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

Read more...

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

Read more...

Immunoassays

Immunoassays
By:Diane C. Jette, Ph.D.
BioMedica Diagnostics Inc.

Test Menu
* Infectious Diseases
* Cardiac Markers
* Cancer Screening
* Fertility / Hormones
* Drugs of Abuse
* Other Tests
o IgE
o Rh Factor
o Occult Blood
o TSH, T3, T4
o Blood Grouping

Test Formats Available
* Latex Agglutination
* DIPSTICK
* Card Format
* Cartridge Format
* ELISA Format with Plate Reader

Structure of IgG
(150,000 Daltons)
Antigen
Binding Site
Fab
Fc

Structure of IgM
(900,000 Daltons)
Binding Sites
Sandwich ELISA Procedure
* Antibody coated on plate.
* Incubate with sample containing antigen.
* Wash away unbound material.
* Incubate with antibody-enzyme conjugate.
* Wash away unbound conjugate.
* Add substrate.
* Enzyme acts on substrate and produces color change.
* Measure amount of color produced.
* Amount of antigen present is proportional to the amount of color produced.

ELISA: Sandwich Format
Sandwich Assay Results
Color Intensity (OD)
Antigen Concentration
Linear Range
ELISA: Inhibition Assay Procedure
* Antigen coated on plate.
* Incubate with sample containing antigen and enzyme-antibody conjugate.
* Antigen in solution binds to enzyme-antibody conjugate preventing binding to antigen on plate.
* Wash away all unbound material.
* Add substrate.
* Enzyme acts on substrate to produce a color change.
* Measure the amount of color.
* The amount of antigen present is inversely proportional to the amount of color produced.
ELISA: Inhibition Format
Inhibition Assay Results
Color Intensity (OD)
Antigen Concentration
Linear Range
Agglutination Assay Procedure
Antibody Detection
* Antigen coated on latex particles.
* Incubate with sample containing antibody.
* Antibody binding to antigen causes cross-linking of latex beads.
* Agglutination is observed.
Antigen Detection
* Antibody coated on latex particles.
* Incubate with sample containing antigen.
* Antigen binding to antibody causes cross-linking of latex beads.
* Agglutination is observed.
Latex Agglutination Assay
Latex Agglutination
Negative Result
Smooth gray appearance
Positive Result
Large aggregates in the center or periphery of the test circle
DIPSTICK Format
Absorbing Pad
Test Area
Maximum Level
Sample Pad
Negative Result
Positive Result
CARD Format
CONTROL
TEST
SAMPLE
Negative Result
CONTROL
TEST
SAMPLE
Positive Result
Cartridge Format
Negative Result
S T C
Positive Result

Immunoassays.ppt

Read more...