Dried Blood Spots

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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 (100L 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

				
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posted:11/24/2011
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