New Born Screening || NBS

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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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.

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

1. Flat Calibration curve and low fluorescence
2. Reagents are deteriorated due to contamination or improper storage
3. Reagents are not warmed up to room temperature
4. Incubation time of reaction is too short
5. Fluorometer wavelength setting not correct.
6. Poor precision
7. Liquid handling devices are not properly calibrated
8. Improper wasing due to blocking of washer tips by the filter paper dust
9. The plate is allowed to stay too long after washing (drying of the plate)
10. NBS samples
11. 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

1. Flat Calibration curve and low fluorescence
2. Reagents are deteriorated due to contamination or improper storage
3. Reagents are not warmed up to room temperature
4. Incubation time of reaction is too short
5. Fluorometer wavelength setting not correct.
6. Poor precision
7. Liquid handling devices are not properly calibrated
8. Improper wasing due to blocking of washer tips by the filter paper dust
9. The plate is allowed to stay too long after washing (drying of the plate)
10. NBS samples
11. 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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