4 Invalid Samples addressed in this study Insufficient Quantity 1.Insufficient Quantity Appears Scratched or abradedNot dried prior to mailingSupersaturatedDiluted, discolored, or contaminated Exhibits serum rings Clotted or layered No blood addressed in this study188.8.131.52.6.7.8.
5 Invalid Samples: NYS 2004* Quantity insufficient 492 15.4 % Invalid TypeSamplesPercentageQuantity insufficient49215.4 %Not dry before mailing150.47 %Diluted, discolored, or contaminated2156.7 %Exhibits serum rings101531.8 %Appears clotted or layered54317.0 %*Note, all specimens on card were identified as invalid, list does notinclude all invalid types counted.
6 MethodologyEvaluate a single patient sample with valid/invalid DBS PairsPunch sample pairs from invalid and valid samplePerformed CAH, TSH, T4, Hgb, Biotinidase, Galactosemia, and MS/MS (AA/AC) testsAnalyze data with Excel (Mean, Std. Dev., t-test, etc.)Compare data to valid sample pairsCAH, TSH, T4 – immunoassayPKU, MCADD – msmsHgb – electrophoresisGalactosemia – BeutlerBiot. Def – Wolff testExample for invalid/valid pair:diluted, discolored or contaminated
7 Study Method: Pros and Cons Dependent on available samplesNot all invalid types were available for this studySometimes difficult to assign invalid sample typeUnlikely for there to be both an abnormal baby and invalid sample type on same specimenUse real world specimensInvalid and valid samples collected at same time from same specimenRandom sampling of DBSInvalid/valid sample pairs analyzed at same time
8 Testing Summary Qualitative tests Quantitative tests HeminoglobinopathiesBiotinidaseGalactosemiaQuantitative testsCAH (17-OHP)CH (T4* and TSH)MCADD (C8*)PKU (phenylalanine*)* Example data shown, other tests showed similar data and are not presented on slides.
9 Invalid Samples - Study Invalid TypeSamplesInsufficient quantity24Diluted, discolored, contaminated69Exhibits serum rings61Specimen clotted or layered180
10 Insufficient quantity Valid SampleInvalid SampleNote: on front side of card, all samples look acceptable
11 Insufficient quantity Phenylalanine High negative bias
12 Insufficient quantity MCADD/C8 High negative bias
13 Insufficient quantity Thyroxine High negative bias
14 Insufficient quantity summary Marker% BiasPhenylalanine-29.8MCADD/C8-15.3CAH-25.8TSH-19.5Thyroxine-25Overall negative biasExpected result with less bloodT-test indicates high probability that invalid data sets are different from valid data sets (P<0.05)Potential for qualifying abnormal specimen as normal (opposite for T4)
15 Insufficient quantity Decision making MarkerValidInvalidCutoffReportValid InvalidCAH10.67.5≥ 50Normal NormalPKU1.491.05≥ 4Normal NormalMCADD0.130.08≥ 0.8T4 (CH)12.52.8≤ 5Normal AbnormalTSH (CH)19.714.9≥ 18Retest Normal
19 Diluted, discolored, contaminated Thyroxine Ave Std Dev large for V/I pair
20 Diluted, discolored, contaminated Summary No difference in average bias for any markerMS/MS analytes – Valid/Invalid pairs indistinguishableThyroxine: large variation in bias (-66 to 46), potential for false positives and negatives
26 Serum Rings SummaryMS/MS analytes – Valid/Invalid pairs indistinguishableThyroxine: large variation in bias for V/I pair compared to V/V pair (see min/max)No major difference in average bias for any marker
27 Serum Rings Decision making MarkerValidInvalidCutoffResultValid InvalidCAH49.515.4≥ 50Normal NormalPKU1.031.73≥ 4MCADD0.160.11≥ 0.8T410.316.9≤ 5TSH (CH)19.111.9≥ 18Retest Normal
28 Clotted or LayeredInvalid Sample(shows layered blood)Valid Sample
29 Clotted or layered Phenylalanine Note T-test show V/I data sets high probability of data sets being different
30 Clotted or layered MCADD/C8 Note T-test show V/I data sets high probability of data sets being different
32 Clotted or layered Summary Overall Positive biasSome specimens w/ large neg. bias resultsPotential to have false positives and false negativesExpected due to layering of bloodMarkerAverage % Bias17-OHP9.8Phenylalanine8.8MCADD/C86.7TSH14.2Thyroxine9.3
33 Clotted or layered: Decision making MarkerValidInvalidCutoffResultValid InvalidCAH50.239.3*≥ 50Retest NormalPKU0.760.96≥ 4Normal NormalMCADD0.090.11≥ 0.8T418.520.1≤ 5TSH7.719≥ 18Normal Retest19.111.9Retest Normal*Invalid result lower than valid specimen result
35 Qualitative tests: valid/invalid samples indistinguishable N - Normal, AC - C-trait , AF - A>F , AFE - fetal variant ,AS - Sickle trait , FST - J, N, or Bart’s variant
36 Quantitative Data: Effect on Decision Making Test17-OHPPheC8T4TSHGalacBiotinHgbsignal↑↓P/AQNSFNFPN/ADilutedFN/FPRingsClotted/layeredP/A = present/absent, FN = False Neg., FP = False Positive
37 Decision making data summary: ConclusionsQuantity InsufficientBias low results, may be false negative or false Positive (T4)Qualitative results were unaffected (however, only 24 points and no positive specimens were tested)Decision making data summary:
38 Decision making data summary: ConclusionsDiluted, discolored, or contaminatedMS analytes largely unaffectedQualitative results unaffected (however, no positive specimens were tested)T4 and TSH results show large variation in measured bias for V/I pairDecision making data summary:
39 Decision making data summary: ConclusionsSerum RingsMS analytes largely unaffected, however there are affected samples (see below)Qualitative results unaffected (however, no positive specimens were tested)T4 and TSH results show large variation in measured bias for V/I sample pairDecision making data summary:
40 Decision making data summary: ConclusionsClotted or layeredBias high results, may be false negative (T4) or false PositiveQualitative results were unaffected (however, no positive specimens were tested)Decision making data summary:
41 Conclusions Quantity Insufficient Diluted, discolored, or contaminated Bias Low results for quantitative tests (false neg. - except T4)Qualitative tests unaffectedDiluted, discolored, or contaminatedMS analytes largely unaffectedT4 and TSH results show large variation in bias for V/I pair (+/-)Qualitative results unaffectedSerum RingsClotted or layeredGenerally bias high results, may be false negative (T4) or false positiveSome bias low results (where blood has not been layered)
42 Urban Legend vs. Reality Invalid okGalactosemiaHgbBiotinidaseInvalid NOT okCHCAHMCADPKU
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