# Presentation: 673693

## Replacement of HIV-1 p24 Antigen Screening with HIV-1 RNA Nucleic Acid Testing (NAT) for Whole Blood Donations

- S.L. Stramer, R.A. Porter, J.P. Brodsky, K.J. Waldman, R.Y. Dodd, T.A. Armstrongand G.D. Griffin

## Background

- Since the implementation of p24 HIV-1 antigen (Ag) screening in March, 1996, only 5 Ag positive window-case donations have been identified
- Due to low Ag yield and the improved sensitivity of NAT screening for HIV-1, replacement of Ag with HIV-1 NAT should be possible

## HIV-1 p24 Antigen Testing Experience

- During 2 years of testing 14 million donations (ARC), 136 samples were HIV-1 p24 Ag confirmed positive
- 58 were false positive (based on RNA negativity, lack of seroconversion, and nonreproducible Ag neutralization) = 1:241,379
- 74 were HIV Ab positive
- 4 were recently infected seroconverting donors
- As of November 1999, 5 seroconverting donors identified in 23 million donations
- 1:4.6 million (ARC)
- 1:9.2 million (US)

## Samples for Testing

- p24 Ag and HIV NAT reactive yield samples (N=4)
- Tested undiluted and diluted 1:128 in RNA-negative defibrinated plasma
- Commercial seroconversion samples (N=25)
- Tested undiluted and diluted 1:128
- Diluted p24 Ag “External Control” sample that is tested on all NAT runs (N=437)
- 3 Ag+/Ab– units (S/CO 1-2 Coulter) pooled
- Pool diluted 1:16

## Comparison of HIV-1 p24 Ag Positive Window-Case U.S. Blood Donors

***Comparison of HIV-1 p24 Ag Positive Window-Case U.S. Blood Donors***

- 1	Index	**6 x 10****5*******	**14.0***	95	0.1		12	**3 x 10****4**	**1.2**	95	**2.3**	POS – minus p31	22	**1 x 10****5**	0.3	 	**5.6**	POS – all bands
- 2	Index	**2 x 10****6*******	**28.5***	94	0.8		20	**1 x 10****5**	0.7	 	**1.6**	POS – minus p17, p31	43	**1 x 10****4**	0.1	 	**5.3**	POS – all bands
- 3	Index	**1 x 10****5**	**8.1**	95	0.1		13	**5 x 10****5*******	**26.5***	90	**8.8**	POS – minus p31
- 4	Index	**1 x 10****5**	**2.4**	95	0.0		13	**1 x 10****6*******	**17.5***	89	**2.7**	IND – p66	75	**5 x 10****4**	0.1	 	**17.4**	POS – all bands
- 5	Index	**3 x 10****5**	**18.6***	99	0.3		10	**4 x 10****5*******	0.2		**4.3**	POS – minus p31	17	**1 x 10****5**	0.9	 	**12.5**	POS – minus p31	24	**3 x 10****4**	0.7	 	**8.5**	POS – minus p31	31	**2 x 10****4**	0.1	 	**13.1**	POS – minus p31

**SC**

**Sample ****Collection****(days)**

**WB**

**RNA ****Copies/mL**

**p24 Ag ****S/CO**

**Percent****Neut.**

**HIV-1/****HIV-2 Ab ****S/CO**

## Detection of HIV-1 p24 Ag and NAT Yield Samples by HIV-1 NAT (1:128 Dilution)

***Detection of HIV-1 p24 Ag and NAT Yield Samples by HIV-1 NAT (1:128 Dilution)***

- 	ARC 2	Ag	6x105	+	(16.08)
- 	ARC 3	Ag	1x105	+	(16.06)
- 	ARC 5	Ag	3x105	+	(29.59)
- 	Milwaukee	NAT	2x104	+	(7.86)

**Case**

**Index****Detected By**

**Index RNA****Conc. Undiluted****(copies/mL)**

**Multiplex****1:128*****(S/CO)**

- * Diluted in RNA screened negative defibrinated plasma

## HIV Panel 6240 – Virologic/Serologic Profile

***HIV Panel 6240 – ******Virologic/Serologic Profile***

- Days

- S/CO

- HIV PCR Quantitation

- 	PCR

- 5 Days

- 2 Days

## HIV Panel 6240 – Virologic/Serologic Profile

***HIV Panel 6240 – ******Virologic/Serologic Profile***

- Days

- S/CO

- HIV PCR Quantitation

- 2 Days 1:128

- 7 Days Neat

*E992750C  *

- 	PCR

- 5 Days

## HIV Panel PRB932 –Virologic/Serologic Profile

- 	PCR

- Days

- S/CO

- HIV PCR Quantitation

***HIV Panel PRB932 –******Virologic/Serologic Profile***

***Cutoff***

*E992750C  *

## HIV Panel PRB932 –Virologic/Serologic Profile

- 	PCR

- Days

- S/CO

- HIV PCR Quantitation

***HIV Panel PRB932 –******Virologic/Serologic Profile***

***Cutoff***

*E992750C  *

- 14 Days

## HIV Panel PRB943 –Virologic/Serologic Profile

***HIV Panel PRB943 –******Virologic/Serologic Profile***

- Days

- S/CO

- HIV PCR Quantitation

- 	PCR

***Cutoff***

*E992750C  *

- 12 Days

- 2 Days

## HIV Panel PRB943 –Virologic/Serologic Profile

- 	PCR

***HIV Panel PRB943 –******Virologic/Serologic Profile***

- Days

- S/CO

- HIV PCR Quantitation

***Cutoff***

*E992750C  *

- 5 Days

- 7 Days

- 2 Days

## Clinical Sensitivity, HIV Panels, Gen-Probe

***Clinical Sensitivity, ******HIV Panels, Gen-Probe***

- Multiplex Assay S/CO (Neat)

- p24 Antigen S/CO

***Cutoff***

***Lot 809355******HIV Seronegative Bleeds****** *****(92 samples from****25 individuals)**

- 29 NAT +/p24 Ag –

## Clinical Sensitivity, HIV Panels, Gen-Probe

***Cutoff***

***Clinical Sensitivity, ******HIV Panels, Gen-Probe***

***Lot 809355******HIV Seronegative Bleeds*****(92 samples from****25 individuals)**

- Multiplex Assay S/CO (1:128)

- p24 Antigen S/CO

- 21 NAT +/p24 Ag – 1:128

## Effect of Dilution on NAT Reactivity (Gen-Probe HIV-1/HCV Assay)

***Effect of Dilution on NAT Reactivity (Gen-Probe HIV-1/HCV Assay)***

**Lot 355**

**HIV-1 (N=25 Panels)**

**Lot 356**

**HCV (N=22 Panels)**

**Lot 355**

**Lot 356**

- Undiluted	162	167	162	160
- Diluted 1:128	145 (90%)	148 (89%)	151 (93%) 	151 (94%)
- p24 Ag	82 (57%)	82 (55%)	—	—
- ALT (60)	—	—	50 (44%)*	49 (43%)**

-   * Of 114 tested;** Of 113 tested

## Gen-Probe Linked Study – Phase I External Control Panel

***Gen-Probe Linked Study – Phase I ******External Control Panel***

**Member**

- HIV-1	300 ± 150
- HCV	 300 ± 150
- NEG	—
- Weak HIV-1 p24 Ag(S/CO 1-2 Coulterdiluted 1:16)	6,800

**Target****Copies/mL**

## Gen-Probe Linked Study – Phase IExternal Control Distributions

***Gen-Probe Linked Study – Phase I******External Control Distributions***

- Frequency

- S/CO

- 50

- 100

- 150

- 200

**Control****	****N****	****      Mean S/CO****	****    Range**
- HIV-1	435	18.66*	8.9 –	37.6
- HCV	438	8.27*	2.1 –	10.4
- NEG	436	0.28	0.1 –	0.9
- HIV-1/p24 Ag	437	20.06*	12.1 –	25.2
- *p<0.05     (437 runs total)

*E992750C  *

## Conclusions

- High RNA titers (105 copies/mL) correspond with p24 Ag positivity
- No p24 Ag reactive sample was found NAT nonreactive even using a pool size of 128
- NAT, even using pools of 128, is more sensitive than screening with the currently licensed tests for HIV-1 p24 Ag. According to these data, the Ag test could be replaced by the use of licensed pooled NAT

## Linear Relationship Between HIV RNA Levels and p24 S/CO Ratios

***Linear Relationship Between HIV RNA Levels and p24 S/CO Ratios***

**4.03 log ****(596-190,108 copies/mL) = 1 **

## 95% Prediction Limits for  Relationship of HIV RNA Levels and p24 S/CO Ratios

***95% Prediction Limits for  Relationship of HIV RNA Levels and p24 S/CO Ratios***

## HIV-1 RNA Levels During “RNA Only” and “p24 Positive” Window Periods

- 1

- 2

- 3

- 4

- 5

- 6

- 7

- 8

- Log HIV RNA Level [copies/mL]

**RNA Only (N=61)**

**p24 Pos (N=85)**

**P = <0.0001***

- * Mann-Whitney Test

***HIV-1 RNA Levels During “RNA Only” ******and “p24 Positive” Window Periods***

## Linear Relationship Between HIV-1 RNA Levels and p24 S/CO Ratios in 32 Cases

- 1

- 2

- 3

- 4

- 5

- 6

- 7

- 8

- Log RNA Level [copies/mL]

- -2

- -1.5

- -1

- -.5

- 0

- .5

- 1

- 1.5

- 2

- Log p24 S/CO

***Linear Relationship Between HIV-1 RNA Levels and p24 S/CO Ratios in 32 Cases***