A Guide To Fluorescent Spot Testing For G6PD Deficiency

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A guide to fluorescent spot testingfor G6PD deficiencyApril 2014

ContentsI.Introduction . 3II.Purpose of this guide . 3III. Overview: FST workflow . 3IV. Blood sampling and storage . 4V.Hardware and supplies required for an FST for G6PD . 4VI. Cost of G6PD FST equipment . 6VII. Illustrated instructions (Trinity Biotech kit 203-A G-6-PDH Screening Kit) . 6VIII. Buffer and reagent storage . 6IX. Formulating reagents in your laboratory . 18X. References . 18XI. Checklist for G6PD deficiency fluorescent spot testing . 19Acknowledgments and publication informationThis material is based on research funded by the Bill & Melinda Gates Foundation, grant number OPP1034534; as well as the UK Departmentfor International Development (DFID), grant number 204139. The findings and conclusions contained within are those of the authors and donot necessarily reflect positions of the Bill & Melinda Gates Foundation, or DFID.This guide was written and edited by PATH staff Nicole LaRue, Scott Wittet, and Dr. Sampa Pal. It was reviewed by Dr. Gonzalo Domingo, PATH;Dr. Ari Winasti Satyagraha, Eijkman Institute, Indonesia; Mrs. Woyneshet Gelaye, Regional Health Research Laboratory Center, Ethiopia; andDr. Daniel Bridges, AKROS Research, Zambia. Design and illustrations: Shawn Kavon. Proofreading: Jake Abrahams.Suggested citation: PATH. A Guide to Fluorescent Spot Testing for G6PD Deficiency. Seattle: PATH; 2014.Cover photo: PATH/Patrick McKern

I. IntroductionII. Purpose of this guideGlucose-6-phosphate dehydrogenase (G6PD) deficiencyis a genetic disorder, and is the most commonhuman enzyme deficiency.1 Individuals with severeG6PD deficiency are vulnerable to serious healththreats—hemolysis of red blood cells and hemolyticanemia—when exposed to certain anti-malarial drugs(compounds in the 8-aminoquinoline group, such asprimaquine) or other substances, including fava beans.This guide was produced to assist laboratory managersand personnel to assemble the necessary equipment andimplement fluorescent spot testing for G6PD deficiency.The fluorescent spot test (FST) is widely used for invitro diagnosis of G6PD deficiency using whole blood ordried blood spots. The test is affordable and producesqualitative, visual results within minutes.Principle of the testG6PD catalyzes a reaction in the pentose phosphatepathway, a metabolic pathway that supplies reducingenergy to cells such as erythrocytes by maintainingthe level of the reduced form of the co-enzymenicotinamide adenine dinucleotide phosphate (NADPH).G6PD is essential for protecting erythrocytes in thepresence of oxidizing agents.The FST for G6PD deficiency involves this reaction:Glucose-6-Phosphate NADP(Not fluorescent)G6PDH6-Phosphogluconate NADPHThe guide focuses specifically on setting up thelaboratory and conducting G6PD tests. It does notcover broader issues such as rationale and strategiesfor G6PD testing, population sampling, external qualityassurance processes, or other topics.In several sections, this guide mentions specificproducts; the products are listed as examples ofequipment and materials that meet FST specifications.Other suitable products that meet FST specificationsare available through a variety of vendors. This listingof specific product examples is not an endorsement byPATH of those products.III. Overview: FST workflowNot all FSTs are the same, but the basic workflow is as follows:1. Prepare controls and samples Prepare the G6PD reagent, controls, andsample aliquots. Prepare filter papers. Prepare heat block or water bath. Add controls and blood samples (whole blood ordried blood spots) to the G6PD reagent aliquot.(Fluorescent)To perform the test, a small amount of blood isincubated with glucose-6-phosphate and NADP in thesubstrate reagent, and then is spotted on filter paper.Once dried, the spots are viewed under long-waveultraviolet (UV) light—the by-product of the reaction(NADPH) is fluorescent. NADPH fluorescence is directlyproportional to G6PD activity and lack of fluorescencesignals G6PD deficiency.The FST was first developed by E. Beutler in the 1960s andis sometimes called the Beutler test.2 The test can be usedto screen for galactosemia in addition to G6PD deficiency.32. Spot controls and samples on filter paper afterspecified incubation period Spot controls (mixed with G6PD reagent) ontofilter paper for “time zero.” Incubate controls in a water bath or heat block. Repeat using blood samples. Allow filter papers to dry.3. View fluorescence View the spots under long-wave UV light. Classify samples as “normal,” “intermediate,”or “deficient.” Dispose of or store filter papers.PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

IV. Blood sampling and storageSpecimen handling Before receiving and processing samples, ensurethat they were properly stored after collection andduring shipping (if applicable).V. Hardware and supplies requiredfor an FST for G6PDNote: Make certain that the test you select will workwell in the ambient temperature of your laboratory. Samples must have been refrigerated between2 C and 8 C.General equipment Single-use dropper pipet No clotting observed. 1- to 2-mL disposable snap-top tubesLess than 1 week since draw date. 10-uL, 200-uL, and 1,000-uL pipets and tipsWear gloves and other appropriate personalprotective equipment. Timer Biohazard waste containers for disposal ofpotential hazardous or infectious wasteCollection Currently all protocols recommend use of venousblood. Pen There are not sufficient data to recommend the useof capillary blood in fluorescent spot assays. Use of ethylenediaminetetraacetic acid (EDTA) or acidcitrate-dextrose (ACD) anticoagulants is acceptable.Storage Can be stored refrigerated between 2 C and 8 C forup to 1 week with no impact on activity output.4Personal protective equipment Gloves when handling all biological and potentiallyhazardous specimens Coat or gown Eye protection (i.e., goggles or glasses) General lab safety (i.e., wearing pants and closedtoe shoes)PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

specialized equipmentproduct examples*Long-wave UV light365 nm, 6 watts Do not use a short-wave UV lightWater bath or heat blockset to 37 CSpectroline long wave (365nm)/short wave(254nm) UV long life filter; Model ENF-260C(cost: 350). Cole-Parmer rechargeable long wave 365nm UVlight; Cat. No. YO-97603-00. VWR microprocessor water bath; Cat. No.97025-130; Model 5L; Serial No. 2H1221341WR. VWR digital 2 block heater 120V; Cat. No. 12621088; Model No. 949302; Serial No. 100617007.Dark box or darkened roomto view fluorescence Spectroline fluorescence analysis cabinet;Model CM-10.Filter paper Whatman No. 1 filter paper; Cat. No. 1001-150.ReagentsNote: As shown below, it is possible to buy FST reagents and buffers for G6PD deficiency, but some laboratories preferto prepare their own reagents (as described in section IX).G6PD substrate Trinity Biotech G-6-PDH Screening Test Kit – SpotTest; Cat. No. 203-A. R&D Diagnostics G-6-PD qualitative kit (UV lampmethod); Cat. No. SQMMR500 and SQMMR1250. See page 18 below for information on producingG6PD substrate in your laboratory.Dilution buffer(for rehydration of G6PD substrate) Buffer is included in the above kits from TrinityControls and R&D Diagnostics.Trinity Biotech sells each control separately: G-6PDH control normal—Cat. No. G6888; G-6-PDHcontrol intermediate—Cat. No. G5029; G-6-PDHcontrol deficient—Cat. No. G5888. Pointe Scientific sells all three controls as a set—Cat. No. G7583-CTL-SAM.* These specific products are listed as examples of equipment and materials that meet FST specifications. Other suitable products that meet FST specifications areavailable through a variety of vendors. This listing of specific product examples is not an endorsement by PATH of those products.5PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

VI. Cost of G6PD FST equipment After incubating each control and sample for 5minutes at 37 C.Based on prices found in catalogs for 2013, PATHestimates that the overall cost of equipment for an FSTbased G6PD testing program in the United States wouldbe approximately: After incubating each control and sample for anadditional 5 minutes at 37 C (for a total of 10minutes of incubation).Equipment costs (non-recurring):Long-wave UV light 300–350Water bath or heat block 830–880Dark box 250Subtotal 1,380–1,480G6PD-specific supply costs (recurring):Whatman filter paper 30Test kit with substrates 50–150Controls 340(100 sheets)(45 to 50 individual tests per kit)(for approximately 300 control batches)The spots are then viewed in a dark box under UV lightand rated for G6PD deficiency based on the amount offluorescence observed.VIII. Buffer and reagent storage forthe Trinity 203-A kit Before reagents and controls are rehydrated, ifrefrigerated between 2 C and 8 C, they are gooduntil the expiration date on the kit. Trizma buffer (rehydration agent for G6PD reagent)can be stored at room temperature (18 C to 26 C)or refrigerated (2 C to 8 C) until expiration. Once reconstituted, G6PD reagent can be kept upto four hours at room temperature (18 C to 26 C)and for up to one week refrigerated (2 C to 8 C) orup to 2 weeks frozen (-20 C). G6PD controls, once reconstituted, can be kept upto one week refrigerated (2 C to 8 C) or for up to 2weeks frozen at -20 C with three freeze-thaw cycles.PATH estimates that recurring costs range from 5.74 to 13.74 per individual tested.Additionally, the laboratory will require generalequipment as noted on page 4.VII. Illustrated instructions for theTrinity Biotech kit 203-A G-6-PDHScreening Test KitFollowing are the illustrated details for performingthe Trinity test:There are a number of FSTs available on the market,and one or more of them may meet the needs of yourprogram. PATH has not evaluated all available tests andcannot recommend one test over another.As an example of the workflow for performing an FSTfor G6PD deficiency, a detailed illustrated procedure isshown below for Trinity Biotech Kit 203-A, as performedin the PATH laboratories.For this test: Each control and each blood sample is spottedon filter paper three times at three different time points: 6Immediately after adding the control or sample tothe reagent.PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

step 1 of 17STEP 1Gather materials and heat the waterbath or heat block1.Open the Trinity kit (Cat. No. 203-A) andremove the Trizma buffer bottle and the vials oflyophilized reagent.2.Each rehydrated reagent vial has enough volumefor nine tests. Only rehydrate the number of vialsnecessary for the number of tests being performed.3.Each kit contains enough supplies for 45 to 50 testsif all the reagents are used before they expire.4.Test only blood samples that have been stored in arefrigerator for less than one 1.5mL DropperG6PDEppendorfreagenttubeG-6-PDH CoCONTROL NHeat the water bath or heat block to 37 C inpreparation for incubation later.BloodsampleLyophilizedG6PD controls(3 types: N, I, D)Filter paperNo. 1-150mmPipetstep 2 of 17STEP 2Rehydrate the G6PD reagent1.Add 2 mL of Trizma buffer (included in the kit) toeach vial of lyophilized G6PD reagent (included inthe kit) to rehydrate the G6PD substrate.2.Swirl and invert the mixture and let it sit for 2minutes until completely resuspended.3.Label the top of the reagent vial with today’s date.Check the manufacturer’s recommendation for theexpiration date or shelf life of the solution. Storethe solution as recommended by the manufacturer.72mL trizma bufferG-6-PDH2mlPATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

step 3 of 17STEP 3Rehydrate the lyophilized controlsNote: lyophilized control samples of G6PD-normal,-intermediate, and -deficient blood are not included in the kit andmust be ordered separately.500µl deionized H201.To rehydrate the controls, add the specified amountof deionized water to the lyophilized powder. Allowthe solution to sit until all the powder has beendissolved into solution.2.Label each vial of control solution with today’s date.Check the manufacturer’s recommendation for theexpiration date or shelf life of the solution. Store thesolution as recommended by the manufacturer.G-6-PDH CoG-6-PDH CoG-6-PDH CoCONTROL NCONTROL ICONTROL DG6PDHsubstratestep 4 of 17STEP 4Prepare control and blood sample aliquots1.Make aliquots by pipetting 200 uL of the suspended G6PDreagent (from step 2) into 1.5-mL Eppendorf tubes.2.Make one aliquot for each sample and each control.You will need three control aliquots and one aliquotfor each blood sample.3.Label the control aliquots:5.You may find it efficient to make all neededaliquots at once. The aliquots can be kept at roomtemperature for up to four hours at 18 C to 26 C.After four hours, they should be discarded.8403Label the blood sample aliquots with your samplenumbers. The image shows an aliquot labeled forblood sample number “403.”D4.STEP 4Ifor the G6PD “normal” controlfor the G6PD “intermediate” controlfor the G6PD “deficient” controlNNID200µl suspended reagentPATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

step 5 of 17STEP 5Prepare filter papersTake out Whatman No. 1 filter papers. 150-mmfilter paper works well, but other sizes can be used.2.One of the papers will be used for the threecontrols, the other for blood samples and a normalcontrol (so that you can easily compare the controlwith the samples on the same piece of filter paper).3.The number of spots that can fit on each filter paperis defined by desired spacing between spots and sizeof filter paper. One 150-mm filter paper can typicallyhold up to five samples (15 spots) comfortably.Tim1.5 min10mineroeZfor controls4.5 minTimNote: An alternate approach—if you have larger filter papers—isto run all three controls (normal, intermediate, and deficient) oneach filter paper along with the blood samples. This provides amore robust internal control for all samples run. In that case, youwill not need a separate filter paper for the three controls.10mineroeZDivide each filter paper into three columns and addheadings—time zero, 5 minutes, and 10 minutes—above each column as shown in the illustration.for blood samples andnormal control9PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

step 6 of 17STEP 6Add the normal control to thenormal aliquot1.Beginning with your “normal” control, use a pipetteto add 10 uL of the control to the aliquot labeled “N.”Vortex briefly or “flick mix” the aliquot to ensure thatit is well mixed.10µl control NNote: Do not mix the other controls or your blood samples withreagent yet.STEP 4NNote: The reaction between the control or blood sample and thereagent happens very rapidly. Therefore, to obtain an accurate“time zero” spot, it is important to spot mixed controls or bloodsamples as soon as possible after mixing.Mix one control with reagent first (step 6), then spot it for “timezero” (step 7). Mix and spot the remaining controls (step 8). Beginincubating the control batch (step 9).After you have finished incubating and spotting the controlsamples for “time zero,” “5 minutes,” and “10 minutes,” you canmix, spot, and incubate your first batch of blood samples.step 7 of 17STEP 7Spot “time zero” normal control ontoall filter papers and labelUsing a disposable plastic drop pipet, take up asmall amount of the solution and drop a single droponto the filter paper being used for the controls,in the “time zero” column. The spot should beapproximately one-half inch in diameter.2.Label this drop “N.”3.Follow this procedure for the filter papers you will usefor your blood samples. Only the normal control willbe spotted alongside the blood samples. One aliquotof normal control should provide enough liquid tospot a total of four filter papers (twelve “N” spots).105 min10 minN1.0PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

05 min10 minstep 8 of 17STEP 8Add controls to the intermediate anddeficient aliquots and spot them1.10µl control I10µl control DOnce you have spotted your “normal” control, usea pipette to add 10 uL of the control to the aliquotlabeled “I.” Vortex briefly or “flick mix” the aliquotto ensure that it is well mixed.DFollow the same process with the “D” controland aliquot.D3.IUsing a disposable plastic drop pipet, take up asmall amount of the solution and drop a singledrop onto the filter paper being used for thecontrols, in the “time zero” column.I2.STEP 4Note: Do not spot the “I” and “D” controls on the filter papers thatwill be used for blood samples (only the “N” control is spotted onthose filter papers). Do not mix your blood samples with reagent yet.step 9 of 17STEP 9Incubate all three controls in a waterbath or heath block for 5 minutes1.Once all three “time zero” controls have been spottedon filter paper, place the control batch of aliquots ina water bath or heat block already heated to 37 C (asinstructed in step 1).2.Set a timer for 5 minutes.010 minN115 minIDPATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

0STEP 105 minstep 10 of 1710 minSpot “5 minute” controls onto filterpaper and label2.Using a disposable plastic drop pipet for each control,place a single drop onto the filter paper being used forthe controls, in the “5 minutes” column.3.Be sure to place the 5 minutes “N” control next to thetime-zero “N” control for easy comparison.4.Do the same for the “I” and “D” controls on the“control filter paper.”5.Not shown in the illustration:Spot the 5 minutes “N” control next to the time-zero“N” control on the “sample filter papers” as well. Donot spot the “I” and “D” controls on those papers.DAfter incubating the three controls for 5 minutes,remove the control aliquots from incubation.IN1.step 11 of 17STEP 11Incubate controls for 5 more minutes1.010 minIncubate the control aliquots again for an additional 5minutes (for a total of 10 minutes of incubation).N125 minIDPATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

05 minstep 12 of 1710 minSTEP 12Spot “10 minute” controls onto filterpaper, label, and dispose of aliquotsBe sure to place the “N” control next to the time-zeroand 5 minutes “N” controls.3.Do the same for the “I” and “D” controls on the“control” filter paper.4.Not shown in the illustration:Spot the 5 minutes “N” control next to the time-zero“N” control on the “sample filter papers” as well. Donot spot the “I” and “D” controls on those papers.D2.IAfter incubating for five more minutes (for a totalof 10 minutes), remove the control batch from thewater bath or heat block. Using a separate, disposableplastic drop pipet for each control, place a single droponto the filter paper being used for the controls, in the“10 minutes” column.N1.Dispose of the aliquots containing solution into abiohazard waste container.STEP 13step 13 of 17Repeat steps 6 through 12 with thefirst batch of blood samples1.Once you have finished mixing, incubating, andspotting the controls, you can begin processing yourblood samples, using the same process.2.Gather the “sample” filter papers you have alreadyspotted with the time zero, 5 minutes, and 10 minutes“N” controls.continued on the next page13PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

step 13 continued010 minAdd first blood sample to aliquot andspot “time zero.”3.5 minAdd 10 uL of a whole blood sample in EDTA (or ACD orheparin) to the aliquot tube labeled with the samplenumber (in the image we are using “sample number403” as an example). Mix.10µl blood sampleNote: Only mix enough blood samples to fit on a single piece of filterpaper—typically up to four samples (plus the “N” control) on 150-mmdiameter paper.STEP 44034.403Immediately after mixing the first blood sample, andusing a separate, disposable plastic drop pipet, takeup a small amount of the solution and drop a singledrop onto the filter paper in the “time zero” columnunder the time zero “N” control.Repeat for up to three more bloodsamples (not shown in the image).Incubate samples for five minutes05.Once the “time zero” blood samples have beenspotted on filter paper, place the batch of bloodsample aliquots in a water bath or heat block alreadyheated to 37 C (as instructed in step 1).6.Set a timer for 5 minutes.5 min10 mincontinued on the next page40314PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

step 13 continued7.After incubating the blood samples for 5 minutes(for a total of 10 minutes), remove the bloodsample aliquots from incubation.8.Using a disposable plastic drop pipet for eachblood sample, place a single drop onto the filterpaper being used for the blood samples, in the “5minutes” column under the 5 minutes “N” control.9.Be sure to place the first “5 minute” blood samplespot next to the “time zero” blood sample spot foreasy comparison.403Spot “5 minute” blood samples ontofilter paper and label.05 min10 min10. Repeat this process for the remaining blood samples.Incubate blood samples for fivemore minutes.11. Once the “5 minute” blood samples have beenspotted on filter paper, place the batch of bloodsample aliquots in a water bath or heat block alreadyheated to 37 C (as instructed in step 1).40312. Set a timer for 5 minutes.Spot “10 minute” blood samples ontofilter paper, label, and dispose ofaliquots.40313. After incubating the blood samples for 10 minutes,remove the blood sample aliquots from incubation.14. Using a disposable plastic drop pipet for each control,place a single drop onto the filter paper being used forthe controls, in the “10 minutes” column under the 10minutes “N” control.15. Be sure to place the first “10 minutes” blood samplespot next to the “time zero” and “5 minute” bloodsample spot for easy comparison.16. Dispose of the aliquots containing solution into abiohazard waste container.15PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

step 14 of 17STEP 14Dry filter papers10minTim1.05 mineroeZAllow each filter paper to dry at room temperaturefor 15 to 20 minutes.NN5 min10 min15-20 mindry timeN30 minIIIDDD5 min10minTimeroeZSTEP 15NNN403403403step 15 of 17View spots under UV light1.16View the filter papers under a long-wave UV light(365 nm) after spots have dried completely. Use aviewing box with UV tempered glass to protect youreyes. View in a dark room so that you can see thefluorescence.PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

step 16 of 17STEP 16Classify samples as “N,” “I,” or “D”5 min10min1.Classify each sample based on level of fluorescence:TimeroeZNNormal (N) G6PD activity: moderate tostrong fluorescence after 5 minutes, strongfluorescence after 10 minutes.IINIIntermediate G6PDenzyme activityDeficient G6PDenzyme activityIntermediate (I) G6PD activity: weakfluorescence after 5 minutes, moderatefluorescence after 10 minutes.DDD5 min10mineroeZTimDeficient (D) G6PD activity: weak or nofluorescence after both 5 and 10 minutes.NNormal G6PDenzyme activityNormal controls2.The bottom image shows the results for normalcontrols and a deficient blood sample.3.Record the test results.4.You may find it useful to take photos of thefluorescing filter paper in the dark box and save thephotos with your records for future reference.STEP 17NN403403NDeficient bloodsample results403step 17 of 17Dispose of or store filter papers1.Filter paper can be disposed of after use in a biohazardwaste container or stored at 2 C to 8 C in a sealedplastic bag with desiccant for up to 2 weeks.2.If you intend to keep the filter papers, retain both thesample papers and control papers (for reference).17PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

IX. Formulating reagents in yourlaboratoryX. References1.It may be simplest to buy reagents on the market, butif you would like to create your own reagents, here aretwo formulas for doing so:2.1. Modified Beutler formula2: Glucose-6-phosphate TPN (triphosphopyridine nucleotide) 0 .1 mL of 0.0075 M0.1 mL of 0.01 M Saponin0.2 mL of 1% Tris-HCL buffer, pH 7.80.3 mL of 0.75 M GSSG (oxidized glutathione)0.1 mL of 0.008 M Distilled water0.2 mL3.Beutler E. Review article: G6PD Deficiency. Blood.1994;84(11)3613–3636. 84/11/3613.longBeutler E, Mitchel lM. Brief report: specialmodifications of the fluorescent screening methodfor glucose-6-phosphate dehydrogenase deficiency.Blood. 1968;32:816–818.Jiang J. et al. Using the fluorescence spot test forneonatal screening of G6PD deficiency. SoutheastAsian Journal of Tropical Medicine and Public Health.2003;34 Suppl 3:140–142. Available at: /southeast-2003-vol-34-supp-3-p-140.pdf.2. Formula used by Jiang et al.3: Glucose-6-phosphate1 mL of 0.01 M Coenzyme II1 mL of 0.0075 M Glutathione, oxidized1 mL of 0.008 M Digitalis2 mL of 0.027 M Tris-HCL buffer, pH 7.83 mL of 0.75 M Distilled water3 mL18PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

XI. Checklist for G6PD deficiencyfluorescent spot testing Promptly transfer drop of solution to filter papernext to original spot (or time zero). Add 5 minutes “N” control alongside. Prepare the reagents: Add 2 mL of Trizma bufferfrom the kit to each vial of lyophilized G6PDreagent (included in the kit) to rehydrate the G6PDsubstrate. Swirl and invert the mixture and let it sitfor 2 minutes until completely resuspended. Heat the water bath or heat block to 37 C inpreparation for incubation later. Prepare the controls: Add 0.5 mL water to vialof G-6-PDH control lyophilized powder. Swirlintermittently and allow the solution to sit for 5 to10 minutes until all the powder has been dissolvedinto solution. Identify spot with controls or sample number andtime: 5 minutes. Place tube in 37 C water bath for 5 minutes. After 5 minutes of incubation, remove tube fromwater bath. Promptly transfer drop of solution to filter papernext to other two spots. Identify spot with controls or sample numberand time: 10 minutes. Add 10 minutes “N” controlalongside. Whole blood specimens are run using the sameprocedure as controls. Allow each filter paper to dry at room temperaturefor 15 to 20 minutes. Procedure: Add 200µl of G6PD substrate solution in 1to 2-mL disposable snap-top tubes. View the filter papers under a long-wave UV light(365 nm) in a dark room. (Compare filter paperwith controls to that of samples to determinefluorescence levels.) Add 10 µL of controls or whole blood (withanticoagulant) to tube containing 200 µl of G6PDsubstrate solution and mix. Promptly transfer drop of solution to filter paperwith a single-use dropper pipet. Identify spot with controls or sample number andtime zero. Add 0 minutes “N” control alongside.(Note: For each sample paper a normal control willbe spotted alongside blood samples. Spots shouldbe approximately 0.5 inch in diameter.) Place tube in 37 C water bath for 5 minutes. Compare G6PD activity by visually comparingfluorescence levels with the expected visualfluorescence levels provided by Trinity Biotech. Record fluorescent intensity (absent, weak,moderate, or strong) of each sample at each timepoint and classify as deficient, intermediate, ornormal. Store filter paper at 2 C to 8 C in a sealed plasticbag with desiccant for up to 2 weeks or Dispose offilter papers in a biohazard waste container. After 5 minute incubation remove tube fromwater bath.19PATH A Guide to Fluorescent Spot Testing for G6PD Deficiency

3 PATH A Guide to Fluorescent Spot Testing for G6PD Deflciency I. Introduction Glucose-6-phosphate dehydrogenase (G6PD) deficien

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