Download - HUSMHEMA-UPT STM C7

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


2/6




ST ART ANALYSER

VERSION NO. 1




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.


3/6




ST ART ANALYSER

VERSION NO. 1




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


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.


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


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)


4/6




ST ART ANALYSER

VERSION NO. 1




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


5/6




ST ART ANALYSER

VERSION NO. 1




- 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.


6/6




ST ART ANALYSER

VERSION NO. 1




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