NEOPLASTINE CI PLUS

Determination of Prothrombin Time (PT)

NEOPLASTINE®CI PLUS ②

Kit lor Approx . 60 Tests Containing :

-8 x 2ml Vials of Reagent 1 ( Néopiastine® Cl Plus 6 x 2-ml Vials of Reagent 2 (Solvent) (REF
00374) NEOPLASTINE.CI PLUS ⑤

* Kit for Approx. 150 Tests Containing: -8 x 5-mi Vials of Reagent 1 (Néoplastine CI Plus - 6 x
5-mi Vials of Reagent 2 (Solvent) ) (REF 00375)

NEOPLASTINE® CI PLuS 10

Kit for Approx. 00 Tests Containing:

-12 x 10 - ml Vials ofReagert 1 ( Néoplasinea Cl Plus ⑩ )

- 12 x 10-ml Vials of Reagent 2 (Solvent) (REF 00376)

March 2018

1/INTENDED USE

The Ndoplastine CI Plus kits provide reagents tor the determination of the prothrombin time (PT)
in plasma.

2 / SUMMARY AND XPLANATION .

 The prothrombin time is a coagulation screening test. It measures test. As a whole , the
activity ol the coagulation factors ll , V , VII ,X and I

 A prolonged PT has been observed in the lollowing clinical stetes:

- congenital or acquired deliciencies of factor II, V, VII, X or librinogen (1).

- liver failure (cirrhosis, hepattis) (1).

- treatments with vitamin K antagonists ( 1 ).

- hypovtaminosis K: nutritional intake deficiency, disordens in absorption or metabolism

of vitamin K (hemorrhagic disease of the newborn cholestasis, treatment with
antibiotics) (5).
 - fibrinolysis (1).

- DIC (1).

 The PT is commonly used for monitoring vitamin K antagonist therapy (3) because of its
senshivity to variations in the concentration of the vitamin K dependent factors B, VIll
and X. Consequently, the comparablity of results of this test is essential for linding the
therapeutical range. It is well known that the PT value of a plasma may vary according to
the origin of the thromboplastin reagent and to the instrument used to measure it (4). A
solution for standardization adopted by the-World Health Organization is a "system of
international reference standards for thromboplastins permitting the definition of an
internatonal scale for the intensity of anticoagulant therapy (4), In this system the PT
ratio is converted into the international Normalized Ratio (NR). The INR value
corresponds to the value of the ratio of the patient's PT to that of the standard PT raised to
the ISI (international Sensitivity Index) power ol the thromboplastin used:

INR = Patients PT IS Mean Normal PT ISI

The ISI value of a given thromboplastin is determined by testing normal plasmas and
coumadin-trealed patient plasmas with that thromboplastin and with the international
Relerence Preparation for thromboplastin. The PT values obtained with the two
thromboplastins are plotied on log-log graph paper, and the orthogonal regression line is
drawn. The siope of this line multipied by the iSi value of the referance thromboplastin
represents the ISI value of the thromboplastin of interest (3).

The use of the INR is recommended for the assassment of the vitamin K antagonist
therapy in patients (6, 8).

3/TEST PRINCIPLE

The principle of the test consists of the use of calcium thromboplastin to easure the
clotting time of the patients plasma and to compare it with that of a normal standard (for
example Etaloquick or Unicalibrator). The test measures, as a whole, the activity of the
coagulation factor II (prothrombin), factor V (proaccelerin), factor VII (procevertin),
factor X (Stuart factor) and factor I (fibrinogen)
4 KIT REAGENTS

 Reagent 1: lyophilized thromboplastin prepared trom fresh rabbit cerebral tissues. The
ISl value of Néoplastine Cl Plus is determined against a secondary standard of the RBT
(rabbit brain thromboplastin- see the Assay Value insert provided in the box for the
escact iSI value of the lot being used). Once reconstituted, Néoplastine Ci Plus exhibits a
low turbidity and does not sediment.

The Néoplastine Cl Plus reagent contains a specific heparin inhibitor. Any prolongation
ol the prothrombin time is, therefore, related to a real deficiency of factor ll, V, Vil, X
and/or fibrinogen (see section 11).

 Reagent 2: aqueous solvent containing calcium, 2-ml per vial (REF 00374), 5-mi per vial
(REF 00375) or 10-mi per vial (REF 00376).

Reagen 2 contains nickel sullate . At the concentration provided ( < 0.1 % ) , this roagent
i Reegent 2 oontains nickel ciasalfied as sanaising.

Warning

May causs an alargic akin reaction, Wagr protective gioves/protactive clothinghye

protectiontace protection. IF ON 8KIN: Wash with plenty of sosp and water.

Reagent 2 contains sodium szide t< 1 gt) as a preservative.

Resgents containing sodiumm azide should be discarded with care to prevent the
formation of explosive metaille azldes. if waste materiale are dumped Into sinks, use
coplous quantier of water to lusn plumbing thoroughhy.

WARNING POTENTIAL BIOHAZARDOUS MATERIAL

Some reagents provided in trase ktts contain matartals ol huaman andlar a Whensves
human plaama is raquirad for h praperation of these reagents approved nuthods are used
to tert the piasma for the antibodies to Hiv 1, HIV 2 and Hcv, and for hepatinis 8 surface
antigan, and reauRs are tound to be negative can offer oompiets eesurance that Infectious
agents ars abeent. Therelore, users of reegents of these types must sxercise axtreme care
in full complianos with safety precautions in the manipulation al thoss biologkcal
materlals ea t they ware infectious
5/ CAUTION

Store at 2-8 °C. For in vitro diagnostic use only. These reagents are to be used only by certified
madical laboratory personnef authorized by the laboratory.

Exercise great care in the handling of these reagents and of patient samples. The disposal of
waste materials must be carried out according to current local regulations.

in the USA, wherever appropriale, observe CLIA-88 requirements.

6/ SPECIMEN COLLECTION AND TREATMENT

Sample coliaction must be in contormity with the recommendations for hasmostasis tests. : Bod (
gvoty ts - collected in 0.109 M ( i.e. , 3.2 % ) trisodium citrate auiant ( vol) lin the USA folow
CLSI guidelines H3-A6 (9) and H21-A5 (10) Centrifugation: 15 minutes at 2000-2500 g. Plasma
storage: 8 hours at 20 5°C (7). Do not store plasma at 2-8 °C (2).

7/ REAGENT PREPARATION AND STORAGE

 Preparation

Transfer the entire contents ot one vial of Reagent 2 (R2) into one vial Reagen at room
temperature (18-25 °C) for 30 minutes. Then, swirl the Reagent vial gently to oblain a
homogeneous suspension.

 Stronge

The reagents in intact vials are stable until the expiration date indicated on the box label,
when stored at 2-8 °C.

Once reconstituted, Reagent 1 remains stable for:

8 hours at 37°C"

48hours at 20 ± 5 ° C

8days at 2-8 ° C .

Do not freeze.
8 / REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED

 Coag Control NP (REF 00621) or System Control P (REF 00617): control plasmas,
normal and abnormal levels.

 For calibration (PT expressed as a percentage of normal activity): Etaloquick (REF

00496): three different levels of activity are provided, or Unicalibrator (REF 00625). -
STA " . Owren - Koller ( REF 00360 )

 Common clinical laboratory equipment and materials (instrurnent such as ST arf...).

9/ PROCEDURE

9.1. Celibration

Only PT results expressed in percontages need to have standards for Only a alibration
curve (called the Thivolle line).

Assy callbration can be carried aut with Elaloqulek or with Unloalibrator

Etaloqulck: use the difterent levals of Eta loquick undiluted

Unicalbrator (UC)y use it undiluted and 1:2, 1:3, 1:a diluted in plastlic test tubes in
Owren-Koller bulffer solution. By detinition, undiluted Unicallbrator represants the assay velue
Indicated in the UC box

1:2 dilution repreconts halt the assay value indicated in the UG bo

1:3 dilution represents one-third the assay value indicated in the UC bax,

14 dilution represents ons-lourh the assay value Indicaled in the UC bax

It ls possible to use the sama calibration curve as long es the same batch identified by the
Lot No.) is in use, and on the condition that a dally quality control is performed.

9.2 Patlenla Samples

The pationts samples are used undiluted.

9.3. Quality Control

It is recommended that controls bs includad in a lost-run once a day. They are used undiluted

9,4 Aasay
Follow the instructions ol the instrument being used. In case ol tostino by the manual method,
irst bring the Reagent 1 (i.e. thi teconstituted thrombopiastin reagenl) to 37 0.5"C

In a glass test tube or cuverte at 37 "C

Plasma (sandard, patients or control) .......................0,1 ml

Incubate at 37 ‘’C.........................................................2 min

Starting e atopwaich, add Reagent 1 ….......................0,2 ml

Mlx. Note the clotting time (seconds).

10 RESULT

 Depending on how PT results should be reported, proceed as lollows:

- In seconds: report out the observed cloting times (sample and mean normal PT0

-As a ratio: calculate the ratio of the patient's PT to the mean normai PT

- In INR: calculate the ratio of the patient's PT to the mean norma get the ratio value;
reler to the enclosed Assay Value insert, which provides pre-calculated INR values for the
ISI of the lot ot thromboplastin being used, and read out the appropriate INR.

-As a percentage ol the normal PT activity · Construct the Thivolile ine on linear graph
paper by plotting the reciprocal of each standard dilution (i.e., the dilution numbers 1, 2. 3,
4) on the x- axis, and the corresponding clotting time (in seconds) on the y-axis. Alternatively,
use the special graph paper provided in the kit. From this calibration line, determine the
percentage PT values of the plasmas being tested.

The result is to be interpreted according to the patient's clinical and biological states.

 In the event that control values are not reproduced, check that all components of the test
system are lunctioning correctly , i.e. , assay conditiogs, reagents, integrity of the plasmas
being tested, etc. If necessary, repeat the tests
11 / LİMITATIONS .

 Sample

The slightest coaguiation (micro-clots) will induce considerable shortening of the times
measured (autocatałytic activation of all the factors) whereas extensive coagulation will prolong
the clotting times use of consumption of factors and tibrinogen. Do not keep plasmas at 2-8 °C
because in this temperature range the factor Vil may be activated by the kailikrein system (2).

 Anticoagulant

Maintain the correct anticoagulant/blood sample volume ratio of 1:9. I there is any considerable
variation In hematocrit, modity the quantihy of anticoagutant accordingly.

 Equipment

Use only clean and disposable labware. Ise

Thetemperature of the instrument is to be kept stable ( 37 ± 0.5 " C )

Do not allow the reagent to be contaminated by any trace of piasma.

 Heparina

The Néopiastine Cl Plus test is insensitive to unfractionated heparin levels up to 1 IU/mi and to
low molecular weight heparin leveis up to 1.5 anti-Xa IU/ml

 Thrombin Iihlbitors

Thrombin inhiibitors (e.g., hirudin, argatroban,.) present Intha ape be tested may lead to a
prolonged prothrombin tlime for this sempel

12/ REFERENCE INTERVAL

Normal values vary from one taboratory to the nen, depending on reagents, instrumentation and
technique. So, each laboratory must determine own expected values based on technique and
instrumentation in use.

If PT results are axpressed in parcentage of normal activity, normal expected values should be
greater than 70 % ( 5 ) . Values greater than 100 % have no pathological significance .
13/ VITAMIN K ANTAGONIST THERAPY .

 Vitamin K antagonists will depress plasma levels of factors li (prothrombin). Vil

(proconwartin), X (Stuart factor) and IX (antihemophilic factor B). .

 For the assessment of the vitamin K antagonist therapy, refer to the current
recommendations.

14/ PERFORMANCE CHARACTERISTICS

Diterent samples were used for the intra - assay and inter - assay reproducibility studies. Results
obtained with Néoplastine CI Plus, by ST ar or KC10 are shown below .
REFERENCES

1. CAEN J., LARRIEU M.J., SAMAMA M.: "Lhémostase. Méthodes d'exploration et 1.

pratique". Paris: L'Expansion scientifique, 344-347, 1975

2. GJONNAESS H FAGRHOL M. K : “Students on coagulation and fibriolysis in

pregnancy”. Acta Obstet. Gynecol. Scand., 54, 363-367, 1975.

3. BEESER H.: "Crtical evaluation of the so tar experience using the WHO model of
orothrombin tiine calibration and outlook for further development. Haemosiasis, 18,
suppl. 2, 181-182, 1988.

4. VAN DEN BESSELAAR A.M.H.P: "The significance of the international normalized ral
(INR) for oral antikoagulant therapy. JIFCC,3 ,4, 146-153, 1991

5. SAMPOL J., ARNOUX O., BOUTIERE B: "Manuel d'hémostape”. Paris: Editions

scientifiques et mediaceles Eisevier, 174-163, 19995

6. BCSH: "Guideines on oral anticoeguletion: third edition". Br. J. Maemato., 101, 374-387,
1998

7. NEQFOTISTOS D, OROPEZA M., TSAO CH: “Stability of plasma tor add-on PT and
APTT tests” Am. J Clin, Pathcl, 109, 6, 758-763, 1998

8. SCHVED J.F.,, DE MOERLOOSE P, JUDE B, TOULON P: Utilisation des normaizad

reto (INA) for orei anicoagulant therapy. JJFCC, 3, 4, 146-153, 1991. scientifiques et
médicales Eisevier, 147-163, 1995. 374-387, 1998, and APTT tests". Am. J. Clin. Pathcl.,
109, 6, 758-763, 1998. antivitamines K an pratique médlicale couranta". Sang Thromb.
Vaiss, 12, Recommandat ons du Groupe d'Eludes sur lHemostase et la Thrombose
(GEHT), 3d edition, 26-39, 2000.

9. CLSI Document H3-AB: "Procedures for the collection ol diagnostic blood specimens by
venipuncture; approved standard: Sith edition, 27, 26, 2007.

10. CLSI Document H21-A5: "Collection, tr ansport, and processing of blood specimens for
testing plasma-based coagulation assays and molecular haamostasis assays; approved
guideline." Fith edition, 28, 5, 2008.