# Presentation: 045423

## Alternate Matrices:  Analysis of Initial Test Results for Cannabinoids and Opiates

- John M. Mitchell, Ph.D.
- Drug Testing Advisory Board
- March 11, 2003

## Bayesian Analysis of Immunoassay Results

- Utilizes the Number of True Positives, True Negatives, False Positives and False Negatives to determine four parameters:
- Sensitivity
- Specificity
- Positive Predictive Value (PPV) and
- Negative Predictive Value (NPV)

## Definitions

**True positive**- A sample containing drug analyte(s) at concentration(s) equal to or greater than the defined cutoff.
**True negative**- A sample containing drug analyte(s) at concentration(s) less than the defined cutoff.
**False positive**- A sample containing drug analyte(s) at concentration(s) less than the defined cutoff (or no drug at all) but identified as positive.
**False negative**- A sample containing drug analyte(s) at concentration(s) equal to or greater than the defined cutoff but identified as negative

## Sensitivity

**Sensitivity         =**

- ________**True Positives_________**_
**True Positives + False Negatives**

**Sensitivity reflects the portion of the true positive samples that have been identified as positive.**

**(1- Sensitivity) - reflects the portion of the true positive samples that have been identified as negative**
** ****(False Negatives).**

**Notes:**

Multiply this value by 100 to obtain the percent of  the true positives identified 

## Specificity

**Specificity         =**

- ________**True Negatives_________**_
**True Negatives + False Positives**

**Specificity reflects the portion of the true negative**
**samples that have been identified as negative.**

**(1- Specificity) - reflects the portion of the true negative **
**samples that have been identified as positive **
**(False Positives).**

**Notes:**
Multiply this value by 100 to obtain the percent of  the true negatives that were identified.

## Positive Predictive Value (PPV)

**PPV         =**

- ________**True Positives_________**_
**True Positives + False Positives**

**PPV reflects that portion of the identified positives **
**that are true positives.**

**(1- PPV) - reflects that portion of the identified **
**positives that are false positives.**

**Notes:**
Multiply (1-specificity) by 100 to obtain the false positive rate

## Negative Predictive Value (NPV)

**NPV         =**

- ________**True Negatives_________**_
**True Negatives + False Negatives**

**NPV reflects that portion of the identified negative **
**samples that are true negatives.**

**(1- NPV) - reflects that portion of the identified  **
**negative samples that are false negatives.**

## Analysis of EMIT Results for THCA in Urine; PT Occasion 61 & 62

|  | Parameter | Value |
| --- | --- | --- |
| DATA | True Positives | 153 |
|  | True Negatives | 507 |
|  | False Positives | 3 |
|  | False Negatives | 0 |
| Bayesian
Analysis | Sensitivity | 1.00 |
|  | Specificity | 0.99 |
|  | Positive Predictive Value | 0.98 |
|  | Negative Predictive Value | 1.00 |

- 3 False positives due to presence of Glutaraldehyde

**Notes:**
3 False positives due to presence of Glutaraldehyde

Sensitivity is 1.00 because all of the true positives were identified.

Specificity is 0.99 because ~ 1% of the true positives were identified as false positives

PPV is 0.98 because 3 of the identified positives were false positives

NPV is 1.00 because it reflects the true neg/(true neg+false neg) and there were no false negatives

## Analysis of FPIA Results for Opiates in Urine; PT Occasion 61 & 62 

|  | Parameter | Value |
| --- | --- | --- |
| DATA | True Positives | 6 |
|  | True Negatives | 19 |
|  | False Positives | 1 |
|  | False Negatives | 0 |
| Bayesian Analysis | Sensitivity | 1.00 |
|  | Specificity | 0.95 |
|  | Positive Predictive Value | 0.86 |
|  | Negative Predictive Value | 1.00 |

## Results of Initial Tests For Cannabinoids in Urine; PT Occasion 61 & 62 

| Results | CEDIA | DRI | EMIT | FPIA | KIMS |
| --- | --- | --- | --- | --- | --- |
| True Positive | 90 | 30 | 153 | 6 | 66 |
| True Negative | 300 | 110 | 507 | 20 | 220 |
| False Positive | 0 | 0 | 3 | 0 | 0 |
| False Negative | 0 | 0 | 0 | 0 | 0 |
| Total Tests | 390 | 143 | 663 | 25 | 286 |

- MPT Occasion 61-62 Cutoffs: IT= 50 ng/mL; Confirm= 15 ng/mL)

**Notes:**
3 false positive probably the result of the presence of glutaraldehyde

## Analysis of Results from Initial Tests for Cannabinoids in Urine; PT Occasion 61 & 62 

| Parameter | CEDIA | DRI | EMIT | FPIA | KIMS |
| --- | --- | --- | --- | --- | --- |
| Sensitivity | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
| Specificity | 1.00 | 1.00 | 0.99 | 1.00 | 1.00 |
| PPV | 1.00 | 1.00 | 0.98 | 1.00 | 1.00 |
| NPV | 1.00 | 1.00 | 1.00
 | 1.00 | 1.00 |

- MPT Occasion 61-62      Cutoffs: IT= 50 ng/mL; Confirm= 15 ng/mL)

**Notes:**
All parameters .98 or better

## Results of Initial Tests For Opiates in Urine; PT Occasion 61 & 62 

| Results | CEDIA | DRI | EMIT | FPIA | KIMS |
| --- | --- | --- | --- | --- | --- |
| True Positive | 63 | 27 | 186 | 6 | 66 |
| True Negative | 210 | 90 | 620 | 19 | 220 |
| False Positive | 0 | 0 | 0 | 1 | 0 |
| False Negative | 0 | 0 | 0 | 0 | 0 |
| Total Tests | 273 | 117 | 806 | 26 | 286 |

- MPT Occasion 61-62  Cutoffs: IT = Confirm =2000 ng/mL

## Analysis of Results from Initial Tests for Opiates in Urine; PT Occasion 61 & 62 

| Parameter | CEDIA | DRI | EMIT | FPIA | KIMS |
| --- | --- | --- | --- | --- | --- |
| Sensitivity | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
| Specificity | 1.00 | 1.00 | 1.00 | 0.95 | 1.00 |
| PPV | 1.00 | 1.00 | 1.00 | 0.86 | 1.00 |
| NPV | 1.00 | 1.00 | 1.00
 | 1.00 | 1.00 |

- MPT Occasion 61-62  Cutoffs: IT = Confirm  = 2000 ng/mL

**Notes:**
All parameters 0.86 or higher.

## Cannabinoids and Opiates in Hair 

- PT Cycles 1-4
- Cutoffs for analysis
- THCA
- Initial Test- 1 pg/mg;
- Confirmation- 0.05 pg/mg
- Opiates
- Initial and Confirmation Tests – 200 pg/mg

## Analysis of Initial Tests For Cannabinoids in Hair

| Parameter | Analyte | EMIT | ELISA 1 | ELISA 2 | ELISA 3 | KIMS | RIA |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sensitivity | THCA |  | 0.43 | 0.63 | 0.54 | 0.62 | 0.67 |
|  | THC |  | 0.40 | 0.80 | 0.60 | 0.67 | 0.87 |
|  | Cannabinoids |  | 0.42 | 0.67 | 0.50 | 0.54 | 0.67 |
| Specificity | THCA |  | 0.81 | 0.95 | 0.93 | 0.94 | 0.77 |
|  | THC |  | 0.94 | 0.94 | 0.98 | 0.98 | 0.94 |
|  | Cannabinoids |  | 0.82 | 0.93 | 0.89 | 0.95 | 0.77 |

- Cutoffs: IT = 1 pg/mg; Confirm = 0.05 pg/mg

**Notes:**
THCA-  
True positives samples with THCA> 0.05
THC
True positives samples with THC > 1.0
Cannabinoids
True positives samples with THCA > 0.05 or THC > 1.0  

Bolded values: Arbitrarily set 0.8 or greater as an index of performance.
Sensitivity- 80% of true positive identified
Specificity – 80% of true negatives identified

## Analysis of Initial Tests For Cannabinoids in Hair

| Parameter | Analyte | EMIT | ELISA 1 | ELISA 2 | ELISA 3 | KIMS | RIA |
| --- | --- | --- | --- | --- | --- | --- | --- |
| PPV | THCA | ___ | 0.48 | 0.83 | 0.76 | 0.73 | 0.55 |
|  | THC | ___ | 0.60  | 0.75 | 0.90 | 0.86 | 0.76 |
|  | Cannabinoids | ___ | 0.50 | 0.80 | 0.67 | 0.78 | 0.55 |
| NPV | THCA | ___ | 0.78 | 0.85 | 0.83 | 0.90 | 0.84 |
|  | THC | ___ | 0.86 | 0.94 | 0.90 | 0.95 | 0.95 |
|  | Cannabinoids | ___ | 0.87
 | 0.95 | 0.91 | 0.95 | 0.97 |

- Cutoffs: IT = 1 pg/mg; Confirm = 0.05 pg/mg

**Notes:**
Bolded values are 0.8 or greater
PPV- 80% of the positives identified were true positives
NPV-  80% of the negatives identified were true negatives

## Analysis of Initial Tests For Opiates in Hair

| Parameter | Analyte | EMIT | ELISA 1 | ELISA 2 | ELISA 3 | KIMS | RIA |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sensitivity | Morphine | 0.47 | 0.93 | 1.00 | 0.87 | 0.79 | 0.93 |
|  | 6-AM | 0.46 | 0.92 | 1.00 | 0.85 | 0.91 | 1.00 |
|  | Codeine | 0.45 | 1.00 | 1.00 | 0.82 | 0.89 | 1.00 |
|  | Opiates | 0.41 | 0.94 | 1.00 | 0.82 | 0.71 | 0.88 |
| Specificity | Morphine | 1.00 | 0.94 | 0.95 | 0.97 | 0.96 | 0.98 |
|  | 6-AM | 0.99 | 0.93 | 0.96 | 0.96 | 0.99 | 1.00 |
|  | Codeine | 0.99 | 0.91 | 0.94 | 0.91 | 0.99 | 1.00 |
|  | Opiates | 1.00 | 0.95 | 0.94 | 0.95 | 0.93 | 0.97 |

- Cutoffs:  IT = Confirm = 200 pg/mg

**Notes:**
Question?  How could EMIT have such high values for Specificity?  This reflects that it identified the true negatives

## Analysis of Initial Tests For Opiates in Hair

| Parameter | Analyte | EMIT | ELISA 1 | ELISA 2 | ELISA 3 | KIMS | RIA |
| --- | --- | --- | --- | --- | --- | --- | --- |
| PPV | Morphine | 1.00 | 0.76 | 0.82 | 0.87 | 0.92 | 0.87 |
|  | 6-AM | 0.86 | 0.69 | 0.80 | 0.79 | 0.91 | 0.80 |
|  | Codeine | 0.83 | 0.63 | 0.71 | 0.60 | 0.73 | 0.71 |
|  | Opiates | 1.00 | 0.84 | 0.80 | 0.82 | 0.93 | 1.00 |
| NPV | Morphine | 0.89 | 0.98 | 1.00 | 0.97 | 0.96 | 0.98 |
|  | 6-AM | 0.90 | 0.98 | 1.00 | 0.97 | 0.99 | 1.00 |
|  | Codeine | 0.92 | 1.00 | 1.00 | 0.97 | 0.99 | 1.00 |
|  | Opiates | 0.85 | 0.98 | 1.00 | 0.95 | 0.93 | 0.97 |

- Cutoffs:  IT = Confirm = 200 pg/mg

**Notes:**
Question:  How could EMIT rate so low for sensitivity and so high for PPV.  Sensitivity reflect the fraction of true positives that are identified.  PPV reflect the percent of the identified positives that are true positives.  There fore if itit only identified 40% of the true positives and had no false positives the PPV would be high.  

Data MOR  TP= 7 TN= 65 FP =0  FN =8
        COD  TP= 5 TN= 67 FP= 1  FN = 6
        6-AM TP= 6 TN= 66 FP= 1  FN =7

## Cannabinoids and Opiates in Oral Fuids

- PT Cycles 1-3
- Neat Oral Fluids
- Cutoffs for analysis
- THCA
- Initial Test- 4 ng/mL
- Confirmation- 2 ng/mL
- Opiates
- Initial and Confirmation Tests – 40 ng/mL

## Analysis of Initial Tests For THC in Oral Fluids 

| Parameter | Analyte | ELISA 1 | ELISA 2 | ELISA 3 | EIA | LF |
| --- | --- | --- | --- | --- | --- | --- |
| Sensitivity | THC | 0.33 | 1.00 | 0.70 | 0.11 |  __ |
| Specificity | THC | 1.00 | 0.98 | 0.98 | 1.00 | __  |
| Positive Predictive Value | THC | 1.00 | 0.90 | 0.86 | 1.00 | __  |
| Negative Predictive Value | THC | 0.89 | 1.00 | 0.95 | 0.86 |  __ |

- Cutoffs:  IT = 4 ng/mL; Confirm = 2 ng/mL

**Notes:**
Question  How could ELISA 1 and EIA have such low sensitivity and all other parameter be high.
Sensitivity tells us that few true positives were identified at the prescribed cutoff.
Specificity tells us that the all true negatives were identified.  
PPV tell us that all of the identified positives were true positives
NPV reflects the False negative results .  
It would appear that the tests had a higher cutoff than 4 ng/mL

## Analysis of Initial Tests For Opiates in Oral Fluids 

| Parameter | Analyte | ELISA 1 | ELISA 2 | ELISA 3 | EIA | LF |
| --- | --- | --- | --- | --- | --- | --- |
| Sensitivity | Morphine | 1.00 | 0.50 | 1.00 | 1.00 | 1.00 |
|  | Codeine | 1.00 | 0.75 | 1.00 | 0.75 | 1.00 |
|  | Total Opiates | 1.00 | 0.70 | 1.00 | 0.70 | 0.70 |
| Specificity
 | Morphine | 0.84 | 0.90 | 0.83 | 0.91 | 0.88 |
|  | Codeine | 0.88 | 0.93 | 0.86 | 0.93 | 0.91 |
|  | Total Opiates | 0.98 | 1.00 | 0.96 | 1.00 | 0.96 |

- Cutoffs:  IT =  Confirm = 40 ng/mL

## Analysis of Initial Tests For Opiates in Oral Fluids 

| Parameter | Analyte | ELISA 1 | ELISA 2 | ELISA 3 | EIA | LF |
| --- | --- | --- | --- | --- | --- | --- |
| PPV | Morphine | 0.18 | 0.14 | 0.17 | 0.29 | 0.22 |
|  | Codeine | 0.36 | 0.43 | 0.33 | 0.43 | 0.44 |
|  | Total Opiates | 0.91 | 1.00 | 0.83 | 1.00 | 0.78 |
| NPV | Morphine | 1.00 | 0.98 | 1.00 | 1.00 | 1.00 |
|  | Codeine | 1.00 | 0.98 | 1.00 | 0.98 | 1.00 |
|  | Total Opiates | 1.00 | 0.94 | 1.00 | 0.94 | 0.94 |

- Cutoffs:  IT = Confirm = 40 ng/mL

**Notes:**
How could assays have high Sensitivity and Low PPV’s?  Sensitivity reflects the portion of the true positives identified.  PPV reflect the portion of the positives that were true positives. This would mean that there were a number of false positives.
Data