====SOP IDENTIFICATION AND QUANTIFICATION OF VARIOUS HEMOGLOBIN BY VARIANT II HEMOGLOBIN TESTING SYSTEM====

**PURPOSE OF EXAMINATION**:

The variant II β thalassaemia short program is intended for the separation and area percent determination of haemoglobin A2 and F, and as an aid in the identification of abnormal haemoglobins in whole blood using ion exchange high performance liquid chromatography.(HPLC).

The VARIANT II β thalassemia short program is intended for use only with the Bio-Rad VARIANT II HEMOGLOBIN TESTING SYSTEM.

**INTRODUCTION:**

The variant II haemoglobin Testing system is a fully automated ,high throughput haemoglobin analyzer. It consist of two modules-

1) The variant II chromatographic system(VCS)

2) The variant II sampling station (VSS).

In addition ,a personal computer is used to control the variant II system using clinical data management software.

**PRINCIPLE OF THE VARIANT II HAEMOGLOBIN TESTING SYSTEM :**

**The VARIANT II utilizes principles of ion exchange high performance liquid chromatography** for separation and determination of relative percentage of specific haemoglobin in whole blood sample.

Two dual piston pumps located in the VCS deliver a buffer solution to the analytic cartridge and the detector.

Primary sample tubes are mixed in sampling station, a sample is then drawn, diluted and introduced to the analytic flow path using automatic injection.

Between sample injection, the sample needle is rinsed with wash solution to minimize sample carry-over.

The sample is carried by the buffer through the analytic cartridge where the sample is separated into its individual components based on their ionic interaction with the cartridge material.The separated component then pass through the dual wavelength detector, where absorbance of the sample component is measured at 415nm.Background noise is reduced with the use of a secondary wavelength at 690nm.The absorbance data are transmitted from the detector to the PC and displayed by CDM(clinical data management)as a real time chromatogram.(graph of Time vs Absorbance).

The processed data is incorporated into a printed report, which contains the following:

1 )a complete summary of samples detected components(i.e.peak identification ,retention time, relative percentage,area).

2) the sample’s chromatogram

3) date and time of analysis

4) vial number and sample identification

**PERFORMANCE SPECIFICATION:**

^**Specific CV**^**Hb A<sub>2</sub>**^**Hb F**    ^
|               |**3.3-4.2%**        |**2.7-5.4%**|

**REQUIREMENTS:**

**Kit components**

-Elution buffer 1 (each bottle contains 1900ml of a sodium phosphate buffer contains <0.05%sodium azide as a preservative).

-Elution buffer 2 (each bottle contains 1800ml of a sodium phosphate buffer contains <0.05%sodium azide as a preservative).

-HbA2/F Calibrator/ Diluent (one set consisting of 6 vials of calibrator and 1 bottle of calibrator diluent. The calibrator vials contain lyophilized human red blood cell hemolysate with gentamicin, tobramycin and EDTA as preservative.

Calibrator diluent contains 100ml of deionized water with <0.05%sodium azide as a preservative.

-Analytic cartridge:cation exchange cartridge(250 tests),4.6mm ID x30mm.

-Whole blood primer :each pack contains 10 vials of lyophilized human red blood cell hemolysate with gentamicin, tobramycin and EDTA as preservative.

-Sample vials: polypropylene vials with pierceable caps,1.5ml.

-Wash/Diluent solution:each bottle contains 1800ml of deionized water with <0.05%sodium azide as a preservative).

-CD-ROM with variant II β thalassaemia short program parameters.

-Microvial adapter

-Paediatric adapter

-Lyphochek Haemoglobin A2 control 4x0.5ml

-Lyphochek Haemoglobin A2 control 2x0.5ml.

Monthly control

-pipettes: 5µl,0.5ml,1ml,1.5ml,10ml.

-deionized water

-sample rack

-disposable gloves

**SAMPLE COLLECTION :**

**Specimen type**: whole blood

**Specimen additives, preservatives :**whole blood should be collected in a vacuum collection tube containing EDTA.

**Specimen storage:** whole blood specimens are stable for 7 days when stored at 2-8 C OR 48 hours at ambient temperature(22-24 C).

**SAMPLE PREPARATION**:

**Whole blood primer:**

-reconstitute with 1 ml ofdeionized water

-allow to stand for 10min,swirl gently.

-stable for 21 days at 2-8c.

-empty contents of vial into microvial.

**Calibrator:**

-one level of calibrator run in duplicate.

-reconstitute with 10ml of cold calibrator diluent.

-allow to stand for 5-10min,swirl gently.

-stable for 10 days at 2-8 c.

-add 1ml to 1.5ml microvial.

**Control:**

**-**reconstitute with 0.5ml of distilled water.

-allow to stand for 10 min,swirl to dissolve.

-diluted 1:200 prior to analysis.(5 µl control to 1.0ml of wash diluent)

-suggest running controls at the beginning and end of run.

-stable for 21 days at 2-8 C.

**Whole blood sample:**

**-**samples should be collected in a vacuum collection tube containing EDTA.

-do not have to be pre diluted unless volume is <1.5ml.

-IF <1.5ml,then pre-diluted sample 1:200 in a 1.5ml microvial.(5 µl sample to 1.0ml of wash diluent).

**DAILY MAINTAINANCE**

**PRE-RUN CHECKLIST:**

Start up

-Switch on main power supply

-Switch on UPS

-Start CPU and Monitor

-Open CDM 5.1 program

-Switch on VCS(VARIANT II CHROMATOGRAPHIC SYSTEM)

-Switch on VSS(VARIANT II SAMPLING STATION)

-Click on variant II β thal Inactive mode

- Click on variant II β thal Inactive mode,it prompt return to active,then click yes

-So,the variant II β thal comes on ready mode,ok it by cliking on it

-Click Run

-In Run click worklist

-Wait upto green light

-When shows green light,click on start option

-Then machine is ready to run,but before it, check following instruction:

-check that correct method is installed.

-check buffer/wash levels/lot no and line position.

-check cartridge injection count and lot no.

-check pump pressure on both pumps.(flow rate should be programmed according to the installed kit.-β thal runs at 20.ml/min).The expected pressure range for each cartridge lot is listed in the cartridge insert,pressure average 18-25kg/cm<sup>2</sup>.

Pumps should not fluctuate >5%.

-Check for leaks during pressure check.

-check waste disposal.

-check printer paper supply.

-perform piston seal flush.

**ROUTINE SAMPLE RUN:**

-load sample into the system racks

-Insert rack adapter if using small diameter tubes.Use the appropriate adapter for prediluted samples or pediatric tubes.

-Load the racks into the VSS .Make sure all racks are aligned properly with the barcode facing the back and the numeric label facing the front.

-Position the stop adapter(with appropriate barcode) or empty microvial so it is the last sample,or insert empty rack after the last patient sample.

-Samples may be added to the run during operation by loading samples into the system racks and loading them onto the right side of VSS.

-During the run there will be six racks on the right and four racks on the left side.Do not attempt to place a fifth rack on the left during a run.This will cause a rack to jam.

-place the following in the sample rack:

^Tube position^Adapter label  ^Reagent                ^
|1            |Primer         |Whole blood primer     |
|2            |Blank          |Distilled water        |
|3            |Blank          |Distilled water        |
|4            |Cal 1          |Variant A2/F calibrator|
|5            |Control level 1|Low control            |
|6            |Control level 2|High control           |
|7 to N       |Patient sample |-                      |
|N+1          |Control level 1|Low control            |
|N+2          |Control level 2|High control           |
|N+3          |Stop           |-                      |

-Make sure barcodes are facing the back of the rack and place rack on the VSS belt on the right side of instrument.

-Go to set up/sample type/calibrator screen and verify that ‘Enable delta factor’ is selected and that ‘stop work list’ is selected under ‘Action if outside limits.

-Verify that the system is in ready state and go to run/worklist screen and press start/stop icon to initiate run.

-Once the run is finished ,note retention time of A2 and temperature.

-After making the temperature adjustment,wait 10-15 min for temperature to equilibrate and start sample run.

**Shut down:**

-Once the run is finished,as indicated by 'STOP' barcoded tube in rack

-Close CDM 5.1 program

-Switch turn off on both monitor and CPU

-Switch turn off on VCS and VSS

-Switch turn off on UPS

-Switch turn off on main power supply.

**POST RUN CHECKLIST:**

**1)Discard any remaining samples:**\\

-Properly dispose of any remaining samples. sample waste should be considered as potentially biohazardous material and must be disposed of in the appropriate manner.\\

2)Wipe up all spills and Decontaminate surface area:\\

-Remove all racks from the sampling station\\

-Prepare decontamination solution of your choice\\

-Dampen a disposable towel with the decontamination solution\\
-From the MAINTAIN/Instrument screen under execute commands, select the cleaning belts option from the combination box. Press start button.\\

-Wipe the conveyer belts with the damp towel.\\

-After decontamination, allow the belts to air dry before use.\\

**QC REQUIREMENTS:**\\

Daily Control: A set of low and high control are run at the beginning of run on daily basis.\\

Monthly control: A set of low and high control are run at the beginning of run once a month.\\

**GUIDELINES FOR INTERPRETATION OF RESULTS:**\\

For an analysis to be considered acceptable,observe following guidelines:\\

-Check for proper baseline construction\\

-Peak shape is sharp and symmetrical\\

-Unknown peak immediately prior to P2 peak is normal\\

__all other unknown peaks require further evaluation__\\

-Calibration of the VARIANT II is complete. The retention time for the hemoglobin A2 in the calibrator is 3.65+/-0.10.The calibration response factor for hemoglobin A2 and F must be greater than 0.7 and less than 1.30(refer to current cartridge insert for typical retention times)\\

-The normal adult range for hemoglobin A2 is typically between 1.75 and 3.25%of total hemoglobin while heterozygous β thalassemia conditions yield HbA2 levels 4.0 to 9.0%.The normal adult range for Hb F is typically less than 1% of the total hemoglobin .Heterozygous and homozygous conditions of β thalassemia yield HbF ranges of 1-5 and 80-100%.\\

-Total area of each analysis should range from 10,00,000 to 30,00,000 µvolt.seconds. Results should not be reported if the area is outside this range.\\

-Although the linearity studies demonstrate a wide linear range.The reportable range for HbA2 is 1-13% and the reportable range for HbF is 1-40%.These were chosen to be consistent with a commerecially available reference method.To be consistent with the established reportable ranges,HbA2 and Hb F values that are lower than the reportable range should be reported as <1.0%.Values in excess of the reportable ranges for HbA2 and HbF should be greater than 13% and 40% respectively.\\

-Diabetic specimen typically exhibit an elevated P2 peak.An additional peak,identified as unknown,may also be present between the normal position of HbF and P2.If this additional peak elutes within the HbF window when there is no HbF present,it may be identified as HbF.Careful comparison of the sample's hemoglobin F retention time with control samples can help differentiate this unknown peak for HbF.\\

**INTERFERENCE**

-Lipemia interference study(triglyceride levels upto 4600mg/dl,Anticoagulant concentration interference study(EDTA concentration 100-1100%) and icterus interference study(conjugated bilirubin concentration in whole blood upto 20 mg/dl do not interfere with HbA2 or HbF measurements using the VARIANT II β thalassemia short program.\\

-Hemolyzed sample study showed that HbA2 and HbF measurenents with the VARIANT II β thalassemia short program are not affected by sample hemolysis.However,Bio Rad lab recommends that samples hemolyzed prior to sample preparation should not be used for analysis.\\

**REFERENCES:**\\

-Reference manual of the VARIANT II β thalassemia short program\\

-Reference guidelines on operation of Variant II and Interpretation of hemoglobin variants\\

-Reagent insert of VARIANT II β thalassemia short program\\