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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.

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Presentation on theme: "Dried Blood Spots. Acknowledgements Dr. Rachanee Chiengasong Kyle Bond Dr. Marie Downer Dr. Joanne Mei Debbie Kuehl Debbie Candal Dr. Mark Rayfield Dr."— Presentation transcript:

1 Dried Blood Spots

2 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

3 Dried Blood Spots (DBS) AKA Guthrie cards Filter paper disks Applications Antibody testing DNA/RNA Amplifications AKA Guthrie cards Filter paper disks Applications Antibody testing DNA/RNA Amplifications

4 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 Easy to collect, store, and transport Stable Adaptable to a variety of techniques Quality protocols already developed Centralized testing Whole blood matrix Safety

5 Disadvantages of DBS Skin puncture required Small sample volume Dilution for analysis Suitability for confirmatory method Clinical sanction of data Skin puncture required Small sample volume Dilution for analysis Suitability for confirmatory method Clinical sanction of data

6 Analytes Measured in Dried Human Blood on Filter Paper ~ 1 Acarboxyprothrombin Acylcarnitine Adenine phosphoribosyl transferase Adenosine deaminase Albumin  -fetoprotein Amino Acids profiles arginine (Krebs cycle) histidine/urocanic acid homocysteine phenylalanine/tyrosine tryptophan Andrenostenedione Antipyrine Arabinitol enantiomers Arginase Benzoylecgonine (cocaine) Biotinidase Biopterin C-reactive protein Carnitine Carnosinase CD4 Ceruloplasmin Chenodeoxycholic acid Chloroquine Cholesterol Cholinesterase Conjugated 1-  hydroxycholic acid Cortisol Creatine kinase Creatine kinase MM isoenzyme Cyclosporin A D-penicillamine De-ethylchloroquine Dehydroepiandrosterone sulfate DNA (PCR) acetylator polymorphism alcohol dehydrogenase  1-antitrypsin cystic fibrosis Duchenne/Becker muscular dystrophy glucose-6-phosphate dehydrogenase hemoglobinopathies A,S,C,E D-Punjab beta-thalassemia hepatitis B virus HCMV HIV-1 HTLV-1

7 Leber hereditory optic neuropathy MCAD mRNA PKU Plasmodium vivax sexual differentiation 21-deoxycortisol Desbutylhalofantrine Dihydropteridine reductase Diptheria/tetanus antitoxin Erythrocyte arginase Erythrocyte protoporphyrin Esterase D Fatty acids/acylglycines Free  -human chorionic gonadotropin Free erythrocyte prophyrin Free thyroxine (FT4) Free tri-iodothyroine (FT3) Fumarylacetoacetase Galactose/gal-1-phosphate Galactose-1-phosphate uridyl transferase Gentamicin Glucose Glucose-6-phosphate dehydrogenase Glutathione Glutathione perioxidase Glycocholic acid Glycosylated hemoglobin Halofantrine Hemoglobin variants Hexosaminidase A Human erythrocyte carbonic anhydrase I 17-  hydroxyprogesterone Hypoxanthine phosphoribosyl transferase Immunoreactive trypsin (CF) Lactate Lead Lipoproteins (a) B/A-1  Lysozyme Mefloquine Netilmicin Phenobarbitone Phenytoin Phytanic/pristanic acid Progesterone Prolactin Prolidase Analytes Measured in Dried Human Blood on Filter Paper ~ 2

8 Purine nucleoside phosphorylase Quinine Reverse tri-iodothyronine (rT3) Selenium Serum pancreatic lipase Sissomicin Somatomedin C Specific antibodies adenovirus anti-nuclear antibody anti-zeta antibody arbovirus Aujeszky’s disease virus dengue virus Dracunculus medinensis Echinococcus granulosus Entamoeba histolytica enterovirus Giardia duodenalisa Helicobacter pylori hepatitus B virus herpes virus HIV-1 IgE (atopic disease) influenza virus Leishmania donovani leptospira measles/mumps/rubella Mycobacterium leprae Mycoplasma pneumoniae Onchocerca volvulus parainfluenza virus Plasmodium falciparum poliovirus Pseudomonas aeruginosa respiratory syncytial virus rickettsia (scrub typhus) Schistosoma mansoni Toxoplasma gondii Trepenoma pallidium Trypanosoma cruzi/rangeli vesicular stomatis virus Wuchereria bancrofti yellow fever virus Spectic antigens hepatitis B virus HIV-1 Succinylacetone Sulfadoxine Theophylline Thyrotropin (TSH) Throxine (T4) Thyroxine-binding globulin Trace elements Transferrin UDP-galactose-4-epimerase Urea Uroporphyrinogen I synthase Vitamin A White blood cells Zinc protoporphyrin Analytes Measured in Dried Human Blood on Filter Paper ~ 3

9 Applications of DBS for HIV testing Surveillance Quality control for HIV rapid testing Quantitation of HIV viral load Identification of HIV infected infants Surveillance Quality control for HIV rapid testing Quantitation of HIV viral load Identification of HIV infected infants

10 Dried Blood Spots (DBS) - Characteristics Fingerstick or whole blood draw Placed onto special collection papers Dried properly Stored appropriately Inspected for quality Fingerstick or whole blood draw Placed onto special collection papers Dried properly Stored appropriately Inspected for quality

11 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.

12 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.

13 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. 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. Instructions for Specimen Collection:

14 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. 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.

15 Collection Problems

16 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 Quality of collection Antibody elution Optimization of enzyme immunoassay (EIA) -- washing, temperature, elution, mixing Development of miniaturized Western blot for confirmation

17 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 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

18 DRIED BLOOD SPOT PUNCHES ARE VOLUMETRIC MEASUREMENTS DRIED BLOOD SPOT PUNCHES ARE VOLUMETRIC MEASUREMENTS Require the Same Accuracy and Precision EQUALS

19 POTENTIAL ALIQUOT VARIABILITY WITH DRIED-BLOOD SPOT SPECIMENS = 10 µL  6 mm punch

20 1.Add Kit Diluent (150 uL, 1:30) 2.Cover plate, incubate overnight at 4 o C 3.Shake plate gently to mix 4.Add Diluent to assay plate (125 uL) 5.Transfer DBS eluate (25 uL) to assay plate (1:150 final serum dilution) Elution Plate Assay Plate 1.Cover plate, incubate plate 90 min at 37 o C 2.Wash plate 4x 3.Add IgG-Enzyme Conjugate 4.Cover plate, incubate 30 min at 37 o C 5.Add Substrate (150 uL) 6.Incubate 10 min at 25 o C 7.Add Stop Solution (150 uL) 8.Read plate at 405 nm Typical EIA Assay Procedure for Dried Blood Spots Punch 3 or 6 mm disks into microwell plate

21 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 Homogeneity within a production lot Homogeneity among production lots Variance among manufacturers Variance within a collection card Cutting and printing process

22 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 Handling and storage of paper Humidity condition of paper Volume of blood collected Hematocrit level of blood donor Absorption time for blood

23 S&S / Whatman Comparison Hematocrit Effect Percent Hematocrit (100  L blood spot)  L Blood per 1/4 inch punch S&S Lot W961 Whatman Lot 6411

24 Spot Volume S&S / Whatman Comparison Blood Spot Voulme (  L) (55% hematocrit)  L Blood per 1/4 inch punch S&S Lot W961 Whatman Lot 6411

25 W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W Schleicher and Schuell Grade 903 Filter Paper Lysed Red Blood Cells Schleicher and Schuell Grade 903 Filter Paper Lysed Red Blood Cells Lot Numbers In Chronological Order Serum Volume per 1/8” Punch (  L) 99% 95% X X _ _ 99% W W W W

26 Optimization of DBS EIA N=108

27 DBS EIA Performance Sensitivity % Specificity % False positive rate % Repeat reactive specimens WB confirmed -- 47%

28 Establishment of performance characteristics Sensitivity = A / A+C Specificity = D / B+D PVP = A / A+B NVP = D / C+D Efficiency = A+D / A+B+C+D Prevalance = A+C / A+B+C+D Sensitivity = A / A+C Specificity = D / B+D PVP = A / A+B NVP = D / C+D Efficiency = A+D / A+B+C+D Prevalance = A+C / A+B+C+D Gold Standard New Test Positive Negative Positive Negative True positives A False positives C False negatives B True negatives D

29 DBS EIA problems Insufficient washing Spot quality Incubation temperature Elution/air bubbles Splashing Insufficient washing Spot quality Incubation temperature Elution/air bubbles Splashing

30 DBS Western Blot Performance Miniaturization of methods for low sample volumes Reduction of background reactivity Assay optimization Presence of non-viral bands Miniaturization of methods for low sample volumes Reduction of background reactivity Assay optimization Presence of non-viral bands

31 Detection of HIV antibodies on miniaturized Western blot gp160 gp120 p66 p55/51 gp41 p31 p24 p17

32 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 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

33 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 4 o C and long-term and –20 o C 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 4 o C and long-term and –20 o C

34 Initial EIA Blank wells for serum controls (must follow configuration of plate as directed by EIA kit) If spots are not white after elution, insufficient elution may have occurred. Record observations. Dried Blood Spot (DBS) controls in duplicate: High Positive (HPC) Low Positive (LPC) Negative (NC) Elution Plate with Initially REACTIVE EIA Samples Retrieve eluates of each DBS control from each plate Retrieve reactive eluates from each plate Transfer eluates to microtubes; store at 4 o C 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.

35 REPEAT EIA RESULTS INTERPRETATION AND ACTION Both DBS LPC’s reactive, both sample replicates nonreative Report as NEGATIVE Both DBS LPC’s reactive, one or both replicates from same elution plate reactive One or both DBS LPC’s from original elution plate nonreactive; results of eluates from this elution plate may be unreliable Repeatedly reactive Suspect repeat EIA IMMUNOBLOT ALL repeatedly reactive eluates ANY eluates with suspect repeat EIA results CONTROLS DBS HPC eluates from oldest elution plate Serum HPC, LPC and NC on each membrane

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42 DBS Collection Forms

43 Detection of antibodies to other viruses in DBS eluates HTLV-I Hepatitis A, B, and C Measles, mumps and rubella Dengue HTLV-I Hepatitis A, B, and C Measles, mumps and rubella Dengue

44 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 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

45 Detection of Viral Nucleic Acid on DBS - Applications HIV-1 Subtypes CMV Hepatitis C HTLV-I HIV-1 Subtypes CMV Hepatitis C HTLV-I


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