Use of Dried Blood Spots (DBS)
Specimens for Measuring
HIV-1 Viral Load in Argentina
Centro Médico Huésped
Fundación Huésped
“Partially funded by
Fogarty International Center / NIH”
(Grant#D43TW1037)
M. Lorena Vázquez
Inés Zapiola
Ana Gun
Silvia Gómez
Alejandro Krolewiecki
Omar Sued
Sara Kaufman
Pedro Cahn
What are DBS (Dried Blood Spots)?:
Whole blood
collected and dried
on filter paper
About DBS and HIV, we know…
DBS are useful for qualitative detection of HIV antibodies, DNA and RNA.
Samples dried on filter paper remain stable for HIV-1 RNA detection.
DBS are currently used to determine HIV-1 viral load in some African
countries.
CDC Recomendations for DBS:
Dried blood spot specimens can be shipped or transported by mail
or other carriers with no reasonable expectations of occupational
exposure to blood or other potentially infectious material.
"Universal precautions" are to be followed in collecting and
preparing these specimens for shipment. Standard filter paper
collection kits should be enclosed and sealed in high quality paper
mailers ideally, extra-strong, tear-proof, air-permeable, and
water-resistant envelopes. These steps provide reasonable safety
against occupational exposure and maintain optimal specimen
integrity.

DBS can be shipped by regular mail or other carriers

DBS must be enclosed in high quality paper mailers
(htpp://www.cdc.gov/od/ohs/biosafety/driblood.htm)
Why Dried blood spots?
Laboratory tests are essential for monitoring the efficacy of
antivirals and for the optimal management of treatment.

Trained personnel

Centrifugation of samples
plasmatic

Storage of plasma in cryotubes, in freezers
viral load

Transport on dry ice

High transportation costs
HIV-1
Such conditions are often unsuitable for resource-limited countries
In Argentina
 HIV-1 viral load costs around USD 74
 Shipment of a sample from Orán
(Salta) to Buenos Aires by a private
courier costs around USD 60
(an additional cost equivalent to the
75% of the value of the viral load)
Hypothesis:
 In resource-limited settings, dried blood spots
represent an easy and suitable alternative to plasma
for the determination of HIV-1 viral load.
Objective:
 To validate a real-time determination of HIV-1 RNA
viral load in dried blood spots by comparison with
standard plasma viral load.
Study design:
82 HIV-1
infected patients
Whole blood
Whole blood to
to get DBS
get Plasma
Storage of plasma
at -70ºC
1 week
Viral load
Storage of DBS at
room temperature
vs
1 week
Viral load
At the laboratory, patients were asked to authorize the use of their
samples for this study and, in this case, they signed a consent.
Population
n= 82 patients
Mean Age
38 years (9-55)
Mean Hematocrit
40 % (25-49)
Mean CD4 (total)
424 cells/ml (0-1257)
Mean CD4 (%)
23 % (0-56)
On treatment
38 (46%)
Without treatment
44 (54%)
Specimen Preparation:
We take two whole blood samples in EDTA tubes.
Plasma: 1000 ul.
Cryopreserved until being used
PLASMATIC
Viral Load
Dried blood spots: 6 spots x 50 ul
each one. (Whatman 903® Collection Paper)
Air dried overnight.
Stored at room temperature in
specimen collection bags with
dessicant during one week.
DBS
Viral Load
DBS: Pre-Extraction Procedure





Cut out the complete blood spots (2 spots of 50 ul per
extraction) from the filter using a pair of scissors.
Add the spots into a Nuclisens® Lysis Buffer 2 ml tube.
Incubate the tubes on a roller mixer for 2 – 3 hours at room
temperature.
Centrifugate the tubes during 15 seconds at 1500 g to spin
down the fluid.
Transfer the lysate for nucleic acid extraction into a new
tube.
RNA isolation

(Nuclisens® miniMagTM)
Addition of an internal calibrator
Sample +
Internal calibrator

Incubation with magnetic silica

Washings

Elution
HIV-1 viral load
(Nuclisens® HIV-1 EasyQ)
At the same time:

Isothermical molecular amplification at 41ºC

Detection of the amplified product by “molecular beacons”
Results:
Number of undetectable and detectable cases in both types of specimens
60
50
40
Plasma
DBS
30
20
10
0
<50 copies/ml
>50 copies/ml
N
Min
Max
Mean Std. Dev Median
log plasma VL
47
2.43
5.7
4.42
0.73
4.30
0.93
log DBS VL
47
2.49
5.7
4.19
0.82
4.28
1.28
Dif log(Plasma-DBS)
47
-1.18
1.1
0.23
0.48
0.29
0.67
1 copy/ml = 1 IU/ml
IQ
Correlation between both types of specimens
Spearman`s
Rho
0.946
P<0.001
n=47
VL>50 copies/ml
0.810
P<0.01
n=31
VL<50 copies/ml
Indetectable by
two methods
6.0
5.0
4.0
3.0
Averages
2.0
Log VL Plasma
Bland Altman´s Analysis
log plasma VL – log DBS VL
n=82
2.0
3.0
4.0
Log VL DBS
5.0
6.0
Results according to range of values
45
4 samples
30
25
20
15
>50 in plasma
35
<50
IU/ml
31
35
50-400
3
1
10
401-3000
6
4
5
>3000
42
42
0
<50
51-400
401-3000
Copies/ml
>3000
n DBS
Total
82
82
n Plasma
SENSITIVITY: 92.2%
SPECIFICITY: 100%
POSITIVE PREDICTIVE VALUE: 100%
NEGATIVE PREDICTIVE VALUE: 88.6%
4 samples
n DBS
<50 in DBS
n Plasma
40
Conclusions:
1. This study demonstrates that DBS performed well as
compared to plasma in monitoring viral load.
2. Since no false positive and only 4 false negatives were found
(all with VL<3000 copies/ml), we conclude that DBS is a
simple and cheaper strategy that may be considered as an
alternative to standard VL quantification in resource-limited
settings.
3. Although sensitivity was reduced in samples with low – level
viremias, DBS would be capable of identifying patients in need
of switching to second-line ART.
Acknowledgements:

All the patients who contributed with this study

Fogarty AIDS International Training and Research Program

Fundación Huésped

bioMerieux Argentina
"We have no solutions in our hands to the world's problems.
But to face the world's problems we have our hands."
P. Menapace
Thank You very much!!!