Fondazione Emo | Final test Lab Tecnhcian

1) About the sample collection:

Is crucial the use of test tubes that do not activated the contact phase (siliconized glass or polypropylene)

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Peripheral vein sampling using needles of adequate caliber between 23 and 26 gauge

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When using collection systems with vacuum tubes it is not necessary to discard a few ml collected with a first tube

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Collection with minimal stasis using the tourniquet for all the time necessary to find the vein

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2) About transport and preanalytic phases of the sample to the laboratory:

The ambient temperature (15-22 °C) is ideal for transport in case of long distance

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In case of long distance the whole sample must be centrifuged and the plasma, separated and frozen prior to transport, must arrive frozen at its destination.

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In the laboratory the sample must be centrifuged after two hours from sample collection and the plasma processed immediately or alquotated and frozen immediately

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The ambient temperature induces losses of labile factors (FVIII, FV)

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3) Centrifugation of capped tubes

Is possible with plasma filtration with any type of filter

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Must be at 2000 rpm for 20’ at controlled temperature to obtain a LPP with platelet count <10×109/L

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Must be in noncontrolled temperature centrifuges

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Must be discarded badly stored samples

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4) About the maturation of coagulation proteins from birth to childhood:

FVIII is generally the same from the birth to adulthood

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FVII maturation is observed at 1-1.5 years of age

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FXIII is increased at birth and decreased in the childhood

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FXI reached maturity at 6 months of age

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5) What reagents are required to determine aPTT?

Platelet reach Plasma from the patient and Control Plasma

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Phospholipid or partial Thromboplastin containing an activator

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CaCl2 0.5 M solution freshly prepared

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Only control sample abnormal

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6) What are the hemorrhagic syndromes with normal screening?

Hemophilia A and B

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vonWillebrand disease

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Deficiency of Vitamin K

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Scurvy, Factor XIII deficiency and α2antitripsina deficiency

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7) Factor XIII defect:

Autosomal Dominant with an incidence between 1:2500000-1:5000000

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Is generally asymptomatic > 5%

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Bleeding is observed very fast(after 6-12 h) in case of post-traumatic hematoma and bleeding

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Minimum hemostatic level is obtain for levels between 5-10 U/dl

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8) What are the factors that can influence the results of one stage test?

The use of vitamin K with reagents for test

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Reach plasma of coagulation factors

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Calculation protocol and calibration material

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The photometrically analysis of the quantity of generated FXa

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9) For laboratory monitoring of modified coagulation factors:

However, with some products, discrepancies may be found not depending on the method used.

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The literature or clinical practice provides specific information, it is advisable to use the type of assay or procedure that has been shown to be effective in minimizing differences between assays.

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The method used for the power labeling of the generical product used is the most practical solution

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The solution could be use only chromogenic method in all cases

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10) For setting and monitoring of prophylaxis the laboratory:

Allow the evaluation of factor recovery in vivo in post-infusion kinetics tests (different products chosen for different patients)

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Not allow monitoring the effectiveness of the treatment (maintaining the level established for the individual patient)

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"It is not useful to tailor prophylaxis

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Is the only parameter to evaluate to tailor prophylaxis

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11) Screening tests for patients suspected of haemophilia show:

Abnormal complete blood count

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No correction on mixing test aPTT/patient Prothrombin time (PT), normal

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Abnormal prothrombin time (PT)

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Prolonged activated partial thromboplastin time(aPTT)

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12) Diagnostic tests for the diagnosis of hemophilia

Require measurement of FVIII / FIX using a one stage assay (OSA) or with chromogenic assay (CrA).

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Show reduced or absent factor antigen levels with normal activity.

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Search for mutations in the FVIII / FIX gene on the autosomal chromosome XII

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Search for platelets disfunction based on PFA test

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13) Chromogenic assay

Is based on the activation of exogenous Factor VIII under experimental conditions, where all coagulation factors (except the one being measured) are added at optimal concentrations externally.

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Measures the generated FXa photometrically using a specific synthetic chromogenic substrate. The intensity of the color is directly proportional to the FXa produced and, consequently, to the level of FVIII present in the plasma sample

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Measures a pool of very high plasma levels of clotting factors, and together with the patient's plasma, it allows the calculation of the results in terms of IU/dL

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Shows more interference than the one-stage assay

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14) The advantage of one stage assay are:

Wide availability of reagents with high stability on board, ideal for automation on available coagulometers

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More reproducible

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Less variability in reagents

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More accurate and precise measurements

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15) How to impact discrepancies of the on stage and chromogenic tests on diagnosis?

For patients with severe haemophilia the results are higher with one stage than chromogenic test

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In patients with mild / moderate haemophilia, differences between tests are systematically detected and are found in more than 10% of patients with mild degree

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Discrepancies that detect lower levels with the chromogenic prevail

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Bleeding symptons correlate more with one stage test results

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16) Are the following indicators of genetics predispositions to inhibitor development?

Moderate Haemophilia

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Familiar History of inhibitor development

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Switch to different clotting factors in the first 50 EDs

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Prophylaxis

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17) When did you suspect inhibitor development:

Ineffectiveness of therapy with same changes of bleeding phenotype of patients

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Further PT elongation from patient baseline

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Complete correction of mixing test

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Increase recovery after clotting factor administration

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18) About Inhibitor titration:

If a lower titer inhibitor is present, it will be necessary to make scalar dilutions of the sample with the buffer and incubate them at 37°C for 2h.

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The principle of the test is to measure the inhibition level in vitro by serial dilutions of the patient's plasma on FVIII activity, using a fixed amount of normal plasma, with which it is mixed in a 1:1 ratio.

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The only test useful for the measure of inhibitor is the Chromogenic Bethesda test

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The most sensitive test for low titer inhibitor is the Bethesda test

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19) What are the main target plasma factor levels for personalized treatment in haemophilia?

Plasma factor level 1-3% is the target level for patients on prophylactic treatment with low bleeding phenotype or no bleeds

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Plasma factor level 5-15 % is the target level for patients on prophylactic treatment with low bleeding phenotype or no bleeds

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Plasma factor level 1-3% is the target level for active patients

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Plasma factor level 1-3% is the target level for young children to safely prevent joint bleed and subsequent joint damage

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20) Population pharmacokinetic:

Can be obtained with a few samples, allowing the evaluation of a more heterogeneous and larger group of patients (eg, pediatric or elderly patients)

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Has only a predictable variability

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Has the following unpredictable variability: body weight, age, haemorrhagic phenotype

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Requires a long washout, with potential bleeding risks for patients

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21) About inhibitors:

Inhibitor development renders replacement therapy more effective in most cases.

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The eradication of the inhibitor is not essential because patients may use bypassing agents and non-substitutive treatments

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Eradication of inhibitors is currently best achieved through prophylaxis treatment

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Patients who develop inhibitors should have access to ITI and to suitable hemostatic agents for control of bleeding as well as surgical interventions, if needed, at specialized centres with relevant experience

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Final test Lab Tecnhcian

08 Mag Final test Lab Tecnhcian

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