coag practical - heme II lab (cls

steps of primary hemostasis

1. tissue injury

2. platelet adhesion—attach to exposed collagen via circulating VWF & GpIb receptor

3. shape change—plts are activated & expose GpIIb/IIa

4. aggregation—GpIIb/IIIa binds fibrinogen

5. secretion—release of alpha & dense granules

6. surface for coag cascade made (unstable plt plug)

lab tests for identifying platelet disorders

• screening: bleeding time & PFA

• confirmatory: plt aggregometry

platelet count values & associated bleeding symptoms

• 100,000 plts = generally normal

• 50,000 plts = bruise easily

• 10,000-50,000 = severe risk of bleeding

• little to no platelets (<10K) = mucosal bleeding

petechiae vs purpura vs ecchymoses

• petechiae = 1-2 mm in size

• purpura = >3 mm

• ecchymoses = >1-2 cm

  • NOT due to trauma

primary vs secondary hemostasis

• primary = involves platelets and formation of unstable plt plug

• secondary = involves coag cascade and ends with formation of fibrin clot

steps of the intrinsic pathway

1. contact factors exposed to neg charged surface

2. XII → XI

3. XI → IX

4. IX + VIIIa + Ca2+ + PLs form the intrsinc Xase

5. intrinsic Xase cleaves factor X

steps of the extrinsinc pathway

1. vessel injury

2. TF (III) + VII

3. addition of Ca2+ forms the extrinsic Xase which can cleave factors X & IX

steps of the common pathway

1. X → Xa by intrinsic/extrinsic Xases

2. Xa + Va (cofactor) + PL + Ca2+ form prothombinase complex

3. prothrombin → thrombin

4. thrombin cleaves XIII & fibrinogen

5. fibrin formed

6. fibrin stabilized by XIIIa → crosslinked fibrin

vitamin K dependent factors

2, 7, 9, 10

• aka II, VII, IX, X

what are the X-linked factors

8 (VIII) & 9 (IX)

what are the contact factors?

XI, XII, HMWK, PK

• 11, 12, high molecular weight kininogen, prekallikrein

what coag factors are serine proteases?

all of the factors EXCEPT I, V, VIII, XIII (1, 5, 8, 13)

what pathways do the PT and APTT test?

• PT = extrinsic + common

• APTT = intrinsic + common

what substance/structure is the endpoint of the majority of coagulation testing?

FIBRIN CLOT

principle of bleeding time test

amount of time required for bleeding to stop after an incision is made under standardized conditions

• incision 5 mm long x 1mm deep is made in the forearm 3 finger widths below elbow

• constant pressure of 40 mmHg maintained above elbow

• blood is blotted w filter paper every 30 sec; bleeding stops after plt plug is made

sources of error for bleeding time test

• bleeding time is affected by plt count, function, VWF, and physician technique

• plt count <10,000 give no reliable correlation between bleeding time & plt function

• aspirin use can prolong bleeding time—pt should be off aspirin meds for 7-10 days prior to test

normal range for bleeding time test

2-8 minutes; if bleeding does not stop at 20 mins, report >20 min

• >8 mins = impaired plt function/uremia/VWD

• times are shortened in ITP or bone marrow recovery after chemo

principle of PFA test

instrument and test cartride where the process of plt adhesion & aggregation are stimulated in vitro

• plts adhere to the collagen-coated membrane & become activated

• plts secrete granules upon contact with a membrane coated with ADP/epinephrine or ADP/collagen

• plt thrombus is made at the aperture & arrests flow of blood & instrument determines the closure time (CT)

3 most common causes of platelet dysfunction

1. related to uremia

2. von Willebrand disease (vWD)

3. exposure to acetyl salicylic acid agents (ASA; ex aspirin)

(PFA) specimen + normal reference ranges

• specimen = citrated whole blood ; must test w/in 2 hours but do not test until 30 mins AFTER collection

• normal reference ranges

  • COL/EPI = 80-192 sec (mean 128 s)

  • COL/ADP = 60-112 sec (mean 98 s)

(PFA) closure times for patients with VWD and aspirin ingestion

• VWD = both COL/EPI & COL/ADP are abnormal

• aspirin = COL/EPI abnormal ; COL/ADP normal

limitations of the platelet function analyzer (PFA)

• hemolysis will interfere

• certain fatty acids & lipids are known to inhibit platelet function

  • avoid fatty meals before testing

anticoagulant of choice for coagulation studies

sodium citrate (3.2%)—blue top

what kind of plasma is used for coagulation studies?

platelet POOR plasma (<10,000 plts)

• can store specimens for 24 hrs for PT and 4 hrs for APTT

(fibrometer) principle of clot detection

uses electromechanical clot detection methodology

• no interference from lipemia, hemolysis, or bilirubin

• stationary + moving electrode that picks up fibrin threads

• fibrin thread completes electrical potential = enables a current to reach detection circuit which stops the fibrometer

sources of error for coagulation tests performed on the fibrometer

• reagent contamination = decrease clotting times

  • times will get shorter and shorter

• dirty probes = drop & stop

• bad coordination (user error + skill issue tbh)

• temp error (failure to turn fibrometer back on) = increase clotting times

principle of prothrombin time (PT) test

amount of time required for a fibrin clot to form after adding reagent containing TF + Ca2+ to plasma

• pt plasma added to recomboplastin

• time measured w fibrometer

• reflects formation of fibrin via extrinsic + common pathway

clinical significance of the PT test

PT/INR is a global screening test w 3 major applications:

• detection of single/combined deficiencies of extrinsic pathway, hereditary/acquired coag disorders, liver disease, or vit K deficiency

• monitoring coumadin/warfarin therapy

• assay for specific extrinsic coag factor

what coagulation factor(s) are detected by the PT?

• extrinsic = only VII (since reagent has TF)

• common = X, V, II, I

contents of PT reagent

thromboblastin/recomboplastin—has tissue factor + calcium ions

sources of error in PT test by the fibrometer

• reagent contamination = decreases time

• dirty probes = drop & stop

• bad coordination (loser)

• temp errors (>37 C) increase clotting times

normal range for PT test

• 10.5-14.5 seconds

• INR normal range = 0.8-1.2

therapeutic range for the INR of pts on warfarin

INR = 2-3 (1.8-2.6)

(PT) how to calculate the INR

(patient PT/mean normal range PT)^ISI

• mean normal range = 12 s

• ISI comes from reagent lot—in lab use 1.02

• read INR off of INR conversion table

principle of activated partial thromboplastin time (APTT)

amount of time required for a fibrin clot to form following the addition of a platelet-like PL substance, activating agent, and Ca to plasma

• pt plasma incubated w a platelet-like phospholipid substance + activator

• activator allows for max activation of contact facotrs

• after incubation, Ca added back as CaCl2

• time required to clot done by fibrometer

• time reflects function of clotting factors in intrinsic pathway (excluding plts + XIII)

cogulation factors detected by the APTT

XII, XI, IX, VIII, PK, HWMK

what reagents are used for the APTT test?

synthasil + CaCl2

specimen sources of error for APTT

• short draw = falsely increase APTT

  • increases anticoagulant/plasma vol ratio

  • excess citrate binding of Ca

• Hct >55% = falsely increase APTT

  • less volume of plasma—seen in smokers & PV

• clotted sample = falsely decreases APTT

• heparin contamination = APTT >200 s

normal range for APTT

25-46 seconds

• can also use 24-40 sec range

• if no clot by 100 sec, report >100 seconds

what anticoagulant does the APTT monitor?

heparin

• patients on heparin should have a value 1.5-2.5 times the normal

• heparin contamination results in APTT >200 s

fibrinogen general info

trimodular glycoprotein; 2% of total plasma proteins

• cleaved by thrombin to form fibrinopeptides

• acute phase protein

• functions:

  • substrate for clot formation

  • binds to plts to support aggregation

  • promote wound healing

  • template for thrombin + fibrinolytic system

principle of fibrinogen test

measure of the amount of fibrinogen present in the plasma expressed as mg/dL

• dilution of pt plasma incubte w excess of thrombin (so that the results only depend on amt of fibrinogen)

• time required for clot measured w fibrometer

• time reflects rate of conversion of fibrinogen → fibrin via final step of common pathway

• amt of fibrinogen gathered by standard curve

normal value for fibrinogen in plasma

200-400 mg/dL

sources of error for quantitave fibrinogen by the fibrometer

• same as the other errors for using a fibrometer

• messed up dilution/inaccurate pipetting = erroneous results

• calculation of fibrinogen errors

how to measure fibrinogen values that are GREATER than the highest value on the curve?

1:20 dilution of the original sample should be made and the test should be repeated

• **MULTIPLY THE END VALUE BY FACTOR OF 2 (bc original dilution was 1:10)

• occurs when clotting times are abnormally short—a lot of fibrinogen present, so it makes sense that the final value would need to be multiplied

how to measure fibrinogen values that are LESS than the lowest value on the curve?

original sample should be diluted 1:2 and the test repeated

• **DIVIDE THE FIB VALUE BY 5 (original dilution is 1:10)

• general rule: if the clotting time is too long, it means that there is not enough fibrinogen, so it makes sense that the final value would need to be divided (made smaller)

reagent for fibrinogen quantitative assay

thrombin reagent

principle of 5M urea test

qualitative determination of presence of factor XIII activity—catalyzes cross-linking of fibrin (last step of coag cascade)

• pt plasma is re-calcified & incubated at 37 C to form a clot

• clot is placed in 5M urea, which dissolves non-covalently bonded fibrin clots

(5M urea) factor XIII deficiency

• affects 1 in 5 million

• XIII is a transglutaminase—forms covalent bonds in fibrin monomers

  • made in megakaryocytes

• symptoms: excessive bleeding after invasive procedures (umbilical cord removal, bleeding in intercranial joint/muscles, recurrent miscarriage) + impaired wound healing

• detection threshold = <2% factor XIII activity

normal/abnormal value for 5M urea test

• normal: clot persists after 24 hrs & is not dissolved

• abnormal: clot dissolves after 24 hrs (actually w/in 2-3 hrs)

sources of error for 5M urea test

plasma containing as little as 1% of factor XIII will still be insoluble in 5M urea

• basically there has to be NO factor XIII whatsoever for the clot to dissolve in 5M urea

principle of fibrin-degradation products (FDP) test

uses latex particles coated w monoclonal anti-FDP ABYs, forming macroscopic clumps in the presence of 2.5 ug/mL of FDP

• detects X, Y, D, and E degradation products

• monoclonal ABY does not react w native fibrinogen

normal level of FDP in adults

plasma from normal individuals should have <5 ug/mL of FDP

sources of error for the FDP test

• clotted specimens produce false positive rxn

• presens of rheumatoid factor (RF) causes false positives

• FDP plasma test insensitive to heparin up to 2 IU/mL

high levels of FDPs are found in what conditions?

• ecclampsia

• cancers

• post-operative complications

• cardiac, renal, hepatic disorders

• fibrinolysis

• pulmonary embolism ; DVT

• leukemias (esp M3 AML)

principle of the D-dimer test

latex agglutination test that detects presence of D-dimer from plasmin degradation of factor XIIIa cross-linked fibrin (XDP)

• degree of agglutination is proportional to the conc of D-dimer

• determined by measuring decrease of transmitted light cause by the aggregates (turbidimetric immunoassay)

clinical significance of D-dimer test

• aids in the diagnosis of thromboembolic events

  • elevated conc have been reported in DVT & PE

• used to exclude VTE (venous thrombosis)

• also used as part of the DIC workup along with PT/PTT, fibrinogen, plt count, RBC morphology (schistocytes)

  • can be used to monitor DIC

normal levels of D-dimer in adults

<230 ng/mL DDU

• reportable range is 150-69,000 ng/mL DDU

limitations for D-dimer test

• detection limit is 21 ng/mL DDU

• not affected by hgb up to 500 mg/dL, bilirubin up to 18 mg/dL, TGs up to 1327 mg/dL

• rheumatoid factor up to 1400 IU/mL

FDP & D-dimer results for secondary fibrinolysis vs primary fibrinolysis

• primary fibrinolysis: FDP pos & D-dimer neg

• seconary fibrinolysis: FDP + D-dimer positive