Average G6PD activity (U/gHb) Trinity G6PD activity (U/gHb)
Di�
eren
ce in
G6P
D a
ctiv
ity
(U/g
Hb)
+ 1.96 SD
- 1.96 SD
Mean
Pearson correlation coe�cient [0.7585]
Linear fit equation: y = 3.84 +0.89xR2 = 0.575
0 2 4 6 8 10 12 14 16 18 20 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
R&D
G6P
D a
ctiv
ity
(U/g
Hb)
20
18
16
14
12
10
8
6420
4
2
0
-2
6
8
10
conclusions• AcomparisonoftwoquantitativeG6PDtestsshows:
– BothtestsequallyclassifysevereG6PDdeficient samples.
– Referencevaluesvarywidelydependingonthereferencetestused.
• TheBinaxNOW®testaccuratelydetectedallseverelydeficientpatients.
• ThefluorescentspottestonlyaccuratelydetectsallseverelydeficientspecimensifintermediatesareconsideredasG6PDdeficient.
• Bycytochemicalstaining,itwaspossibletodetectheterozygousfemalesinthepopulation.Onlythreeofthesewouldhavebeenexcludedfromtreatmentusingamoderatelydeficientcut-offof60%normalactivity.
w w w . p a t h . o r g
introductionTreatmentwithanti-malarialdrugsinthe8-aminoquinolonegroup(e.g.,primaquine,pamaquineandtafenoquine)cancauseacutehemolysisinpeoplewithglucose-6-phosphatedehydrogenase(G6PD)deficiency.Assuch,thereisaneedforatestthatisappropriateforG6PDdeficiencyscreeninginordertoensuresafeadministrationofthesedrugsforradicalcureofPlasmodium vivax.
WeevaluateddifferentassaysandplatformsfordeterminingtheG6PDstatusofapatient.
methodsAssays used to measure G6PD activity Trinity Biotech spectrophotometer test:AquantitativekineticassayexpressingG6PDactivityinunitspergramofhemoglobin.
R&D Diagnostics Ltd® enzymatic colorimetric method:AquantitativekineticcolorimetricassayexpressingG6PDactivityinunitspergramofhemoglobin.
Trinity Biotech Fluorescent spot test:Mostcommonqualitativeassay,inwhichfluorescenceofnicotinamideadeninedinucleotidephosphate(NADPH)isdirectly observed.
BinaxNOW® G6PD Test:Aqualitativeassaythatusesalateralflowtestplatform.
Cytofluorometric assay:Allowsobservationofmosaicredbloodcellpopulationsinfemales.
evaluation of diagnostic platforms for g6pd deficiency Nicole LaRue, Maria Kahn, Michael Kalnoky, Brandon T. Leader, Pooja Bansil, Sarah McGray, Gonzalo J. Domingo
table 1: trinity Biotech reference Summary of g6pD activity (N = 214)
reference values total Female Male adjusted male
Number of cases 214 107 107 101Mean (U/g Hb) 7.17 7.70 6.63 6.99Standard deviation (U/g Hb) 7.295 7.69 7.06 7.18Median (U/g Hb) 7.295 7.69 7.06 7.18
Range (U/g Hb) 0.12–14.04 1.03–14.04 0.12–12.26 0.84–12.26
table 2: Number and percent distribution of BinaxNow® and trinity Biotech Fluorescent Spot test results (N = 214)
Number (N) percent (%)
BinaxNow® test
Normal (N=201) 182 90.5
Deficient (N=201) 19 9.45
Untreated (N=214) 13 6.07
trinity Biotech Fluorescent Spot test (FSt)
Normal 189 88.32
Deficient 13 6.07
Intermediate 12 5.61
Deficient + intermediate 25 1.68
Normal + intermediate 201 93.93
figure 2: Comparison of g6pD activity classified samples by the trinity Biotech quantitative assay to two qualitative tests. the shading in the table indicates discrepancies in definition of phenotype between the tests within intermediate ranges.
Classification by trinity Biotech Quantitative
trinity Biotech Fluorescent Spot
testBinaxNow®
trinity Biotech Quantitative
Calculated activity
Sample #147 (female) NORMAL 7.50
Sample #141 (male) NORMAL 8.24
Sample #110 (female) INTERMEDIATE
3.40
Sample #105 (male) DEFICIENT 0.12
Sample #92 (male) DEFICIENT
0.66
Sample #145 (male) DEFICIENT
0.69
figure 1: pearson’s correlation coefficient and a Bland altman scatterplot describing the relationship between the trinity Biotech quantitative and r&D Diagnostics Ltd® quantitative test for g6pD activity.
results
figure 3: g6pD zygosity measured by mean FItC fluorescence compared to g6pD activity measured by trinity Biotech.
0 5 10 15250
300
350
400
450
500
550
600
650
10% 20% 30% 60%
Mea
n FI
TC C
hann
el In
tens
ity
mea
sure
by
Cyto
met
er
G6PD Zygosity measured by mean FITC Fluorescence and Trinity Zygosity determined by Flow Cytometry & Percent Normal determined by Trinity
G6PD activity measured by trinity (U/(g/dl)Hb)
NormalHeterozygousDeficient60% normal30% normal20% normal10% normal
results continued results continued
We thank Amanda Vilbrandt and Scott Brown for their support in the development of this poster. This poster is based on research funded by the Bill & Melinda Gates Foundation. For further information please contact [email protected], PATH, Seattle, WA.
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table 3: Clinical performance of the BinaxNow® and trinity Biotech Fluorescent Spot screening tests for detection of deficient g6pD activity*
Cut-off value (% of adjusted normal male value, 7.18)
10% 20% 30% 60%
Cut-off value (U/gHb) 0.718 1.436 2.154 4.308
Number of samples with G6PD levels below cut-off (%)
6 (2.80) 16 (7.48) 18 (8.41) 23 (10.75)
BinaxNow g6pD test
Sensitivity % (95%Cl) 100.0 (54.1, 100.0)
100.0 (79.4, 100.0)
100.0 (81.5, 100.0)
82.6 (61.2, 95.0)
Specificity % (95%Cl) 93.3 (88.9, 96.4)
98.4 (95.3, 99.7)
99.5 (97.0, 100.0)
100.0 (97.9, 100.0)
trinity Fluorescent Spot testa
Sensitivity % (95%Cl) 100.0 (54.1, 100.0)
100.0 (79.4, 100.0)
100.0 (81.5, 100.0)
91.3 (72.0, 98.9)
Specificity % (95%Cl) 90.9 (86.1, 94.4
95.5 (91.5, 97.9)
96.4 (92.8, 98.6)
97.9 (94.7, 99.4)
trinity Fluorescent Spot testb
Sensitivity % (95%Cl) 83.3 (35.9, 94.6)
75.0 (47.6, 92.7)
72.2 (46.5, 90.3)
56.5 (34.5, 76.8)
Specificity % (95%Cl) 96.2 (92.6, 98.3)
99.5 (97.2, 100.0)
100.0 (98.1, 100.0)
100.0 (98.1, 100.0)
*Gold standard: Trinity Biotech test a Trinity Biotech spot test intermediate test results combined with deficient test resultsb Trinity Biotech spot test intermediate test results combined with normal test results
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