8/4/2019 HUSMHEMA-UPT STM C7
1/6
HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA
TITLE:DETERMINATION OF LUPUS
ANTICOAGULANT USING HPE KIT BY
ST ART ANALYSER
VERSION NO. 1
PROCEDURE NO.HUSM/HEMA-UPT/STM-C7
VERSION DATE. 24.03.2011
Page 1 of 6 STANDARD TEST MANUAL
APPROVED BY :
...
ASSOC. PROF. DR ROSLINE HASSANHEAD OF HAEMATOLOGY DEPARTMENT
CONTROLLED COPY NO : 3
REGISTERED HOLDER
HAEMATOLOGY LABORATORY
RECORD OF REVIEW/AMMENDMENT
DATE VERSION NO. DETAIL OF AMMENDMENT BY
8/4/2019 HUSMHEMA-UPT STM C7
2/6
HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA
TITLE:DETERMINATION OF LUPUS
ANTICOAGULANT USING HPE KIT BY
ST ART ANALYSER
VERSION NO. 1
PROCEDURE NO.HUSM/HEMA-UPT/STM-C7
VERSION DATE. 24.03.2011
Page 2 of 6 STANDARD TEST MANUAL
PREPARED BY : MASETA ISMAIL
DESIGNATION : MEDICAL LABORATORY TECHNOLOGIST U32
CHECKED BY : ASSOC. PROF DR WAN ZAIDAH WAN ABDULLAHDESIGNATION : DEPUTY QUALITY MANAGER/HAEMATOLOGIST
AUTHORISED BY : ASSOC. PROF. DR ROSLINE HASSANDESIGNATION : 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) andwithout (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 ofclotting time of Tube 2. By comparing the difference between clotting time of Tube 1 and Tube2, the presence of LA antibodies in the test plasma can be identified. The prolongation of theclotting time due to factor deficiencies that might be present in the plasma can be corrected byaddition of normal plasma (Reagent 3) to the test system.
4. REQUIREMENTS
4.1 EQUIPMENT
4.1.1 ST art4.1.2 Centrifuge
4.1.3 Micropipettes (25l, 50 l, 1000 l )4.1.4 Pipette tips
4.2 REAGENT4.2.1 STACLOT LA (REF 00600)
4.2.1.1 Reagent 1 (Buffer)
1. Ready to use.
2. Allow the reagent to reach room temperature before use if it is refrigerated.3. Swirl the reagent vial gently to obtain a homogeneous suspension.
4. Once opened, the reagent remains stable for:8 hours at 205 C24 hours at 2-8 C
5. Do not freeze the reagent.
8/4/2019 HUSMHEMA-UPT STM C7
3/6
HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA
TITLE:DETERMINATION OF LUPUS
ANTICOAGULANT USING HPE KIT BY
ST ART ANALYSER
VERSION NO. 1
PROCEDURE NO.HUSM/HEMA-UPT/STM-C7
VERSION DATE. 24.03.2011
Page 3 of 6 STANDARD TEST MANUAL
4.2.1.2 Reagent 2 (Phospholipids - Hexagonal phase
PhosPhosphatidylethanolamine)
1. Reconstitute each vial of Reagent 2 with 0.250 ml of distilled water.2. Swirl the vial gently until complete dissolution.3. Allow the reconstituted reagent to stand at room temperature (18 25 ) 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- 24 hours at 2-8 C
6. 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 beforeuse.
4. Once reconstituted, the reagent remain stable for: 8 hours at 205 C24 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)
8/4/2019 HUSMHEMA-UPT STM C7
4/6
HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA
TITLE:DETERMINATION OF LUPUS
ANTICOAGULANT USING HPE KIT BY
ST ART ANALYSER
VERSION NO. 1
PROCEDURE NO.HUSM/HEMA-UPT/STM-C7
VERSION DATE. 24.03.2011
Page 4 of 6 STANDARD TEST MANUAL
Reagent 1 ( STA- Control LA 1) Control Negative
Reagent 2 ( STA- Control LA 2) Control Positive1. 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 citrateanticoagulant (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 at2500 g for 15 minutes.
4. Plasma stability: 4 hours at 205 C1 month at -20 C
6 month at -70 C
(Frozen plasma must be thaweddirectly at 37 C for 15 minutes).
5. PROCEDURE
NO ACTIVITY RESPONSIBILITY
5.1 Pre-warm the cuvette-strips at incubation area (37C) for at least 3
minutes.
In pre-warmed cuvettes (37C)
Incubation Area Cuvette 1 Cuvette 2
1.Dispense:
- Plasma (patients or control)- Reagent 1 (R1) ...- Reagent 2 (R2)
25 l
25 l
-
25 l
-
25 l
2.Start the timer for incubation 600 secs
- Shake the cuvette-strip immediate after addition of R1 and R2.
- Place back the cuvette-strips into incubation areaST art start to beep at 510 seconds incubation time- At 540 seconds mark, add Reagent 3 25 l 25 l
MLT/SO
8/4/2019 HUSMHEMA-UPT STM C7
5/6
HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA
TITLE:DETERMINATION OF LUPUS
ANTICOAGULANT USING HPE KIT BY
ST ART ANALYSER
VERSION NO. 1
PROCEDURE NO.HUSM/HEMA-UPT/STM-C7
VERSION DATE. 24.03.2011
Page 5 of 6 STANDARD TEST MANUAL
- 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 50 l
- Second incubation period 300 seconds
- Shake the cuvette-strips immediately after addition theReagent 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.
50 l
50 l
5.2 Result Entry
1. Result HPE is transferred to LA worksheet for the individual patientand send result for interpretation to MO/Lecturer in-charge.
2. 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 LAprocedure.
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 toprolongation of the sample to be test. An appropriate test for anti-factor antibodies shouldbe 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 negativeand positive for LA.
8/4/2019 HUSMHEMA-UPT STM C7
6/6
HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA
TITLE:DETERMINATION OF LUPUS
ANTICOAGULANT USING HPE KIT BY
ST ART ANALYSER
VERSION NO. 1
PROCEDURE NO.HUSM/HEMA-UPT/STM-C7
VERSION DATE. 24.03.2011
Page 6 of 6 STANDARD TEST MANUAL
9. INTERPRETATION
HPE T1 and T2 results should be interpreted with other LA tests.(see Table 1 in guideline for LA detection)
10. REFERENCES
1. Diagnostica Stago Haemostasis brochure MHT/DL/102
2. 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. STA Compact Protocols- Tests Settings MHT/DL/106
4. STA Compact User Operation & Training Manual MHT/DL/109
5. STA Compact Operators Manual MHT/DL/110
6. Insert package STACLOT LA - Revised May 2009
7. Insert package STA Control LA 1 + 2 - Revised June 2006
8. Insert package STA- CaCl2 Revised June 2008
9. Journal Collection of Coagulation Specimens by Sterling T. Bennett
10. National Committee for Clinical Laboratory Standards. Preparation and Testing of ReagentWater in the Clinical Laboratory, Third Edition, NCCLS Document C3-A3: Vol. 17 No.18
END OF DOCUMENT
Top Related