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Symposium : Newer Diagnostic Tests

Laboratory Studies in Coagulation Disorders Renu Saxena, Meganathan Kannan and Ved P Choudhry Department of Hematology, All India Institute of Medical Sciences, New Delhi, India

ABSTRACT It is important to go in a stepwise approach to diagnose spectrum of bleeding disorders, so that minimum tests are undertaken to make a definitive diagnosis and to avoid unnecessary tests and laboratory load. Depending on the abnormalities observed in the short screening, extended screening tests can be performed followed by specialized diagnostic tests. Bleeding time is prolonged in thrombocytopenia and platelet function disorders (PFD). If platelet count is normal, extended screening tests such as RVVT, PF3 availability and clot retraction can be performed. Russel viper venom directly activates FX, in presence of PF3, is an indicator of common pathway of coagulation. However, if there is deficiency of PF3 as obtained in PFD and APTT PT are normal, its prolongation indicates PFD. These can be tested invitro by performing RVVT with and without inosithin it is highly suggestive of underlying PFD. In such cases, diagnostic tests for PFD such as platelet aggregation with ADP, ADR, AA, Collagen and Ristocetin can be performed followed by electron microscopy if possible. Few of the interesting cases also have been discussed in the text. [Indian J Pediatr 2007; 74 (7) : 649-655]

Key words : Coagulation disorders; Screening tests; Laboratory approach; Case studies

Bleeding disorders constitute an important group of disorder in haematology. At AIIMS, over the past five yr, it has been seen that of a total of 600 coagulation disorders, 70.8% are due to Hemophilia A, 14.1% due to Von Willibrand Disease (vWD), 8.4% are due to Haemophilia B and the rest due to rare coagulation disorders like FX/V/XIII deficiency etc. In diagnosis of hemostatic disorders, it is important to have relevant clinical information before interpreting the results. From the history, it is important to differentiate hereditary disorders from acquired. Hereditary disorders generally start early in age, or recurrent in nature and may have a positive family history. On the other hand, acquired disorders occur at any age and have an underlying predisposing cause. History of medication should include replacement therapy or PRP, acitrom or 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.

Correspondence and Reprint requests : Dr. Renu Saxena, Professor and Head, Department of Hematology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029. India, Phone : 01126594670(off), Fax : 91(0)1126588663, E-mail : [email protected]

1. Laboratory Approach to bleeding disorders Since a large number of tests are essential to diagnose spectrum of bleeding disorders, it is important to go in a stepwise approach so that minimum tests are undertaken to make a definitive diagnosis and to avoid unnecessary tests and laboratory load. In addition, such an investigative approach becomes cost effective and patient friendly. A short screening coagulation should be the starting point (Table 1). Depending on the abnormalities observed in the short screening, extended screening tests can be performed followed by specialized diagnostic tests. It is essential to understand the coagulation system, which has extrinsic, intrinsic and common pathways. Coagulation is a continuous process which has been subdivided in to pathways for our better understanding only. (Fig. 1) TABLE 1. Short Screening Test Screening Test

Normal

Defective

Platelet count (PC) Bleeding time (BT)

150-450/cumm •P.C. 205’ • Platelet function • Vascular function Prothrombin time (PT) 12-14s • Extrinsic pathway • (FVII def.) • Common Path. (FX, V, II, I def. Activated Partial 35-45s • Intrinsic Thromboplastin time • Common path (FX, V, (APTT) II, I def) Clot stability (CS) Stable at 2 hr, •F, XIII def. 24 hrs

[Received June 20, 2007; Accepted June 20, 2007]

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R. Saxena et al TABLE 2. PPT (N) APTT (High) Hereditary defect (VIII, IX, XI, XII) Mixing studies (APTT) NS (corrected) Ads(not correct) Diagn.

NS (not corrected) Ads(corrected)

FIX def.

NS (not corrected) Ads. (not corrected)

FVIII def.

F XI, XII def

VWD (↑BT) Diagnostic test Fig. 1. Normal coagulation pathway

Bleeding time is prolonged in thrombocytopenia and platelet function disorders(PFD). If platelet count is normal, extended screening tests such as RVVT, PF3 availability and clot retraction can be performed. Russel viper venom directly activates FX, in presence of PF3, is an indicator of common pathway of coagulation. However, if there is deficiency of PF3 as obtained in PFD and APTT PT are normal, its prolongation indicates PFD. These can be tested invitro by performing RVVT with and without inosithin it is highly suggestive of underlying PFD. In such cases, diagnostic tests for PFD such as platelet aggregation with ADP, ADR, AA, Collagen and Ristocetin can be performed followed by electron microscopy if possible. Abnormalities in PT/APTT The following possibilities of the prolongation of PT & APTT Exist: • Prolonged PT • Normal PT • Prolonged PT

+ + +

Normal APTT Prolonged APTT Prolonged APTT

Isolated prolongation of prothrombin time (PT) may be hereditary as in FVII deficiency or acquired as in mild liver disease or warfarin effect. Isolated prolongation of APTT This is seen in inherited defects of deficiency of intrinsic factors like FVIII, IX, XI. In the absence of facilities for factor assay, mixing studies of APTT with normal serum or aluminium hydroxide adsorbed plasma (Ads) can be performed. Normal serum contains factors FIX, X, XI and XII. Adsorbed plasma contains FV, FVIII, XI, XII. The interpretation is given in (Table 2). In case prolonged APTT is accompanied by prolonged bleeding time, possibilities of VWD is suspected and this can be conformed using RIPA, VWF Ag and VWF RiCoF assays and multimeric analysis. Sometimes, however in VWD variants, BT may not be prolonged. Acquired cause of isolated prolongation of APTT includes presence of lupus anticoagulant, intrinsic factor inhibitors and heparin therapy. Lupus anticoagulant can be confirmed 650

Factor Assay

by Kaolin clotting assay and dilute russel veiper venom test. Factor inhibitors can be confirmed by screening tests of APTT followed by inhibitor assay (Bethesda assay). Prolongation of both PT and APTT This indicates deficiency in common pathway of coagulation (FV, FX, FII and FI) or multiple coagulation factor defects which may be inherited or acquired. When hemostasis suggests hereditary defects, mixing studies with normal serum or adsorbed plasma are helpful in differential diagnosis is given in Table 3. TABLE 3. PPT (High) APTT (High) Hereditary Defect (V, X, II, I) Mixing studies (APTT/PT) NS (corrected) Ads (not correct) Diagn.

FIX def.

Diagn. test

NS (not corrected) Ads(corrected) FV def.

Factor Assay

NS (not corrected) Ads. (not corrected)

TT n ↑ FII Def Hypofibrinogenemia Dysfibrinogenemia

Acquired conditions like DIC, Fibrinolysis, Liver disease, vitamin K deficiency may result in prolongation of both PT and APTT. DIC is confirmed by estimation of D Dimer, FDP. Fibrinolysis show normal D Dimer but abonormal FDP. Liver disease can be confirmed by liver function tests (SGOT/SGPT). Vitamin K deficiency can be confirmed by repetition of PT, APTT after giving 5mg of vitamin K injection for 3 days. Despite such extensive testing, normal screening test can be obtained in the following conditions. Disorders with normal screening tests • • • • • •

Mild clotting factor deficiency/Mild Platelet functional defects Simple purpura Senile purpura Henoch schonlein purpura Scurvy Hereditary hemorrhagic telengiectasia

Mild Von Willebrands Disease If these conditions are suspected strongly, it is suggested Indian Journal of Pediatrics, Volume 74—July, 2007

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Laboratory Studies in Coagulation Disorders that above tests are repeated to confirm the diagnosis 2. Collection of blood samples from patients and control As in all laboratory testing, ‘the results are only as good as the samples’. In coagulation , this cannot be stressed enough. Blood should not be collected through cannulae if they have been indwelling for more than 30 minutes or if heparin has been used to keep them patent. 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. 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 with in 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 3 Tests For coagulation disorders 3.1 Bleeding Time(Template method) 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.1-3 METHODS 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.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. Immediately a stop watch started. The sides of the Indian Journal of Pediatrics, Volume 74—July, 2007

incision are gently blotted with a filter paper every 30 seconds to remove the blood oozing out, until the incision is dry (avoid touching the wound itself with the filter paper). Stop the stop watch and note the reading. Clean the wound with spirit. Oppose wound edges and put adhesive plaster for at least 48 hr. Normal Range: 2-5 minutes Each laboratory should establish its own normal range. Interpretation: Bleeding time is a measure of capillary function, platelet number and Platelet function. It is prolonged in conditions such as Thrombocytopenia and Platelet function defectsVon Willebrand’s Disease, Capillary defect 3.2 Prothrombin Time (PT) Principle: The prothrombin time test belongs to a group of blood tests that assess the clotting ability of blood.4, 5 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 prothrombin to 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 prothrombin time. 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. A prolonged prothrombin time indicates a deficiency in any of factors VII, X, V, prothrombin, or fibrinogen. It may mean that the patient has a vitamin K deficiency, a liver disease, or disseminated intravascular coagulation (DIC). The prothrombin time of patients receiving warfarin therapy will also be prolonged-usually in the range of one and one half to two times the normal PT time. PT time that exceeds approximately two and a half times the control value (usually 30 seconds or longer) is ground for concern, as abnormal bleeding may occur. Limitations: Spurious results may occur if the blood to anticoagulant ratio is not 9:1. The PT clotting times may be prolonged by substances including corticosteroids, EDTA, oral contraceptives, asparaginase, clofibrate, erythromycin, ethanol, Tetracycline and anticoagulants such as heparin and coumarin. The PT may be shortened by substances including antihistamines, butabarbital, 651

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R. Saxena et al caffeine, oral contraceptives, phenobarbital and vitamin K. 3.3 Activated Partial Thromboplastin 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: Every laboratory should determine its own normal range. In our laboratory with commercial reagents it is 29-35 seconds. 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. 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. 3.4 Clot Stability 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.6 Normal – Clot stable If clot dissolves : Factor XIII deficiency

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.7-9 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 70 yr) Limitations : Rheumatoid factor (RF), if present, may lead to false positive d-dimer test result. When suspected, RF presence should be confirmed with an independent procedure for RF.Do not test suspect samples, especially those in which the coagulation process may already have started, since these tend to produce false positive reaction with the D-Dimer test. 3.7 APTT Mixing Study with normal serum / 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.

3.5 Thrombin Time (TT)

3.8 Factor VIII assay ONE STAGE F VIII CLOTTING ASSAY

Principle : Thrombin is added to the plasma and clotting time measured. The clotting of citrated plasma is affected by the concentration and reaction of fibrinogen, and also by the presence of inhibitory substances. The normal range is 15 – 17 seconds.

Principle : The assay is based o the ability of the patient’s plasma to shorten the abnormal activated partial thromboplastin time of a severe F VIII deficient plasma as compared to the corrections obtained from normal pool plasma.

Interpretation : Thrombin time is prolonged in the presence of heparin, hypofibrinogenemia, dysfibrinogenemias and fibrin degradation products.

Normal Range : Factor VIII concentration range from 50 to 200% but varies widely in a given population and also in the same subject at different times, therefore normal range must be established in each laboratory.

3.6 D-Dimer Test 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 652

Interpretation : Decreased values of F VIII activity may be due to A. Congenital deficiencies 1. Haemophilia A Indian Journal of Pediatrics, Volume 74—July, 2007

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Laboratory Studies in Coagulation Disorders 2. Von Willebrand’s Disease 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. 3.9 Screening for FVIII inhibitors The acquired coagulation disorders occur, by definition, in previously normal individuals, secondary to underlying disease which are associated with loss of coagulation factors, due to circulating inhibitors. An unexplained abnormal PTT that is not corrected by addition of normal plasma indicates the presence of a circulating anticoagulant.

Ristocetin AA 400uM

Ristocetin Correction

ADR 20uM ADP 2.5uM Fig. 3. Aggregation chart showing Bernard Soulier Syndrome

ADP 1.25

ADF

ADP

AA collagen

3.10 Platelet aggregation tests 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). Diagnosis : Absent aggregation with all agonist Glanzmann Thrombasthenia (Fig. 4), afibrinogenemia. Reduced aggregation with Ristocetin : vWD, Bernard Soulier (Figs. 2 and 3)

Fig. 4. Aggregation chart showing Glanzmann Thrombasthenia

a. Platelet Aggregation with Ristocetin (RIPA) Principle: A sample of platelets rich plasma is stirred at a constant speed in a thermostatically heated block and an aliquot of ristocetin is added. The pattern of aggregation is followed on a potentiometric chart recorder which traces the change in optical density of the plasma sample which occurs when platelets aggregate. Interpretation : An antibiotic ristocetin derived from the bacterium Nocardia purida was withdrawn from clinical use because of the high incidence of thrombocytopenia in patient’s treated with the drug. It was found that ristocetin causes platelet aggregation in vitro and in vivo, and later it was shown that platelets from a majority of patient’s with von Willebrand’s Disease fail to aggregate with this chemical. (b) Von Willebrand’s Factor (vWF) Antigen

Fig. 2. Aggregation chart showing von Willebrand disease

Reduced aggregation with all agonist : Storage pool disease, Arachidonic acid pathway defect. Reduced aggregation with any one or 2 agonist only: Unclassified PFD 3.11 Tests for vWD Indian Journal of Pediatrics, Volume 74—July, 2007

Principle of the test VWF: Ag assay is a sandwich ELISA. A capture antibody specific for human vWF is coated to 96-microwell polystyrene plates. Diluted patient plasma is incubated in the wells, allowing any available vWF: Ag to bind to the anti-human vWF antibody on the microwell surface. The platelets are washed to remove unbound proteins or other plasma molecules. Bound vWF:Ag is quantitated using horseradish peroxidase (HRP) conjugated anti-human vWF detection antibody. Following incubation, unbound conjugate is removed by 653

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R. Saxena et al washing. A chromogeneic substrate of tetramethybenzidine (TMB) and hydrogen peroxide (H2O2) is added to develop a colored reaction. The intensity of the color is measured in optical density (OD) units with a spectrophotometer at 450nm. Platelet vWF:Ag in relative percent concentration is determined against a curve made from the reference plasma. Expected values 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 47197% (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. C Assay of VWF: RCo (von Willebrand Ristocetin cofactor activity) Principle: The degree of aggregation of washed platelets induced by ristocetin is related to the vWF level present in the system. The level of vWF in the plasma that are being tested at the appropriate dilution is obtained by comparing the speed of platelet aggregation in the test plasma with that of the standard obtained from a diluted normal human plasma pool. Normal range: of vWF RCo (in adults = 60-150%) 4. Case Studies Case 1 •A 5-yr-old male child presented with history of spontaneous swelling of right calf for 2 days, Recurrent joint swelling, past 2 years, and Prolonged bleed in elder brother. Earlier patient responded to FVIII but now no effect on swelling was seen. Examination revealed that he had mild Pallor , diffuse swelling of right calf. Contracture of left knee joint with marked restriction of joint movements Wasting of Left thigh muscle was present Investigations • Hb: 12g/dl • TLC: 9000/uL • Platelet count 5.3 lac/uL • Bleeding Time: 3’ (Normal 2- 5’) • Prothrombin Time : 15”/12” • Activated partial prothrombin time: 90”/30” • Clot Solubility: Normal • Factor VIII level: 5 Bethesda Units

Diagnosis: Haemophilia A with Inhibitors Case 2 A 15-yr female presented with history of menorrhagia, gum bleeding off and on, for 8 years. History of recurrent epistaxis and menorrhagia i was present in her mother Examination revealed mild pallor and systemic examination was essentially normal. There were few ecchymotic spots only. Investigations • • • • • • • • • •

Hb: 9.3 g/dl TLC: 9800 Platelet count: 240x103/ul, Bleeding Time: > 15’ Prothrombin Time: 15”/12” APTT: 65”/ 30” CS: Normal RIPA: Absent vWF Ag: 3% (low) (Normal 50 to 150%) RiCoF: 0.3% (low) (Normal 60 to 150%)

Diagnosis: Type II vWD Case 3 A 7-yr-old female child presented with history of epistaxis off and on since birth, along with peticheal hemorrhages . Her elder sister had similar complaints from early childhood Examination revealed mild pallor, few peticheal hemorrhages over the high. Systemic examination was essentially normal. Investigations • • • • • • • •

Hb: 9.9 g/dl TLC: 11 000 /ul Platelet: 5.5 lakh Bleeding Time: > 15’ PT: 14”/13” APTT: 32”/30” CS: Normal Platelet aggregation study: Absent with all agonists except with Ristocetin • Flow Cytometery: Absent GPIIb/IIIa • Diagnosis: Glanzmann Thrombasthenia Case 4 A 10-yr-female presented with history of recurrent gum bleeds and high grade fever of 5 days duration. She complained of breathlessness and bodyaches. Examination revealed that she had moderate pallor along with gum bleeds and generalized oozing from multiple puncture sites. She had sternal tenderness and no lymph node enlargement Hepatosplenomegaly of 2 and 3 cms Indian Journal of Pediatrics, Volume 74—July, 2007

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Laboratory Studies in Coagulation Disorders were present

• Plt: 2.3 lakhs

Investigations

• PT: 20”/13”

• Hb: 5.3 g/dl • TLC: 600/ul • DLC: P20 L17 Promyelocytes 60 Myeloblasts 3% • Platelet count: 5000/ul • PT: 23”/12” • APTT 45”/30” • TT: 23”/17” • Fibrinogen: 100mg/dl • D dimer: >0.5 ug/ml • Peripheral blood smear revealed : Micro angiopathic Haemolytic anemia bone marrow was compatible with AML M3 • Diagnosis: DIC secondary to AML M3 Case 5 • One month male child presented with history of spontaneous ecchymosis following intramusculor injection for past 10 days. This baby was born premature in village and was delivered by village dai. Baby was in breast fed. There was no history of fever joint swelling, hematuria or prolonged bleeding from umbilical stump during first week of life. Examination revealed only ecchymotic patches, while rest of examination, was essentially normal. Investigations • Hb: 11.9 g/dl • TLC: 12000/ ul

Indian Journal of Pediatrics, Volume 74—July, 2007

• APTT: 40”/30” • CS: Normal • Injection vitamin K 2mg IV given • Repeat PT, APTT after 72 hr of vitamin K PT 14”/13” APTT: 33”/30” •Diagnosis: Vitamin K deficiency REFERENCES 1. Mielke CH, Jr., Kaneshiro MM, Maher IA et al. The standardized normal Ivy bleeding time and its prolongation by aspirin. Blood 1969; 34 : 204-215. 2. Mielke C. H. Measurement of the bleeding time. Thrombosis and Haemostasis 1984; 52 : 210-211. 3. Bowie, EJW et al. Mayo clinic laboratory manual of hemostasis, Philadelphia; W.B. Saunders and Co., 1971; 29-33. 4. Miale JB, Laboratory Medicine: Hematology, 4th ed. St. Louis, C.V. Mosby Co., 1972; 1268-1269. 5. Rizza, Charles R and Walker, William. One stage Prothrombin time techniques. In Bang NU, et al. eds. Thrombosis and Bleeding Disorders, New York, Academic press, 1971; 92- 100. 6. Sirridge M. Laboratory evaluation of the bleeding patient. Clin Lab Med 1984 Jun;4(2) : 285-301. 7. Wells PS, Anderson DR, Rodger M, Forgie M, Kearon C, Dreyer J, Kovacs G, Mitchell M, Lewandowski B, Kovacs MJ. Evaluation of D-dimer in the diagnosis of suspected deep-vein thrombosis. N Engl J Med 2003 Sep 25; 349(13) : 1227-1235. 8. 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. 9. Saxena R, Gupta PK, Ahmed R, Vinita B, Suresh K D – dimer test :Diagnostic role in clinical and sub – clinical DIC. IJPM 2003; 46(3) : 425-426.

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