# Presentation: 739966

## Invalid Specimen Study

- Newborn Screening Program NYSDOH

- Joe Orsini, PhD
- Ken Pass, PhD

## Joe Orsini, PhD

**Joe Orsini, PhD**

## Valid Sample

- Completely fills circle
- Un-layered
- Un-smeared
- Homogeneous

## Invalid Samples

- Insufficient Quantity 
- Appears Scratched or abraded
- Not dried prior to mailing
- Supersaturated
- Diluted, discolored, or contaminated 
- Exhibits serum rings 
- Clotted or layered 
- No blood
-   **addressed in this  study**

- 1.

- 2.

- 8.

- 7.

- 6.

- 5.

- 4.

- 3.

## Invalid Samples: NYS 2004*

| Invalid Type | Samples | Percentage |
| --- | --- | --- |
| Quantity insufficient | 492 | 15.4 % |
| Not dry before mailing | 15 | 0.47 % |
| Diluted, discolored, or contaminated | 215 | 6.7 % |
| Exhibits serum rings | 1015 | 31.8 % |
| Appears clotted or layered | 543 | 17.0 % |

- *Note, all specimens on card were identified as invalid, list does not
- include all invalid types counted.

## Methodology

- Evaluate a single patient sample with valid/invalid DBS Pairs
- Punch sample pairs from invalid and valid sample
- Performed CAH, TSH, T4, Hgb, Biotinidase, Galactosemia, and MS/MS (AA/AC) tests
- Analyze data with Excel (Mean, Std. Dev., t-test, etc.)
- Compare data to valid sample pairs

- Example for invalid/valid pair:
- diluted, discolored or contaminated

- CAH, TSH, T4 – immunoassay
- PKU, MCADD – msms
- Hgb – electrophoresis
- Galactosemia – Beutler
- Biot. Def – Wolff test

## Study Method: Pros and Cons

- Use real world specimens
- Invalid and valid samples collected at same time from same specimen
- Random sampling of DBS
- Invalid/valid sample pairs analyzed at same time

- Dependent on available samples
- Not all invalid types were available for this study
- Sometimes difficult to assign invalid sample type
- Unlikely for there to be both an abnormal baby and invalid sample type on same specimen

_**Pros**_

_**Cons**_

## Testing Summary

- _Qualitative tests_
- Heminoglobinopathies
- Biotinidase
- Galactosemia

- _Quantitative tests_
- CAH (17-OHP)
- CH (T4* and TSH)
- MCADD (C8*)
- PKU (phenylalanine*)

***** Example data shown, other tests showed similar data and are not presented on slides.

## Invalid Samples - Study

| Invalid Type | Samples |
| --- | --- |
| Insufficient quantity | 24 |
| Diluted, discolored, contaminated | 69 |
| Exhibits serum rings | 61 |
| Specimen clotted or layered | 180 |

## Insufficient quantity

- Valid Sample
- Invalid Sample

- Note: on front side of card, all samples look acceptable

## Insufficient quantityPhenylalanine

## Insufficient quantity MCADD/C8

- High  negative bias

## Insufficient quantityThyroxine

- High  negative bias

## Insufficient quantitysummary

- Overall negative bias
- Expected result with less blood
- T-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)

| Marker | % Bias |
| --- | --- |
| Phenylalanine | -29.8 |
| MCADD/C8 | -15.3 |
| CAH | -25.8 |
| TSH | -19.5 |
| Thyroxine | -25 |

## Insufficient quantity Decision making

| Marker | Valid | Invalid | Cutoff | Report
Valid    Invalid |
| --- | --- | --- | --- | --- |
| CAH | 10.6 | 7.5 | ≥ 50 | Normal  Normal |
| PKU | 1.49 | 1.05 | ≥ 4 | Normal   Normal |
| MCADD | 0.13 | 0.08 | ≥ 0.8 | Normal  Normal |
| T4 (CH) | 12.5 | 2.8 | ≤ 5 | Normal  Abnormal |
| TSH (CH) | 19.7 | 14.9 | ≥ 18 | Retest    Normal |

## Diluted, Discolored, Contaminated Sample

- Valid Sample
- Invalid Sample

## Diluted, discolored, contaminated Phenylalanine

## Diluted, discolored, contaminated: MCADD/C8

## Diluted, discolored, contaminated Thyroxine

- Ave Std Dev large for V/I pair

## Diluted, discolored, contaminated Summary

- No difference in average bias for any marker
- MS/MS analytes – Valid/Invalid pairs indistinguishable
- Thyroxine: large variation in bias (-66 to 46), potential for false positives and negatives

## Diluted, discolored, contaminated Decision making

| Marker | Valid | Invalid | Cutoff | Result
Valid  Invalid |
| --- | --- | --- | --- | --- |
| CAH | 24.9 | 20.3 | ≥ 50 | Normal  Normal |
| PKU | 0.97 | 1.21 | ≥ 4 | Normal  Normal |
| MCADD | 0.17 | 0.14 | ≥ 0.8 | Normal  Normal |
| TSH (CH) | 10.3 | 5.8 | ≤ 5 | Normal  Normal |
| TSH (CH) | 17.4 | 20.6 | ≥ 18 | Normal  Abnormal |

## Serum Rings

- Invalid Sample
- Valid Sample

## Serum ringsPhenylalanine

## Serum ringsMCADD/C8

## Serum ringsThyroxine

## Serum RingsSummary

- MS/MS analytes – Valid/Invalid pairs indistinguishable
- Thyroxine: 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

## Serum RingsDecision making

| Marker | Valid | Invalid | Cutoff | Result
Valid  Invalid |
| --- | --- | --- | --- | --- |
| CAH | 49.5 | 15.4 | ≥ 50 | Normal  Normal |
| PKU | 1.03 | 1.73 | ≥ 4 | Normal  Normal |
| MCADD | 0.16 | 0.11 | ≥ 0.8 | Normal  Normal |
| T4 | 10.3 | 16.9 | ≤ 5 | Normal  Normal |
| TSH (CH) | 19.1 | 11.9 | ≥ 18 | Retest   Normal |

## Clotted or Layered

- Invalid Sample
- (shows layered blood)
- Valid Sample

## Clotted or layeredPhenylalanine

- Note T-test show V/I data sets high probability of data sets being different

## Clotted or layeredMCADD/C8

- Note T-test show V/I data sets high probability of data sets being different

## Clotted or layeredThyroxine

## Clotted or layeredSummary

- Overall Positive bias
- Some specimens w/ large neg. bias results
- Potential to have false positives and false negatives
- Expected due to layering of blood

| Marker | Average % Bias |
| --- | --- |
| 17-OHP | 9.8 |
| Phenylalanine | 8.8 |
| MCADD/C8 | 6.7 |
| TSH | 14.2 |
| Thyroxine | 9.3 |

## Clotted or layered: Decision making

- *Invalid result lower than valid specimen result

| Marker | Valid | Invalid | Cutoff | Result
Valid  Invalid |
| --- | --- | --- | --- | --- |
| CAH | 50.2 | 39.3* | ≥ 50 | Retest Normal |
| PKU | 0.76 | 0.96 | ≥ 4 | Normal  Normal |
| MCADD | 0.09 | 0.11 | ≥ 0.8 | Normal  Normal |
| T4 | 18.5 | 20.1 | ≤ 5 | Normal  Normal |
| TSH  | 7.7 | 19 | ≥ 18 | Normal  Retest |
| TSH | 19.1 | 11.9 | ≥ 18 | Retest   Normal |

## Summary

## 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

## Quantitative Data: Effect on Decision Making

| Test | 17-OHP | Phe | C8 | T4 | TSH | Galac | Biotin | Hgb |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| signal  | ↑ | ↑ | ↑
 | ↓
 | ↑ | ↑ | ↑ | P/A |

| QNS | FN | FN | FN | FP | FN | N/A | N/A | N/A |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Diluted | FN/FP | FN/FP | FN/FP
 | FN/FP
 | FN/FP
 | N/A
 | N/A
 | N/A |
| Rings | FN/FP | FN/FP | FN/FP
 | FN/FP
 | FN/FP
 | N/A
 | N/A
 | N/A |
| Clotted/layered
 | FN/FP | FN/FP | FN/FP | FN/FP | FN/FP | N/A
 | N/A
 | N/A |

- P/A = present/absent, FN = False Neg., FP = False Positive

## Conclusions

**Quantity Insufficient**
- Bias 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:

## Conclusions

**Diluted, discolored, or contaminated**
- MS analytes largely unaffected
- Qualitative results unaffected (however, no positive specimens were tested)
- T4 and TSH results show large variation in measured bias for V/I pair

- Decision making data summary:

## Conclusions

**Serum Rings**
- MS 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 pair

- Decision making data summary:

## Conclusions

**Clotted or layered**
- Bias high results, may be false negative (T4) or false Positive
- Qualitative results were unaffected (however,  no positive specimens were tested)

- Decision making data summary:

## Conclusions

- Quantity Insufficient
- Bias Low results for quantitative tests (false neg. - except T4)
- Qualitative tests unaffected
- Diluted, discolored, or contaminated
- MS analytes largely unaffected
- T4 and TSH results show large variation in bias for V/I pair (+/-)
- Qualitative results unaffected
- Serum Rings
- MS analytes largely unaffected
- T4 and TSH results show large variation in bias for V/I pair (+/-)
- Qualitative results unaffected
- Clotted or layered
- Generally bias high results, may be false negative (T4) or false positive
- Some bias low results (where blood has not been layered)
- Qualitative results unaffected

## Urban Legend vs. Reality

- _Invalid ok_
- Galactosemia
- Hgb
- Biotinidase

- _Invalid __**NOT**__ ok_
- CH
- CAH
- MCAD
- PKU

## Thank you.