2 What Is Blood?Slightly alkaline fluid made up of water, cells, enzymes, proteins, glucose, hormones, organic and inorganic substancesCirculates throughout bodySupplies nutrients and oxygen to bodyRemoves wasteAlso carries hormones, vitamins, antibodies
3 Blood Cells Blood cells are made in the bone marrow Hemopoiesis Starts as a “stem cell” (blasts)Some immature blood cells stay in the bone marrow to matureOthers travel to other parts of the body to mature (lymph nodes, spleen, liver)
4 Blood CellsCells mature and differentiate into several classes of cells:Red blood cellsWhite blood cellsPlatelets
5 Red Blood Cells (Erythrocytes) Have no nucleusNot useful for DNA analysis6-8 µm in size~45% total volume of bloodMost abundant cell in the blood
6 Red Blood CellsDisk shaped cells which make up 99% of the cells in the bloodPrincipal carriers of the red colored hemoglobin molecules.Hemoglobin is an iron containing protein and binds about 97% of all oxygen in the body.
8 Hemoglobin Contains 4 subunits Globular proteins with heme Heme groups contain iron which binds O2Arterial Blood: bright red when hemoglobin carries O2Venous Blood: dark red (veins appears blue in color due to optical effects of light when hemoglobin lacks O2)Heme is a non-protein
10 Heme With Iron Iron is held in this heterocyclic porphyrin ring. The iron is bonded equally to all 4 nitrogens. 2 additional bonds are perpendicular to the plane – one is connected to the protein and the other is available to bind oxygen.
11 White Blood Cells (WBC) (Leukocytes) Produced in bone marrowWBCs have a nucleusUseful for DNA analysisVital source of defense against external organismsWhite blood cells also clean up dead cells and tissue debris that would otherwise accumulate and lead to problems.
12 White Blood Cells Five classes of leukocytes: Neutrophil Eosinophil BasophilMonocyteLymphocyte
13 Red and White Blood Cells image courtesy of the U.S. National Institutes of Health
14 PlateletsIrregularly-shaped, colorless bodies produced in the bone marrowTheir sticky surface lets them, along with other substances, form clots to stop bleedingOnly active when damage occurs to the circulatory system walls
16 Plasma Liquid portion of blood Composed of water, proteins, electrolytesblood cells and platelets are suspended in plasmaRegulates osmotic pressureThe transport medium for:GlucoseLipidsHormonesOxygen (not as much as heme)Clotting factorsWaste
17 Serum Clear liquid that is left after blood coagulates Plasma without the clotting factors
18 Forensic Significance Of Blood Hemoglobin (RBC)Peroxidase-like activity can cleave H2O2Blood Group Antigen (RBC)Bound to RBC membrane (ABO groups)DNA (WBC)Found in cells with nucleusProteins (PLASMA)Serum used in species testing
19 Forensic Testing Presumptive tests Indicates a substance is present Not specificConfirmatory testsConfirm a substance is presentSpecific
20 Presumptive Tests Kastle-Meyer (Phenolphthalein) Leucomalachite Green (LMG)HemastixLuminolOther tests:Tetramethylbenzidine (TMB)BenzidineOrtho-tolidineOrtho-toluidine
21 Presumptive Tests CATALYTIC TESTS Based on the fact that hemoglobin (and some of its derivatives) exhibit a peroxidase activityTests based on this property are generally named after the compound undergoing oxidation (benzidine, TMB, etc.) or the discoverer (Kastle-Meyer, etc.)Oxidant (hydrogen peroxide) oxidizes a colorless reagent to a colored reagentHeme catalyzes this oxidation by cleaving an oxygen from hydrogen peroxide (H2O2)
23 Kastle-Meyer Test HOW TO MAKE: Phenolphthalein + Potassium Hydroxide + ZincReflux – boil / condenseReduces phenolphthalein to phenolphthalinOxygen removed and combined with OH from potassium hydroxide – boils off as waterBecomes a colorless solutionWorking solutionPhenolphthalin + ethanol + zinc pelletsRequires a lot of time to prep ~2-3 hours for refluxing
24 Kastle-Meyer Test HOW TO STORE: Amber bottle Light affects stability Zinc pelletsBinds free oxygen, prevents oxidationRoom temp (working solution)Refrigerate (stock solution)
25 Kastle-Meyer Test HOW TO PERFORM: Place a small cutting, swabbing, or extract of the suspected bloodstain on filter paper- OR -Swab the stain using a slightly moistened swab
26 Kastle-Meyer Test 3 STEP TEST STEP 1: Add 2-3 drops of ethanol to the stain or swabbingNote: This will increase sensitivity by cleaning the area around the hemoglobin, better exposing the heme
27 Kastle-Meyer Test STEP 2: Add 2 drops of reagent Wait for ~5 seconds Note: This step aids in ruling out false positives due to the presence of chemical oxidants such as rust.
28 Kastle-Meyer Test Add 2-3 drops of 3% H2O2 STEP 3:Add 2-3 drops of 3% H2O2If immediate color change to PINK – the test is POSITIVE for the possible presence of bloodIf no color change – blood is not present or is in too limited quantity for the test to detect.Note: swab will eventually turn pink (even if negative) over time due to nature of oxidation reactions.A color change should occur in 10 seconds or less
29 Kastle-Meyer Test LIMITATIONS Sensitivity 1 in 105 to 1 in 106 on dried stainsSpecificityCan weed out false positives between steps 2 and 3Chemical oxidants, vegetable peroxidasesWill not detect differences in animal or human bloodStabilityRelatively stable if the reagents are stored separately and refrigerated.Km one of the most widely used tests due to its safety factor & sensitivitySensitivity differs in articles. I saw ~1:1000 in my training samples. Would be a good exercise to do in your own lab.Note: With all catalytic tests, high levels of bleach can cause false positives
32 Leucomalachite Green HOW TO MAKE: Malachite green + acetic acid + water + ZincReflux – boil / condenseBecomes a colorless solutionWorking solutionLMG reagent + zinc pelletsSimilar to KM prep – requires time
33 Leucomalachite Green HOW TO STORE: Amber bottle Zinc pellets Relatively stable at room temp
34 Leucomalachite Green HOW TO PERFORM: Place a small cutting, swabbing, or extract of the suspected bloodstain on filter paper- OR -Swab the stain using a slightly moistened swab
35 Leucomalachite Green HOW TO PERFORM: STEP 1: Add 1-2 drops of LMG reagentNote color change, if there is a color change, the test is considered inconclusive
36 Leucomalachite Green HOW TO PERFORM: STEP 2: Add 1-2 drops of H2O2 Note resultsIf color change to deep green-blue, the test is positive for the possible presence of bloodIf no color change the test is negative
37 Leucomalachite Green LIMITATIONS: Sensitivity ~1:1000 Specificity Chemical oxidants, vegetable peroxidasesWill not detect differences in animal or human bloodStabilitySimilar to KMLess specific then KM b/c doesn’t rule out vegetable peroxidases as well.
39 Hemastix Reagent strips Bottle of 50 reagent strips Store at room temp Test is based on the peroxidase activity of hemoglobinOriginally designed to detect blood in urine
40 HemastixReagent on hemastix is diisopropylbenzene dihydroperoxide and ,3’,5,5’-tetramethylbenzidine (TMB)Color change ranges from orange to greenPossibly blue with higher concentrations of blood
41 Hemastix HOW TO PERFORM: STEP 1: Slightly moisten the pad on the tip of the strip with waterSTEP 2:Rub the damp pad on the stain in questionNote color change within 60 seconds and compare to the chart on the bottlemore green indicates more hemoglobin
42 Hemastix LIMITATIONS Sensitivity = 0.015-0.062 mg/dL free hemoglobin SpecificityChemical oxidants, vegetable peroxidasesWill not detect differences in animal or human bloodStabilityStable for ~ 1 year – date stamped expiration date on bottle
45 LuminolHOW IT WORKS:The iron in hemoglobin acts as a catalyst to cause a reaction between the luminol and H2O2.Luminol loses nitrogen and hydrogen and gains oxygen.This results in 3-aminopthalate which is energized and emits light
46 Luminol HOW TO MAKE: Reagents needed: Luminol (3-aminophthalhydrazide) Sodium PerborateDistilled waterSodium Carbonate
47 Luminol HOW TO STORE: Spray bottle works best for testing Make each time you use it (daily)
48 Luminol HOW TO PERFORM: STEP 1: Spray the luminol directly onto the stain in questionNote: This test needs to be done in the dark to see the luminescence reaction which can last for approximately 15 secondsIf the stain emits a light then the test result is POSITIVE for the possible presence of bloodIf there is no reaction the result is NEGATIVE
49 Luminol LIMITATIONS: Sensitivity 10-6 to 10-8 – most sensitive presumptive testSpecificityMany false positives – bleach, metals, chemical oxidants, vegetable peroxidasesWill not detect differences in animal or human bloodStabilityVery unstable ~8 hour limitMostly used at crime sceneCan dilute out stain (possibly too much for DNA analysis)Used more for blood spatter, crime scene reconstructionIf a stain is invisible to the naked eye and then you douse it with luminol, will dilute out any remaining stain. Not much hope for obtaining enough sample to get a DNA profile.
51 Tetramethylbenzidine (TMB) HOW TO MAKE:3,3’, 5,5 ‘-tetramethylbenzidine (TMB) + glacial acetic acidEasy to make compared to KM and LMG reagents
52 Tetramethylbenzidine (TMB) HOW TO STORE:Amber bottleLight affects stabilityRefrigerate between uses – only good for ~ 1 week.Best to make fresh for each use - will decrease in sensitivity
53 Tetramethylbenzidine (TMB) HOW TO PERFORM:Place a small cutting, swabbing, or extract of the suspected bloodstain on filter paper- OR -Swab the stain using a slightly moistened swab
54 Tetramethylbenzidine (TMB) HOW TO PERFORM:STEP 1:Add one drop of TMB solutionSTEP 2:Add one drop of 3% H2O2Detect color change:If the stain turns blueish green, the test result is POSITIVE for the possible presence of bloodNEGATIVE if no color change
55 Tetramethylbenzidine (TMB) LIMITATIONSSensitivity1:10,000 on dried stainsSpecificityNot as specific as KM testFalse positives to vegetable peroxidases, bleach, potassium permanganateWill not detect differences in animal or human bloodPossibly unsafe – labs beginning to eliminate use and use LMG or hemastix
56 Tetramethylbenzidine (TMB) LIMITATIONSStabilityVery unstable – 1 week maximumLoses sensitivity by a factor of 10 after 1 daySafetyMutagen
57 Other TestsBenzidineOrtho tolidine (o-tolidine) - 3,3'-DimethylbenzidineIncreased sensitivity, decreased specificity, and same stability when compared to Kastle-MeyerRarely used due to safety concerns- carcinogenic
58 Species Testing Definitions: Antigen (Ag) – a soluble protein that causes the production of an antibody. Usually foreign to the body.Antibody (Ab) – A “Y” shaped immunoglobulin produced by B-lymphocytes in the bone marrow.Produced in response to an antigen.
60 Species TestingAg and Ab bind together to create a lattice network of moleculesThis becomes insoluble and precipitates out of solution
61 Species Testing To make the antibodies: Host animal Goat, rabbit Inject with human serumMakes antibodies in responseCollect heart blood and test for AbSample extracts are made from stained areas of interest, and from nearby unstained areas (substrate controls). Note that the use of unstained controls is a fundamental principle in forensic immunologic testing.
62 Tube MethodThe original precipitin reaction was carried out by layering a solution of antibody on top of a solution of stain extract in a tubeThis mixture was left for a period of time to allow the development of a precipitin band at the interface.This is referred to as the tube method, and is still used in a few laboratories today
63 OuchterlonyMethod published in 1948 and uses radial diffusion of the antigen and antibody through agar gel.How to perform:Extract the stain in a microcentrifuge tube with saline. Extract should be straw colored.Stain and controls samples are loaded in the outer wells and a drop of anti-human antiserum is loaded into the center well. The process is repeated for antisera to other species, such as dog, cat, and cow; this may include the species from which the antiserum was obtained (e.g., rabbit).
64 OuchterlonyThe plates are left at room temperature or 37°C for a suitable period (which can range from a few hours to overnight) and the serum proteins and antibody molecules diffuse outward from the wells. A precipitin band is formed when the diffusing stain contains proteins that are recognized by IgG molecules in the diffusing antiserum. The precipitin band is sometimes clearly visible to the naked eye, but it is normal to stain the plates with amido black, Coomassie Blue, or other general protein stain, to enhance sensitivity and clarity.
65 OuchterlonyIf a white line occurs between the samples, then the test sample is positive for human antigen activity (or whatever species is being tested)If no white line occurs then the test sample is negative for human antigen activity (or whatever species is being tested)
66 Ouchterlony Sensitivity 10-1 Specificity False positives: Detergents Tannic AcidFalse negatives:Excess Ab or Ag – the lattice will not form, no precipitate
68 OuchterlonyPunch holes in the plate with 1 in the center and 6 around it. Add the Ab to the center well and the stain extract and controls to the outer wells. Should use antibodies from the animal used to create the anti-human antibodies to ensure there would be no cross reaction with human blood.
70 Cross-over Electrophoresis A variant of the Ouchterlony test, which uses an electric field rather than diffusion to move the extract and antibody through the gel.Cross-over electrophoresis for species identification is conducted using agar at a pH of 8.6. Stain extracts are loaded into wells arranged in a line at the cathode end of the plate and the antiserum is loaded into wells at the anode end.
71 Cross-over Electrophoresis During electrophoresis, the electric field drives the serum proteins towards the anode, but the IgG molecules, which are essentially neutral at this pH, are driven to the cathode by the process of electroendosmosis. The antigen-antibody precipitation occurs at the interface between the two rows of wells.
72 Cross-over Electrophoresis Electroendosmosis occurs because the supporting medium acquires a net negative charge.If free, the negatively charged molecules would migrate to the anode, but this is not possible because the agar is immobilized on the plate. Instead, the effect is countered by positively charged water molecules migrating to the cathode. The migrating water molecules carry any dissolved neutral molecules (such as IgG) with them.
73 ABAcard® Hematrace® Tests for human hemoglobin (Hb) If human Hb is present – reacts with a mobile monoclonal anti-human HB antibodyForms a mobile Ag-Ab complexThis migrates to the “T” zone
74 ABAcard® Hematrace®In the “T” zone – polyclonal antihuman HB antibodies.Forms Ab-Ag-Ab complexAntibodies tagged with pink dye – upon aggregation at “T” zone – pink lineControl zone – has immobile anti-immunoglobulin which binds excess antihuman HB antibodies – form a pink line
75 ABAcard® Hematrace® To perform: Extract cutting from stain in ~300 µl bufferLeave 1-5 minutesAdd 150 µl of sample to the “S” well of the test cardWait 10 minutes – read resultsPink line in “T” and “C” zones = POSITIVEPink line in only “C” zone = NEGATIVE
76 ABAcard® Hematrace® Limitations: High Dose Hook Effect – can give a false negativeOccurs with excess hemoglobin which binds to the stationary antihuman HB Antibody in the “T” areaThis prevents the mobile Ab-Ag complex from bindingResults in no pink line = NEGATIVEFALSE NEGATIVENot a confirmatory test – gives positive result with ferret blood
79 Seratec HemDirect Hemoglobin Assay Tests for human hemoglobin (Hb)Similar to the ABAcard® Hematrace® test
80 Hematin Crystals Teichmann published method in 1853 Blood is treated with a solution of potassium bromide, potassium chloride and potassium iodide in glacial acetic acid, and is heated to react with hemoglobin. The reaction first converts the hemoglobin to hemin, and then the halides react with the hemin to form characteristic brownish-yellow rhomboid crystals.The test went through many modificationsTest did not always work even when the presence of blood was known
81 Hemochromagen Crystals Many variations were usedTakayama described his method in 1912, and it became the preferred method. His name is commonly used to describe the reagents and procedure he devised and also the hemochromogen crystals obtainedBlood is treated with a saturated dextrose solution, sodium hydroxide, pyridine and water. Ferrous iron from hemoglobin reacts with pyridine to produce red feathery crystals of pyridine ferroprotoporphyrin
82 Takayama Crystals Red feathery appearance image courtesy Steve Gilbert, M.F.S., PhD -- SUNY at Canton
83 Crystal Tests Crystals are unique to blood Not human specific Not widely used anymoreCombo KM and ABA cardTime/money constraintsDNA testing is higher primate specificUse of qPCR
85 Positive Controls Used to determine if the tests are working properly a quality control checkThis is documented in the case fileExample: Known blood on cloth or a swab
86 Negative ControlsUsed to determine if the reagents are contaminant free and working properlya quality control checkThis is documented in the case fileExample: unstained cloth or a swab
87 Substrate ControlsUsed to determine if the substrate is interfering with the testtroubleshootingThis is documented in the case fileExample: unstained area adjacent to the questioned stain being tested
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