Presumptive blood tests

Presumptive vs confirmatory blood tests Presumptive Tests (Also known as preliminary tests, screening tests or field tests) Indicates a substance is present Not specific Provide initial information to determine what test to perform next used in combination with confirmatory tests Confirmatory Tests Confirm a substance is present specific May be more expensive, require additional equipment, and take longer

tests Kastle‐Meyer (Phenolphthalein) Leucomalachite Green (LMG) Hemastix® Tetramethylbenzidine (TMB) Luminol Fluorescein

Swab the stain using a slightly moistened (with distilled water) swab Kastle‐Meyer Test The Kastle-Meyer solution is a phenolphthalein solution which has been reduced, usually by reacting it with powdered zinc. The basis of the test is that the heme in blood reacts with the colorless phenolphthalein into bright pink phenolphthalein. Place a small cutting, swabbing, or extract of the suspected bloodstain on filter paper ‐OR ‐ Swab the stain using a slightly moistened (with distilled water) swab Add two drops of reagent and wait for about five seconds. Add two to three drops of 3% H2O2. If immediate color change to PINK–the test is POSITIVE for the possible presence of blood If no color change –blood is not present or is in too limited quantity for the test to detect. Note: The swab will eventually turn pink (even if negative) over time due to nature of oxidation reactions.

Kastle-Meyer limitations Sensitivity: 1:1000 on dried stains Specificity Can weed out false positives between steps 2 and 3 False positive in presence of cauliflower or broccoli Will not detect differences in animal or human blood https://www.youtube.com/watch?v=sF0Z 0jJGwX8

Leucomalachite Green –How to Perform Hemoglobin catalyzes the reaction between LMG and hydrogen peroxide, converting the colorless LMG into malachite green. Therefore, the appearance of a green color indicates the presence of blood Place a small cutting, swabbing, or extract of the suspected bloodstain on filter paper ‐OR ‐ Swab the stain using a slightly moistened swab Add one to two drops of LMG reagent. Note color change: If there is a color change, the test is considered inconclusive. 2. Add one to two drops of H2O2. Note the results. If color change to deep green‐blue, the test is positive for the possible presence of blood. If no color change the test is negative.

Leucomalachite Green Limitations Sensitivity: 1:1000 Specificity: Chemical oxidants, vegetable peroxidases Will not detect differences in animal or human blood

Hemastix® Reagent strips React with hemoglobin in blood Reagent on Hemastix® is diisopropylbenzene dihydroperoxide and 3,3’,5,5’‐tetramethylbenzidine (TMB) •Color change ranges from orange to green to Possibly blue with higher concentrations of blood 1. Slightly moisten the pad on the tip of the strip with water. 2. Rub the damp pad on the stain in question. Note any color change within 60 seconds and compare to the chart on the bottle. More green indicates more hemoglobin

Hemastix limitations Sensitivity = 0.015 ‐0.062 mg/dL free hemoglobin Specificity: Chemical oxidants, vegetable peroxidases Will not detect differences in animal or human blood https://www.youtube.com/watch?v=Uax90QN91Z c

Tetramethylbenzidine (TMB) Reacts with heme to produce a color change Place a small cutting, swabbing, or extract of the suspected bloodstain on filter paper ‐OR ‐ Swab the stain using a slightly moistened swab Add one drop of TMB solution. Add one drop of 3% H2O2. Detect color change: If the stain turns blue‐ish green, the test result is POSITIVE for the possible presence of blood NEGATIVE if no color change

TMB Limitations Sensitivity1:10,000 on dried stains Specificity: Not as specific as Kastle-Meyer test False positives to vegetable peroxidases, bleach, potassium permanganate Will not detect differences in animal or human blood

Luminol The iron from the hemoglobin in the blood starts the chemiluminescence reaction that causes luminol to glow, so a blue glow is produced when the solution is sprayed where there is blood. Only a tiny amount of iron is required to catalyze the reaction. Spray the luminol directly onto the stain in question. If the stain emits a light then the test result is POSITIVE for the possible presence of blood If there is no reaction the result is NEGATIVE Note: This test needs to be done in the dark to see the luminescence reaction, which can last for approximately 15 seconds.

Luminol limitations Sensitivity: (10-6 to 10-8 ) Specificity: Many false positives –bleach, metals, chemical oxidants, vegetable peroxidases Will not detect differences in animal or human blood •Mostly used at crime scene Can dilute out stain (possibly too much for DNA analysis) Used more for blood spatter, crime scene reconstruction https://www.youtube.com/watch?v=u73H-5-RP5Y

Fluorescein Reacts with red blood cells to glow Needs an alternative light source to visualize Reveals trace amounts of blood Spray the fluorescein directly onto the stain in question. If the stain emits a light then the test result is POSITIVE for the possible presence of blood If there is no reaction the result is NEGATIVE Note: This test needs to be done in the dark to see the luminescence reaction, which can last for approximately 5-7 minutes.