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Dried Blood Spots
Acknowledgements
Dr. Rachanee Chiengasong Kyle Bond
Dr. Marie Downer Dr. Joanne Mei
Debbie Kuehl Debbie Candal
Dr. Mark Rayfield Dr. Bharat Parekh
Dr. Harry Hannon Steve Soroka
Dr. Rich Respess Trudy Dobbs
Dried Blood Spots (DBS)
AKA
Guthrie cards
Filter paper disks
Applications
Antibody testing
DNA/RNA
Amplifications
Advantages of DBS
Easy to collect, store, and transport
Stable
Adaptable to a variety of techniques
Quality protocols already developed
Centralized testing
Whole blood matrix
Safety
Disadvantages of DBS
Skin puncture required
Small sample volume
Dilution for analysis
Suitability for confirmatory method
Clinical sanction of data
Analytes Measured in Dried Human Blood
on Filter Paper ~ 1
Acarboxyprothrombin Cholesterol
Acylcarnitine Cholinesterase
Adenine phosphoribosyl transferase Conjugated 1-b hydroxycholic acid
Adenosine deaminase Cortisol
Albumin Creatine kinase
a-fetoprotein Creatine kinase MM isoenzyme
Amino Acids Cyclosporin A
profiles D-penicillamine
arginine (Krebs cycle) De-ethylchloroquine
histidine/urocanic acid Dehydroepiandrosterone sulfate
homocysteine DNA (PCR)
phenylalanine/tyrosine acetylator polymorphism
tryptophan alcohol dehydrogenase
Andrenostenedione a 1-antitrypsin
Antipyrine cystic fibrosis
Arabinitol enantiomers Duchenne/Becker
Arginase muscular dystrophy
Benzoylecgonine (cocaine) glucose-6-phosphate
Biotinidase dehydrogenase
Biopterin hemoglobinopathies
C-reactive protein A,S,C,E
Carnitine D-Punjab
Carnosinase beta-thalassemia
CD4 hepatitis B virus
Ceruloplasmin HCMV
Chenodeoxycholic acid HIV-1
Chloroquine HTLV-1
Analytes Measured in Dried Human Blood
Leber hereditory optic
on Filter Paper ~ 2
Glutathione perioxidase
neuropathy Glycocholic acid
MCAD Glycosylated hemoglobin
mRNA Halofantrine
PKU Hemoglobin variants
Plasmodium vivax Hexosaminidase A
sexual differentiation Human erythrocyte carbonic anhydrase I
21-deoxycortisol
17-a hydroxyprogesterone
Desbutylhalofantrine
Hypoxanthine phosphoribosyl transferase
Dihydropteridine reductase
Diptheria/tetanus antitoxin Immunoreactive trypsin (CF)
Erythrocyte arginase Lactate
Erythrocyte protoporphyrin Lead
Esterase D Lipoproteins
Fatty acids/acylglycines (a)
Free b-human chorionic B/A-1
gonadotropin b
Free erythrocyte prophyrin Lysozyme
Free thyroxine (FT4) Mefloquine
Free tri-iodothyroine (FT3)
Netilmicin
Fumarylacetoacetase
Phenobarbitone
Galactose/gal-1-phosphate
Galactose-1-phosphate uridyl transferase Phenytoin
Gentamicin Phytanic/pristanic acid
Glucose Progesterone
Glucose-6-phosphate dehydrogenase Prolactin
Glutathione Prolidase
Analytes Measured in Dried Human Blood
Purine nucleoside phosphorylase on Filter Paper ~ 3
Quinine parainfluenza virus
Reverse tri-iodothyronine (rT3) Plasmodium falciparum
Selenium poliovirus
Serum pancreatic lipase Pseudomonas aeruginosa
Sissomicin respiratory syncytial virus
Somatomedin C rickettsia (scrub typhus)
Specific antibodies Schistosoma mansoni
adenovirus Toxoplasma gondii
anti-nuclear antibody Trepenoma pallidium
anti-zeta antibody Trypanosoma cruzi/rangeli
arbovirus vesicular stomatis virus
Aujeszky’s disease virus Wuchereria bancrofti
dengue virus yellow fever virus
Dracunculus medinensis Spectic antigens
Echinococcus granulosus hepatitis B virus
Entamoeba histolytica HIV-1
enterovirus Succinylacetone
Giardia duodenalisa Sulfadoxine
Helicobacter pylori Theophylline
hepatitus B virus Thyrotropin (TSH)
herpes virus Throxine (T4)
HIV-1 Thyroxine-binding globulin
IgE (atopic disease) Trace elements
influenza virus Transferrin
Leishmania donovani UDP-galactose-4-epimerase
leptospira Urea
measles/mumps/rubella Uroporphyrinogen I synthase
Mycobacterium leprae Vitamin A
Mycoplasma pneumoniae White blood cells
Onchocerca volvulus Zinc protoporphyrin
Applications of DBS for HIV testing
Surveillance
Quality control for HIV rapid testing
Quantitation of HIV viral load
Identification of HIV infected infants
Dried Blood Spots (DBS) -
Characteristics
Fingerstick or whole blood draw
Placed onto special collection papers
Dried properly
Stored appropriately
Inspected for quality
Tenderfoot® Lancet for Heel Sticks
Safe for obtaining blood
samples from heels of infants.
A surgical blade incises to a
standardized depth and length
An incision created to allow
blood to flow freely
A higher quality blood sample
is collected and bruising is
diminished.
Blade permanently retracts
after use for safety
Available in three incision
depths for preemies, toddlers
and full term infants.
Tenderlett® Lancet for Finger Sticks
Incision device with blade that
cuts to a controlled,
standardized depth.
Shallow incision created which
cuts more of the capillary bed
without cutting too deeply.
Blood flows more freely
providing a higher quality
blood specimen.
Blade permanently retracts
after use for safety
Available in three depths for
the appropriate patient
population.
Instructions for Specimen Collection:
Do not touch any of the filter paper circle before or after collection.
Select puncture site and cleanse with 70% isopropanol.
Use a sterile, disposable lancet with 2.0 mm, or less, point
Keep heel in down position at or below heart level.
Wipe away first blood drop.
Use second LARGE blood drop to apply to surface of filter paper
circle.
If not completely filled, add a second LARGE drop immediately.
FILL all required circles completely. FILL from only one side of the
filter paper.
Dry specimen at room temperature 3-4 hours in HORIZONTAL
position.
IMPROPERLY COLLECTED SAMPLES MUST BE REJECTED.
Tips for Specimen Collection:
Complete each item on the collection form.
Closely follow the collection instructions on the request
form.
Warm heel with a warm towel and hold heel at or below the
heart.
Fill one circle at a time.
If capillaries are used to transfer blood from heel to paper:
Capillaries must be heparinized (Do not use EDTA).
Mix capillaries well before applying blood to filter paper.
Apply blood to filter paper immediately after filling.
Do not touch capillary to filter paper.
Collection Problems
DBS -- HIV Antibody detection
Quality of collection
Antibody elution
Optimization of enzyme immunoassay (EIA) --
washing, temperature, elution, mixing
Development of miniaturized Western blot for
confirmation
Assay Optimization
Optimal antibody elution
Determination of effective specimen dilution
Assessment of antibody detection
Strongly reactive samples
Weakly reactive samples, seroconversion
Non-reactive samples
Others – HIV-2, subtypes?
Assay modifications
Number of tests to perform
DRIED BLOOD SPOT PUNCHES
ARE VOLUMETRIC MEASUREMENTS
EQUALS
Require the Same Accuracy and Precision
POTENTIAL ALIQUOT VARIABILITY
WITH DRIED-BLOOD SPOT SPECIMENS
10 µL 10 µL
=
6 mm punch 6 mm punch
Typical EIA Assay Procedure for Dried Blood
Spots
Punch 3 or 6 mm disks into microwell plate
Elution Plate Assay Plate
1. Cover plate, incubate plate 90 min at 37oC
1. Add Kit Diluent (150 uL, 1:30) 2. Wash plate 4x
2. Cover plate, incubate overnight at 4oC 3. Add IgG-Enzyme Conjugate
3. Shake plate gently to mix 4. Cover plate, incubate 30 min at 37oC
4. Add Diluent to assay plate (125 uL) 5. Add Substrate (150 uL)
5. Transfer DBS eluate (25 uL) to assay plate 6. Incubate 10 min at 25oC
(1:150 final serum dilution) 7. Add Stop Solution (150 uL)
8. Read plate at 405 nm
Variable Affecting Measurements for
Specimens Collected on Filter Paper
Homogeneity within a production lot
Homogeneity among production lots
Variance among manufacturers
Variance within a collection card
Cutting and printing process
Variable Affecting Measurements for
Specimens Collected on Filter Paper
Handling and storage of paper
Humidity condition of paper
Volume of blood collected
Hematocrit level of blood donor
Absorption time for blood
Hematocrit Effect
12
i n
Hematocrit Effect
l o
11 S&S / Whatman Comparison
4
S&S / Whatman Comparison
B
/ 10
L Blood per 1/4 inch punch
1
15
15
L
14 9
/
14
u
13
13
d
8
30 40 50 60
o
1212
% Hematocrit
l o
30 1111 40 50 60 70
% Hematocrit
B
10
10
S&S Lot W961
S&S Lot W961 Whatman Lot 64
S&S Lot W961 Whatman Lot 6411
Whatman Lot 6411
L
99
u
88
30
30 40
40 50
50 60
60 70
70
% Hematocrit
Percent Hematocrit (100L blood spot)
S&S Lot W961 Whatman Lot 6411
Spot Volume
l
11
i n c h
Spot Volume
B
S&S / Whatman Comparison
10
L Blood per 1/4 inch punch
15
S&S / Whatman Comparison
L
14
1 /4 9
u
14
8
/
13 30 40 50 60
l o o d
13
% Hematocrit
30 40 50 60 70
12 % Hematocrit
12
S&S Lot W961
S&S Lot W961 Whatman Lot 6
B
11 S&S Lot W961 Whatman Lot 6411
Whatman Lot 6411
u L
11
10
25
25 50
50 75
75 100
100 125
125
Spot Volume (uL)
Blood Spot Voulme (L)
(55% hematocrit)
S&S Lot W961 Whatman Lot 6411
Schleicher and Schuell Grade 903 Filter Paper
Lysed Red Blood Cells
1.7
1.6
Serum Volume per 1/8” Punch (L)
99%
1.5
95%
1.4
_
1.3
X
1.2
1.1 95%
99%
1.0
0.9
W W W W W W W W W W W W W W W W W
2 2 3 3 4 5 8 8 8 8 8 9 9 9 9 9 9
1 2 1 2 1 2 5 7 7 8 9 0 2 3 4 6 8
3 1 2 1 1 1 1 2 1 1 1
Lot Numbers In Chronological Order
Optimization of DBS EIA
Sample distribution DBS vs. Serum
Serum 45
40
DBS 35
30
Frequency
25
20
15
10
5
0
0.1 0.3 0.5 0.99 2 5 >10
Signal/Cutoff Ratio
N=108
DBS EIA Performance
Sensitivity -- 100%
Specificity -- 99.8%
False positive rate -- 0.15%
Repeat reactive specimens WB
confirmed -- 47%
Gold Standard
Positive Negative
True positives False negatives
Positive
A B
New
Test
False positives True negatives
Negative C D
Sensitivity = A / A+C
Specificity = D / B+D
Establishment of
PVP = A / A+B
performance NVP = D / C+D
characteristics Efficiency = A+D / A+B+C+D
Prevalance = A+C / A+B+C+D
DBS EIA problems
Insufficient washing
Spot quality
Incubation temperature
Elution/air bubbles
Splashing
DBS Western Blot Performance
Miniaturization of methods for low sample volumes
Reduction of background reactivity
Assay optimization
Presence of non-viral bands
Detection of HIV antibodies on
miniaturized Western blot
gp160
gp120
p66
p55/51
gp41
p31
p24
p17
Quality Control for DBS
Evaluation of kit controls
DBS controls performed in duplicate
All DBS controls must be properly classified
Low DBS controls monitor EIA performance and eluate
stability
High DBS controls may be used as Western blot controls
High DBS controls should be included with frozen specimens
to monitor long-term stability
Quality Control for DBS (Cont.)
Specimens should be separated from the filter
paper if testing will not be completed immediately
Specimens should be stored in microvolume tubes
with caps equipped with rubber gaskets.
Store short-term at 4oC and long-term and –20oC
Blank wells for serum controls
(must follow configuration of plate
If spots are not white after
as directed by EIA kit)
elution, insufficient elution may
have occurred. Record
Initial EIA observations.
Dried Blood Spot (DBS) controls
in duplicate: High Positive (HPC)
Low Positive (LPC)
Negative (NC)
Elution Plate with Initially
REACTIVE EIA Samples
Retrieve reactive
Retrieve eluates of each DBS
eluates from each plate
control from each plate
Transfer eluates to microtubes; store at 4oC
Repeat EIA in duplicate on all reactive
Samples and on DBS LPC in tandem
If EIA plate consists only of repeat
DBS samples, a set of new DBS
controls is not required; load serum
controls as directed by kit.
REPEAT EIA
RESULTS INTERPRETATION AND ACTION
Both DBS LPC’s
reactive, both sample Report as NEGATIVE
replicates nonreative
IMMUNOBLOT
Both DBS LPC’s
reactive, one or both
Repeatedly reactive ALL repeatedly reactive eluates
replicates from same
elution plate reactive
One or both DBS LPC’s from
original elution plate
ANY eluates with suspect
nonreactive; results of Suspect repeat EIA
repeat EIA results
eluates from this elution plate
may be unreliable
CONTROLS
DBS HPC eluates from
oldest elution plate
Serum HPC, LPC and NC
on each membrane
DBS Collection Forms
Detection of antibodies to other
viruses in DBS eluates
HTLV-I
Hepatitis A, B, and C
Measles, mumps and rubella
Dengue
Detection of Viral Nucleic Acid
on DBS
Extraction procedures
Boiling
Phenol/Chloroform
Glass beads/powder
Amplification protocols
Single step DNA PCR
Nested DNA PCR
RT-PCR
Precautions
Contamination
Removal of inhibitors
Chelex 100
Proteinase K
Detection of Viral Nucleic Acid
on DBS - Applications
HIV-1
Subtypes
CMV
Hepatitis C
HTLV-I
