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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

LA SCREEN / LA CONFIRM FOR DETECTION OF LUPUS ANTICOAGULANT (DRVVT BASED)

TITLE:

VERSION NO.

1

PROCEDURE NO.

HUSM/HEMA-UPT/STM-C6

VERSION DATE.

24.03.2011

APPROVED BY:

VERSION DATE. 24.03.2011 APPROVED BY: ………… ………………………………… ASSOC

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

CONTROLLED COPY NO : 3

REGISTERED HOLDER

HAEMATOLOGY LABORATORY

RECORD OF REVIEW/AMMENDMENT

VERSION DATE DETAIL OF AMMENDMENT BY NO.
VERSION
DATE
DETAIL OF AMMENDMENT
BY
NO.

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

LA SCREEN / LA CONFIRM FOR DETECTION OF LUPUS ANTICOAGULANT (DRVVT BASED)

TITLE:

VERSION NO.

1

PROCEDURE NO.

HUSM/HEMA-UPT/STM-C6

VERSION DATE.

24.03.2011

PREPARED BY

: WAN SORIANY WAN MD ZAIN

DESIGNATION

: QUALITY OFFICER / SCIENTIFIC OFFICER

CHECKED BY

: ASSOC PROF DR WAN ZAIDAH WAN ABDULLAH

DESIGNATION

: DEPUTY QUALITY MANAGER / HAEMATOLOGIST

AUTHORISED BY DESIGNATION

: ASSOC PROF DR ROSLINE HASSAN : HAEMATOLOGIST / LAB DIRECTOR

1. OBJECTIVE

For the detection of lupus anticoagulants (LA) in plasmas by the diluted Russell’s 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 Russell’s 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 STA Compact

4.1.2 Centrifuge

4.1.3 Micropipettes (5000 µ l, 1000 µ l)

4.1.4 Pipette tips

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.

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

LA SCREEN / LA CONFIRM FOR DETECTION OF LUPUS ANTICOAGULANT (DRVVT BASED)

TITLE:

VERSION NO.

1

PROCEDURE NO.

HUSM/HEMA-UPT/STM-C6

VERSION DATE.

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

distilled water.

and

Reagent 2

with

1

ml

of

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 20±5 ° 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 x10 9 /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 20±5 ° C 1 month at -20 ° C (Frozen plasma must be thawed directly at 37° C for 15 minutes)

5. 0

.

PROCEDURE (DRVV SCREEN AND DRVV CONFIRM)

NO

ACTIVITY

RESPONSIBILITY

5.1

Loading Reagent @ Control in Product Drawer

MLT/SO

1. From Test Panel menu, Press “F2” to open product drawer

@

1. From Main Menu, select “ Loading”

2. Product Drawer

Page 3 of 8

STANDARD TEST MANUAL

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

LA SCREEN / LA CONFIRM FOR DETECTION OF LUPUS ANTICOAGULANT (DRVVT BASED)

TITLE:

VERSION NO.

1

PROCEDURE NO.

HUSM/HEMA-UPT/STM-C6

VERSION DATE.

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

 

MLT/SO

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

 

MLT/SO

1. Press “ ESC” key to access Main Menu

2. Calib/Control

 

3. “ Enter”

Page 4 of 8

STANDARD TEST MANUAL

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

LA SCREEN / LA CONFIRM FOR DETECTION OF LUPUS ANTICOAGULANT (DRVVT BASED)

TITLE:

VERSION NO.

1

PROCEDURE NO.

HUSM/HEMA-UPT/STM-C6

VERSION DATE.

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

MLT/SO

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.

Page 5 of 8

STANDARD TEST MANUAL

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

LA SCREEN / LA CONFIRM FOR DETECTION OF LUPUS ANTICOAGULANT (DRVVT BASED)

TITLE:

VERSION NO.

1

PROCEDURE NO.

HUSM/HEMA-UPT/STM-C6

VERSION DATE.

24.03.2011

5.4ii

Immediate mixing for DRVV SCN @ DRVV CFM

 

MLT/SO

Prepare 1ml normal pool plasma in an eppendorf tube with adaptor.

 

1. Open sample Drawer.

  1. Open sample Drawer. F8 for microtube

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:

 

MLT/SO

 

1.

Access to Test Panel screen or Patient Files Screen

2.

Highlight the desired patient file

 

3.

‘ Enter’

4.

Press ‘F5’ insert test

2.

Select the DRVV SCN @ DRVV CFM and press ‘ Enter’

 

3.

‘ Esc’

4.

Press ‘F10’ to save

5.

‘ Esc’

 

6.

REFERENCE RANGE

 

LA are absent from normal human plasmas. Cut off values for LA is determined for

DRVVTs and DRVVTc (see below).

 
 

7.

RESULT AND INTERPRETATION

7.1

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.

 

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

LA SCREEN / LA CONFIRM FOR DETECTION OF LUPUS ANTICOAGULANT (DRVVT BASED)

TITLE:

VERSION NO.

1

PROCEDURE NO.

HUSM/HEMA-UPT/STM-C6

VERSION DATE.

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 99 th 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: 1737–1740)

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
DRVV s
DRVV c
pt PNP ÷ DRVV s DRVV c
pt
PNP
÷
DRVV s
DRVV c

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: 1737–1740)

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

LA SCREEN / LA CONFIRM FOR DETECTION OF LUPUS ANTICOAGULANT (DRVVT BASED)

TITLE:

VERSION NO.

1

PROCEDURE NO.

HUSM/HEMA-UPT/STM-C6

VERSION DATE.

24.03.2011

8.

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: 1737–1740

3. Insert package STA- DRVV SCREEN 2 – Revised May 2007

4. STA Compact Protocols- Tests Settings MHT/DL/106

5. STA Compact User Operation & Training Manual MHT/DL/109

6. STA Compact Operator’s Manual MHT/DL/110

7. 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

8. Quality Assurance For Coagulation Lab. HUSM/58/PK/80

9. Collection of Coagulation Specimen. HUSM/58/PK/88

END OF DOCUMENT