Chapter 3 ForChem.docx

**Chapter 3: Blood and its Analysis** **Blood** Slightly alkaline (pH: 7.2-7.4) Components: Red Blood cells, White Blood cells, Plasma, Platelets Made in the bone marrow as stem cells **Red Blood Cells** Carries oxygen from the lungs to all cells inside the body as well as transport other nutr...

**Chapter 3: Blood and its Analysis** **Blood** Slightly alkaline (pH: 7.2-7.4) Components: Red Blood cells, White Blood cells, Plasma, Platelets Made in the bone marrow as stem cells **Red Blood Cells** Carries oxygen from the lungs to all cells inside the body as well as transport other nutrients and wastes. Makes 40% of the blood's volume. Contains hemoglobin which gives its red color and enables it to carry oxygen. a.k.a. eryhtrocytes **White Blood Cells** Helps the body fight infection as part of the immune system. a.k.a. leukocytes. 1 in every 600-700 RBCs. Main types of white blood cell: Neutrophils, eosinophils and basophils Lymphocytes Monocytes **Plasma** Liquid component of blood where red blood cells, white blood cells, and platelets are suspended. Constitutes more than half of the blood's volume and is made up of mostly water that contains electrolytes and proteins. **Platelets** Helps in blood clotting, they stick together to stop the bleeding in an injury. a.k.a. thrombocytes 1 in every 20 RBCs. **Immune System** Antigens: any material that can provoke an immune response. It can be a protein/carb/lipid and are attached to surface of red blood cells. Antibodies: proteins that bind tightly to specific antigens and remove them from the bloodstream. Example: Allergic reaction antigen: pollen antibodies: IgE antibodies **Blood Types** Based on the works of **Karl Landsteiner** (1868-1943) in his research for the reason why it is not possible to transfuse blood from any one human to any other human. Chemical nature of the antigens responsible for the blood types (ABO blood group) was first discovered by **Walter Morgan** and **Winifred Watkins** in 1953. **Compatibility:** **How does all this information about blood typing help to solve crimes?** **History:** Dr. Edmond Locard (1877-1966) Pioneer in forensic science who became known as Sherlock Holmes of France. Formulated the basic principle of forensic science: "Every contact leaves a trace" Locard's exchange principle **Locard's Exchange Principle** "Wherever he steps, whatever he touches, whatever he leaves, even unconsciously, will serve as a silent witness against him. Not only his fingertips, but his hair, the fibers from his clothes, the glass he breaks, the tool mark he leaves, the paint he scratches, the blood or semen he deposits or collects. All of these and more, bear mute witness against him...Physical evidence cannot be wrong, it cannot perjure itself, it cannot be wholly absent. Only human failure to find it, study and understand it can diminish its value". -Dr. Edmond Locard **Rh Blood Typing System** **Punnet Square: Recall** **Predicting blood types:** **Predict the blood type of the offsprings from the ff parents:** A father who has a heterozygous genotype for B and a mother with blood type AB. A mother who has heterozygous genotype for A and a father who has blood type O. Cross the genes of a Rh positive (homozygous) and Rh negative parents. Use letter D and d. **Blood as evidence** Guide questions for determining the nature of a crime: Is the sample, blood? Is the sample animal blood? If the sample is animal blood, from which specie did it come from? If the sample is human blood what type is it? Can the sex, age, and race of the source of blood be determined? **Can determine the nature of crime:\ Murder\ Accidental deaths\ Sexual crimes\ Blood of pregnancy/abortion\ ** **Bloody cases:** Death due to Asphyxia: Dark fluid blood in heart chambers. Death due to drowning: Alteration in Chloride content Death due to poisoning: Carbon monoxide poisoning results in cherry pink color of blood. Cyanide poisoning: Brick red color of blood Heavy metal poisoning (i.e. lead): anemia **Blood splashes** Classified by John Glaister (1930s) into 6 distinct types: Drops on a horizontal surface. Splashes, from blood flying through the air and hitting the surface at an angle. Pools around the body, which can show if it's been dragged **Blood splashes** 4\. Spurts from a major artery or vein. 5\. Smears left by movement of a bleeding person. 6\. Trails, either in the form of smears when a bleeding body is dragged, or in droplets when it is carried. **Blood Splatter:** **Projected Blood Stain Analysis** 2 important determinations: Direction of splatter Angle of impact **Arterial or Venous Blood?** Arterial blood: Copious Bright red in colour Spurting Venous blood Dark red Oozes out gradually **Blood as evidence: Collection** Blood stains: Blood in liquid pools should be picked up on a gauze pad or other sterile cotton cloth and then allowed to air dry at room temperature. Refrigerate as soon as possible then brought to the laboratory as quickly as possible. Delays beyond 48 hours may make the samples useless. **Blood as evidence** Dried Blood stains: On clothing: if possible wrap the item in clean paper, place the article in a brown paper bag, box, seal and label container. Do not attempt to remove stains from the cloth. On small object: send the whole stained object to the laboratory after proper labeling and packaging. **Blood as evidence** Dried Blood stains: On large solid objects: scrape the stain onto a clean piece of paper, which can be folded and placed in an envelope. Do not mix dried stains. Place each stain in a separate envelope. Never attempt to wipe dried stains from an object using a moistened cloth or paper. **Standard Blood Specimens** Autopsy Blood samples: Request that pathologist obtain the sample directly from the heart into a yellow (ACD) or purple stoppered vacutainer (some labs request both). In rare cases when no liquid blood is available, ask pathologist to collect a section of liver, bone, and/or deep muscle tissue and freeze for typing **Blood as evidence** Blood samples from live individuals: If the victim is injured to the extent that a transfusion is necessary, make an effort to obtain or begin necessary procedures to obtain the pre-transfusion sample collected by the hospital. These samples are not retained for long periods by the hospital, so it is important to act promptly. Also, make sure that some bloodstained garment worn by the individual has been air dried and frozen to serve as a secondary standard. **Problems in Blood Analysis** Difficulty in searching, collection, preservation, packaging and transportation Degradation Contamination Hemolysis **Blood Examinations** Preliminary Tests: To determine whether the stain contains blood or another substance. It is used to demonstrate the presence of blood. Confirmatory Tests: To determine whether bloodstain really contains blood. Precipitin Tests: Determines whether blood is human or non-human origin, and if non-human, the specific animal from which it originated **Preliminary Tests** Benzidine Test Phenolphthalein Test Guaiacum Test Leucomalachite **Benzidine Test** Developed in 1904 by Oskar & Rudolf Adler. Most commonly used preliminary test for blood. Reagent: Benzidine solution and 3% Hydrogen peroxide. Positive result: Intense blue color Limitation: Not specific for blood. False positives from other substances like sputum, pus, nasal secretion, plant juices, and formalin can be obtained. **Phenolphthalein Tests** Alternative to benzidine test. It can detect blood in a dilution of 1:80, 000, 000 parts. Reagents: Phenolphthalein solution and 3% solution of hydrogen peroxide. Positive results: Rose color or deep pink color. Limitation: False positives can be given by copper salts and potatoes. **Luminol Test** Presumptive identification test for blood when blood evidence is wiped. Reagent: Luminol reagent Positive result: Display luminescence when viewed with black light. **Confirmatory Tests** Microscopic Tests Microchemical Test **Microscopic Tests** It differentiates mammalian, avian, piscine, and reptilian blood. **Microchemical Test** Takayama Test: can detect the presence of hemoglobin. Limitations: Less specific than some modern tests, and the crystal formation may not always occur if the sample is degraded. Wagenhaar Test: Another confirmatory test for blood presence. Limitations: It requires careful preparation, and the crystal formation may vary depending on the age and condition of the blood sample. **Determination of Sex, Race and Age** Sex determination: Presence of Davidson's body in leukocytes. Can be identified as early as 7 weeks into pregnancy. Sex chromosome. (XX-female, XY-males). Race: Contains racial genetic markers involving protein and enzyme tests that can help establish race. Age determination: Biomarkers can correlate with aging and disease and can be used to compute overall biological age. Forensic Chemistry Lecture Notes: Chapter 3. Semen and Seminalysis **Content:** - *Importance of Study in Forensic Chemistry* - *Composition and function of seminal fluid* - *Physiology of the male reproductive organ* - *Stages of semen production* - *Morphology, Physical and Chemical& Confirmatory test for seminal fluid.* **1. *Importance of Study in Forensic Chemistry*** - Treatment for infertility - Useful for assault cases where stains are left in cloth - Isolation and detection of seminal stain found on various suspected evidence i.e. clothes, bed sheet, pillow, blanket etc. - Analysis of various proteins present in semen/seminal stain - Seminal fluids are examined during rape cases, sodomy, bestiality, buccal coitus and in cases of sexual perversions ***2. Composition and function of seminal fluid*** **Semen:** Organic fluid that may contain spermatozoa. Excreted by the gonads (sexual glands) and other sexual organs of male or hermaphroditic animals which can fertilize female ova. In humans, it contains several components besides spermatozoa: proteolytic and other enzymes as well as fructose. *Haworth projection of Fructose*: Glucose versus fructose chemical structure. (A) The hemiacetal group of\... \| Download Scientific Diagram *Fisher projection of Fructose:*  Conversion: Converting Fischer, Haworth, and Chair forms of Carbohydrates - Chemistry Steps **3. *Physiology of the male reproductive organ***  **1. Penis**: the male organ for sexual intercourse. It contains many sensitive nerve endings and has 3 parts: a. Root-the base of the penis and it attaches to the wall of your abdomen. b. Body/shaft- shaped like a tube/cylinder. It consists of three internal chamber: 2 large chambers (corpora cavernosa) and corpus spongiosum. The ***corpora cavernosa*** run side by side, while the corpus spongiosum surrounds your urethra. There's a special, sponge-like erectile tissue inside these chambers. The erectile tissue contains thousands of spaces. During sexual arousal, the spaces fill with blood, and your penis becomes hard and rigid (erection). An erection allows you to have penetrative sex. The skin of the penis is loose and stretchy, which lets it change size when you have an erection. c. Glans/head- cone-shaped tip of the penis. A loose layer of skin usually covers the glans and is removed during circumcision. **2. Scrotum:** the loose, pouch-like sac of skin that hangs behind the penis. It holds the testicles (testes) as well as nerves and blood vessels. For normal sperm development, the testes must be at a temperature that's slightly cooler than body temperature. The Scrotum - Contents - Nervous Supply - TeachMeAnatomy **3. Testicles:** oval-shaped organs that lie in your scrotum. A spermatic cord holds the testicles in place and supplies them with blood. It makes the testosterone and produces sperm. Within the testicles are coiled masses of tubes that produce sperm cells through spermatogenesis.  **4. Epididymis:** A long, coiled tube that rests on the back of each testicle. It carries and stores the sperm cells. It also brings the sperm to maturity-the sperms that emerge from the testicles are immature and incapable of fertilization. Epididymitis: Causes, Symptoms, Diagnosis & Treatment **5. Vas deferens:** A long muscular tube that travels from the epididymis into the pelvic cavity just behind the urinary bladder. It transports mature sperm to the urethra in preparation for ejaculation.  **6. Ejaculatory ducts:** Each testicle has a vas deferens that joins with seminal vesicle ducts to form ejaculatory ducts. The ejaculatory ducts move through the prostate, where they collect fluid to add to semen. They empty into the urethra. **7. Urethra**: Is the tube that carries pee from the bladder outside of the body. If you have a penis, it also ejaculates semen when you reach orgasm. **8. Seminal vesicles:** are sac-like pouches that attach to the vas deferens near the base of the bladder. It makes up to 80% of ejaculatory fluid, including fructose/ Fructose is an energy source for sperm and helps them move (motility). **9. Prostate gland:** is a walnut-sized gland that rests below the bladder, in front of your rectum. It adds additional fluid to ejaculate, which helps nourish sperm. **10. Cowper glands (Bulbourethral):** Are pea-sized on the sides of the urethra, just below the prostrate. They create a clear. Slippery fluid that empties directly into the urethra. It lubricates the urethra and neutralizes any acids that may remain from urine. ***4. Stages of Semen Production*** How does sperm formation take place? Sperm production occurs in the testicles. Upon reaching puberty, a man will produce millions of sperm cells every day, each measuring about 0.002 inches (0.05 millimeters) long. There is a system of tiny tubes in the testicles. These tubes, called the seminiferous tubules, house the germ cells that hormones --- including testosterone, the male sex hormone --- cause to turn into sperm. The germ cells divide and change until they resemble tadpoles with a head and short tail. The tails push the sperm into a tube behind the testes called the epididymis. For about five weeks, the sperm travel through the epididymis, completing their development. Once out of the epididymis, the sperm move to the vas deferens. When a man is stimulated for sexual activity, the sperm are mixed with seminal fluid --- a whitish liquid produced by the seminal vesicles and the prostate gland --- to form semen. As a result of the stimulation, the semen, which contains up to 500 million sperm, is pushed out of the penis (ejaculated) through the urethra. ***5. Morphology, Physical and Chemical& Confirmatory test for seminal fluid.*** **Stages of Seminal Fluid Collection:** 1. 2. 3. 4. 5\) If semen is on an immovable surface, collect sample using an absorbent medium such as a new, sterile cotton gauze pad. 6\) Seal the container/s with evidence tape. 7\) Store the item in a secure, temperature-controlled location such as a refrigerator or climate-controlled evidence room until you are able to transport to the laboratory. **Morphology of Sperm**  **Types of sperm:** 1\. Normal sperm: A normal sperm will have an oval-shaped head, an intact midpiece and an uncoiled single tail. Sperm with normal morphology are able to swim well and in a straight line. Normal sperm will also contain healthy genetic information rather than having too many or too few chromosomes, which are common in abnormally shaped sperm. 2. Macrocephaly refers to when the sperm has a giant head. These types of sperm often carry extra chromosomes and have problems fertilizing the woman's egg. Macrocephalic sperm may be caused by homozygous mutation of the aurora kinase C gene. This means that because this kind of sperm abnormality may be genetic, fathers may be able to pass the condition on to their sons. 3\. Microcephaly is when the sperm's head is smaller than normal, also known as small-head sperm. Small-head sperm may have defective acrosome (a pocket of enzymes in the sperm head used to enter the egg) or reduced genetic material. 4. Pinhead sperm, a variation of the small-head sperm, is when the head appears as a pin with minimal to no paternal DNA content. The presence of pinhead sperm may point to a diabetic condition. 5\. Tapered head sperm are sperm with "cigar-shaped" heads that may indicate the presence of varicocele in the male or constant exposure of the scrotum to high temperature locations such as daily hot sauna. These tapered head sperm often contain abnormal chromatin or packaging of the paternal DNA genetic material. Abnormal number of sperm chromosomes called aneuploidy has been shown in tapered head sperm. 6. Thin narrow head sperm: An extreme variation of the tapered head sperm is the "thin narrow" head sperm with pathologies seemingly different from the tapered head sperm. Thin head sperm are seldom identified and they may be due to broken DNA, varicocele or disrupted head formation. 7\. Globozoospermia, or round headed sperm condition, is an abnormal sperm morphology that indicates either there is an absence of the acrosome or the sperm is missing inner parts of its head responsible for "activating" or turning on the egg and starting the fertilization process. A variation of this type of sperm is the Decondensing head sperm seen when the sperm prematurely starts to break down its nucleus and the unraveling DNA material fills the entire sperm head. 8. Headless sperm have no head at all and are called acephalic sperm or decapitated sperm syndrome. They do not have genetic material or chromosomes. This type of sperm may look like a pinhead sperm but upon close examination, one can see that there is no tiny sperm head giving the sperm the appearance of a loose piece of string. 9\. Tail-less sperm are called acaudate sperm, and these sperm are often seen during necrosis, the death of most or all of the cells in an organ. 10. Nuclear vacuoles sperm have two or more large vacuoles (cyst-like bubbles) or multiple small vacuoles in the sperm head. These nuclear vacuoles are visible under high magnification microscopy. While some studies show this type of sperm has low fertilization potential, other studies have shown no effect. Studies are still in progress. 11\. Multiple parts sperm can have multiple heads or tails. When the sperm has two heads, it is called a duplicate sperm, a condition linked to exposure to toxic chemicals, heavy metals like cesium, smoke or high prolactin hormone in the male. 12. Large swollen midpiece or sperm neck may be related to defective mitochondria, the energy-making parts of the sperm cell. It may also be a sign that the centrioles, the guidance system for moving chromosomes stored at the sperm neck, may be missing or broken. 13\. Coiled-tail sperm have been exposed to either incorrect seminal fluid conditions or the presence of bacteria. These sperm cannot swim, as their tails are damaged. Heavy smoking has been linked to the presence of coiled-tail sperm. 14. Sperm cells with short tails often have low or no motility and are called stump tail or Dysplasia of Fibrous Sheath (DFS) sperm. This tail defect happens during late spermiogenesis or sperm cell formation in the testis. Some patients with DFS also have chronic respiratory disease associated with immotile cilia syndrome. Stump-tail or DFS is an autosomal recessive genetic disease connected with abnormal genes that encode for proteins such as the sperm neck centrin 1 protein. DFS has been linked to a higher percentage of sperm aneuploidies or abnormal chromosomes. ***How to keep your sperms healthy:*** - Exercising regularly - Avoiding self-medicated testosterone steroid use, heavy tobacco use, drinking or illegal drugs - Reducing the amount of caffeine consumed - Losing weight, if needed - Avoiding hot tubs - Decreasing stress - Wearing loose, cotton boxer shorts - Eating foods or supplements rich in antioxidants every day. **Physical Properties of Seminal Fluid** Forensic semen analysis \| PPT **Chemical Properties of Seminal Fluid**  **Examination of Semen** 1\. Physical/Visual Examination: Color: Thick, yellowish-white, glairy, opalescent secretion with a characteristic seminal odor. Semen is normally a whitish-gray color. Changes in semen color might be temporary and harmless or a sign of an underlying condition that requires further evaluation. Possible causes, by color, include: Red semen. This could be caused by inflammation of the prostate or the glands that help produce semen. While red semen can be alarming, the cause is usually harmless (benign), especially in younger men. Yellow or green semen. This could be caused by an infection, jaundice, or the presence of vitamins or medication in the semen Texture: On touch, seminal stains are starchy. Appearance: Garment stains are usually dirt, in natural light some stains are reddish colored while others are brown, yellow or faint grey in color. 2\. Microscopic Examination of Semen Sperm hunting on optical microscope slides for forensic analysis with deep convolutional networks -- a feasibility study - ScienceDirect 3\. Presumptive Test: a\. Acid Phosphate Test: This technique is widely employed for the identification of semen because of the high acid phosphatase activity of semen. The enzyme acid phosphatase of semen reacts with phosphoric acid to give a purple color.  b\. Thin Layer Chromatography (TLC): used for detection of choline and spermine. It is possible to detect both simultaneously in seminal stains. And is even possible to detect both in old seminal stain events after 5 years. There are 2 types of reagents used: a. Dragedroffs reagent b. Potassium iodoplatinato reagent Thin Layer Chromatography (TLC) c. UV Light Examination: classified into bright (white, bright blue, and pink), medium (blue, green, yellow, orange, red), dark (black, brown). 4\. Confirmatory Tests: a\. Cross-over electrophoresis: Detection of p30 semen-specific protein. Pulsed-field gel electrophoresis of semen samples DNA from fertile\... \| Download Scientific Diagram b\. Florence Test Florence reagent (Potassium Iodide+Iodine+water) is applied to the slide which produces rhomboidal shape dark crystals of choline periodide. Limited by concentration of choline which could give inaccurate result.  c\. Berberio's Test: Sample is reacted with picric acid which leads to the formation of yellow needle shaped spermine picrate crystals. Uploaded image