HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. LA SCREEN / LA CONFIRM FOR DETECTION VERSION NO.

OF LUPUS ANTICOAGULANT (DRVVT BASED) HUSM/HEMA-UPT/STM-C6 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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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO.

PREPARED BY DESIGNATION CHECKED BY DESIGNATION AUTHORISED BY DESIGNATION 1. OBJECTIVE

LA SCREEN / LA CONFIRM FOR DETECTION VERSION NO. OF LUPUS ANTICOAGULANT (DRVVT BASED) HUSM/HEMA-UPT/STM-C6 VERSION DATE.

1 24.03.2011

: WAN SORIANY WAN MD ZAIN : QUALITY OFFICER / SCIENTIFIC OFFICER : ASSOC PROF DR WAN ZAIDAH WAN ABDULLAH : DEPUTY QUALITY MANAGER / HAEMATOLOGIST : ASSOC PROF DR ROSLINE HASSAN : HAEMATOLOGIST / LAB DIRECTOR

For the detection of lupus anticoagulants (LA) in plasmas by the diluted Russells viper venom performed with analyzers of the STA Compact or STA line. These tests are indicated for use in patients suspected to have LA /Anti phospholipids Syndrome. . 2. METHOD Mechanical clot detection 3. PRINCIPLE The Russells viper venom is present in the STA-Staclot dRVV Screen reagent. The venom acts in presence of calcium as an activator of Factor X and thereby triggers the coagulation cascade downstream from Factor X, thus eliminating the influence of coagulation factors acting upstream. The test is not affected by the contact factor anomalies or by Factor VIII and IX deficiencies or inhibitors. The test is performed with a low concentration of phospholipids. If lupus anticoagulants (LA) are present, the clotting time will be lengthened. The STA-Staclot dRVV Confirm contains a higher concentration of phospholipids which neutralize the LA present in the plasma to be tested. 4. REQUIREMENTS 4.1 EQUIPMENT 4.1.1 4.1.2 4.1.3 4.1.4 4.2 REAGENT 4.2.1 STA-Staclot dRVV Screen (REF 00339) 1. Reconstitute each vial with 2ml of distilled water. 2. Allow the reconstituted reagent to stand at room temperature (18 25 ) for 30 minutes. 3. Swirl the reagent vial gently to obtain a homogeneous suspension. 4. Once reconstituted the reagent stable for 72 hours on STA Compact.
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STA Compact Centrifuge Micropipettes (5000l, 1000l) Pipette tips

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. LA SCREEN / LA CONFIRM FOR DETECTION VERSION NO. OF LUPUS ANTICOAGULANT (DRVVT BASED) HUSM/HEMA-UPT/STM-C6 VERSION DATE. 1 24.03.2011

4.2.2. STA-Staclot dRVV Confirm (REF 00334) 1. Reconstitute each vial with 2ml of distilled water. 2. Allow the reconstituted reagent to stand at room temperature (18 25 ) for 30 minutes. 3. Swirl the reagent vial gently to obtain a homogeneous suspension. 4. Once reconstituted the reagent stable for 72 hours on STA Compact.

Note: STA-Staclot dRVV Screen and STA-Staclot dRVV Confirm reagents are insensitive to unfractionated heparin (UFH) levels up to 0.8 IU/ ml.

4.3 CONTROL 4.3.1 STA Control LA 1+2 (REF 00201) Reagent 1 ( STA- Control LA 1) Reagent 2 ( STA- Control LA 2) 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 on STA Compact - 8 hours at 205 C 4.4 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 2500g for 15 minutes. 4. Plasma stability: 4 hours at 205 C 1 month at -20 C (Frozen plasma must be thawed directly at 37 C for 15 minutes) . 5. 0 NO 5.1 PROCEDURE (DRVV SCREEN AND DRVV CONFIRM) ACTIVITY Loading Reagent @ Control in Product Drawer 1. From Test Panel menu, Press F2 to open product drawer @ 1. 1. From Main Menu, select Loading 2. Product Drawer
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RESPONSIBILITY MLT/SO

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. LA SCREEN / LA CONFIRM FOR DETECTION VERSION NO. OF LUPUS ANTICOAGULANT (DRVVT BASED) HUSM/HEMA-UPT/STM-C6 VERSION DATE. 1 24.03.2011

3. Enter 4. Scan reagent/control barcode label * Note: Change the product mode to Micro Volume F8 if reagent/control has been Transferred into microcup. 5. Press Enter ( the cursor will move to Pos column ) 6. Load reagent/control into the product drawer within 10 seconds *Note: Please ensure the beep sound is heard. 7. If different lot number of reagent/control/calibrator is detected, following window will displayed:

LOT NUMBER CHANGED All concerned calibrations will be definitively invalid

_______________________

Accept (YES / NO)

8. Type YES and Press Enter 9. Scan the barcode sheet 10. Press Esc when end of the data processing 11. Continue loading of other reagents @ control 12. If not , close the Product Drawer 5.2 CALIBRATION Calibration should be performed before each sample run in batch. Reference Pool clotting time / Reference Time is corresponds to the clotting time of pool of normal plasma, which is pool from at least 20 fresh healthy donors. OR else commercial pool normal plasma, Pool Norm (REF 00539) can be used. 1. Press ESC key to access Main Menu 2. Calib/Control 3. Enter 4. Calibration 5. Enter 6. Choose DRVV SCN @ DRVV CFM 7. ENTER 8. Press ESC key 9. Choose Modify Ref.Time 10. Enter 11. Type password: CQ 12. ENTER 13. Key in Ref.Time 14. ENTER 15. Press ESC key ( 2 times) 5.3 Run Quality Control 1. Press ESC key to access Main Menu 2. Calib/Control 3. Enter
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MLT/SO

MLT/SO

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. LA SCREEN / LA CONFIRM FOR DETECTION VERSION NO. OF LUPUS ANTICOAGULANT (DRVVT BASED) HUSM/HEMA-UPT/STM-C6 VERSION DATE. 1 24.03.2011

4. Quality Control 5. Enter 6. Choose DRVV SCN @ DRVV CFM 7. Press F1 to select the test 8. Press F10 to run selected control 9. Type password : HUSM 10. Enter 11. Press ESC key (2 times) To View Quality Control Result 1. From Main Menu, Select Calib/Control 2. Enter 3. Quality Control 4. Enter 5. Highlight the DRVV SCN @ DRVV CFM 6. Enter View Levey-Jennings graph 1. Press F1 to view the results in tabular form 2. Press F2 to access other levels (Level 1 @ Level 2) 5.4i Run Patient Sample @ Reference Pool Plasma If QC results OK, proceed with patient testing 1. From Test Panel menu, Press F1 to open sample drawer @ 1. From Main Menu, select Loading 2. Sample Drawer 3. Enter 4. Press F12 for STAT sample F8 for microcup sample *Note: Pediatrics sample tube need to transferred into adaptor 5. Scan sample barcode label @ Key in Patient ID 6. Press Enter ( The cursor will move to Pos) 7. Load the sample tube ( Ensure the beep sound is heard) 8. Select DRVV SCN @ DRVV CFM 9. Press F10 to validate 10. Continue loading other sample tube ( follow step 6-10) 11. If not, close the sample drawer. *Note: The clotting time of reference pool plasma will be used as reference time for DRVV SCN @ DRVV CFM calibration. MLT/SO

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

5.4ii

LA SCREEN / LA CONFIRM FOR DETECTION VERSION NO. OF LUPUS ANTICOAGULANT (DRVVT BASED) HUSM/HEMA-UPT/STM-C6 VERSION DATE.

1 24.03.2011
MLT/SO

Immediate mixing for DRVV SCN @ DRVV CFM Prepare 1ml normal pool plasma in an eppendorf tube with adaptor. 1. Open sample Drawer. F8 for microtube 2. Type manually the below information: ID: POOL F1 for diluents Name: NORMAL Vial: 1 ml Stability: 4 h 3. Press Enter 4. Put pool plasma tube in any position.

5.4.iii

Patient sample is present in Sample Drawer: 1. 2. 3. 4. 2. 3. 4. 5. Access to Test Panel screen or Patient Files Screen Highlight the desired patient file Enter Press F5 insert test Select the DRVV SCN @ DRVV CFM and press Enter Esc Press F10 to save Esc

MLT/SO

6.

REFERENCE RANGE LA are absent from normal human plasmas. Cut off values for LA is determined for
DRVVTs and DRVVTc (see below).

7. 7.1

RESULT AND INTERPRETATION Result interpretation : DRVV Screen The DRVV Screen is expressed in ratio: Screen Ratio = screen clotting time of tested plasma screen clotting time of reference pool NP (see appendix 1 on results tabulation) The DRVV Screen(secs) > cut off point: Potentially suggestive of LA The DRVV Screen (secs) < cut off point : LA is unlikely.

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. LA SCREEN / LA CONFIRM FOR DETECTION VERSION NO. OF LUPUS ANTICOAGULANT (DRVVT BASED) HUSM/HEMA-UPT/STM-C6 VERSION DATE. 1 24.03.2011

Note: DRVV Screen result should be interpreted with other LA tests. (see Table 1 and Table 2 in guideline for LA detection)

Determination of cut off value: DRVV Screen is performed on plasmas from healthy donors. Take cut off value above the 99th percentile of the distribution.
(Ref: 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)

7.2

Result interpretation for LA confirmatory test using DRVV screen/DRVV confirm. The DRVV screen/DRVV confirm. is expressed LA ratio. LA ratio : DRVV s DRVV c Normalization of results = DRVV s DRVV c
pt

DRVV s DRVV c

PNP

LA ratio > local cut off value confirmed the presence of LA in the tested plasma. LA ratio < local cut off value excludes LA. (see appendix 1 on results tabulation) and LA guideline on interpretation of LA tests. Note: DRVV confirmatory test for LA should be interpreted with others LA tests (see table 1) in guideline for LA detection. Determination of local cut off value: clotting times for DRVVc and DRVVs are obtained from healthy donors (20-40 subjects). LA ratio = (DRVVs / DRVVc) healthy donor cut off value for LA ratio = mean of LA ratio derived from healthy donors (Ref: (Ref: 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)

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. LA SCREEN / LA CONFIRM FOR DETECTION VERSION NO. OF LUPUS ANTICOAGULANT (DRVVT BASED) HUSM/HEMA-UPT/STM-C6 VERSION DATE. 1 24.03.2011

8.

REFERENCES 1. 2. 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. Insert package STA- DRVV SCREEN 2 Revised May 2007 STA Compact Protocols- Tests Settings MHT/DL/106 STA Compact User Operation & Training Manual MHT/DL/109 STA Compact Operators Manual MHT/DL/110 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 Quality Assurance For Coagulation Lab. HUSM/58/PK/80 Collection of Coagulation Specimen. HUSM/58/PK/88

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