Forensic Anthropology

Forensic Anthropology
Why Forensic Anthropology

* Forensic pathologists are trained to analyze soft tissue and organs. Their experience with hard tissue (bone) is limited.
* The forensic anthropologist specializes in hard tissue morphology, structure and variability. In those cases in which soft tissue has been degraded by time, temperature, environment or other external forces, the only tissue remaining more or less intact is bone.
* Physical Anthropologists and Archeologists study human remains-and have become part of solving crimes.

What Questions Can Forensic Anthropology Answer?

* What is the race of the individual?
* What is the sex of the individual?
* What is the age of the individual?
* What is the stature of the individual?
* What pathologies did the individual have?
* What traumas did the individual have?
* What individual traits did the individual have?

Identity of Decomposed or Skeletal Remains

* Are the remains human or animal? (butchers remains and skeletal remains of dead pets etc. may be found in unlikely places)
* Are they really bones? (wood, stones)
* Are they human?
* How many bodies?
* How long dead? - recent or ancient (e.g. construction or digging at an old burial site)
* Cause of death?
How does this Work?

* Forensic anthropologists use regression equations to determine sex, age, stature, and race of skeletal remains.
* Regression equations are mathematical equations developed from studies of bones of individuals of known sex, age, race, and stature, and are used to predict such things of even fragmentary skeletal remains."

Sex Estimation
* The sex of an individual is determined, when soft tissue is not present, by a number of skeletal indicators.
* The more indicators used to determine sex, the more accurate the results.
* A forensic anthropologist is analytically limited by the bones present and the condition of the bones.
* In general, the muscles in a man are stronger and more developed than in a woman.
* Bones of men are larger and more robust than bones of women.
* Some bones display specific features which can be used to help determination of the sex of the skeleton. The best indicators are the:
o Skull
o Pelvis
o Head of the Femur
Sex Estimation – Adult
* Usually related to size in adult long bones
* Male bones: usually larger, longer in a single population – be cautious if different populations are involved
* Maximum diameter of head of humerus and head of femur may be used (Bass).
* Much more difficult to estimate sex in children’s skeletons.
Sex Estimation: Skull

* Good area for sex determination
* Generalization: male skull more robust, muscle-marked than female: ABSOLUTE
* DIFFERENCES SELDOM EXIST (Bass)
* Sex estimation: face, mandible, vault

Sex Estimation: Face
1. Supraorbital (Brow) ridges: more prominent in males
2. Superior orbital margin: sharper in females
3. Palate: larger in males
4. Teeth: larger in males (Bass)
5. Mastoid process: more prominent and rugged in males.
6. Orbit (Eye socket): Rounder in females, more rectangular in males
7. Chin: more pronounced in males and larger jaws.

Pelvis
* Women give birth. For this reason, the pelvis of a woman is larger than the pelvis of a man.
* The pelvis of a woman is wide and circular whereas the pelvis of a man is narrow and heart-shaped.
* Two angles, the sub-pubic angle and the sciatic notch, cause the differences in the shape of the pelvis.
* In women, the sub-pubic angle and sciatic notch are wide. In men, the sub-pubic angle and sciatic notch are narrow.

Male Pelvis Subpubic Notch
Female Pelvis Subpubic Notch
Pubis Bone Traits Related to Sex
Subpubic angle (degrees) angle made by the inferior borders of the articulated pubis bone
Pubis body width (mm)
Ventral arc: a roughened projection of bone visible on the anterior surface of the pubis bone
Head of the Femur
* In men, the diameter of the head of the femur is larger than 51 mm.
* In women, the diameter of the head of the femur is less than 45 mm.

Determining Ages of Skeletons
* Bone growth stops at about 20 yrs. of age in humans.
* Adult bone continuously adapts to prevailing stresses by appropriate deposition and resorption.
* Deposition and resorption are under hormonal control - integrated with regulation of blood calcium levels.
Skeletal Age
* Skeletal age is the estimated age at which a person died. Skeletal age can be determined by looking at the following:
o sutures of the skull
o teeth
o ribs
o vertebrae
o growth areas of the long bones: epiphyses
Sutures of the Skull
* When a baby is born, the skull is still growing.
* To accommodate this growth, the different bones of the skull are separate.
* By the age of 7, all the different bones have finished growing and the fontanelles have disappeared.
Skull Sutures

The Teeth
* The teeth are arranged in upper and lower arches. Those of the upper are called maxillary; those of the lower are mandibular.

Dental Tissues.
* Enamel. The protective outer surface of the anatomic crown. It is 96% mineral and is the hardest tissue in the body.
* Dentin. Located in both the crown and root, it makes up the bulk of the tooth beneath the enamel and cementum. It lines the pulp cavity.
* Cementum. This substance covers the surface of the anatomic root.
* Pulp. The central, innermost portion of the tooth. It has formative, sensory, nutritive, and functions during the life of the tooth.

* There are four types of teeth with very different shapes:
* Incisors (2)
* Canines (1)
* Premolars (bicuspids) (2)
* Molars (2-3)
* Individual teeth are quite distinct, even when lost from a jaw.

Dental Formula (from the midline)

* Primary (deciduous) teeth.
* It is said as: incisors, two upper and two lower; canines, one upper and one lower; molars two upper and two lower equals ten per side.
* Permanent teeth.
* It is said as: incisors, two upper and two lower; canines, one upper and one lower; premolars, two upper and two lower; and molars, three upper and three lower.

Teeth
* The first teeth to appear are the incisors, which are followed by canines and molars.
* When chewing food, teeth grind down.
* Comparing different teeth gives an idea of how long the teeth have been used.
* Eventually teeth may be lost, due to caries or attrition.

X-Rays Are Used to Date Skulls
* This is the side view of the dentition of a six year old boy.
* There is still some variation from person to person in the order in which the teeth erupt.

Baby Teeth Permanent Teeth
Dental Disease - Cavities, Abscesses, and Attrition
Ribs
* Because of breathing, the front part of the ribs is constantly moving.
* As a person gets older, the front part of the ribs begin to change and form bony spikes.
Vertebrae
* As a person gets older, bony spikes can also start growing on the vertebrae.
* This starts at approximately 40 years of age.
Growth areas of the long bones
(epiphysis)
* From birth to ą25 years of age, a person grows at a relatively constant rate.
* Growth takes place at the ends of the long bones.
* At a certain age, growth is completed and this can also be seen on the bone.

Epiphyseal Fusion
* The pattern of fusion of bone ends (epiphysis) to bone shaft (metaphysis) in each bone indicates age.
* Charts & tables are used.
* The upper arm stops growing at the shoulder at approximately age 20 and at the elbow at approximately age 14.5.
* The upper leg stops growing at the hip at approximately age 17.5 and at the knee at approximately age 18.

Determining Ages of Skeletons
* Cranial suture fusion is less reliable.
* Pubic symphysis changes slightly with age.
* Arthritic changes and osteoporosis give further clues.

Arthritic changes and osteoporosis give further clues to the ages of skeletons.
Ossification Centers
* Useful only in fetuses and babies.
* May be determined radiologically or by cutting into ossification centers.
* May be confirmed histologically.
* Most important center in medico-legal work is the distal center of the femur.
* This is present at birth and indicates a full term baby.
Age Determination from Skeleton
* Long bone length (femur, tibia, humerus) is proportional to height.
* Tables are used.
* Fairly reliable up to the age of epiphyseal fusion.
* There are sex, race, nutrition and personal variations to consider.
Individual Characteristics
Fractures
Head Injuries
Individual Characteristics
* Bone disease (Paget's disease, tumors)
* Previous injury to bone (fracture callus, prosthesis, metallic fragments).
* Comparison of trabecular pattern of bone.
* Pattern of skull's frontal air sinuses. Outline is unique and comparisons with clinical X-rays are useful.
Forensic Dentistry
* Teeth are commonly used to establish identity of deceased.
* Dental X-rays and dental casts are available often for 10 years after a patient visits the dentist last..
Height
Body Type
Race or Ethnic Group Determination
Minimum Number of Individuals
Dating of Human Skeletal Remains
Age of Human Remains
* Naked eye appearance is unreliable:
* Tags of soft tissue, periosteum, ligaments etc, indicate less than 5 years old.
* Soapy texture of surface indicates age less than a few decades.
* Light, crumbling bones are likely to be a century or more old.

Laboratory Tests Can Help
* Immunological reaction between bone extract and anti human serum ceases within months of death.
* If blood pigments are present bones are usually less than 10 years old.
* Up to 20 amino acids may be identified in bones less than a century old.
* Fluorescence of freshly sawn bone surface under UV light diminishes after 100 years.
* New bones contain 4.0 - 4.5 gms% nitrogen; 2.5 gms% indicates approximately 350 years.
* Radioactive carbon dating indicates which century.
Taphonomy
* Coined from the Greek words taphos, for "burial," and nomos, for "law."
* Forensic Taphonomy : The Postmortem Fate of Human Remains
* Skeletal trauma, decomposition, and dispersal of remains.
* Weathering, a taphonomic process, is very useful in determining the elapsed time since death.
Facial Reconstruction
* Skull can be scanned into a computer and "fleshed" by computer reconstruction to give likely facial appearance in life.
* Unfortunately eye color, hair color and lips are independent of bony structure.
* Pearl was a female who died in her early forties approximately three hundred years ago.
* She was Caucasian, of European ancestry and stood about 5'1".
* Her dental health was extremely poor and she had lost 63 per cent of her teeth prior to death. She had no teeth on either side of her jaw. This was most important as the loss of those teeth would evidence themselves in the final reconstruction as sunken cheeks. Of her remaining teeth, the condition was poor and she had several abscesses.
* During her lifetime, there are indicators that she also suffered from acute infections, rickets, sinusities, an upper respiratory infection, arthritis, and gout. Whew--all this in an era when aspirin didn't exist!
* On the other hand, it was determined that she was very muscular, as the ridges on her long bones were very developed.

Various items (glasses, clothing, hats) may be applied to better accentuate the features of the individual.
This method can be very successful.
Cause of Death
* Anthropologists can distinguish between marks from the result of a weapon attack and those resulting from the gnawing and biting of bones by scavenging animals.
* They can also determine the exact kind of weapon and animal, and they can tell if a wound is old or if it occurred at death.
* They can be called upon to testify as to the type of weapon used (saw vs. knife).

Lizzie Bordon’s Father’s skull: Ax Whacked.

* Two outstanding cases of the use of forensic anthropology to successfully solve unsolved mysteries are the cases of Francisco Pizarro.

Pizarro conquered the Incas.
* Pizarro was hated by the Peruvians because he was a brutal ruler. On June 26, 1541 (at age 66), he was stabbed to death by a crowd of angry subjects and in view of many witnesses (Dickerson 1993).
* His brutal death is not questioned due to the well documentation at the time. It was his remains that were questioned.
* In the 1890's, Peruvian officials decided to put Pizarro's remains on exhibit. "They asked officials at the Cathedral of the Plaza de Aramis in Lima for Pizarro's body and were directed to a mummy, which they put on view." (Dickerson 1993)
* In 1978 workers discovered a secret niche that had been walled over in the cathedral, and on a shelf in the niche was a box with a skull and an inscription that identified it as the head of Pizarro.
* Another box was found containing the bones of several unidentified individuals (Dickerson 1993).

* An investigation of the bones in the second box led to the discovery that the postcranial bones matched the skull in the first box.
* These bones and the skull were then placed together and prepared for study to determine if they had marks consistent with sword or knife wounds.

Forensic Anthropology.ppt

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Vitamins

Definition and Classification
* Non-caloric organic nutrients
* Needed in very small amounts
* Facilitators – help body processes proceed; digestion, absorption, metabolism, growth etc.
* Some appear in food as precursors or provitamins

Definition and Classification
* 2 classes, Table 7.1
o Fat soluble:
o Water soluble:
* Fat soluble vitamins
o Found in the fats and oils of food.
o Absorbed into the lymph and carried in blood with protein transporters = chylomicrons.
o *Stored in liver and body fat and can become toxic if large amounts are consumed.
* Water soluble vitamins
o Found in vegetables, fruit and grains, meat.
o Absorbed directly into the blood stream
o Not stored in the body and toxicity is rare. Alcohol can increase elimination, smoking, etc. cause decreased absorption.

Fat Soluble Vitamins
* Vitamin A (precursor – beta carotene)
o 3 forms: retinol (stored in liver), retinal, retinoic acid
o Roles in body:
+ Regulation of gene expression
+ Part of the visual pigment rhodopsin, maintains clarity of cornea (yes eating carrots is good for your eyesight)
+ Required for cell growth and division - epithelial cells, bones and teeth
+ Promotes development of immune cells, especially “Natural Killer Cells”
+ Antioxidant
* Vitamin A
o Deficiencies cause:
+ Night blindness, xerophthalmia (keratin deposits in cornea), macular degeneration.
+ Skin and mucous membrane dryness and infection, keratin deposits.
+ Anemia
+ Developmental defects – bones, teeth, immune system, vision

o Toxicities (RetinA/Accutaine, single large doses of supplements, eating excessive amounts of liver) cause:
+ Fragile RBCs, hemorrhage
+ Bone pain, fractures
+ Abdominal pain and diarrhea
+ Blurred vision
+ Dry skin, hair loss
+ Liver enlargement
o DRI: 700(women)-900(men) micrograms/day, UL 3000 micrograms
o Sources, see snapshot 7.1

* Vitamin D – precursor is cholesterol, converted by UV from sunlight exposure, therefore is a “non-essential” vitamin.
o Roles:
+ Increases calcium absorption in bone, intestines, kidney. Promotes bone growth and maintenance.
+ Stimulates maturation of cells – heart, brain, immune system, etc.

o Deficiencies: rickets (children), osteomalacia (adults). What are some of the causes of deficiencies?
o Toxicities (5X DRI)
+ Loss of calcium from bone and deposition in soft tissues.
+ Loss of appetite, nausea and vomiting, psychological depression.

Bowed legs – Characteristic of rickets

Beaded ribs – Characteristic of rickets
* Vitamin D
o DRI – 5 micrograms/day for ages 19-50, 10 for ages 51-70, 15 for ages >70.
o Sources, see snapshot 7.2

Fat Soluble Vitamins
* Vitamin E – tocopherol, *alpha-, beta -, gamma-, and delta-
o Roles:
+ Antioxidant (protects polyunsaturated fats)
+ Prevention of damage to lungs, RBCs, WBCs (immunity), heart
+ Necessary for normal nerve development
* Vitamin E
o Deficiencies (decreased absorption of fats- liver disease, low fat diets)
+ Premature babies – fragile RBCs (hemolysis)
+ Loss of muscle coordination, vision, immune functions
o Toxicities (more than 1000 milligrams/day)
+ Increases the effects of anticoagulants (Coumadin, Warfarin)
o DRI 15 milligrams/day (alpha-tocopherol)
o Sources, see snapshot 7.3
* Vitamin K – produced by bacteria in large intestine
o Roles
+ Promotes synthesis of blood clotting proteins (**Interferes with Coumadin)
+ Bone formation
o Deficiencies are rare but seen in infants, after prolonged antibiotic therapy, and in patients with decreased bile production.
o Toxicities (>1000 mg/day): rupture of RBCs and jaundice

o DRI: 90(women) – 120(men) micrograms/day
o Sources, see snapshot 7.4

Water Soluble Vitamins
* 8 B vitamins – Tender Romance Never Fails with 6 to 12 Beautiful Pearls (Thiamin, Riboflavin, Niacin, Folate, B6, B12, Biotin, and Pantothenic acid)
o Aid in metabolism of and energy release from carbohydrates, lipids, amino acids.
o Mode of action – coenzymes or parts of coenzymes that are necessary for the proper activity of enzymes, Without the coenzyme, compounds A and B don’t respond to the enzyme.

With the coenzyme in place, compounds A and B are attracted to the active site on the enzyme, and they react.
The reaction is completed with the formation of a new product. In this case, the product is AB.
Muscles and other tissues metabolize protein.
Brain and other tissues metabolize carbohydrates.
Bone tissues make new blood cells.
Liver and other tissues metabolize fat.
Digestive tract lining replaces its cells.

* Thiamin and Riboflavin
o Roles – energy metabolism in cells, part of nerve cell membranes.
o Deficiencies
+ Beri beri, edema &/or muscle weakness
+ Alcohol abuse – Wernicke-Korsakoff syndrome
o DRI thiamin:1.1(women) – 1.2(men) mg/day; riboflavin 1.1(women) – 1.3(men) mg/day
o Sources: All food groups except fats and oils
* Niacin – can be produced from the amino acid tryptophan.
o Roles: energy metabolism
o Deficiencies: Pellagra – dermatitis, diarrhea, dementia, death
o Toxicities (2 - 3X DRI): *prevents blood clotting, causes liver damage, enhances action of Coumadin
* Niacin
o DRI 14(women) -16(men) mg/day
o Sources, snapshot 7.8
+ Meats
+ Some vegetables and grains
* Folate
o Role: required for synthesis of DNA - ***pregnancy
o Deficiencies (drug interactions, smoking)
+ Anemia
+ Decreased immunity
+ Decreased digestive and cardiovascular function
+ Colon and cervical cancers
+ *Neural tube defects, ?other birth defects
o Toxicities (>1000 mg/day): rare, interferes with anticancer drugs.
o DRI 400 milligrams/day
o Sources, snapshot 7.8

* Vitamin B12 (requires intrinsic factor for absorption)
o Roles: works with folate, part of insulating sheath around nerves.
o Deficiencies:
+ Pernicious anemia
+ Paralysis
+ Nerve damage in fetus
o DRI 2.3 micrograms/day
o Sources, see snapshot 7.9
+ Meat and dairy
+ Implications for vegans??
* Vitamin B6
o Roles:
+ Conversion of amino acids to other amino acids
# Ex.: Tryptophan to niacin
+ Synthesis of hemoglobin and neurotransmitters
+ Release of glucose from glycogen
+ Immune function
+ Promotes steroid hormone activity
+ Development of nervous system
o Deficiencies
+ Anemia
+ Dermatitis
+ Muscle weakness
+ Behavioral problems
+ ?Heart disease
o Toxicities (>100 mg/day) – muscle weakness, nerve damage
o DRI 1.3 milligrams/day
o Sources, see snapshot 7.10
+ Meat and dairy
+ Vegetables and fruits
* Biotin and Pantothenic acid
o Roles:
+ Metabolism of carbohydrates, fats and proteins
+ Synthesis of lipids, neurotransmitters, steroid hormones, hemoglobin.
* “Non-B vitamins”: choline, carnitine, inositol, lipoic acid, etc. No beneficial effects proven!!

* Vitamin C, ascorbic acid – history of controversy
o Roles:
+ Connective tissue development, collagen
+ Antioxidant
+ Promotes iron absorption, immunity?
+ Protects vitamin E

o Deficiency – Scurvy (skin and mucous membrane damage), anemia.
o Toxicity (> 2grams/day) – pro-oxidant, activates oxidizing agents.
o DRI – 75(w) – (90(m) milligrams/day. Increased for smokers.
o Sources, see snapshot 7.11
o Notes: can interfere with diagnostic tests for diabetes, and blood clotting
o ??Prevents colds

Vitamin/Mineral Supplements

* Who needs them?
* Who does not need them?
* Oyo read - Controversy

Vitamins.ppt

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Coagulation Testing

Coagulation Testing
By:Diane Jette
BioMedica Diagnostics Inc.

Composition of Blood
* Formed Elements
o Erythrocytes (RBC)
o Leukocytes (WBC)
+ Neutrophils
+ Eosinophils
+ Basophils
+ Lymphocytes
+ Monocytes
o Thrombocytes (Platelets)
* Plasma
o 92% water
o 7 to 9 % of solutes are proteins
+ 55 to 60% Albumin, 15% Globulins, 4% Fibrinogen
o Non-protein nitrogen substance, Enzymes, Antibodies, Electrolytes, etc.
o Serum: No fibrinogen or Factors II, V and VIII

Hemostasis is the arrest of bleeding from an injured blood vessel
* Vasoconstriction and compression of injured vessels
* Platelets adhere to the site of injury and form a platelet plug
* Platelets release factors to augment vasoconstriction and initial vessel wall repair
* Platelets provide surface membrane sites and components for the formation of enzyme/cofactor complexes in blood coagulation reactions

Coagulation Reactions Lead to the Formation of a Blood Clot
* Two pathways: Intrinsic and Extrinsic - Coagulation Cascade
* Formation of a prothrombin activator - complex of Factor Xa, Factor Va and procoagulant phospholipid on surface of platelets.
* Prothrombin activator cleaves prothrombin into two fragments to give Thrombin.
* Thrombin cleaves small peptides from fibrinogen to form fibrin monomers that polymerize.
* Thrombin activates Factor XIII to cross-link the fibrin to form an insoluble clot.

Coagulation Cascade
* Intrinsic Pathway: (APTT)
o Factors VIII, IX, XI, and XII.
o Activated on surface of exposed endothelium.
o Complexes form on platelet phospholipids.
* Extrinsic Pathway: (PT)
o Factors IV, V, VII, X
o Activated by Tissue phospholipids (Tissue Factor or Tissue thromboplastin) released into blood as a result of tissue damage.
* Common Pathway (Thrombin Time)
o Factors I and II
o Leads to the formation of Fibrin Clot
o Thrombin time does not measure deficiencies in Intrinsic or Extrinsic pathway

The Role of Calcium
* Ca ions are needed for most of the reactions in the Coagulation Cascade
* Ca-chelating agents are used in vitro as anticoagulants (Citrate, EDTA, Oxalate)
* When Coagulation Factors are synthesized without Vitamin K they cannot bind Ca and lose enzymatic function

Regulatory Mechanisms
* Inhibition of Factor Activity
o Plasma protease inhibitors: anti-thrombin III (ATIII), *2-macroglobulin, *1 - antiprotease
o Heparin converts ATIII from a slow acting inhibitor to an instantaneous inhibitor of Thrombin, Factor Xa and Factor IXa
o Protein C and Protein S are serine proteases that cleave Factors VIII and Factor Va rendering them inactive

Fibrolysis
* Fibrin clot is degraded by protolytic enzymes and fragments dissolved in blood
* Process is catalyzed by Plasmin
* Plasminogen is converted to Plasmin
* Activation by tissue plasminogen activator (tPA) and urokinase
* Fibron degrades into large fragments X and Y then smaller fragments D and E

Regulation of Fibrolysis
* Plasminogen activator inhibitors (PAIs) and plasmin inhibitors slow the fibrolysis process
* tPA and urokinase have short half-lives and are rapidly cleared through the liver
* Unbound plasmin is instantaneously neutralized by 2-antiplasmin

Hereditary Coagulation Disorders
* Hemophilia A
o Factor VIII deficiency
o 80% of all Hemophilia cases
* Hemophilia B
o Factor IX deficiency
* Prolonged ATPP
o Recovered by dilution 1:1 with normal plasma
* Normal PT and Normal Bleeding Time
* Factor XI Deficiency
o 5 to 9% of European Jews
* 2-antiplasmin Deficiency

Acquired Coagulation Disorders
* Liver Disease
o Impaired clotting Factor synthesis
o Increased fibronolysis
o Thrombocytopenia
* Desseminated Intravascular Coagulation (DIC)
o Something enters the blood that activates factors
o Complication of obstetrics, infection, malignancy, shock, severe brain trauma
o Elevated PT, APTT, D-Dimer and other fibron degradation products

Circulating Anticoagulants
* Antibodies that neutralize clotting factor activity
* Factor VIII Anticoagulants
o Antibody
o Same profile as Hemophilia A
o Clotting time not restored by mixing with normal plasma
o Life-threatening condition

Lupus Anticoagulants
* Antibodies to phospholipid binding sites on clotting factors
* Prevent factors from accumulating on phospholipid surfaces
* Elevated APTT clotting times not corrected with mixing with normal plasma
* PT normal or slightly elevated.
* Non-specific depression of clotting factor activities (Factors VIII, IX, XI, XII)
* Test sensitivity increased by using diluted reagent
o Dilute ATPP reagent, Russell’s viper venom time, Kaolin time
o Clotting times corrected with the addition of phospholipids

Oral Anticoagulant Therapy
* Coumadin or Warfarin
* Inhibitor of Vitamin K dependant Factor synthesis
* Oral anticoagulant
* Dose regulated by therapeutic effect
* PT assay to measure INR
* INR range established for optimum therapeutic effect (typically 2.0 to 3.0)

Prothrombin Time: PT
* PT reagent contains Calcium ions and Thromboplastin from brain tissue (Rabbit).
* Thromboplastin (Tissue Factor) protein-lipid complex found in tissues outside blood vessels.
* Measures the function of the Extrinsic Pathway.
* Sensitive to Factors IV, V, VII, X.
* Provided as a lyophilized reagent.
* Used to monitor oral anticoagulant therapy (Warfarin / Coumadin).

PT Reagent Calibration
* Reagents are calibrated against standard PT reagent established by the WHO.
* ISI = International Sensitivity Index.
* ISI is assigned by the manufacturer for each lot of reagent using reference material traceable to WHO.
* The lower the ISI the more sensitive the Reagent
o ISI of 1.8 to 2.4 = Low sensitivity (North American Standard PT)
o ISI of 1.4 to 1.8 = Average sensitivity
o ISI 1.0 to 1.4 = High Sensitivity

PT: INR Values
* INR = International Normalised Ratio.
* MNP = Mean Normal Plasma.
* INR = (PT / MNP)ISI
* An INR of 1.0 means that the patient PT is normal.
* An INR greater then 1.0 means the clotting time is elevated.

INR Calculation
* Example 1
o MNP = 12.0 s
o ISI = 1.25
o Patient Plasma = 20 s
o INR = (20.0 / 12.0)1.25 = 1.9
* Example 2
o MNP = 12.0 s
o ISI = 1.85
o Patient Plasma = 17 s
o INR = (17.0 / 12.0)1.85 = 1.9
* Example 3
o MNP = 12.0 s
o ISI = 1.4
o Patient Plasma = 20 s
o INR = (20.0 / 12.0)1.4 = 2.0
* Example 4
o MNP = 12.0 s
o ISI = 2.0
o Patient Plasma = 20 s
o INR = (20.0 / 12.0)2.0 = 2.8

Expected PT Values
* Mean Normal Plasma = 10 to 14 seconds.
* Mean Normal Plasma value varies with PT sensitivity. A high sensitivity (Low ISI) PT will give a high normal PT value (13 to 15 seconds).
* Oral anticoagulant monitoring = Target INR of 2.0 to 3.0.
* INR of greater than 5 or 5.5 = unacceptable high risk of bleeding.
* %CV between duplicates less than 5%

Performing a PT test
* Pre-warm PT reagent and sample to 37 oC
* Add 100 L sample to cuvette
* Add 200 L of PT reagent to cuvette
* Start timer
* Record time to clot in seconds
* Calculate INR
* see product insert for PT

Activated Partial Thromboplastin Time
* APTT or PTT
* Reagent contains phospholipids and a ‘surface activator’; (Ellagic Acid, Micronized Silica)
* Calcium Chloride reagent added to start the reaction.
* APTT reagent mimics the surface of a platelet.
* Measures activity of clotting factors in the Intrinsic Pathway, factors VIII, IX, XI and XII
* No WHO calibration standard

Uses of APTT
* Sensitive to 30 to 40% deficiencies of all factors except VII and XIII.
* Heparin inhibits the activity of clotting factors in the Intrinsic Pathway.
* A standard curve (APTT time vs Heparin concentration) is prepared using a heparin standard.
* APTT is also sensitive to other non-specific Factor inhibitors such as Lupis Anticoagulant.
* Can be influenced by Vitamin K deficiency and Coumadin therapy.
* Negative APTT result usually rules out Hemophilia.

Expected APTT Values
* Normal Range: 26 to 40 seconds
* Slightly Elevated: 45 to 65 seconds
* Extremely Elevated = > 70 seconds
* %CV less than 7%

Performing an APTT Test
* Pre-warm Calcium Chloride reagent to 37 oC.
* Add 100 microL of sample to cuvette.
* Add 100 microL of APTT to cuvette and incubate for 3 minutes.
* Add 100 microL of Calcium Chloride reagent and start timer.
* Record the time to clot in seconds.
* See APTT product Insert

Heparin Monitoring
* Prepare Heparin Standards
o Prepare stock heparin (10 USP units/mL)
o Prepare working heparin dilutions (0.1 to 0.8 U/mL) by diluting stock in normal plasma
o Run heparin dilutions as samples in APTT assay
* Plot the results as U/mL vs Log Clotting times
* Run patient samples in APTT assay and determine heparin concentration from the plot.

Heparin Calibration Curve
Factor Substitution Tests:
PT and APTT
* Dilute patient sample one to one with adsorbed plasma and serum to determine if normal clotting time is restored.
* Serum: Source of Factors IX, X, XI & XII
* Adsorbed Plasma: Source of Factors VIII, V, XI, & XII

Factor Plots: PT and APTT
* Dilute a normal plasma sample or control 1 to 10 in saline
* Dilute in saline: 100%, 50%, 25%, 12.5%. 6.25%, 3.12%,
* Mix one to one with a factor deficient plasma.
* Measure PT or APTT and plot Log % Factor vs Log clotting time.
* Dilute patient plasma 1 to 10 in saline.
* Mix one to one with a factor deficient plasma.
* All factors will be restored except the deficient factor. If the factor is present in test sample no reduction in clotting will be seen. (Same clotting time as the 100% standard)
* Determine percent factor from standard curve.

Factor Calibration Curve
Plasma Controls
* Used to monitor assay performance (QC)
* Made from pooled normal human plasma with some factors selectively adsorbed.
* Three levels available:
o Level 1 control represents a normal plasma
o Level 2 represents a slightly elevated plasma (INR ~ 1.5)
o Level 3 represents a severely elevated plasma (INR ~ 2.5)
* Each laboratory should establish expected ranges for PT and APTT.

Thrombin Time (TT or TAT)
* Measures common pathway.
* Fibrinogen --> Fibron Clot
* Not sensitive to deficiencies in Intrinsic or Extrinsic pathways.
* Reagent consists of animal thrombin.
* Normal Clotting time is 15 seconds.
* If elevated sample is mixed one to one with normal and re-tested. If normal clotting is not restored then an anti-coagulant is present.
Bleed Time Test
* An incision is made
* Time to stop bleeding is measured
* Normal clotting time is 7.5 minutes

D-Dimer Test
* D-Dimer is a fibrin monomer
* Product of fibrolysis
* Latex agglutination assay is used
* Anti-D-Dimer antibody coated on micro-latex beads
* Cardiac Infarction Marker

Activated Clotting Time (ACT)
* Clotting time of whole blood in the presence of silica based activator.
* Normal clotting times = 90 to 170 sec.
* Used to monitor heparin doses from 1 to 10 U/mL (APTT is sensitive to heparin at 0.2 to 1 U/mL).
* Used with invasive procedures that require on-site adjustment of heparin and protamine dosage. (ex. Cardiopulmonary bypass surgery).
* Not amenable for use with an optical instrument, too cloudy.
* Also called HMT, Heparin Management Test

Fibrin-1 Clot Detection
LIGHT SOURCE
CUVETTE
DETECTOR
Clot Detection
Time in seconds
Optical Density
Change in slope > Threshold = CLOT

Coagulation Testing.ppt

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