Standard Operating Procedures

SOP 1-FILLING THE NBS CARD

An Institutional Ethics Committee approval has been obtained by all participating institutions locally. The selected centers will receive and process filter paper dried blood spots submitted by hospitals, birth centers, physicians, clinics and local health officials. The processing includes entering and maintaining demographic data and screening data from the submitted filter paper specimens.

The NBS Card has three leaflets :

• The first leaflet is white in color. The left side of this leaflet has a filter paper (mounted from the back side of the white leaflet) with five circles printed on it. This portion of the NBS Card is meant for collection of blood from the enrolled neonates. The detailed information on how to collect blood on this filter paper is provided in the section of blood collection procedure.


• The right side of this leaflet contains 21 items, for which you need to furnish and enter information about the enrolled neonate.


• The second (pink in color) and third (yellow in color) leaflets are made up of self inking paper. Whatever information you write on the white leaflet, the same will be copied on the pink and yellow leaflets on its own. There is no need to place a carbon paper for this purpose.

Important precautions

• Make sure that you get familiarized with this card well before you get started. Read and understand carefully all the 21 items. Seek help from Principal Investigator, if you have any doubt.


• Place NBS Card on some hard surface (such as a hard bound book) before you enter information in the Card. Use a ball pen ONLY (not an ink or gel pen) to enter the information on the card, else the information written on the white leaflet will not be copied on the pink and yellow leaflets.


• There are small boxes for writing. Make sure that you enter one letter or one digit in a single box. If the number of boxes falls short, bring this to the attention of PI for solution.


• Make sure that you spend sufficient time in filling up of the form. Never fill a form in haste.


• Make sure that your writing is legible. Use BLOCK letters

How to fill the card

• Centre Code: enter the centre code of your centre. Inquire from the Principal Investigator (PI) of your centre regarding the code of your centre. The Center codes for Chennai, Delhi, Hyderabad, Kolkata and Mumbai are 1,2,3,4 and 5 respectively.


• Study ID No: this is the identification no of the enrolled newborn. Ask the PI of your centre to let you know the series of number you need to enter.


• Baby registration No: this number is the HOSPITAL registration no. You can find it on the hospital case file of the baby or mother. Make sure that you do not enter the mother's registration number.


• Name of Mother & Father: this item has four lines to be filled in:



a. First two lines are for the mother's first name and her surname

b. The third and fourth lines are for father's first name and his surname



• Complete postal address: enter complete postal address. Always confirm the address from the parents/other relative, if you copy it from the hospital case file. Make sure that you record the address which belong to the family (sometimes family gives some one else's address at time of hospital admission). Make sure that you record complete address and confirm it from family that a letter would reach them if we write this address on the envelope. Never forget to record PIN.


• This item is about recording telephone nos:



a. Telephone no (with STD code): take the landline number and STD code. Enter STD code and then landline separated by a hyphen. For example:

0

1

2

3

4

5

6

7




If the family does not have a landline-enter landline number some relative. If no landline number is available: keep it blank




b. Enter mobile number of ten digits.



• Email ID: record email ID of the mother or father or some close relative, if available. Make sure that you take care of upper/lower case and spaces, hyphen or underscore. Once entered, you can show it to the family for making sure that it is written correctly.


• Date & Time of birth: record date of birth in ddmmyyyy format at designated places. Record time of birth in two digits with minutes also recorded in two digits. Circle AM or PM, as the case may be (do not tick/cross it).


• Sex: enter 1 if male, 2 if female and 3 if the sex is ambiguous. Confirm with PI before you enter sex as ambiguous (3).



a. Are parents of baby blood related: inquire from the family if the mother and father have any blood relation (consanguineous marriage). Sometimes this question may not be understood well by the family as what exactly you are trying to ask. In that case you can give appropriate example such as marriage between cousins. Enter 1 if yes and 2 if no.

b.If yes degree of consanguinity: you need to fill this item if answer to previous question was 1 (yes). If answer to previous item was 2 (no): enter 9 (which means not applicable). Degree of consanguinity - Write 1 for second degree consanguinity ( Uncle niece marriage), write 2 for first cousin marriage and 3 for more distant consanguinity.

• Birth weight: enter birth weight in gram in four digits such as



3

4

1

2



• Gestation: write gestation in completed weeks. For example if the gestation of the pregnancy was 39 weeks and 2 days (sometimes written as 39+2 or 39+2,), you need to enter only 39. Sometimes information about the gestation may not be easily available or recorded differently at different places; in that case you must consult the PI, regarding correct gestation.


• Age at sampling: record age at blood sampling (for taking spots on the filter paper). You have to calculate it by the date & time of birth and date & time of blood sampling. You need record completed hours only and ignore the minutes.


• Age at first feed: ask the mother when she offered first feed to the baby- breastfeeding or any other milk feeding. By date & time of birth and date & time of first feed you can calculate age at first feed. Record in completed hours, ignore minutes.


• Find out if this pregnancy was singleton or twin or higher order. Sometime only one of the twin may be with mother (other twin may have died/ or in nursery), therefore do not assume singleton if the mother has one baby. Record 1 if singleton; 2 if twin and 3 if triplets or quadruplets or even higher.


• Mode of delivery: record 1 if normal delivery, 2 if LSCS and 3 if baby has been delivered by forceps, vacuum or breech extraction.


• 5 minutes APGAR: see the resuscitation notes on the hospital case file of the baby and find out 5 minute score. Keep in mind that APGAR scores are also recorded at 1 minute and 10 minutes and sometimes at 15 or 20 minutes as well. You just need to find 5 minutes score. If any difficult in ascertaining, consult PI.



a. Gross malformation: if the baby has some gross malformation such as tracheo-esophageal fistula, neural tube defect, then record 1. Record 2 if there is no gross malformation.




b. if yes, specify the malformation: consult the PI, to know the exact nature of malformation. Record this information on the provided space.



• Drug to baby: it asks you about three types of drugs namely antibiotics, dopamine, and steroids.Ask the treating physician or nurse, if the baby is getting any of these drugs. If baby is getting any of these three drugs, put a tick mark against the relevant box. If the baby is getting any antibiotics, ask the treating physician, the name of antibiotics and record this information in the relevant space.


• Antenatal steroids to mother within 7 days: ask the treating physician if the mother has received antenatal steroids during past 7 days. If yes, record 1 else record 2.


• Name of the collecting person: record in capital latter the name(s), who filled the form and collected the blood sample.


• Supervised by: record the name in capital letter of the person who has supervised the procedure.

SOP 2-Blood Sample Collection

Dried blood spots will be collected by heel-prick on the blood sample collection cards. At least three (preferably five) blood spots will be collected from each neonate. Venous sample should only be collected if sampling is being done for some other test so that two pricks can be avoided.

Device

Surgicare lancets for newborn screening (FDA approved ) have been supplied by Perkin Elmer and will be used for this study. Procedure (Figure 3)

Following items are required :

• A sterile, disposable lancet or automated lancet device (maximum tip length 2.4 mm)


• 70% alcohol swab


• 3 sterile cotton wool balls or gauze swabs


• Disposable gloves


• Blood collection card


• Baby's medical record
  

Following universal precautions must be observed when collecting samples :



• Put on gloves


• Ask the mother/care taker to cuddle the baby on her knee to assist you and comfort the baby with legs hanging down or keep the baby against her shoulder.


• Place a paper towel on the lap of the person holding the baby to protect any blood staining


• Ensure that the heel is warm. Hold the heel in warm hands or rub gently for 3 minutes or dip heel in warm (not hot; dip your own finger to check the temperature of water before you emerse baby's foot into it) water or cover it with a small towel soaked in warm water for few minutes. Warming the heel and keeping the feet below the level of heart will enhance the blood flow and will help to collect a good specimen.


• Wipe the heel with 70% isopropyl alcohol/spirit. Do not leave any alcohol on the skin as this may dilute the sample and adversely affect the test results. Never use Vaseline or any other material on the collection site.


• Ensure that the heel is warm. Hold the heel in warm hands or rub gently for 3 minutes or dip heel in warm (not hot; dip your own finger to check the temperature of water before you emerse baby's foot into it) water or cover it with a small towel soaked in warm water for few minutes. Warming the heel and keeping the feet below the level of heart will enhance the blood flow and will help to collect a good specimen.


• Encircle the heel with fingers and thumb and squeeze gently until skin looks like taut (tight) and suffused with blood.


• Puncture the heel with a firm deliberate stab with lancet on the medial or lateral aspect (Figure 1).


• The depth of the puncture should not exceed 2.4mm which is ensured by using a lancet which will not puncture beyond the certain depth.


• If a second puncture is necessary, make this a few millimeters away from the first or in other foot.


• After the puncture, wait for five seconds as vasoconstriction occurs initially. Then gently apply intermittent pressure with your thumb to the area surrounding the puncture site. Avoid excessive pressure as this may graze or bruise the site.


• Always wipe away the first drop of blood. The first drop of blood is often diluted with tissue fluid. This could result in a false negative test result. ICMR – Task Force on Inborn Metabolic Disorders 33.


• Just touch the circle marked on the card gently to the hanging drop so that blood soaks through the other side. Do not ever try to put drop on both sides of a circle. It is very important that blood should soak through the other side.


• Do not press the card on to the skin.


• Do not apply multiple drops to fill each circle or one drop on another blood drop.


• Do not touch blood spot with finger


• Press clean cotton wool firmly on to wound until bleeding stops. It is not advisable to place adhesive bandages over skin puncture sites in newborns.


• Dry the sample at room temperature in a horizontal position. Specially designed stands can be used for drying (Fig 2). Do not use lamp, dryer or sunlight to dry.


• Dry for 4 hrs before sending to laboratory for testing.

Skin punctures must never be performed on the fingers of newborns.
Never prick bruised heel.
Dried blood spot specimen drying and storage It is important to dry blood spot specimens before storage or transport. (Moisture may harm the specimen by inducing bacterial growth or altering the elution time of the specimen.)
The filter paper containing the dried blood specimen should be protected by a sturdy paper such as glassine paper. The specimens should be protected from moisture by packing them in zip-closure bags.
Dried blood spot specimens protected in this manner can be stored at -20°C for many weeks or years.
Transportation of dried blood spot specimens.
Dried blood spot specimens that have been packed as described above have to be transported through the mail / courier every day if possible or twice a week.If samples are not being transported the same day, the cards should be kept in the collection centers in refrigerators and protected from moisture.
COMMON SAMPLE COLLECTION PROBLEMS
Ensure good quality of samples. Poor quality sample will be labeled as invalid sample for testing and you will get request to send second sample. Poor quality samples cause unnecessary trauma to the newborn (and parents) and could potentially delay the detection and treatment of an affected infant.

Common sampling problems include :

• Insufficient blood (not filling all circles); not enough sample to perform tests or repeat tests.


• Milking or squeezing the puncture site can cause hemolysis and mixing of tissue fluids with blood.


• Layering or applying successive drops of blood (double collection) in the same printed circle causes caking and /or non-uniform concentrations of blood.


• If the blood flow diminishes, such that circles are not completely filled, then repeat the sampling technique in a new circle.


• Do not put many blood spots in same printed circle.


• Ensure that blood soaks through. Do not apply blood on both sides.


• Contamination of sample during collection, drying, or mailing with urine samples will render the results unreliable. Such samples will be labeled invalid and will not be tested.


• Inadequate or inappropriate drying.

• Humidity and moisture adversely affect the quality of sample and analyte recovery


• Excess heat or sunlight bakes the sample

Envelope for carrying samples

Zipfoil pouch for storage		Drying stand

Using dried blood spot specimens for storage and re-use for research purpose :

Out of the five spots two will be used for TSH and CAH , remaining will be stored for future research for upto 20 years at AIIMS New Delhi. Samples are to be transported to AIIMS after TSH and 17 OHP testing. All the samples sent for storage at AIIMS need to be bar-coded and anonymised for the purpose of maintaining the confidentially. The samples may be used for research only after appropriate approvals from the Institutional Ethics committees as well as ICMR.

SOP 3. Analytical Protocol

All Regional Centers will follow the same laboratory methods for the initial screening of the newborn blood samples. Perkin Elmer has installed the required equipment of the study at all Regional study centres. The equipment is "Victor 2D" which uses the Time resolved Flurometry method for assay.

A single protocol shall be followed in all the labs in order to maintain uniformity of results. Analytical protocols are provided with the neonatal screening kits. Perkin Elmer shall also provide the laboratory personnel with required hands on training regarding using the instruments when it installs the equipment at the respective Regional centers.

General Instructions

TIPS TO BE REMEMBERED PRIOR TO START OF ASSAY :

• A thorough understanding of the kit insert is must for successful use of kit Read it carefully


• DBS : Blood collection card should be checked thoroughly and segregated into good ones and those which are unacceptable due to improper collection. A good properly collected dried blood spot is a prerequisite for correct assay result


• Store the DBS cards in suitable paper bag at 2-8oC


• A sample list should be made prior to the day of the assay


• ELISA plate lay out format should be filled keeping in mind that the standards and controls are run in duplicates and samples as single determination


• Calculate the volume of the tracer and antisera to be diluted (always dilute an extra volume for 4-5 wells of these reagents)


• Check the levels of the 'Wash' and 'Rinse' solutions.Always write the expiry date of the wash solution on the container


• Check expiry date of the wash solution if the test is being done after a gap of few days


• All reagents and samples must be brought to room temperature (25-28 C) before use


• Always use sterile, deionized water and disposable plastic wares


• Eppendorf multipipette/Finnpipette is recommended for use


• Please paste the Xerox copies of assay flow charts and tracer and antisera dilution charts on the wall of the working table


• All recommended precaution must be observed for the handling of the blood sample and the kit reagents


• Disposal of waste should be in accordance with local regulations

TIPS TO BE REMEMBERED PRIOR TO START OF ASSAY :

Principle of Assay

Neo hTSH assay is a solid phase, two site fluoroimmunoassay based on sandwich ELISA technique wherein two monoclonal antibodies directed against two separate antigenic sites on the & chain of hTSH are made to react with the sample TSH. The fluorescence measured is directly proportional to the concentration of hTSH present in the samples

Reagent Preparation and Storage

All the reagents and samples must be brought to room temperature (20-25 °C) before use

hTSH Standards :

One sheet of filter paper containing 5 sets of standards. Protect from light and moisture. Store sealed at 2-8 °C

Anti-hTSH-EU Stock tracer (20 μg / ml) : Store at 2-8 °C

Working Tracer solution : Please consult the tracer dilution chart (kit pack insert). Make the required dilution carefully just before use

Wash concentrate (A 25 fold concentrate of Tris-HCL buffered Tween 20). Store at 2-8 °C
Working solution : 100ml of stock is diluted to 2400 ml with DI water
Neo hTSH Assay Buffer : Tris-HCL buffered (pH-7.8). Ready for use
Enhancement solution : Ready for use store at 2-8 °C. Do not expose to direct sunlight

Assay Steps :

• Set the requisite micro titration strips to a strip frame properly and firmly
• Punch out filter paper disks of standards, controls and samples in to the wells by using DBS puncher (remove one set of standards to be used and store it in a separate bag to avoid exposure to the rest of the std.strips to light and moisture)
• Add 200 μl of diluted tracer solution to each well.
• Use only disposable tips. Do not use repetitor for dispensing tracer
• Put the plate on DELFIA plate shaker for 10 min for shaking. It ensures complete extraction of blood components from the filter paper
• Incubate the plate at RT (20-25 °C)
• Alternatively the plate can be incubated overnight in a refrigerator (2-8 °C) without shaking followed by an additional incubation for 1 hour with slow shaking at RT the next day
• Wash the assay plate using DELFIA Washer

Keep an eye on the needles of the washer during dispensing of solution for any blockage. If any of the sample disk gets stuck to the wells remove it very carefully using suction nozzle. Check that the wells are properly dried

• Add 200 μl of enhancement solution
• Avoid touching the edge of the wells or its contents while dispensing enhancement solution
• Shake the plate slowly for 5 min.
• Dry the bottom of the plate before placing it on the platform of victor 2D
• Read the fluorescence within 1 hour of adding the enhancement solution as external factors may cause a decrease in fluorescence with time

Interpretation of Results :

Improper blood collection, contamination of blood spot, deterioration of sample by exposure to heat and humidity and presence of heterophillic antibodies in the samples may interfere with the assay.

The values given in the kit insert should be used as a guideline only.

Ideally, each laboratory should establish its own normal range.

Cut-off value for neonate = 20 µU/L

Flow Chart (TSH Assay)

Arrange requisite number of micro titration strips to a strip frame

Place the plate on the puncher platform and punch out the sample disk into the well

* Add 200 µl of diluted anti-hTSH tracer solution using multipepette

Fast shaking for 10 minutes prior to incubation

Cover the plate and incubate for 4 hours at RT (25-27 °C) with slow shaking or overnight in a refrigerator (2-8 °C) without shaking

* Disk removing and washing

Add 200 µl enhancement solution to each well

Slow shaking for 5 minutes

Read the fluorescence within 1 hour

(* Please refer to detailed assay procedure)

Details of 17–OHP Assay Procedure

Principle of the assay : Neo 17–OHP assay is a solid phase, fluoroimmunoassay based on the competition between Eur-labelled 17-OHP and sample 17-OHP for a limited number of binding sites on 17-OHP specific polyclonal antibodies (raised in rabbit). A second antibody against rabbit IgG coated to the solid phase helps in the separation of antibody bound and free antigen.
The fluorescence for each sample is inversely proportional to the quantity of 17-OHP in the sample.
Reagents Preparation & Storage : Reagents must be brought to room temperature (20-25 °C) before use
Standards & Controls : One sheet of filter paper containing 5 sets of standards. Protect from light and moisture. Store at 2-8 °C
Working Tracer Solution : Please consult the tracer dilution chart. Make the required dilution carefully. Use within one hour 17-OHP antiserum stock solution (Rabbit Polyclonal) : Store at 2-8 °C
17-OHP-EU tracer stock solution : Approximately 250 nmol/L. Store at 2-8 °C
Working antiserum solution : Consult the antiserum dilution chart. Prepare the requisite volume carefully and use within 1 hour
Wash concentrate : A 25-fold concentrate of Tris-HCL buffered tween-20. Store at 2-8 °C
Working solution : 100ml of stock is diluted to 2400 ml with DI water
17-OHP Assay buffer : Tris-HCL buffer (pH 7.8) ready for use. Store at 2-8 C
Enhancement solution : Ready for use. Contains Triton-X-100, acetic acid and chelators. Store at 2-8 °C. Avoid direct sunlight

Assay Steps :

• Set the requisite microtitration strips to a strip frame properly and firmly
• Punch out filter paper disks of standards, controls and samples in to the wells by using DBS puncher. Remove a set of standards to be used and store it in a separate bag to avoid exposure of the rest of the std strips
• Calibrators and controls are run as duplicate determinations
• Add 100 μl of diluted anti 17-OHP antiserum solution to each well
• Shake the plate slowly for 5 min for proper elution of the 17-OHP from disks
• Add 100 μl of 17-OHP-EU tracer solution to each well
• Shake the frame slowly for 1 min by using DELFIA plate shaker
• Incubate the covered plate at RT (25-32 °C) for 3 hours without shaking or overnight in a refrigerator (2-8 °C)
• Wash the assay plate using DELFIA washer
• Keep an eye on the needles of the washer during dispensing of solutions for any blockage in the needles.If any of the sample disk gets stuck to the well remove it very carefully using a suction nozzle
• After washing check that the wells are properly dried
• Add 200 μl enhancement solution to each well and shake slowly for 5 minutes
• Measure the fluorescence within 1 hour

Interpretation of Results :

The concentration of 17-OHP in newborns depends on the age, weight, prematurity and twinning. Ideally, each laboratory should establish its own validated cutoff levels related to gestational age, infant age and birth weight.

The values given in the kit insert should be used as a guideline only.

Cut-off value for neonates =37.5 nmol/L
CDC cut-off value for neonates=50nmol/L

Flow chart (17 -OHP)

Arrange the requisite number of micro titration strips to a strip frame

Place the plate on the puncher platform and punch out the sample disk into the wells

* Add 100 µl of diluted anti 17-OHP anti serum solution with a multipepette

Shake for 5` slowly at RT

Add 100 µl 17-OHP tracer solutions

Shake for 1 minute slowly

Cover the plate and incubate overnight (18-22 h) in a refrigerator or at RT for 3 hrs without shaking

* Disk removing and washing on washer

Add 200 µl enhancement solution

Shake slowly for 5 minutes

Read the fluorescence within 1 hour

(* Please refer to detailed assay procedure)

FLOWCHART FOR OPERATING DELFIA DBS PUNCHER, WASHER AND FLUOROMETER

PUNCHING

Switch on the machine Select mode is displayed

Press S/A

Press Punch

Press Ok

Load plate is displayed

Keep the plate with A1 on the right side

Press OK

Punch the blood spot

When punching is complete check the plate

End punching is displayed

Press Yes

Press Ok

Press Exit

WASHING Switch on the washer

Go to main screen

Select run

Keep the plate

Press In

Press Yes

Select the program (17OHP/TSH

Press Yes

Select the strips to be washed (A to H)

Press Yes

Press Yes

Starts rinsing followed by Washing

Starts rinsing followed by Washing

HOW TO READ ASSAY PLATE Switch on the P.C. Press Enter Select the wells Press Enter Press F6-F7 Measure the plate Enter the pass-word Blank Screen appears Click Next Double click on Wallace soft-ware Wait for initialization Press Enter (make worklist) Next type the Information Click on Multical (DOS functioning) Press Esc. Click Next Main screen appears Press F1 (Quit + Save) Keep the plate Press Esc Press Esc Click Finish Press (F4) for protocol Main Screen appears Click Live display Select the program (TSH/17OHP) Counter mode appears Results saved & printed automatically Press Enter Activate Wallac1420 manager   Press Esc. Select protocol (17OHP/TSH)   Press F1 Click Start   Press F1 (Quit + Save) Click Next       Press Esc         Main Screen appears         Press F3 for work list         Press F2 Create         Select the program (yellow colour)         Press Enter

SOP 4. Quality Control

QUALITY ASSURANCE PROGRAM

Quality assurance (QA) includes all systems, procedures and activities that are performed to fulfill requirements of obtaining desired analytical quality complying with current national & international standards. Quality control includes establishing specifications for the analytical process, monitoring the analytical process to determine conformance to these specifications and taking any necessary corrective actions to bring the analytical process into conformance.

Good Laboratory Practices

As part of good laboratory practices following things are done :

• Procedures selected/performed for usage are unmodified FDA/CE approved manufacturer's procedure.


• IQ, OQ and PQ are performed for the instrument/s used in the conduct of the examinations. All relevant records are maintained.


• Whenever a standard method is modified, it is validated before being used. Each validation exercise is documented for records and for future reference.


• The environmental conditions in the laboratory are always maintained to ensure optimal performance from man and machines.


• All testing is performed as per documented standard operating procedures (SOPs). A review of all procedures by technologists and Heads is undertaken initially, annually and with each revision. These reviews are documented.


• All calibrators/standards used are traceable to national/international reference material. Documentation of traceability as provided by supplier or manufacturer is maintained in the department.


• All equipment to be uniquely labeled; label to mention last date of calibration and next due date


• All the instruments are maintained and calibrated as per manufacturer guidelines; Annual calibration & maintenance plan to be made and complied. Records maintained for all maintenance, calibration and service calls.


• Maintain inventory records. Record the conditions in which reagents are received and utilized. Perform verification of vendor cold chain/vendor evaluation at least once in a year.


• Define the job profile of each technician. Competency of the technical staff is critical to achieving quality in examination procedures. Technologists are trained on procedures performed by them and are assessed for their competence biannually and records maintained.


• Internal Quality Control data is reviewed daily by the technician performing the test, weekly/ monthly by supervisor/HOD. In case of QC failure the whole run is rejected.


• Uncertainty of measurement (parameter, associated with the result of a measurement, that characterizes the dispersion of the values that could reasonably be attributed to the measurand) expressed as CV percentage is monitored on a periodic/monthly basis.


• All vials, kits and aliquots should be labeled with date of opening/date of reconstitution and expiry date and entries initialed.


• Equipment like centrifuges, pipettes etc should be calibrated at least twice a year; thermometers once a year. ICMR – Task Force on Inborn Metabolic Disorders 47


• Eating, drinking and smoking are strictly prohibited in the testing area. Intake of alcohol, drugs etc is strictly prohibited. The testing area is a restricted entry zone and only authorized persons should be allowed to enter.


• Universal safety precautions should be followed when handling specimens/biohazardous material.


• Samples to be retained at appropriate temperature to allow for retesting during the stability period. All specimens to be disposed of as per local Biological Waste disposal guidelines/relevant local regulations.


• Blood collection cards should be checked; segregated into those which are acceptable and those which are unacceptable due to improper collection, layering, serum rings, and clotted, contaminated and insufficient sample. Same DBS cards should be used by all the labs participating in the program.

PRE-EXAMINATION PROCEDURES

• Collect samples at specified age point; any deviation to be recorded


• Clean the area being pricked with an antiseptic wipe


• Use only the prescribed paper for sample collection


• Touch the circle on the card with only one application; prevents layering


• Dry well at room temp


• All specimens with layering, smearing, non-homogenous, insufficient sample to be discarded. Maintain record of all such rejected samples. If needed, initiate re-training of sample collection personnel


• Puncher to cut out filter paper discs of the dried blood spot (DBS) with a diameter of 3.2mm or 1/8th of an inch. Same puncher should be used by all the labs participating in the program.

EXAMINATION PROCEDURES

Analytical quality may be achieved by :

• Calibration traceability


• Instrument calibration & maintenance


• Preparing & following SOPs


• Training of personnel


• Quality control in testing


• Participation in EQA


• Appropriate environmental conditions

Installation of instrument

Perform installation qualification (IQ), Operator qualification & performance qualification (PQ) for each equipment. The IQ and OQ to be performed by the vendor. PQ to be performed by laboratory when the first two are complete. Relevant records to be maintained.

Method Verification (MV) :

Validation is defined as :

"Establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its pre-determined specifications and quality attributes"
FDA's Guideline on General Principles of Process Validation May 11, 1987.

• Accuracy – The closeness of agreement between a test result and the accepted true value. It indicates ability of a measuring instrument/test method to give responses close to true value. Bias can be calculated from EQC data/peer group comparison by the difference between the user's mean values for samples and the grand mean of participants. Bias can also be calculated by running certified material or from method comparison data.


• Precision – No test can be more accurate than it is precise. Analysis of precision by repetitive measurements of a single sample gives rise to a Gaussian distribution with a mean and SD. Imprecision (lack of precision) is the random dispersion of a set of replicate measurements and / or values expressed quantitatively by a statistic, such as standard deviation or coefficient of variation (CV) : CV% = SD/mean x 100 Imprecision is readily demonstrated by monthly QC statistics- CV%. Precision should be measured at different analyte concentrations


• Analytical measurement ranges (AMR) – Defines the ability of the method to obtain test results proportional to the concentration of analyte without any dilution, concentration or other pretreatment that is not part of the usual assay process. The linear range is by inference the range of analyte concentrations over which the method gives test results proportional to the concentration of the analyte. AMR verification need not be performed if analyte can not be diluted.

Verification of a new method :

Verification is a method to perform Error assessment :

• Assess how much error & what type of error might be present in a test result produced by a
  method in laboratory


• Evaluate the ability to detect or minimize these errors
  


• Ensure that these errors won't affect the interpretation of the test result & thereby compromise patient care

Approach to Verification :

• Develop an evaluation plan to verify the precision and accuracy. Specify the application, methodology and performance requirements for the test of interest in the verification protocol.


• dentify the experiments, specify the amount of data to be collected, and identify the decision concentrations or analytical ranges where the data should be collected. Schedule personnel time to carry out the validation studies. Protocol is dated & signed by the Lab Head.


• Prepare a set of worksheets that define the amount of data to be collected in the different experiments.


• All deviations, failures and causes for repeat testing or delays are recorded.


• Appropriate statistical calculations performed on the data collected in different experiments. If method performance is judged acceptable, the reference intervals/cut-offs may be verified.


• A test is considered "verified/validated" if it meets the user's pre-determined requirements.

Experiments in Method Verification (MV) :

• Accuracy Experiment – Run accuracy control or external PT sample, in five runs.


• The Replication Experiment (EP5 CLSI) – A minimum of 20 replicate determinations on at least two levels of control materials whose concentrations are selected to span the analytical range are recommended to estimate the imprecision or random error of the method. Tabulate the number of ICMR – Task Force on Inborn Metabolic Disorders 49 measurements, the mean, and the standard deviation or coefficient of variation for each material.
  Always perform a replication study over at least five days.


• The Linearity or Reportable Range Experiment (EP6 CLSI) – Patient specimens with known or assigned values should be analyzed in duplicate at five linearly related levels, to assess the reportable range. Perform this experiment over at least over three runs.
  If method performance is judged acceptable, the reference intervals/cut-offs may be established for each parameter, by testing the analyte in minimum 240 healthy subjects (120 males; 120 females).

Ongoing Test validation :

Ongoing (at least semi-annual) assessment (or after major instrument repair) of test performance is required to validate the test performance characteristics. This is accomplished through evaluation of data from internal quality control, proficiency testing or by some other system for verifying the inaccuracy and imprecision. Ongoing validation monitors the applicability of the test and may also utilize data from instrument calibration/maintenance, historical data and documentation, competency of testing personnel, etc.

QUALITY CONTROL (QC)

The term quality control (QC) represents largely a statistical approach used to monitor the analytical processes to ensure that the results meet the desired quality requirements. A control procedure is usually implemented by collecting test results on stable control materials, then plotting those control observations on a control chart that has specified control limits or by evaluating those control results by data calculations employing specified decision criteria or control rules.

Quality control is implemented under two broad heads :

• Internal Quality Control
• External Quality Control