Instruction Manual

18
A specimen stability study was conducted to confirm that signal degradation at high temperatures would not occur
during shipping. Contrived samples for this study were prepared by spiking a SARS-CoV-2 remnant positive patient
sample into pooled remnant SARS-CoV-2 negative patient samples at concentrations targeting 2X LoD and 5-10X
LoD. The remnant patient samples used for this study included upper respiratory swabs in two different transport
media: VCM and sterile saline (0.9% NaCl). For each transport media, a total of 20 replicates at 2X LoD and 10
replicates at 5-10X LoD were tested.
This study simulated shipping conditions by cycling the samples through the following temperature excursion:
Storage Temperature
Time at Storage Temp (hours)
Total Time (hours)
40°C
8
8
22°C
4
12
40°C
2
14
30°C
36
50
40°C
6
56
Samples were tested at each timepoint with the Quest SARS-CoV-2 assay. The Ct values at each timepoint were
compared to the Ct values at time zero. All samples for both transport media remained positive at 56 hours after
cycling in and out of high temperatures. Additionally, Ct values remained less than 1 Ct between time 0 and 56 hours,
indicating acceptable specimen stability under simulated shipping conditions.
16) Pooling Validation/
Sensitivity for Pools with One Positive Sample and Three Negative Samples (n = 101)
Quest Diagnostics evaluated the sensitivity of sample pooling using positive samples collected from three different
populations with the following positivity rates: 0-3% (n=30), 3-6% (n=36) and 6-10% (n=35). The samples were
sequentially selected from de-identified specimen remnants that had been previously tested individually using the
Quest Diagnostics SARS-2-CoV RT-PCR molecular assay. Sample pools were made by combining one positive
sample and three negative samples. Each pool was tested, and agreement with the individual sample result was
calculated. Since any pool that does not yield negative results is re-tested individually, the positive percent agreement
includes all pools that were not negative (i.e., positive, inconclusive, and invalid). Of the 30 pools in the 1-3% positivity
rate group, 100% (30/30, 95%CI 88.7-100%) were not negative (30/30 were positive). Of the 36 pools in the 3-6%
prevalence group, 100% (36/36, 95%CI 90.4-100%) were not negative (36/36 were positive). Of the 35 pools in the 6-
10% prevalence group, 100% (35/35, 95%CI 90.1-100%) were not negative (33/35 were positive, and 2/35 were
inconclusive). Overall in the study, none of 101 positive specimens would have been determined to be negative when
tested in a pool of 4 samples (0/101, 95% CI 0.0-3.7%).
Sensitivity for Pools with One Positive Sample and Three Negative Samples (n = 101)
Positivity Rate
Group
n
Results of Pooled Specimens
% Positive Percent
Agreement*
Negative
Inconclusive
Positive
Invalid
1-3%
30
0
0
30
0
100% (30/30)
95%CI: (88.7-100%)
3-6%
36
0
0
36
0
100% (36/36)
95%CI 90.4-100%
6-10%
35
0
2
33
0
100% (35/35)
95%CI 90.1-100%
total
101
0
2
99
0
100% (101/101)
95%CI 96.3-100%
* Since any pool that is not negative is re-tested as an individual sample, the % Agreement
includes all pools that were not negative (i.e., positive, inconclusive and invalid).
17) Pooling Validation – Efficiency with Pooled Negative Specimens (n = 247)
Quest Diagnostics evaluated efficiency of sample pooling using negative samples collected from three different
populations with the following positivity rates: 1-3% (n=103), 3-6% (n=107) and 6-10% (n=37).The samples were
selected sequentially from de-identified specimen remnants that had been previously tested individually using the
Quest Diagnostics SARS-2-CoV RT-PCR molecular assay. Each 4-sample pool contained four negative samples.