4/ KIT REAGENTS • Reagent 3 (R3) is ready for use.

Shake vial well before use to 12/ EXPECTED VALUES

- STACHROM® AT III A bar-code insert is provided in the box. This bar-code contains the reconstitute Reagent 1. • The normal plasma AT level in the adult population is usually in the range
Colorimetric Assay of Antithrombin following information: lot number, kit code number, reagent code numbers NB: Considering the numerous combinations of storage conditions (partly of 80 %-120 % (8). However, each laboratory should determine its own
and expiration date. on board, partly at 2-8 °C), each laboratory should establish its own stability normal range.
• Reagent 1: approximately 11.3 nKat of bovine thrombin per ml after durations according to its practices. These durations should not exceed the • The AT level in women up to the menopause period is a little lower than
- STACHROM® AT III 3 above mentioned figures which have been determined under controlled
reconstitution, lyophilized. that in men (2). A diminution of this level is observed during
• Kit Containing: conditions. pregnancy (9).
– 4 x 3-ml Vials of Reagent 1 (Thrombin)
• Reagent 2: chromogenic substrate CBS 61.50, EtM-SPro-Arg-pNA, In case of storage at 2-8 °C, allow the reagents to stand at room
AcOH, approx. 1.4 µmole per ml after reconstitution, lyophilized. In men, the AT level decreases with age (2).
– 4 x 3-ml Vials of Reagent 2 (Substrate) temperature (18-25 °C) for 30 minutes before use. In children (4), the AT level is normally low until the age of 6 months; at
– 4 x 3-ml Vials of Reagent 3 (Thrombin Solvent)
• Reagent 3: 3-ml vial (REF 00596) or 6-ml vial (REF 00672) of solvent * Each STA® - mini Reducer is used only once. Do not re-use it. this time it reaches the adult level.
containing heparin.
(REF 00596)
The Reagent 3 contains sodium azide (< 1 g/l) as a preservative.
Reagents containing sodium azide should be discarded with care to prevent the formation of 8/ REAGENTS AND EQUIPMENT REQUIRED BUT NOT 13/ PERFORMANCE CHARACTERISTICS
- STACHROM® AT III 6 explosive metallic azides. If waste materials are dumped into sinks, use copious quantities PROVIDED • Specificity
of water to flush plumbing thoroughly.
• Kit Containing: • STA® - Owren-Koller (REF 00360). The AT assay with the STA® - Stachrom® AT III procedure allows to detect
WARNING - POTENTIAL BIOHAZARDOUS MATERIAL
Some reagents provided in this kit contain materials of human and/or animal origin. • STA® - Unicalibrator (REF 00675). the different types of AT deficiencies, particularly that of type II HBS
– 4 x 6-ml Vials of Reagent 1 (Thrombin)
Whenever human plasma is required for the preparation of these reagents, approved which affects the binding of AT to heparin (11).
– 4 x 6-ml Vials of Reagent 2 (Substrate) methods are used to test the plasma for the antibodies to HIV 1, HIV 2 and HCV, and for • STA® - Coag Control N + P (REF 00679) or STA® - System
hepatitis B surface antigen, and results are found to be negative. However, no test method Control N + P (REF 00678): control plasmas, normal and abnormal In the described conditions, the specificity of the method is assured by:
– 4 x 6-ml Vials of Reagent 3 (Thrombin Solvent)
can offer complete assurance that infectious agents are absent. Therefore, users of reagents levels. – the presence of heparin which makes the AT exert its inhibitory action
(REF 00672) IVD of these types must exercise extreme care in full compliance with safety precautions in the fully and instantly, thereby rendering ineffectual any action that may be
manipulation of these biological materials as if they were infectious. • Analyzer of the STA® line suitable with these reagents. exerted by the progressive antithrombins (e.g., α2-macroglobulin);
October 2018 English 2
• STA® - mini Reducer (REF 00797). – the presence of aprotinin which ensures that the activity of plasmin is
5/ CAUTION • Common clinical laboratory equipment and materials. blocked if it is present;
1/ INTENDED USE – the use of bovine thrombin and of a specific solvent which renders any
For in vitro diagnostic use only. Store at 2-8 °C. These reagents are to be interference by heparin cofactor II insignificant.
The STA® - Stachrom® AT III kits are intended for use, with analyzers of used only by certified medical laboratory personnel authorized by the
the STA® line suitable with these reagents, for the quantitative 9/ PROCEDURE • Working Range - Detection Limit
laboratory. Take care to use only the reagents from the same kit or the
determination of the antithrombin (AT) activity level in plasma by the same lot. The disposal of waste materials must be carried out according to 9.1. Calibration On the STA®, the assay linearity is observed up to 140 % AT activity and
synthetic chromogenic substrate method (3). (In the USA, this procedure current local regulations. Assay calibration is performed with STA® - Unicalibrator. Prepare the the value of the detection limit is 9 %.
has been assigned to the high complexity category per CLIA 1988 - CDC The STA® - Stachrom® AT III kits are designed for use with analyzers of the STA® - Unicalibrator and scan the information contained in the bar-
Analyte Code 0456; CDC Test System Codes 4677 and 4875).
• Reproducibility
STA® line suitable with these reagents. Read the Reference Manual of the code of the Assay Value insert to the instrument. The standards are Normal and abnormal samples were used for the intra-assay and inter-
analyzer model carefully before starting. Exercise great care in the automatically prepared by the analyzer by dilution with Owren-Koller assay reproducibility studies. Results obtained with STA® - Stachrom®
2/ SUMMARY AND EXPLANATION handling of these reagents and of patient samples. buffer according to the parameters entered in the instrument for the AT III by the STA® are shown below:
In the USA, wherever appropriate, observe CLIA-88 requirements. assay. The Owren-Koller buffer alone represents the 0 %-point.
• Antithrombin is a glycoprotein of a molecular weight of approximately
The calibration curve can be examined on the screen of the analyzer Intra-Assay Reproducibility Inter-Assay Reproducibility
58,000 daltons, synthesized in the liver (8).
in the “Calibration” menu (see the Reference Manual).
As an inhibitor of thrombin, the activity of AT is dramatically enhanced by 6/ SPECIMEN COLLECTION AND TREATMENT Sample Sample 1 Sample 2 Sample 3 Sample 4
heparin. It also inhibits factor Xa and to a lesser extent the factors IXa, 9.2. Patients’ Plasmas
Sample collection must be in conformity with the recommendations for Patients’ plasmas are tested undiluted. They are loaded in the
XIa, XIIa as well as plasmin and kallikrein (8). _n 21 21 10 10
haemostasis tests. instrument (see the Reference Manual of the analyzer model). X (%) 105 46 89 56
• Since the first report (1965) of a hereditary deficiency of AT and its
• Collect blood (9 vol.) in 0.109 M (i.e., 3.2 %) trisodium citrate Dilutions with Owren-Koller buffer are automatically prepared by the SD (%) 2.72 2.27 4.33 3.78
consequences, AT has been considered an important parameter in anticoagulant (1 vol.) [in the USA follow CLSI guideline documents CV (%) 2.6 5.0 4.8 6.7
instrument.
thromboembolic disorders (1). GP41-A6 (14) and H21-A5 (15)]. Then select the test(s) to be performed.
At present, the AT congenital deficiencies are classified into four
categories whose characteristics are summed up in the table below (10):
• Centrifuge all blood specimens at 2000-2500 g for 15 minutes. 9.3. Quality Control
• Plasmas remain stable for: 8 hours at 20 ± 5 °C It is necessary to run controls in order to ensure accuracy and REFERENCES
Type of Protein Biological 1 month at −20 °C. Place the sample at reproducibility of the results. Two different levels of control should be
Deficiency Amount Activity Remarks
37 °C, the necessary and sufficient time to used. Use STA® - Coag Control N + P or STA® - System 1. EGEBERG O.: “Inherited antithrombin deficiency causing thrombophilia”. Thromb.
I decreased decreased More frequent case obtain the complete thawing. Control N + P . Prepare the controls and scan the information Diath. Haemorrh., 13, 516-530, 1965.
contained in the barcodes printed on their respective Assay Value 2. FAGERHOL M.K., ABILDGAARD U.: “Immunological studies on human antithrombin III.
II RS normal decreased Anomaly of the inserts to the instrument. These controls are used undiluted; the Influence of age, sex and use of oral contraceptives on serum concentration”. Scand. J.
(reactive site) reactive site 7/ REAGENT PREPARATION AND STORAGE dilution with Owren-Koller buffer is automatically prepared by the Haematol., 7, 10-17, 1970.
analyzer. 3. ODEGARD O.R., LIE M., ABILDGAARD U.: “Heparin cofactor activity measured with an
II HBS normal when The reagents in intact vials are stable until the expiration date indicated on amidolytic method”. Thromb. Res., 6, 287-294, 1975.
Anomaly of the AT- the box label, when stored at 2-8 °C.
(heparin binding normal heparin is heparin binding 9.4. Assay 4. TEGER-NILSSON A.C.: “Antithrombin in infancy and childhood”. Acta Paediatr. Scand.,
site) absent • Reagent 1 Refer to the “Standardized Operating Procedures” of the instrument for 64, 624-628, 1975.
II PE Dysfunctional protein and Take one vial of Reagent 3 (R3) and shake it well. Then, pour its entire full details on how to proceed from this point. 5. BICK R.L., DUKES M.L., WILSON W.L., FEKETE L.F.: “Antithrombin III (AT-III) as a
(pleiotropic effect) decreased decreased at reduced quantity contents into a vial of Reagent 1 of the same kit. Allow the reconstituted The AT assay of the plasmas to be tested is automatically carried out diagnostic aid in disseminated intravascular coagulation”. Thromb. Res., 10, 721-729,
1977.
reagent to stand at room temperature (18-25 °C) for 60 minutes. Swirl vial by the analyzer at 405 nm as soon as the samples have been loaded.
The less frequent congenital deficiencies are qualitative (type II gently; then, install an STA® - mini Reducer* (REF 00797) in the vial and 6. KAUFFMANN R.H., VELTKAMP J.J., VAN TILBURG N.H., VAN ES L.A.: “Acquired
If any of the patient results falls outside the working range of the assay, antithrombin III deficiency and thrombosis in the nephrotic syndrome”. Am. J. Med., 65,
deficiency) (12). In this case, it is recommended to look for the gene the perforated cap on top before loading the reagent vial on the analyzer. the instrument automatically retests the sample in question at an 607-613, 1978.
mutation. When the defect involves the AT-heparin binding, the incidence
After reconstitution, the Reagent 1 is stable: appropriate dilution provided that this option has been entered in 7. AUROUSSEAU M.H., D’ANGELI J.L., JOSSO F.: “Antithrombin III versus prothrombin
of thrombosis is low in the heterozygous patients. However, studies – in its original vial with STA® - mini Reducer and perforated cap in place: memory in the test setup (see the Reference Manual). in liver cirrhosis”. Haemostasis, 10, 104-107, 1981.
suggest that this risk is increased when this defect is combined with an - 7 days on STA Compact® and STA-R® 8. SAMAMA M., CONARD J., HORELLOU M.H., LECOMPTE T.: “Physiologie et
other anomaly of hemostasis, such as the presence of the - 8 days on STA Satellite® exploration de l’hémostase”, Paris: Doin, 165-168, 1990.
factor V Leiden (11, 12). – in its original capped vial (remove the STA® - Reducer): 21 days at 10/ RESULTS 9. CONARD J.: “Hémostase et grossesse” in “Manuel d’hémostase”. J. Sampol,
In addition to these congenital deficiencies, a number of acquired 2-8 °C (REF 00596). The AT level (%) of the plasmas being tested is displayed in the “Test D. Arnoux, B. Boutière, Paris: Elsevier, 551-563, 1995.
deficiencies have been described in DIC (5), nephrotic syndromes (6), Panel/Test Status” screen of the analyzer (see Reference Manual). The 10. BONEU B., CAZENAVE J.P.: “Introduction à l’étude de l’hémostase et de la thrombose”,
liver diseases (7) and in L-asparaginase treatments (13).
• Reagent 2
result is to be interpreted according to the patient’s clinical and biological 199, 1997.
Reconstitute each vial with 3 ml (REF 00596) or 6 ml (REF 00672) of 11. BORG J-Y., DURIEZ D., THIRION C., NICHAM F., PERRY D.J., CARRELL R.W.:
states.
distilled water. Allow the solution to stand at room temperature (18-25 °C) “Improvement in the identification of antithrombin deficient patients”. XVIth congress of
3/ TEST PRINCIPLE for 60 minutes. Swirl vial gently; then install an STA® - mini Reducer* Ensure that the values obtained for the controls are within the ranges the International Society on Thrombosis and Haemostasis, Thromb. Haemost., supp.,
(REF 00797) in the vial and the perforated cap on top before loading the stated in the Assay Value inserts provided in the control box. If the control 433, abstract 1768, June 1997.
Antithrombin exerts a powerful and immediate inhibitory action on thrombin
reagent vial on the analyzer. values are outside the stated ranges, check all components of the test 12. LANE D.A., BAYSTON T., OLDS R.J., FITCHES A.C., COOPER D.N., MILLAR D.S.,
when heparin is present. The STA® - Stachrom® AT III test procedure JOCHMANS K., PERRY D.J., OKAJIMA K., THEIN S.L., EMMERICH J.: “Antithrombin
After reconstitution, the Reagent 2 is stable: system to ensure that all are functioning correctly, i.e., assay conditions,
consists of two steps: mutation database: 2nd (1997) update”. Scientific and Standardization Committee
– in its original vial with STA® - mini Reducer and perforated cap in place: reagents, calibration, integrity of the plasmas being tested, etc. If Communication. Thromb. Haemost., 77, 1, 197-211, 1997.
– first, the plasma to be tested is incubated with a known excess of
- 7 days on STA Compact® and STA-R® necessary, repeat the assays. 13. ALBERTSEN B.K., SCHRODER H., INGERSLEV J., JAKOBSEN P., AVRAMIS V.I.,
thrombin (Reagent 1) in the presence of heparin
– the residual thrombin is quantitated by its amidolytic action on the - 8 days on STA Satellite® MULLER H.J., CARLSEN N.T., SCHMIEGELOW K.: “Comparison of intramuscular
– in its original capped vial (remove the STA® - Reducer): 21 days at therapy with Erwinia asparaginase and asparaginase Medac: pharmacokinetics,
synthetic chromogenic substrate CBS 61.50 (Reagent 2) (pNA release 11/ LIMITATIONS pharmacodynamics, formation of antibodies and influence on the coagulation system”.
measured at 405 nm). Since the quantity of thrombin that is neutralized 2-8 °C (REF 00596). Br. J. Haematol., 115, 983-990, 2001.
in the first reaction step is proportional to the AT level present in the Do not freeze. • Thrombin inhibitors (e.g., hirudin, argatroban...) present in the sample to
be tested may lead to an over-estimation of the AT level for this sample. 14. CLSI Document GP41-A6: “Procedures for the collection of diagnostic blood specimens
plasma being tested, it follows that the residual thrombin in the second by venipuncture; approved standard”. Sixth Edition, 27, 26, 2007.
For example, at the maximal doses used during therapy, an over-
reaction step (as measured by the pNA release) is inversely proportional estimation of approx. 10 % can be observed with hirudin. 15. CLSI Document H21-A5: “Collection, transport and processing of blood specimens for
to the AT level of the tested plasma. testing plasma-based coagulation assays; approved guideline”. Fifth edition, 28, 5,
• The STA® - Stachrom® AT III procedure is insensitive to the following 2008.
The test procedure described here is not affected by therapeutic doses of substances: hemoglobin (up to 7 g/l), bilirubin (up to 200 mg/l),
heparin. It is therefore suitable for testing of plasmas collected from patients triglycerides (up to 7.6 g/l). Significant changes are indicated by dotted lines in the margin.
receiving heparin therapy. DIAGNOSTICA STAGO S.A.S. Information and/or pictures contained in this document
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