New Born Screening (NBS) Test : Newborn screening involves testing infant to enable early detection of treatable metabolic disorders so that infants can be identified and treated before problems occur. Newborn Screening tests are done using whole blood drawn from the heel of the babies and collected on a special filter paper. This is a very simple and fairly painless method of pricking the heel of the baby to draw a very small amount of blood.
Instrument use in NBS Lab : Read this operating manual completely before using the equipment for the first time. Where applicable, follow instructions for use of accessories..
- Microplate incubator/shaker ( Thermomixer)
- Microplate washer
- Microplate flurotometer
- Microtiter plate spectrophotometer
- DBS (Dry Blood Spot ) Puncture : The dried blood spot (DBS) method is an approach to blood sample processing whereby a drop of blood is placed onto a designated filter paper (DBS card) from which target compounds are extracted for quantitative determination. 3 MM Dia , 1/8″ inch round punch for the DBS card sample and easy use.
2. Microplate incubator/shaker ( Thermomixer) : We can use the ThermoMixer to perform the simultaneous mixing and temperature control of the sample material.
The thermoblocks can be used with the following lab vessels:
- Micro test tubes with volumes of 0.2 mL to 5.0 mL
- Conical tubes with volumes of 15 mL to 50 mL
- Microplates and deepwell plates with any kind of bottom shape
- PCR plates in 96 and 384-well format
- Tubes with a diameter of 11.0 mm to 11.9 mm
- Cryogenic tubes with volumes of 2.0 mL
In this SOP we can discussed only Microplate and deepwell plate only
When we attach the thermoblock, the device automatically recognizes the mounted thermoblock. The mixing frequency is automatically limited to the maximum value for the thermoblock being used.
First only attach the rear edge of the thermoblock. The writing must face to the front. Push the front edge of the thermoblock down.g the thermoblock :
The thermoblock and the heating/cooling plate can be very hot after heating and cause burns. Allow the thermoblock and the heating/cooling plate to cool down completely before removing the thermoblock.
Inserting the plate:
- When inserting microplates, make sure that the height sensor is not covered.
- Take care that the height sensor does not get contaminated.
- Only use closed plates.
- Insert the plate with the back edge first. Then press it down at the front.
- To ensure uniform temperature control in all wells, place the lid on the thermoblock.
Temperature, speed and time setting:
- The time mode is set to Time Control
- To switch off the mixing function, select the 0 rpm setting with the speed arrow keys
- (▼ before 300 rpm or ▲ after 3 000 rpm).
- Set the temperature control duration (time) with the time arrow keys.
- Set the temperature with the temp arrow keys.
- To start the time counter, press the start/stop key.
- The symbol
flashes on the display
- The temperature control duration is counted down.
- The display shows the remaining temperature control duration and the actual temperature/set temperature.
- A signal sounds after the temperature control duration has elapsed.
NOTE : Polystyrene microplates melt at temperatures above 70 °C. Polypropylene deepwell plates deform at temperatures above 80 °C. Deformed p lates may become detached from the thermoblock or are more difficult to remove. Temper polystyrene microplates up to 70 °C max. If you are heating deepwell plates above 80 °C, do not exceed the mixing frequency of 1000 rpm.
Temperature control
Peltier cooling allows samples to be cooled to 15 °C below ambient temperature.
The temperature range can be set from 1 °C to 110 °C.
Mixing
Depending on the utilized thermoblock, we can select mixing frequencies between 300 rpm and 3000 rpm.
Anti-spill technology prevents lid wetting and cross contamination.
3. Microplate washer :
We use Microplate Strip Washer provides microplate priming and washing for ELISA, fluorescence and chemiluminescence immunoassays, cellular and agglutination assays.
System Startup:
To turn on the on/off switch on the right side of the base. Automatically will perform a self-test, displaying the screens shown below until initialization is complete. During this time, all keys are inactive.
Define Wash Program:
To define a Wash program, follow the menu path shown here. Some of these items are options and some are prompts. The options are described below.
DEFINE “>> CREATE “>> WASH > SELECT WASH PROGRAM (EX. 01) > Method, Dispense, and Aspiration > Method > NO.OF CYCLE (EX. 05) > WASH FORMATE PLATE > SOAK/SHAKE“> > NO > Dispense > 300µL > DISPENSE FLOW RATE (500µL/WELL) > DISPENSE HEIGHT (120MM) > HORIZONTAL DISP POS. (00) > BOTTOM WASH FIRST >NO > PRIME BEFOR START > NO > ASPIRATE HIGHT (024 (3.048MM) > HORIZONTAL ASP POS. (00) > TREVEL PLATE (03(5.8MM/SEC.) > ASPIRATE DELAY (15 SEC.) >SAVE PROGRAM?
Run Wash Program
Before running any program, make sure that the microplate or the holder containing the microstrips is properly seated in the plate carrier. To run a Wash program, follow the menu path shown here. Some of these items are options and some are prompts. The options are described below
RUN > WASH > SELECT WASH PROGRAM >SELECT REAGENT BOTTLE* > PRIME THE WASHER? > SELECT PRIME PROGRAM >FIRST STRIP > NUMBER OF STRIPS > PLACE PLATE IN CARRIER AND PRESS START “KEY” > WASH PROGRAM RUNNING.PRESS “STOP” KEY TO QUIT > WASH PROGRAM COMPLETE. PRESS “ENTER” KEY
Maintenance
Perform Daily, Overnight, and Periodic Maintenance
- Cleaning the manifold tubes and chambers.
- Cleaning the plate carrier.
- Cleaning the supply, rinse, and waste bottles.
- Rinsing and checking the tubing and the check valves.
4. Microplate fluorometer (Microplate multimode reader) :
We use monochromator and filter-based detection techniques for fluorescence microplate detection. For this we use Tecan’s Infinite 200, it is possible to use a full range of leading detection technologies in one easy-to-use in this modular instrument. (For more information please read Tecan’s Infinite 200 manual)
Principle:
Fluorescence Intensity (FI) may also be used to measure Fluorescence Resonance Energy Transfer (FRET). For some microplate applications, FRET offers advantages over FI and TRF, because they simplify assay preparation. These preferably apply for mix and measure binding studies. Compared to Fluorescence Polarization (FP), FRET requires both binding partners to be labeled in a suitable way. On the other hand, FRET may utilize TRF labels for increased sensitivity, then being referenced as HTRF (Homogeneous TRF).
Fluorescent molecules emit light of specific wavelength when struck by light of shorter wavelength (Stokes Shift). In particular, a single fluorescent molecule can contribute one fluorescence photon (quantum of light). This is a part of the energy, which has been absorbed before (electronic excitation), but could not be released quickly enough into thermal energy. The average time it takes between excitation and emission is called the fluorescence lifetime. For many fluorescent molecular species, fluorescence lifetime is on the order of nanoseconds (prompt fluorescence). After excitation, fluorescence emission occurs with a certain probability (quantum yield), which depends on the fluorescent species and its environmental conditions.
Use of Microplate flurotometer :
In NBS lab tecan Infinite 200 PRO is mainly used for fluorescence reading like G6PD, PKU, Galactose, Biotinidase, 17-OHP, IRT, and hTSH etc.
Programming for test reading:
Start the system computer
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Switch on the tecan Infinite 200 PRO before software
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Open megellan software program (A dialogs box open)
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Click to the create/edit a method (A dialogs box open)
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Click NEW (A dialogs box open)
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Select the number of strip (the test being performed)
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Select wavelength
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Select gain and put its value
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Click on choose fluorescent parameter (A dialogs box open)
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Select the plate and lay out to the test, standard and QC.
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Enter conc. value of plate layout (according to the kit insert)
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Click the standard curve (A dialogs box open)
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Select analysis type
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Select analysis type
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Click next
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Save program
Measurement test reading:
Click start measurement (Home menu)
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Click next
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Select save program
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Click start
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Measurement starts and out result with graph
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Click next
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Save result
View to the test results:
Click Evaluate result (Home menu)
Select the drive and click to test and see it.
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We use flowing table for programming:
| Test name | Excitation wavelength | Emission wavelength | Gain | Std. Curve |
| TSH | 320 nm | 400/405 nm | Optimal | Linear regression |
| 17-OHP | 320 nm | 400/405 nm | Manual 30 | Four Parameter |
| IRT | 320 nm | 405 nm | Manual 30 | Cubic Spine |
| BIOTINIDASE | 355/360 nm | 460/465 nm | Manual 30/40 | Linear regression |
| G6PD | 355/360 nm | 460 nm | Optimal | Cubic spine |
| PKU | 390 nm | 485 nm | Manual 30 | Linear regression |
| GALACTOSE | 355 nm | 460 nm | Manual 30 | Linear regression |
5. Microtiter plate spectrophotometer: For MSUD plate Reading we use BioTek’s monochromator-based Eon Microplate Spectrophotometer combines high performance and high value in microplate-based absorbance readings. Eon’s monochromator-based optics allow flexible, filter-free 200-999 nm wavelength selection in 1 nm increments, and spectral scanning for 6- to 384-well microplates.
Programming for test reading of MSUD :
Start the system computer
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Switch on the BioTek’s Eon Microplate Spectrophotometer
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Open Gen 5 software program (A dialogs box open)
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Select experiments than click New (A dialogs box open)
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Select standard Protocol than click ok (A dialogs box open, left side)
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Click on the Procedure (A dialogs box open)
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Click the read (A dialogs box open)
Observe 1. Detection method: Absorbance,
2. Read type: Endpoint/Kinetic,
3. Optical type: Monochromators are selected
Optical type: Monochromators are selected
Select wavelength 1 and fill 550 and then click ok and again click ok (previous page)
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Click Plate layout (A dialogs box open)
Select Standard curves and Sample and then click next (A dialogs box open)
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Fill Concentration of STD/CAL and Unit and click next
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Click Finish (A dialogs box open)
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on STD and select STD and sample column
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Click ok
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Click on Data Reduction (A dialogs box open)
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Click on the Standard curve (A dialogs box open)
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Select X-Axis Concentration and Y-axis 550 and then click ok again click ok (previous page)
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Save program
Measurement test reading:
Click plate option (A dialogs box open)
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Click Read plate 1 option (A dialogs box open)
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Click ok and measurement start
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After measurement Result show as a dialogs box
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Click curve option and see std. curve
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Note the result and save the result.
Lab diagnosis of NBS:
We use following procedure in lab for diagnosis of NBS
- Hospital staff submits NBS samples to the lab staff of the department of biochemistry (D-BLOCK) at the sample accession room, which is labeled by a lab technician and stored in the refrigerator at 40C temperature.
- The laboratory staff of NBS collect to the NBS sample from D-Block and fills all the information (provide on the NBS card) in the NBS master register and then test performed all the parameters as per the number of NBS samples according our SOPs.
- After running the NBS test by the lab, the report are dispatched within 7 to 15 days.
- Must to inform test result (if positive) telephonic on urgent basis to parents for proper treatment without any dealey.
Lab Parameter: The parameters to be applied by the lab are as follows
- G6PD (glucose-6-phosphate dehydrogenase)
- PKU (Phenylketonuria)
- Galactose
- Biotinidase
- 17-OHP (17-OHP-Progesterone)
- IRT ( immunoreactive Trypsinogen )
- hTSH (human thyrotropin/human thyroid-stimulating hormone)
- MSUD (Maple Syrup Urine Disease)
Required instruments and materials:
The equipment and materials below are used in the NBS test.
Non consumable material:
1. Disk puncher with a diameter of 3 mm to cut off paper disk of dried blood spot.
2. Microplate incubator/shaker ( Thermomixer)
3. Microplate washer
4. Microplate flurotometer
5. Microtiter plate spectrophotometer
6. Adjustable micropipettes with disposable tips (10-100/200ul, 200-1000ul, and up to 10ml)
7. Precision pipettes (multi-channel pipettes 50- 300ul)
8. Graduate measuring cylinder for reagent dilution
9. Vials to store the diluted reagents
10. Discarding jar
Consumable Materials :
1. Distilled or deionized water, preferably sterile
2. Absorbent paper (Tissue paper and bloating absorbed paper)
3. Absolute alcohol (ethanol)
4. Round bottom microtiter plate
5. Flat-bottomed microtiter plate
6. Sterile container to prepare reagents
7. Sodium hypochlorite solution
8. Disposable powder free gloves
9. Kits and reagents (available in kits)
10. Sanitizer/ 70% alcohol
Test and method
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G6PD (glucose-6-phosphate dehydrogenase) : This test is done for the determination of the deficiency of glucose-6-phosphate dehydrogenase .
Principle
G-6-P+ + NADP+ ->G-6-PD -> 6-phosphogluconolatone + NADPH + H
NADP+ is reduced by g-6-pd in the presence of G-6-P and the rate of formation of NADPH is proportional to the G-6-PD activity.
Method: (Please read kit insert (kit manual), provided by manufacturer in kit.)
Preliminary Preparations
Bring the reagents to room temperature (20 to 25°C) before starting the assay, except the stopping solution which should be kept at +4°C.
Prestep : Solubilize the substrate vial with 16ml of substrate buffer. This is sufficient for 96 wells.(Stability of opened and diluted reagents at +20C to +80C for one month).
Punch out single 3mm disks from patient samples as well as calibrator and QC into the microplate wells.
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Add 150ul of reconstituted substrate into each well (Make sure that the disks are completely soaked in the liquid before incubation).
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Cover the plate and incubate 30 min. at RT (+20 to +25°C) on Microplate incubator/shaker in dark with shaking speed of 1150 rpm.
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Add 150 µl of cold (+40C stop solution (make sure that the disks are completely soaked in the liquid otherwise remove the floating disks before measurement)
Measure immediately the fluorescence (ex. 355 nm, em 460 nm, and standard curve cubic spine).
Reference Value : >3 IU/gHb blood
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PKU (Phenylalanine): This test is done for the determination of the deficiency of classical phenylketonuria(PKU) or phenylalanine hydroxylase(PAH),oligophrenia phenylpyruvica,Follings disease.
Principle:
Phenylalanine eluted from dried blood spots forms a Fluorescent compound with Ninehydrin.The Flurometric response is greatly enhanced by the prescence of a dipeptide L-leucyl-L-alanine. The pH during the reaction is strictly controlled by succinate buffer at 5.8+/- 0.1 in order to optimize fluorescence and maximize specificity copper reagent is added to stabilize the fluorescent complex and enhance the signal.
Method: (Please read kit insert (kit manual), provided by manufacturer in the kit.)
On a separate plate (provide by lab), Punch out single 3mm disks from patient samples as well as calibrator and QC into the microplate wells.
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Add 80µl of 80% ethyl alcohol to each well, cover the plate and elute phenylalanine for 30 min at room temperature (+20 to +25°C) on a Microplate incubator/shaker with shaking speed 900rpm.
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Transfer 50 µl eluates with a multichannel pipette into a reaction plate (provide with kit).
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Add 50 µl of reaction mixture, seal tightly with a new incubation cover, shake for 1 min at room temperature and incubate 60min at 60°C without shaking.
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Add 200 µl of cold copper reagent to each well.
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Incubate the plate for 15 min at room temperature.
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Read the fluorescence within 15-30 min after addition of the copper reagent (using fluorometer with filters excitation 390nm/emission 485 nm and standard curve linear curve).
Reference Value : <2.5 mg/dl blood
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Galactose: This test is done for the determination of the total galactose for diagnosed of rare genetic disorder called galactosemia.
Principle:
Galactose-1-phosphate + H2O –>AFOS-> galactose + phosphate
Galactose + NAD ->GDH-> galactono-lactone + NADH + H+
1). Alkaline phosphate cleaves galactose from galactose-1-phosphate.
2). In this step glactose serves as a substrate for galactose dehydrogenase which reduces nicotinamidedinucleotide to form a flurogenic product.
The determination is based on two reactions
Fluorescence intensity of the reaction is proportional to the amount of galactose. Cold copper reagent stabilized to the fluorescent product.
Method: Please read kit insert (kit manual), provided by manufacturer in the kit.
Preliminary Preparations
Bring the reagents to room temperature (20 to 25°C) before starting the assay, except the stopping solution which should be kept at +4°C.
On a separate plate (provide by lab), Punch out single 3mm disks from patient samples as well as calibrator and QC into the microplate wells.
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Add 20ul of 80% ethanol into each well and incubate 60 min at +37°C, do not cover the plate (It should be completely dried after incubation).
(Dissolve AFOS in 24 ml & NAD in 24ml of reaction buffer in separate vials & stored at +20C to +80C. This is for 2 plates = 192 wells. (100ul of GDH is ready to use for 192 wells)
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Prepare the reaction mixture just before use. According to the number of wells (calculate amount of AFOS, NAD & GDH to be added).
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Add 200ul of reaction mixture into each well. Make sure that the disks are completely soaked in the liquid before incubation.
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Cover the plate and incubate 30min at 37°C with shaking speed of 900 rpm.
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Add 100ul of cold (+4°C) stopping solution. Transfer 200ul of the final product into a white plate (separate plate provide with kit).
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Measure the fluorescence (ex.355 nm, em. 460 nm) between 15-60 minutes after the transfer and standard curve as linear curve.
Reference Value : <17 mg/dl blood
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Biotinidase: This test is done for the Fluorometric enzyme immunoassay for the determination of the biotinidase for diagnosed of biotinidase deficiency.
Principle:
The Biotinidase enzyme (fount in the blood sample) utilize biotin-6-aminoquinoline(BAQ) as a substrate. During the enzymetic reaction, substrate is cleaved to fluorescent product 6-Aminoquinoline (6-AQ) and biotin is produced and the reaction is terminated by adding ethanol to the wells and the fluorescent measured with microplate fluorometer.
Method: Please read kit insert (kit manual), provided by manufacturer in the kit.
bring the reagents to room temperature (20 to 25°C) before starting the assay
Punch out single 3mm disks from patient samples as well as calibrator and QC into the microplate wells.
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Add 35ul of reconstituted substrate solution to each well.
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Tap the plate or shake slowly (600rpm) for 1min. Make sure that the disks are properly soaked and cover the plate.
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Incubate at +50°C for 4 hrs without shaking.
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Remove the cover and add 225ul of absolute ethenol (not included to the kit) to each well immediately.
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Shake the plate 5min at 600rpm (Do not cover).
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Incubate the plate for 30 min at room temperature (No need to remove the disks)
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Measure immediately fluorescence at ex. 355 em, 460 nm and find linear standard curve
Reference Value : >50 U blood
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17-OHP (17-OHP-Progesterone): This test is done for the Fluorometric enzyme immunoassay for the determination of the 17-hydroxyprogesterone for diagnosed Congenital Adrenal Hyperplasia (CAH).
Principle:
17-OHP-progesterone (from the patient sample) competes with the horseradish peroxidase. 17-OHP-progesterone conjugate, which binding to specific antibodies attached to the polystyrene surface of the microplate wells.
Residual patient sample and unbound conjugate are removed by washing. . The enzymatic reaction is performed with fluorogenic substrate solution ( 3-(p-Hydroxyphenyl)propionic acid , H2O2) is performed , reaction stoped by adding glycine buffer. Measure Fluorescence at 320 em. 405 nm no later than 60 min. standard curve –four parameter logistic curve. The fluorescence intensity is conversely proportional to the concentration of 17-hydroxy-Progestrone.
Method : Please read kit insert (kit manual), provided by manufacturer in the kit.
Preliminary Preparations
Bring the reagents to room temperature (20 to 25°C) at least 30 min. before starting the assay
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Punch out single 3mm disks from patient samples as well as calibrator and QC into the microplate wells
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(Dilute HRP conjugate via-2 with conjugate diluent vial-3 (as ratio 1:100) just before use)
Add 200ul of 17-OHP-HRP conjugate (in dark). Make sure that the disks are properly soaked.
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Cover the plate and incubate 3hrs 30 min at 25°C with shaking 900 rpm.
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Remove the disks and liquid and wash 4 x 300ul. (wash program 3)
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(Dilute HPPA Diluent vial-4b with HPPA substrate vial-4a (as ratio 1:50) just before use)
Add 200ul of substrate solution, cover the plate and incubate 60min. at 4°C in dark without shaking.
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(Dilute stop solution (1:1) with distilled water.)
Add 100ul of stop solution, measure Fluorescence at 320 em. 405 nm no later than 60 min.
standard curve –four parameter logistic curve.
Reference Value : <35 nmol/L blood
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IRT ( immunoreactive Trypsinogen ) : This test is Flurometric enzyme immunoassay for determination of human immunoreactive trypsinogen for primary test for screening cystic fibrosis (CF) in the new born babies.
Principle:
IRT FEIA is a solid phase fluorometric enzyme immunoassay in which IRT is eluated from dried blood discs. It simultaneously forms a sandwich between the solid phase, which is coated with an antibody that recognizes IRT-1 and another antibody, which is labeled with HRP.
Residual patient sample and unbound conjugate are removed by washing. . The enzymatic reaction is performed with fluorogenic substrate solution ( 3-(p-Hydroxyphenyl)propionic acid , H2O2) is performed , reaction stoped by adding glycine buffer and measerd with microplate fluorometer (Ex.320nm,Em.405nm) and found standerd curve as cubic spine curve.
Method: Please read kit insert (kit manual), provided by manufacturer in the kit.
Preliminary Preparations
Bring the reagents to room temperature (20 to 25°C) at least 30 min. before starting the assay.
Punch out single 3mm disks from patient samples as well as calibrator and QC into the microplate wells
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(Dilute HRP conjugate via-2 with conjugate diluent vial-3 (as ratio 1:100) just before use)
Add 150ul of anti-IRT1-HRP conjugate solution. Make sure that the disks are properly soaked.
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Cover the plate and incubate. 3hrs at room temperature in dark with shaking speed of 900 rpm.
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Remove the disks and liquid, then wash 5 x 300ul. (wash program 9)
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(Dilute HPPA Diluent vial-4b with HPPA substrate vial-4a (as ratio 1:50) just before use)
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Add 150µl of substrate solution, Cover the plate and incubate 60 min at room temperature in the dark shaking 650 rpm.
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(Dilute stop solution (1:1) with distilled water. Add 100ul of stop solution.)
Add 100ul of stop solution. Measure immediately fluorescence at ex 320 ,em.405 nm. Standard curve Cubic spline curve.
Reference Value : <70 µg/L blood
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hTSH (human thyrotropin/human thyroid-stimulating hormone) : This test is Flurometric enzyme immunoassay for determination of human thyrotropin for primary test for screening Cogenital hypothyroidism (CH) in the new born babies.
Principle:
IRT FEIA is a solid phase fluorometric enzyme immunoassay in which hTSHT is eluated from dried blood discs. It simultaneously forms a sandwich between the solid phase, which is coated with an antibody that recognizes α-subunit of hTSH and another antibody, which is labeled with HRP.
Residual patient sample and unbound conjugate are removed by washing. . The enzymatic reaction is performed with fluorogenic substrate solution ( 3-(p-Hydroxyphenyl)propionic acid , H2O2) is performed , reaction stoped by adding glycine buffer and measerd with microplate fluorometer (Ex.320nm,Em.405nm) and found standerd curve as linear curve.
Method: Please read kit insert (kit manual), provided by manufacturer in the kit.
Preliminary Preparations
Bring the reagents to room temperature (20 to 25°C) at least 30 min. before starting the assay.
Punch out single 3mm disks from patient samples as well as calibrator and QC into the microplate wells
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(Dilute HRP conjugate via-2 with conjugate diluents vial-3 (as ratio 1:100) just before use)
Add 200ul of anti-TSH-HRP conjugate solution. Make sure that the disks are properly soaked.
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Cover the plate and incubate. 3hrs at room temperature in dark with shaking speed of 650 rpm.
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Remove the disks and liquid, then wash 4 x 300ul. (wash program 01)
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(Dilute HPPA Diluent vial-4b with HPPA substrate vial-4a (as ratio 1:50) just before use)
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Add 200µl of substrate solution, Cover the plate and incubate 60 min at room temperature in the dark shaking 650 rpm.
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(Dilute stop solution (1:1) with distilled water. Add 100ul of stop solution.)
Add 100ul of stop solution. Measure immediately fluorescence at ex 320 ,em.405 nm. Standard curve is linear curve.
Reference Value : <10mIU/L blood
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MSUD (Maple Syrup Urine Disease): This test is done for the determination of Maple Syrup Urine Disease in new born.
Principle
Leucin + NAD+ (Catalysed by Leucin dehydrogenase ) -> α – ketoisocaprote + NAD+
NADH+ + NBT (ox yellow) —————> NAD+ + formazon(violate)
(catalysed by a solution of an intermediate electron receptor, measured at 550 nm)
The leucine and BCAAs (leucine, isoleucin and valine – are often called the branched-chain amino acid) are eluted with trichloroacetic acid (Elution buffer) from dried blood spot. After elution, the eluted sample is combined with the enzyme reagent Leucine dehydrogenase. This enzyme reagent catalyzed the NADA-dependent oxidative determination of leucine and L-BCAAs to α – ketoisocaprote acid. The NADH produced react with a color reagent in which a tetrazolium salt gets reduced producing a distinct color. This color measured by colorimetcally with a photometer at 550 nm. Color is directly proportional to Leucin and BCAAs.
Method: Please read kit insert (kit manual), provided by manufacturer in the kit.
Preliminary Preparations
Bring the reagents to room temperature (20 to 25°C) at least 30 min. before starting the assay.
On a separate U bottom plate, punch out double (two disk in each well) 3mm disks from patient samples as well as calibrator and QC.
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Add 100 µl elution buffer in each well and incubate for 30 minutes at RT with shaking 700 RPM
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Transfer 40 µl elute in another flat bottom microplate
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(Prepare enzyme-coenzyme solution, add 400 µl enzyme, 400 µl co-enzyme and 200 µl diluting buffer for 10 test)
Add 100µl of this enzyme-coenzyme reagent in each well and incubate at RT for 30 minutes without shaking
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(Prepare color reagent mixture for 80 µl color booster and 800 µl color reagent for 10 test)
Add 80µl of this color reagent mixture in each well and incubate at RT in dark mode for 10 minutes without shaking.
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Measure immediately at RT and kipping away from the light at 550 nm optimal endpoint single measurement and find Standard curve is linear curve.
Reference Value: < 4.12mg/dl blood
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Precautions
- Wear disposable gloves while handling sample ant performed test
- Do not mix or interchange reagents of different lots
- Do not use reagents beyond their expiry date
- Use thoroughly clean glassware
- Once test has been started, all subsequent step should be performed without interruption , So run only one test in a time
- Do not pipette reagent by mouth
- Do not smoke, eat, drink during assay
Cause error
- Flat Calibration curve and low fluorescence
- Reagents are deteriorated due to contamination or improper storage
- Reagents are not warmed up to room temperature
- Incubation time of reaction is too short
- Fluorometer wavelength setting not correct.
- Poor precision
- Liquid handling devices are not properly calibrated
- Improper wasing due to blocking of washer tips by the filter paper dust
- The plate is allowed to stay too long after washing (drying of the plate)
- NBS samples
- Uneven distribution of blood in the samples.
Discarding material after use
- NBS Card (Dry blood spot): Discard all NBS sample must be autoclave for at least 1 hour at 1210C, then after that it should be discarded in the yellow color biohazard bag.
- Sodium hypochlorite must be added to liquid wastes to reach a final concentration of 3%, Let the hypochlorite act for at least 30 minutes.
- Wear disposable gloves while handling sample ant performed test
- Do not mix or interchange reagents of different lots
- Do not use reagents beyond their expiry date
- Use thoroughly clean glassware
- Once test has been started, all subsequent step should be performed without interruption , So run only one test in a time
- Do not pipette reagent by mouth
- Do not smoke, eat, drink during assay
Cause error
- Flat Calibration curve and low fluorescence
- Reagents are deteriorated due to contamination or improper storage
- Reagents are not warmed up to room temperature
- Incubation time of reaction is too short
- Fluorometer wavelength setting not correct.
- Poor precision
- Liquid handling devices are not properly calibrated
- Improper wasing due to blocking of washer tips by the filter paper dust
- The plate is allowed to stay too long after washing (drying of the plate)
- NBS samples
- Uneven distribution of blood in the samples.
Discarding material after use
- NBS Card (Dry blood spot): Discard all NBS sample must be autoclave for at least 1 hour at 1210C, then after that it should be discarded in the yellow color biohazard bag.
- Sodium hypochlorite must be added to liquid wastes to reach a final concentration of 3%, Let the hypochlorite act for at least 30 minutes.
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