERTEST
D-Dimers are unique FSPs that are formed when cross-linked
fibrin is lysed by plasmin.
Principle :The latex particles provided in the D-Di Test are coated
with mouse anti-human D-dimer monoclonal antibodies. Test
samples containing D-dimers when mixed with the latex particle
suspension make the particles agglutinate.
At a predetermined concentration of D-dimers that the D-di test is
designed for, the agglutination of the latex particles produces
macroscopic clumps that can be visualized by the naked eye.

34. Results: D-Dimer test results are expressed in initial fibrinogen
equivalent units (FEU), which are exactly the same units, used for
the FDP assay.
By definition, as FEU is the quantity of fibrinogen initially
present that leads to the observed level of D-dimer. In general, the
actual quantity of D. -dimer is approximately half of an FEU.
For practical purposes that positive cut-off value of 0.5 ug/ml
(FEU) is approximately 0.25 ug/ml (actual Ddimer).
Either unit may be used as preferred. For consistency, all results
discussed in this insert are in FEU.
Normal : The d-dimer levels are <0.5 ug/ml (expressed in FEU).
The D-dimer level increases during pregnancy. It also rises with
age ( >70 yr)
D-DIMERTEST

35. The d-dimer is a sensitive test for DIC and likely is superior
to traditional FSP assays for this purpose.
However, d-dimer concentrations are not always elevated in
patients with DIC, and elevated d-dimer levels are certainly
not specific for DIC.
Elevated concentrations have been demonstrated in conditions
with thromboembolism, neoplasia, hepatic disease, renal
failure, cardiac failure, internal hemorrhage, and following
surgical procedures.
It should be considered an ancillary diagnostic test, with the
diagnosis of DIC relying on the appropriate constellation of
clinical findings and abnormal results of hemostatic testing.
D-DIMERTEST

36. FACTORVIII ASSAY
ONESTAGEFVIIICLOTTINGASSAY
Principle:Theassayisbasedontheabilityofthepatient’splasma
toshortentheabnormalactivatedpartialthromboplastintimeofa
severeFVIIIdeficientplasmaascomparedtothecorrections
obtainedfromnormalpoolplasma.
NormalRange:FactorVIIIconcentrationrangefrom50to200%
butvarieswidelyinagivenpopulationandalsointhesame
subjectatdifferenttimes,thereforenormalrangemustbe
establishedineachlaboratory.

37. Interpretation : Decreased values of F VIII activity may be
due to
A. Congenital deficiencies
1.Haemophilia A
2.Von Willebrand’sDisease
B. Acquired deficiencies
1.DIC
2.F VIII Inhibitors
Increased values may be found in:
1. Physical exercises
2. Pregnancy
3. Stress
4. After DDAVP administration
5. Liver disease etc.
FACTORVIII ASSAY

38. PLATELETAGGREGATIONTESTS
Principle: Platelets are disk-shaped blood cells that are also
called thrombocytes. They play a major role in the blood-
clotting process. The platelet aggregation test is a measure of
platelet function. The platelet aggregation test aids in the
evaluation of bleeding disorders by measuring the rate and
degree to which platelets form a clump (aggregate) after the
addition of a chemical that stimulates clumping (aggregation).

39. PLATELETAGGREGATIONWITHRISTOCETIN(RIPA)
Principle:Asampleofplateletsrichplasmaisstirredataconstant
speedinathermostaticallyheatedblockandanaliquotof
ristocetinisadded.Thepatternofaggregationisfollowedona
potentiometricchartrecorderwhichtracesthechangeinoptical
densityoftheplasmasamplewhichoccurswhenplatelets
aggregate.
Interpretation:Anantibioticristocetinderivedfromthe
bacteriumNocardiapuridawaswithdrawnfromclinicaluse
becauseofthehighincidenceofthrombocytopeniainpatient’s
treatedwiththedrug.Itwasfoundthatristocetincausesplatelet
aggregationinvitroandinvivo,andlateritwasshownthat
plateletsfromamajorityofpatient’swithvonWillebrand’s
Diseasefailtoaggregatewiththischemical.

40. DIAGNOSIS:Absent aggregation with all agonist indicates
Glanzmann Thrombasthenia, afibrinogenemia.
Reduced aggregation with Ristocetin : vWD, Bernard
Soulier
Reduced aggregation with all agonist : Storage pool disease,
Arachidonic acid pathway defect.
Reduced aggregation with any one or 2 agonist only:
Unclassified PFD
PLATELETAGGREGATIONTESTS

41. VONWILLEBRAND’SFACTOR(VWF) ANTIGENTEST
PrincipleofthetestVWFAgassayisasandwichELISA.
AcaptureantibodyspecificforhumanvWFiscoatedto96-
microwellpolystyreneplates.Dilutedpatientplasmaisincubated
inthewells,allowinganyavailablevWF:Agtobindtotheanti-
humanvWFantibodyonthemicrowellsurface.Theplateletsare
washedtoremoveunboundproteinsorotherplasmamolecules.
BoundvWF:Agisquantitatedusinghorseradishperoxidase
(HRP)conjugatedanti-humanvWFdetectionantibody.

42. Followingincubation,unboundconjugateisremovedbywashing.A
chromogeneicsubstrateoftetramethybenzidine(TMB)and
hydrogenperoxide(H2O2)isaddedtodevelopacoloredreaction.
Theintensityofthecolorismeasuredinopticaldensity(OD)units
withaspectrophotometerat450nm.PlateletvWF:Aginrelative
percentconcentrationisdeterminedagainstacurvemadefromthe
referenceplasma.
VONWILLEBRAND’SFACTOR(VWF) ANTIGENTEST

43. Normal Range : Plasma VWF:Ag values are generally
expressed in relative percent (%) as compared to pooled normal
plasma.
The normal range when normal plasma samples were tested by
REAADS vWF:Ag assay was 47-197% (mean 105.8% SD
39%).
Available assays (50-160%).
Sample with values above the range of the reference curve may
need to be diluted and retested for accurate results.
VONWILLEBRAND’SFACTOR(VWF) ANTIGENTEST

44. AssayofVWF:RCo(vonWillebrandRistocetincofactoractivity)
Principle:Thedegreeofaggregationofwashedplateletsinduced
byristocetinisrelatedtothevWFlevelpresentinthesystem.The
levelofvWFintheplasmathatarebeingtestedattheappropriate
dilutionisobtainedbycomparingthespeedofplateletaggregation
inthetestplasmawiththatofthestandardobtainedfromadiluted
normalhumanplasmapool.
Normalrange:ofvWFRCo(inadults=60-150%)
VONWILLEBRAND’SFACTOR(VWF) ANTIGENTEST

45. CONCLUSION
Bleedingdisordersconstituteanimportantclassofdiseases
whichshouldbemanagedappropriately
Laboratoryinvestigationsinasystematicmannerisvery
importanttoreachacorrectdiagnosis.
Variousscreeningaswellasconfirmatorytestsshouldbe
performedinanappropriateorderwhichcanhelptheclinician
aswellasthepatienttosavethetimeaswellasforcorrect
treatment

46. REFERENCES
Guyton, Hall. Textbook of Medical Physiology. 11
th
edition.
Edwards CW etal. Davidson’s principle and practice of Medicine.
18th edition.
Hutchinson’s Clinical Methods. 23
rd
edition.
Venkatraman BK. Diagnostic Oral medicine.
Chandler WL.Handbook of diagnostic hemostasis and thrombosis
tests. 3
rd
edition.
Gupta PK, Gupta M, Chatterjee T, Saxena R. Comparative
evaluation of whole blood D-dimer test to plasma D-dimer test for
diagnosis of disseminated intravascular coagulation. Indian J Exp
Biol 2005 Apr;43(4) : 382-384.