"Dried Blood Spots"
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