An Analysis of Parvo B 19In Source Plasma
Andrew Conrad Ph.D.
INTRODUCTION• We are currently testing for Parvo B 19 in an
effort to remove the high titer samples from pools for manufacture.
• 40% of individual samples tested were IgG positive 75% of those had low level viremia.
• Removal of all Parvo B 19 is not feasible and would cause dramatic shortages of plasma.
OBJECTIVE
• We developed a mechanism to eliminate the high titer, antibody negative, (Infectious?) Parvo B19 samples from plasma pools for manufacture.
Robotic Pooling Device
Steps to Build Pool a (8x8x8)512 Member Pool
The 8 Tecan pipettes draw from all (colored) ROWS in COLUMN Y1 in LAYER X1 and makes 3 deposits into secondary pool vials:
A) 8 individual aliquots into ROW (Z1-Z8=colors) pool vials
B) all 8 pipettes aliquots into the single COLUMN Y1 pool vial
C) all 8 pipettes aliquot into the single LAYER X1 pool vial
“Three Dimensional” Plasma Pool Sample Matrix
8X8X8 (512)
Primary Pool X1 Y3 Z3 Found Positive
Y3Z3
X1
“Three Dimensional” Plasma Pool Matrix
• Master Pools are diluted 1:1000 (512,000 fold dilution) Sensitivity 20 copies per/Ml =10 6
• Result = Negative
conclude that all component samples are below the cutoff i.e. “Not Implicated” and
will not cause high level contamination of the pool.
• Master Pool Result = Positive
resolve through testing of diluted primary pools and the implicated sample
Resolution of the Individual Sample
• Primary pools must be diluted or quantitative analyzed or low titer samples will begin to confuse the resolution.
• Up to 30% of the samples could have low level viremia.
• Recommended cutoffs.
Parvo Investigation Algorithm
• Retrieve samples of prior and subsequent bleeds for individual Parvo PCR analysis
• Determine Parvo antibody status
• Determine Parvo DNA levels over time
• Determine prevalence of the high titer samples.
Composite Results D
on
or
1
2
3
4
5
6
7
Week
0 4 8 12 16
Positive
Negative
0 4 8 12 16
Ideal Subject
determine viremic periodand shape of curve for each donor
and
examine antibody status (IgG vs. IgM)throughout viremic window
Summary Parvo Profile
1
10
100
1000
10000
100000
1000000
10000000
100000000
1000000000
10000000000
-10 -7 0 5 31 35 39 48 56 68 73 87 94 105 112 114 136 173 203 224 234 266 381 411 438 471 492
Day
Par
vo V
iral
Cop
ies/
mL
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
IgG
IU/m
L
Parvo Copies/mL
IgG IU/mL
Day 5 IgM POS
Day 136 IgMEquivicol
Day 192 IgMNeg
Summary of 20 donors:
1.0E+00
1.0E+02
1.0E+04
1.0E+06
1.0E+08
1.0E+10
0 50 100 150 200 250 300
First appearance of IgM
First appearance of IgG
0
Summary Points
• IgM Appears within 14 days of Viremia– persists for several months
• IgG Appears after about 3 weeks– Titer: IgG remains elevated for years
• Viremia : There is an immediate burst in viral load . The Average peak viral load is > 1011
– Virus persists for years
• Viral Titers peak at about two weeks and immediately prior to IgM. Viral load then drops rapidly and remains at low levels for years. Highest titers can be above 10 10
Incidence and Prevalence of Parvo B19
• Incidence of high titer infections 2002 was 1/3281 donations. We feel this truly represents new infections because of the short duration of high viremia. (>10 6 copies /ML)
• Prevalence of donors with some viremia is 1/3
• Breakdown according to season.
2002 Parvo Incidence
0
2000
4000
6000
8000
10000
12000
Month
1 in
X
% Incidence High Titer Parvo B19
0.000%
0.010%
0.020%
0.030%
0.040%
0.050%
Month
% P
ositi
ve
2002 Incidence of Parvo B19 High Titer SamplesJan 3663Feb 3296March 2295April 3126May 2490June 2987July 4836Aug 2231Sept 5998Oct 6912Nov 10581