HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO.

DETERMINATION OF LUPUS ANTICOAGULANT USING HPE KIT BY ST ART ANALYSER HUSM/HEMA-UPT/STM-C7 VERSION NO. VERSION DATE. 1 24.03.2011

APPROVED BY :

... ASSOC. PROF. DR ROSLINE HASSAN HEAD OF HAEMATOLOGY DEPARTMENT

CONTROLLED COPY NO : 3 REGISTERED HOLDER HAEMATOLOGY LABORATORY RECORD OF REVIEW/AMMENDMENT DATE VERSION NO. DETAIL OF AMMENDMENT BY

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STANDARD TEST MANUAL

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. PREPARED BY DESIGNATION CHECKED BY DESIGNATION DETERMINATION OF LUPUS ANTICOAGULANT USING HPE KIT BY ST ART ANALYSER HUSM/HEMA-UPT/STM-C7 VERSION NO. VERSION DATE. 1 24.03.2011

: MASETA ISMAIL : MEDICAL LABORATORY TECHNOLOGIST U32 : ASSOC. PROF DR WAN ZAIDAH WAN ABDULLAH : DEPUTY QUALITY MANAGER/HAEMATOLOGIST

AUTHORISED BY : ASSOC. PROF. DR ROSLINE HASSAN DESIGNATION : LAB DIRECTOR/HAEMATOLOGIST 1. OBJECTIVE

Confirmation tests for lupus anticoagulant.

2.

METHOD
Clotting method

3.

PRINCIPLE
The test plasma suspected to contain LA is first allowed to incubate at 37 C with (Tube 2) and without (Tube 1) hexagonal phase phosphatidylethanolamine (HPE). APTT test is carry out on both test tube sample using LA sensitive reagent (Reagent 4). If LA were present in the test plasma, they would be neutralized by HPE in the test tube 2. Thus resulting in shorting of clotting time of Tube 2. By comparing the difference between clotting time of Tube 1 and Tube 2, the presence of LA antibodies in the test plasma can be identified. The prolongation of the clotting time due to factor deficiencies that might be present in the plasma can be corrected by addition of normal plasma (Reagent 3) to the test system.

4.

REQUIREMENTS 4.1 EQUIPMENT

4.1.1 4.1.2 4.1.3 4.1.4 ST art Centrifuge Micropipettes (25l, 50 l, 1000 l ) Pipette tips

4.2

REAGENT
4.2.1 4.2.1.1 1. 2. 3. 4.

STACLOT LA (REF 00600) Reagent 1 (Buffer)

Ready to use. Allow the reagent to reach room temperature before use if it is refrigerated. Swirl the reagent vial gently to obtain a homogeneous suspension. Once opened, the reagent remains stable for:8 hours at 205 C 24 hours at 2-8 C 5. Do not freeze the reagent.
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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. DETERMINATION OF LUPUS ANTICOAGULANT USING HPE KIT BY ST ART ANALYSER HUSM/HEMA-UPT/STM-C7
4.2.1.2 1. 2. 3. 4. 5. 6.

VERSION NO. VERSION DATE.

1 24.03.2011

Reagent 2 (Phospholipids - Hexagonal phase PhosPhosphatidylethanolamine) Reconstitute each vial of Reagent 2 with 0.250 ml of distilled water. Swirl the vial gently until complete dissolution. Allow the reconstituted reagent to stand at room temperature (18 25 ) for 30 minutes. Swirl the reagent vial gently to obtain a homogeneous suspension before use. Once reconstituted the reagent remain stable for: - 8 hours at 205 C - 24 hours at 2-8 C Do not freeze the reagent.

4.2.1.3 Reagent 3 (Normal Plasma with Heparin Inhibitor) 1. Reconstituted each vial of Reagent 3 with 0.5 ml of distilled water. 2. Swirl the vial gently until complete dissolution. 3. Allow the reconstituted reagent to stand at room temperature (1825) for 30 minutes. 4. Swirl the reagent vial gently to obtain a homogeneous suspension before use. 5. Once reconstituted the reagent remain stable for 8 hours at 205 C. 6. Do not freeze the reagent. 4.2.1.4 Reagent 4 (Freeze-dried PTT-LS, Cephalin from rabbit cerebral tissue) 1. Reconstituted each vial of Reagent 4 with 1 ml of Reagent 5. 2. Allow the reconstituted reagent to stand at room temperature (18 25) for minutes. 3. Swirl the reagent vial gently to obtain a homogeneous suspension before use. 4. Once reconstituted, the reagent remain stable for: 8 hours at 205 C 24 hours at 2-8 C 5. Do not freeze the reagent.

4.2.1.5 Reagent 5 (Solvent) 1. The Reagent 5 is ready to use. 4.2.1 STA-CaCl2 0.025 M ( REF 00367) 1. The reagent is ready to use. 2. If the reagent is refrigerated, allow it to stand at room temperature (205 C) for 30 minutes before use. 3. Once opened, the reagent remain stable for 24 hours at 37C

4.3

CONTROL
4.3.1 STA Control LA 1+2 (REF 00201)

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STANDARD TEST MANUAL

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. DETERMINATION OF LUPUS ANTICOAGULANT USING HPE KIT BY ST ART ANALYSER HUSM/HEMA-UPT/STM-C7 VERSION NO. VERSION DATE. 1 24.03.2011

Reagent 1 ( STA- Control LA 1) Control Negative Reagent 2 ( STA- Control LA 2) Control Positive 1. Reconstituted each vial of Reagent 1 and Reagent 2 with 1 ml of distilled water. 2. Allow the reconstituted reagent to stand at room temperature (15-25 C) for 30 minutes. 3. Swirl the vial gently before use. 4. Once reconstituted, Reagent 1 and Reagent 2 remain stable for 8 hours at 205 C

4.3

SPECIMEN COLLECTION AND TREATMENT

Blood (9 volume) is collected in 0.109 M (i.e.3.2%) trisodium citrate anticoagulant (1 volume). Centrifugation: 1. Double centrifugation to obtain platelet-free plasma. The platelet count should be less than 10 x109/l. 2. First centrifugation at 2500 g for 15 minutes. 3. Collect the plasma supernatant and repeat the centrifugation step at 2500 g for 15 minutes. 4. Plasma stability: 4 hours at 205 C 1 month at -20 C 6 month at -70 C (Frozen plasma must be thawed directly at 37 C for 15 minutes).

5.
NO 5.1

PROCEDURE
ACTIVITY Pre-warm the cuvette-strips at incubation area (37C) for at least 3 minutes. In pre-warmed cuvettes (37C) RESPONSIBILITY MLT/SO

Incubation Area
1.Dispense: - Plasma (patients or control) - Reagent 1 (R1) ... - Reagent 2 (R2)

Cuvette 1

Cuvette 2

25 l 25 l

25 l -

2.Start the timer for incubation

25 l

600 secs

- Shake the cuvette-strip immediate after addition of R1 and R2. - Place back the cuvette-strips into incubation area ST art start to beep at 510 seconds incubation time 25 l 25 l - At 540 seconds mark, add Reagent 3
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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. DETERMINATION OF LUPUS ANTICOAGULANT USING HPE KIT BY ST ART ANALYSER HUSM/HEMA-UPT/STM-C7 VERSION NO. VERSION DATE. 1 24.03.2011

- Shake the cuvette-strips immediately after addition the Reagent 3. - Place back the cuvette-strips into incubation area - At 600 seconds, add Reagent 4. 50 l - Second incubation period

50 l

300 seconds

- Shake the cuvette-strips immediately after addition the Reagent 4. - Place back the cuvette-strips into incubation area - At 290 seconds, ST art start to beep again. - Transfer cuvette-strips to test-column area - Press pipette key to activate the Finnpiptte - Dispense: STA-CaCl2 0.025M ( Pre-warmed at 37C) ( Finntips 1.25ml Position 2) .. - Immediately shake the cuvette-strip and place it back to test column. Noted the clotting time for each test tube. 5.2 50 l 50 l

Result Entry
1. 2. Result HPE is transferred to LA worksheet for the individual patient and send result for interpretation to MO/Lecturer in-charge. Record the result in Laboratory Information System (LIS)

MLT/SO

6. LIMITATION 1. Patients plasma with heparin levels greater than 1 IU/ml may interfere with Staclot LA
procedure. 2. Thrombin inhibitor present in the sample may lead to falsely positive results. 3. Presence of anti-factor antibodies in the sample may lead to falsely negative results due to prolongation of the sample to be test. An appropriate test for anti-factor antibodies should be carrying out.

7.

REFERENCE RANGE
The different of T1 T2 < 8 secs is expected for normal plasma.

8.

RESULT
The difference of T1-T2 8 seconds : Positive LA Where T1and T2 correspond to clotting time of cuvette 1 and cuvette 2 respectively. The result obtained for STA Control LA1 and STA Control LA 2 are respectively negative and positive for LA.
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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. DETERMINATION OF LUPUS ANTICOAGULANT USING HPE KIT BY ST ART ANALYSER HUSM/HEMA-UPT/STM-C7 VERSION NO. VERSION DATE. 1 24.03.2011

9.

INTERPRETATION
HPE T1 and T2 results should be interpreted with other LA tests. (see Table 1 in guideline for LA detection)

10.
1. 2.

REFERENCES
Diagnostica Stago Haemostasis brochure MHT/DL/102 Pengo V., Tripodi A., Reber G., Rand J. H., Ortel T. L., Galli M. and De Groot P. G. Update of the guidelines for lupus anticoagulant detection. Journal of Thrombosis and Haemostasis. 2009; 7: 17371740 3. 4. 5. 6. 7. 8. 9. STA Compact Protocols- Tests Settings MHT/DL/106 STA Compact User Operation & Training Manual MHT/DL/109 STA Compact Operators Manual MHT/DL/110 Insert package STACLOT LA - Revised May 2009 Insert package STA Control LA 1 + 2 - Revised June 2006 Insert package STA- CaCl2 Revised June 2008 Journal Collection of Coagulation Specimens by Sterling T. Bennett

10. National Committee for Clinical Laboratory Standards. Preparation and Testing of Reagent Water in the Clinical Laboratory, Third Edition, NCCLS Document C3-A3: Vol. 17 No.18

END OF DOCUMENT

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