Clinical Chemistry Section - Unit 1 PDF

UNIT 1: THE CLINICAL CHEMISTRY SECTION ACTIVITY 01- ENGAGE: IN THE HISTORY OF CLINICAL CHEMISTRY Clinical chemistry is a branch of medical science that involves the analysis of chemical components of body fluids....

UNIT 1: THE CLINICAL CHEMISTRY SECTION ACTIVITY 01- ENGAGE: IN THE HISTORY OF CLINICAL CHEMISTRY Clinical chemistry is a branch of medical science that involves the analysis of chemical components of body fluids. The development of scientific research in medicine and the emergence of organic & physiological chemistry have been identified as the two main origins of Clinical Chemistry. The development of Clinical Chemistry was made possible by significant contributions from biology and natural philosophy. Early beginnings: Attribution of Diseases to Imbalances of Bodily Humors vs. Anatomic Approach Hippocrates A Greek physician considered as the “Father of Medicine” and author of the Hippocratic oath (as introduced in Module 1). He started the belief that diseases are caused by imbalances of humors in the body. This belief sparked an interest among early physicians to observe body fluids. Giovanni Morgagni He introduced the anatomic approach of disease process and explained diseases in terms of localized pathologic anatomy, rather than as attributable to an imbalance of the humors diffused throughout the system. Antoine Laurent Lavoisier He is considered to be the “Father of Modern Chemistry”. He recognized and named two elements, oxygen and hydrogen. He discovered the role of oxygen in the process of combustion and that respiration is a slow combustion process. He started the belief that chemical analysis is a refined type of dissection that sparked a renewal of interest in the examination of body fluids. Before: Urine color as a diagnostic tool for liver or kidney disease Now: Liver and kidney panel tests 8 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  Vitalists, Mechanists, and Darwinists: Opposing figures Vitalists They believed that living organisms contain a “vital force” that was the very essence of life. They also believed that processes within living organisms were unique and could not be duplicated in the laboratory. To them, in vitro synthesis of “organic” compounds is impossible and denied that chemistry has a role in physiology. Vitalism was the popular belief among leading physiologists and physicians including Mari Francois Xavier Bichat, Johannes Muller, and Justus Baron von Leibig. Mechanists Mechanists believed that animals are no more than “machines” and that life could be explained fully by chemical and physical principles and properties alone. Leading figures: Rene Descartes Carl Ludwig Ernst Brucke Emil Du-Bois Reymond Darwinists Believed that man is not unique. The believe that there is continuity between man and the animals as attested by Darwin’s publication “Origin of Species” which is considered to be the foundation of evolutionary biology. Animal Chemistry and How it Slowly Toppled Vitalism Antoine Francois de Fourcroy He was successful in isolating urea from urine samples. He also believed that chemical laboratories should be located near the wards, where chemical analysis of urine excretions of the sick could be carried out. 9 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  Friedrich Wohler He was able to synthesize urea in vitro by evaporating an isomeric solution of ammonium cyanate proving that “organic” substances could be synthesized in vitro without any “vital force” in a living organism. This created a bridge between the “organic” and “inorganic” worlds. In doing so, he gave the first proof that vitalism is wrong. Marcellin Berthelot He was able to synthesize organic compounds such as ethanol, formic acid, and benzene in vitro via chemical treatments of inorganic compounds. Claude Bernard He discovered that glycogen was formed by the liver which contradicted the vitalism belief that only plants can produce complex compounds. John Bostock He was the first to observe that urea and albumin concentration in plasma decreases as their concentration increases in the urine of the patient. Chemistry in Medical Education William Prout Credited as the first to make the true connection between chemistry and medical practice and was a vitalist. However, he advocated the benefits to be derived from the application of chemistry to physiology in the treatment of diseases. Also, he favored the study of physics and chemistry by medical students. 10 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  Henry Bence Jones He stressed the practical diagnostic value of chemistry and urged medical school curriculum to use English as the medium of instruction. “Medical men would be better served if they spent some time in acquiring knowledge about chemistry and physics instead of learning some Latin and Greek.” Thomas Hodgkin “Chemical studies are relevant to clinical medicine.” “It is in the blood that we must look for many important modifications in connection with disease.” During the 19th century, the average medical student or average practitioner barely had a nodding acquaintance with chemistry and could not use a microscope. Massachusetts General Hospital: In 1847, the Hospital recognized the powerful aid that the science of medicine “has received from the study of organic chemistry and knowledge and use of the microscope”, thus authorizing the purchase of a microscope at a cost not to exceed 50 US dollars. In 1851, the position of a “Chemist-Microscopist” was established. To cope with the growing number of chemical tests, the physician would usually enlist the help of chemists orf physicians skilled in chemistry. Otto Knut Folin He proposed that the American hospitals must employ clinical chemists to advance their ability to differentiate between the physiologic and the pathologic. Clinical Chemistry Takes the Center Stage Otto Knut Folin and Donald Dexter Van Slyke Scientists who determined reference intervals of chemicals/ analytes. They correlated variations/ abnormal values with pathologic conditions. They also elucidated metabolic pathways in health and disease. 11 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  Linked chemical variations to disease processes Donald Dexter Van Slyke He invented a volumetric gas-measuring apparatus for the determination of carbon dioxide concentration. Established reference intervals for chemicals Otto Knut Folin Together with Hsien Wu, they developed a method for the production of a protein- free filtrate that can be used for determining blood sugar. He also developed the Duboscq type colorimeter for the measurement of creatinine in urine. Max Jaffe He developed the alkaline picrate method for the determination of creatinine concentration. Early Instrumentation in Clinical Chemistry Preceding the era of automation, colorimetry has been pioneered by Otto Knut Folin after his development of the duboscq-type visual colorimeter. The basic principle of colorimetry involves the observation of the intensity of colored product after chemical reactions. Another development in the early instrumentation in clinical chemistry includes the measurement of light absorbance at selected wavelengths (spectrophotometry) which was initiated by the development of the Beckman DU Spectrophotometer by Cary and Beckman. In an attempt to automate instruments used in a clinical chemistry laboratory, Auto- Analyzer was introduced. This is a continuous-flow instrument that reacted specimen and reagents to produce a measurable color density. The second attempt towards automation involved the centrifugal analyzer introduced by Norman Anderson which was the first clinical analyzer to incorporate a computer. During those times, the number of patients and the number of samples are increasing, and the previous equipment are incapable of running multiple tests. Sequential Multiple Analyzer with Computer (SMAC) was developed to address this concern and is capable of performing multiple tests analyzed one after another on a given clinical specimen. Beckman Astra also introduced the perfected technology of automated pipetting which is the approach of choice for automation in clinical chemistry laboratories even up to these days. Over the last few decades, different laboratories are required to meet very high standards in order to reduce the number of testing errors. Today presents a new world of automation. From liquid handling to releasing of results, many technologies arose with the fundamental purpose of saving time and improving performance through eliminating human errors and reduced risk of cross contamination. Clinical laboratory testing is still an unseen side of medical practice and care for many people. Laboratory results are the basis for many clinical conclusions that doctors and clinicians make regarding a person’s health. Your job as future medical laboratory scientists is to release an accurate and precise laboratory result to aid in the physician’s diagnosis. 12 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  EXPLAIN: ROUTINE TESTS IN CLINICAL CHEMISTRY SECTION Blood sugar testing is one of the most commonly performed assays in the Clinical Chemistry section. This test is ordered by physicians to detect hyperglycemic and hypoglycemic states. Hyperglycemic state refers to an increased level of blood glucose (blood sugar) while hypoglycemic state refers to low level of blood glucose(blood sugar). Blood sugar testing can be in the form of determining RBS (random blood sugar), FBS (fasting blood sugar), or performance of OGTT (Oral Glucose Tolerance Test). Random Blood Sugar test measures the levels of glucose in the blood at any given point in the day. In contrast, Fasting Blood Sugar test prohibits the patient to eat and drink any liquids other than water for at least eight hours before collection of blood. OGTT is often requested by physicians for pregnant patients to rule out or confirm diagnosis of Gestational Diabetes Mellitus. Another laboratory test that is often used for evaluating blood glucose NOTE: HbA1c (Hemoglobin A1c) is also known (blood sugar) level is the HbA1c as Glycated hemoglobin or Glycosylated (Hemoglobin A1c) test. It reflects the hemoglobin average blood glucose levels of the patient over a three-month period. The Lipid Profile Tests are also considered as routine services in the Clinical Chemistry section. It involves a combination of tests conducted together to check for any risks of cardiovascular diseases. Blood lipid profile testing include determining the concentrations of (a) fatty acid, (b) cholesterol, and (c) lipoprotein levels. Fatty acids are the simplest form of lipid but are not usually measured in the clinical laboratory. Triglycerides, on the other hand, is the storage form of fat. Elevated triglyceride levels are observed in obese or diabetic patients and are associated with heart diseases. Cholesterol is a steroid alcohol and is the precursor of hormones, vitamin D, and bile salts. Lipoproteins are special particles made up of fats and proteins. Lipoproteins are often described as carriers of cholesterol and triglycerides. The lipoproteins that are commonly measured in the clinical chemistry section are (a) Low Density Lipoprotein (LDL), (b) High Density Lipoprotein (HDL), (c) Very Low Density Lipoprotein (VLDL), and (d) Chylomicrons. LDL, also known as the “bad cholesterol”, transports cholesterol from the liver to peripheral tissues. In contrast, HDL is also referred to as the “good cholesterol”. HDL transports cholesterol from peripheral tissues back to the liver for metabolism. Once transported by HDL, cholesterol is then broken down by the liver. Chylomicrons transport exogenous triglycerides (triglycerides coming from diet) to the muscles and adipocytes. VLDL, on the other hand, is responsible for transporting endogenous triglycerides. Renal Function Tests are also included in the routine services offered by the Clinical Chemistry section. It includes Creatinine, Blood Urea Nitrogen (BUN), and Blood Uric acid (BUA) testing. Creatinine is a waste product of muscle metabolism that is elevated in impaired renal function. BUN is a waste product of protein catabolism that is also elevated in cases of kidney diseases. Elevation of BUN is termed as Azotemia. If azotemia is accompanied by renal failure, it is called as Uremia. 13 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  UNIT 2: THE HEMATOLOGY SECTION ACTIVITY 03- ENGAGE: IN THE HISTORY OF HEMATOLOGY SECTION William Harvey (1628) He discovered the closed circulation of blood and proved that blood flows into two separate loops, the pulmonary circulation and the systemic circulation. Anton van Leeuwenhoek (1674) He giave the first accurate description of red blood cells. William Hewson (1770-1773) He is considered to be the “Father of Hematology”. He was responsible for the discovery of white blood cells, lymphatic circulation, and fibrinogen (Coagulation Factor I). He also discovered fundamentals of coagulation and Glauber’s salt, which is the first anticoagulant. Anticoagulant is a substance that prevents blood from clotting of blood. Franz Ernst Christian Neumann (1868) He discovered the role of bone marrow in hematopoiesis. Hematopoiesis is the production of the cellular components of blood and blood plasma. Giulio Bizzozero In 1868, he made an independent investigation and subsequent discovery of the role of bone marrow in blood cell production. He also described platelets as “petite plaques” and described the role of platelets in hemostasis and thrombosis in 1888. Hemostasis is a process to prevent and stop bleeding, while thrombosis is the formation of blood clot known as thrombus. 16 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  Paul Ehrlich (1878) He developed the first method of blood cell staining and identified three types of granulocytes, mast cells, and megaloblasts. James Homer Wright (1902) He developed the Wright stain and the refinements thereof such as the Wright’s Romanowsky-type stain remains the foundation of blood cell identification. Milestones Leading to Automated Cell Counting in Hematology The first individual to perform a blood count was Karl Vierordt in 1852. His method involved drawing blood into a capillary tube and spreading a known volume of the collected blood onto a slide, followed by microscopic analysis. In 1896, George Oliver provided an RBC (red blood cell) count without the need The method used by George Oliver in 1896 for manual counting of individual cells. could be considered as the forerunner of His method was based on the visual automated blood count. measurement of light loss by scattering and absorption in a test tube filled with diluted blood. Oliver’s method was refined by Mercandier et al in 1928 by utilizing a photodetector for the measurement of light absorption instead of relying on unaided eyes. Coping in the advancement of instrumentation, Wallace Coulter in 1953 developed cell counting by impedance measurement. This method was based on the fact that cells are poor electrical conductors and that they manifest electrical resistance as they pass through a small aperture (opening). One of the basic building blocks of the minimum database in medicine is the complete blood count. Over the past decades, there has been a tremendous advancement in the technology of hematology analyzers and their availability to the general practitioner. Today, hematology laboratories are heading toward reliable automation to achieve faster turn-around time (TAT), reducing risks of human errors, and any possible risks of cross contamination. 17 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  EXPLAIN: THE BLOOD Hematology is the scientific study of blood and its components. The word heme is the Greek word for blood. The blood is a specialized liquid connective tissue that supplies essential substances such as sugars, oxygen, and hormones around the body. Blood is comprised of two components: the liquid extracellular matrix called plasma and the formed elements. Plasma comprises 55% of the blood volume. The plasma is composed of 91.5% water, 7% plasma proteins and 1.5% other solutes, such as electrolytes, gases and waste products. Within the context of laboratory samples, plasma is different from a serum sample. Plasma Serum Preparation Blood sample is NOT allowed to Blood sample is allowed to clot clot prior to separation from cells before separation from the clot Blood sample used for collection is anticoagulated Blood sample used for collection is NOT anticoagulated Appearance Pale yellow fluid separated from Yellow fluid separated from the after the blood cells via centrifugation blood clot via centrifugation separation Clotting Presence of fibrinogen (Factor I) Absence of fibrinogen (Factor I) factors Presence of all clotting factors Absence of Factor V, VIII, XIII, II Formed elements comprises 45% of the blood volume. The formed/cellular elements of blood are the red blood cells, white blood cells, and platelets. Red blood cells (Erythrocytes) Red blood cells are biconcave disc-shaped cells that are anucleated (have no nucleus). RBCs contain the oxygen-carrying hemoglobin, which is a pigment that gives whole blood its red color. These cells are primarily responsible for physiological gas exchange, specifically transporting oxygen from the lungs to the different parts of the body and carry carbon dioxide back to the lungs. 18 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  WBCs (Leukocytes) are nucleated cells that defend the body against infections. These cells are divided into two main types, granulocytes and agranulocytes. The cytoplasm of granulocytes contains conspicuous and easily observed granules. There are three types of leukocytes that are considered granulocytes: Neutrophils, Eosinophils, and Basophils. On the other hand, agranulocytes are leukocytes containing cytoplasmic granules that are not as obviously observed as those found in granulocytes. The agranulocytes include Monocytes and Lymphocytes. A. Granulocytes: Neutrophils/Polymorphonuclear cells (PMN)/Mature segmenters Nucleus has 2-5 lobes Cytoplasm has fine, pale lilac granules with NEUTRAL affinity for stains (thus, the name neutrophil) Phagocytic; respond to bacterial infection Comprises 50-70% of total WBC population Eosinophils Nucleus usually has 2 lobes connected by thick chromatin strand Cytoplasm contains large, red-orange granules with affinity for ACIDIC stains such as eosin (thus, the name eosinophil) Responds to parasitic & helminthic infection and allergy Also characterized to have phagocytic activity Comprises 1-3% of the total WBC population Basophils Nucleus has 2 lobes; Nucleus is not easily observed because it is often covered by large granules Cytoplasm contains water soluble blue-black granules with affinity for BASIC stains (thus, the name basophil) Involved in allergic and hypersensitivity reactions Comprises 0-2% of total WBC population 19 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  B. Agranulocytes: Monocytes Nucleus is horseshoe or kidney-shaped often with brain-like convolutions Cytoplasm is blue-gray colored and foamy and has very fine azurophilic granules responsible for the characteristic “Ground glass” appearance Are converted to macrophages as they leave the blood circulation and enter peripheral tissues Macrophages are potent phagocytes which defend the body against Mycobacterium species and other bacteria, fungi, protozoa, and viruses Comprises 2-11% of total WBC population Lymphocytes Round or slightly indented nucleus that occupies majority of the cell area Scanty cytoplasm with a characteristic “Robin’s egg blue coloration” Immunocytes Predominant WBC that responds to several viral infections Comprises 18-42% of total WBC population Platelets (Thrombocyte/Cell fragments) Platelets are cell fragments that play significant roles in hemostasis. These cells contain many vesicles but have no nucleus. When your skin is injured puncturing the vascular area, platelets clump together and form clots to stop bleeding. Routine Work: COMPLETE BLOOD COUNT (CBC) A Complete Blood Count or CBC is a commonly performed blood test that is often included as part of a routine checkup. CBC can be used to help in the detection of a variety of disorders including infections, anemia, diseases of the immune system, and blood cancers. This is a panel of tests including (a.) Hemoglobin determination , (b.) Hematocrit determination, (c.) RBC count, (d.) WBC count, (e.) WBC differential count, (f.) RBC morphology examination, (g.) Platelet count, and (h.) RBC indices. Hemoglobin determination is primarily used H/H Test, RBC and WBC counts, for the diagnosis of anemia. Anemia is a Differential count, and RBC condition in which the number of red blood cells or their oxygen-carrying pigment morphology exam are integral parts of hemoglobin is insufficient to meet CBC. physiologic needs. Anemia is defined by Due to the advent of automation in the World Health Organization (WHO) as hematology, modern CBC results also hemoglobin levels of less than 12.0 g/dL in include platelet count and RBC women and less than 13.0 g/dL in men. indices. Another parameter being used in conjunction with hemoglobin determination for the diagnosis of anemia is the hematocrit determination. The resulting tandem is then referred to as the H/H Test. Hematocrit, also known as Packed Cell Volume (PCV) or Erythrocyte Volume Fraction (EVF), is the volume percentage of RBCs in a whole blood sample. 20 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  In contrast to H/H determination, RBC count is not used by physicians for diagnosis of anemia. This is because even automated RBC counts are highly erroneous. WBC count, unlike RBC count, is a clinically significant value. Leukocytosis/ High WBC count is often seen in infections, allergy, and leukemic states. Leukopenia/ Low WBC count, on the other hand, is observed in cases of viral infections that temporarily disrupt bone marrow, autoimmune disorders, and immunodeficiency. Platelet count is the quantification of thrombocytes of the blood samples. WBC differential count (Differential count) is a routine procedure that involves observing a total of 100 WBCs and simultaneously classifying them as either neutrophils, lymphocytes, monocytes, eosinophils, and basophils. RBC morphology examination involves microscopic observation of the size and shape of the red blood cell population of the sample. RBC indices aids in morphological classification of anemia. MCV, MCH, and MCHC are commonly reported indices. Commonly reported RBC indices RBC Indices Other Name Indicative Mean Corpuscular Volume Mean Cell Volume Average volume of a single (MCV) erythrocyte in a given blood sample. Mean Corpuscular Mean Cell Hemoglobin Indicates the average weight of Hemoglobin (MCH) hemoglobin per erythrocyte. Mean Corpuscular Mean Cell Hemoglobin Indicates the average Hemoglobin Concentration Concentration concentration of hemoglobin in (MCHC) the erythrocytes. SAMPLE CBC RESULT FORM: Note that the given reference ranges in the sample form are set by the clinical laboratory. Even though there are reference ranges given in textbooks, clinical laboratories should establish their own reference ranges. 21 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  UNIT 3: THE SEROLOGY SECTION ACTIVITY 05 – ENGAGE: IN THE DEFINITION OF TERMS AND HISTORICAL EVENTS OF SEROLOGY DEFINITION OF TERMS Immunology is the study of an individual’s reactions when foreign substances are introduced into the body. Immunity refers to the condition of being resistant to infection, the state of protection from infectious disease. Antigen is a foreign substance (non-self) that may be specifically bound by an antibody molecule. Antigens range from small, simple intermediary metabolites such as sugars, lipids, and hormones, to macromolecules such as complex carbohydrates, proteins and nucleic acids. Immunogens are molecules that stimulate immune responses. “All immunogens are antigens, but not all antigens are immunogens.” An antibody is a protein (immunoglobulin) that is found in the blood plasma. Antibodies are produced by plasma cells, which are derived from B lymphocytes, in response to a foreign antigen. An antibody is usually specific against the antigen that triggered its production. Serology is a division of immunology that specializes in laboratory detection and measurement of a specific antibody that is produced as a response to exposure to an antigen. This division of immunology studies in vitro antigen-antibody reactions. HISTORICAL BACKROUND Thucydides During an outbreak of plague in Athens in 430 B.C., he observed that only those who had recovered from the plague could nurse the sick because they would not contract the disease a second time. Chinese Started the practice of variolation to prevent acquisition of smallpox. Variolation is a process where dried crusts derived from smallpox were directly inhaled by patients. 23 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  Edward Jenner In 1798, he started the practice of vaccination (vacca, meaning “cow”) in an attempt to produce a therapeutic procedure against smallpox. He observed that milkmaids who contracted the mild disease cowpox were subsequently immune from the deadly smallpox. Jenner then took material from the cowpox lesions of a dairy maid and scratched the said materials into the skin of a boy named James Phipps. Six weeks later, Jenner inoculated Phipps with material from a smallpox lesion. Within days, the boy developed a reaction at the site but failed to show any signs of smallpox. Louis Pasteur He was the first to observe attenuation and coined the term “vaccine”. Attenuation is the process of making something weaker. He was also the first to demonstrate that it was possible to attenuate, or weaken, a pathogen and administer the attenuated strain as a vaccine. He also succeeded in growing the bacterium thought to cause fowl cholera in culture and had shown that chickens injected with the cultured bacterium developed fowl cholera. He observed that old cultures of the causative agent, when injected to chickens, would not cause the disease. He also observed that chickens previously injected with old cultures were completely protected from fowl cholera when he injected them with a fresh culture of the bacterium. Thus, Pasteur hypothesized and proved that aging had weakened the virulence of the causative agent and that such an attenuated (weakened) strain might be administered to protect against the disease. He eventually called the attenuated strain a “vaccine”, in honor of Jenner’s work with cowpox inoculation. He was the first to vaccinate sheep using heat-attenuated anthrax bacillus. Aside from all of these, he also successfully immunized a young boy against rabies. Emil Roux tested the proposed rabies vaccine with success in dogs and observed that all immunized animals survived a rabies exposure. Pasteur administered the untested rabies vaccine in humans to Joseph Meister, a 9-year-old boy, who had been bitten and mauled by a rabid dog. The treatment lasted 10 days and the boy recovered and remained healthy. 24 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  EXPLAIN Humoral Immunity vs. Cellular Immunity: Discovery of their Intertwined Nature Emil von Behring and Demonstrated that serum from animals previously immunized to Shibasaburo Kitasato diphtheria could transfer the immune state to unimmunized animals. Elvin Kabat Demonstrated that that a fraction of serum first called gamma- globulin (now immunoglobulin) was shown to be responsible for immunity. Because immunity was mediated by antibodies contained in body fluids (known then as humors), it was called humoral immunity. Eli Metchnikoff Demonstrated that cells also contribute to the immune state of an animal. He observed that certain WBCs, which he termed as phagocytes, were able to ingest (phagocytose) microorganism and other foreign material. Thus, he hypothesized that cells were the major effector of immunity, becoming the first proponent of cellular immunity. Merril Chase Succeeded in transferring immunity against the tuberculosis by transferring WBCs between guinea pigs, reinforcing the claims of cellular immunity. Improved Cell Culture Lymphocyte was identified as the cell responsible for both Techniques (1950’s) cellular and humoral immunity. Bruce Glick Performed a series of experiments on chickens which indicated that there were two types of lymphocytes. T lymphocytes and B lymphocytes which are derived from thymus and from the bursa of Fabricious, respectively. T lymphocytes mediated cellular immunity, while B lymphocytes mediated humoral immunity. In humans, both B and T lymphocytes are produced by the bone marrow. Immature T lymphocytes migrate to the thymus for further maturation while the B lymphocytes mature in the bone marrow. Innate Immunity vs. Adaptive Immunity Innate immunity is also known as native immunity. It consists of cellular and biochemical defense mechanisms that are already in place even before infection and poised to respond rapidly to infections. Response in innate immunity remains the same for all pathogens or foreign substances to which one is exposed. No prior exposure is required, and the response does not change with subsequent exposures. 25 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  Adaptive immunity, also known as acquired immunity, will only be produced after an antigenic challenge to human host. It is characterized by the ability to remember a prior exposure, which results in an increased response upon repeated exposure. This type of immunity is also characterized by specificity for each individual pathogen, or microbial agent. There are two types of adaptive immune response: (a) cellular Immunity and (b) Macrophages humoral Immunity. Cellular immunity is mediated by T lymphocytes and is a principal defense mechanism against intracellular microbes. It is also responsible for killing infected cells. T- cells Humoral immunity is primarily mediated by antibodies produced by plasma cells, B- cells which are derived from B lymphocytes. This type of immunity is the principal defense Produce mechanism against extracellular microbes. Antibodies LINES OF DEFENSES FIRST LINE OF Anatomic barriers: Intact skin, Mucous membranes DEFENSE Physiologic processes: Sneezing, coughing, vomiting, gag reflex, constant motion of ciliated epithelial cells in the respiratory tract INNATE/ NATURAL Normal microbiota (Normal flora): Nonpathogenic IMMUNITY bacteria that are usually found in certain parts of the Response: immediate body such as the throat and intestines Secretions: Sweat, mucus, earwax (cerumen), saliva, tears, lactic acid in sweat, stomach acid Very low pH of vagina and stomach SECOND LINE OF Phagocytes: Monocytes, Macrophages, Neutrophils, DEFENSE Natural Killer cells Inflammatory reaction Complement System ADAPTIVE/ THIRD LINE OF Cellular components: Lymphocytes- T lymphocytes, ACQUIRED DEFENSE B lymphocytes, Plasma cells IMMUNITY Response: delayed Humoral components: Antibodies, cytokines Screaning test - Specificity Confirmity - sensitivity 26 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  ROUTINE TESTS IN SEROLOGY LABORATORY NON-TREPONEMAL ANTIBODY TESTS Are utilized as diagnostic tools for the detection of syphilis. These tests detect the presence of reagin antibodies which are antibodies against cardiolipin. Cardiolipin is found in the mitochondrial membrane. When the membrane is damaged, cardiolipin will be released into the blood circulation stimulating the production of reagin antibodies. Reagin antibodies are almost always produced by persons with syphilis. However, reagin antibodies are not specific for syphilis and can be produced in other infectious diseases such as leprosy, tuberculosis, malaria, measles, chickenpox, and hepatitis. Non-treponemal tests include Rapid Plasma Reagin (RPR) Test and Venereal Disease Research Laboratory (VDRL) Test. FTA-ABS (fluoroscent treponemal antibody absorption) HBsAg (Hepatitis B Surface Antigen) Test Current infection with Hepatitis B virus is indicated by the presence of HBsAg. Presence of HBs also indicates that the patient is infectious. This test is being utilized as a diagnostic tool for the detection of Hepatitis B infection. Anti-HBs (Hepatitis B Surface Antibody) Test The presence of anti-HBs is generally interpreted as indicating immunity from Hepatitis B virus infection. Anti-HBs are usually produced by individuals who have been successfully vaccinated against Hepatitis B and those that have recovered successfully from Hepatitis B infection. WIDAL TEST Diagnostic tool for the detection of Enteric fever and Typhoid fever. This test detects the presence of antibodies to disease-causing Salmonella organisms. DENGUE IgG/IgM TEST This test is used as screening test for dengue viral infection and as an aid for the differential diagnosis of primary and secondary infection. Results: IgG Positive Only: indicative of past dengue infection IgM Positive Only: indicative of primary (first-time) dengue infection Both IgG and IgM Positive: indicative of secondary dengue infection 27 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  DENGUE NS1 TEST This test detects of the presence of Non-Structural protein NS1 of the dengue virus. The antigen (NS1 antigen) is detectable during the acute phase of dengue virus infection, especially during the first seven (7) days of symptoms. DENGUE DUO KIT Designed to detect both dengue virus NS1 antigen and antibodies to dengue virus (Dengue IgG and IgM). 28 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  UNIT 4: IMMUNOHEMATOLOGY: THE BLOOD BANK SECTION ACTIVITY 07 – ENGAGE: IN THE HISTORICAL BACKGROUND OF BLOOD BANK Immunohematology is a branch of immunology that deals with the uses of immunologic principles to study and identify the different blood groups. Blood bank is an area in the clinical laboratory that collects blood products from donors and is responsible for preparation and storing whole blood and blood components for transfusion. Early history Pope Innocent VII (1492) In the hopes of curing him, he received blood by drinking blood from three young boys. Unfortunately, the pope and all three children died Animal-to-Human Transfusion Jean Baptiste Denis (1667) He performed the first animal-to-human transfusion by bloodletting a 16-year-old boy. He exchanged three ounces of boy’s blood to nine ounces of lamb’s blood. His second patient was Anton Mauroy who received a calf’s blood. Anton Mauroy suffered from a transfusion reaction but survived and became well. Richard Lower He transfused sheep’s blood to a student. Human-to-Human Transfusion Philip Syng Physick Performed the first human-to-human transfusion in 1795 but this was not documented. 30 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  John Henry Leacock Subsequently performed and published a set of animal experiments which proved that the donor and the recipient must be of the same species. James Blundell In 1825, He successfully transfused a woman dying from postpartum hemorrhage with by transfusing blood from the woman’s husband. Emil Ponfick Observed red cell lysis in the blood of a woman who died after receiving a transfusion of sheep blood. He also observed that incompatible transfusion reactions were associated with hemorrhage and congestion of the kidneys, lungs, and liver. Leonard Landois Observed that human red cells would lyse when mixed with the sera of other animals. He set the stage for the study of the immunologic basis of blood incompatibility. Did you know that clotting was the principal obstacle to overcome in the early practices of blood transfusion? To solve this, Braxton Hicks recommended the use of an anticoagulant. Discovery of Blood Groups In 1901, Karl Landsteiner discovered the ABO blood group and explained the serious reactions that occur in humans as a result of incompatible transfusions. He never failed in noticing that human blood mixed in test tubes with other specimens of human blood sometimes resulted in agglutination. Agglutination is the clumping of cells. His observations led to the discovery why some blood transfusions were successful while other could be deadly. He identified three types, called A, B and C (C was later to be re-named O for the German word “Ohne”, meaning “without”). In 1902, Alfred von Descatello and Adriano Sturli discovered the fourth less frequent blood group “AB”. The discovery of Rh blood group by Karl Landsteiner and Alex Weiner would come much later in1940. 31 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  EXPLAIN: THE ABO BLOOD GROUP: INHERITANCE Basic Concepts A. Genes o The units of heredity; Often defined at the molecular level as a DNA sequence and are responsible for expression of a trait B. Allele o One of two or more alternate forms of a gene C. Genotype o The set of genes possessed by an individual organism D. Phenotype o A physical trait whose expression depends on the inherited genes along with environmental factors § The 4 possible phenotypes based on ABO blood group is Blood Type A, Type B. Type AB, and Type O. § In a genotype, one allele is typically inherited from the mother while the other is inherited from the father § There are three alleles associated with ABO inheritance pattern namely A, B, and O o Allele A and Allele B are dominant. § Allele A is expressed whether there is only one copy of A (Heterozygous A) or two copies of A (Homozygous A) in the genotype § Allele B is expressed whether there is only one copy of B (Heterozygous B) or two copies of B (Homozygous B) in the genotype o Allele A and Allele B are codominant. § In genotype AB, both allele A and allele B are expressed by the individual o O allele is recessive § Allele O is only expressed if there are two copies of O in the genotype (Genotype OO) Genotype Phenotype Genotype AA Blood Type A (Homozygous A) Genotype AO Blood Type A (Heterozygous A) Genotype BB Blood Type B (Homozygous B) Genotype BO Blood Type B (Heterozygous B) Genotype AB Blood Type AB Genotype OO Blood Type O 32 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  SAMPLE PROBLEM: 1. Enumerate all possible blood types of an offspring if the mother is heterozygous A while the father is heterozygous B. What is the expression probability of each possible blood type? Analysis: Mother’s genotype is AO while the father’s genotype is BO B O A AB AO O BO OO There are four (4) possible genotypes. Possible Genotypes (Offspring) Phenotype Probability 1 AB AB 25% AB 1 BO B 25% B 1 AO A 25% A 1 OO O 25% O There is a 25% chance that the offspring will have an AB blood type. There is a 25% chance that the offspring will have a B blood type. There is a 25% chance that the offspring will have an A blood type. There is a 25% chance that the offspring will have an O blood type. 2. The mother’s blood type is A while the father’s blood type is AB. What are the possible blood types of the couple’s offspring? Analysis: Mother’s genotype can either be AO or AA Father’s genotype is AB Possible Scenario 1 Possible Scenario 2 Mother: AO Father: AB Mother: AA Father: AB A B A B A AA AB A AA AB O AO BO A AA AB There are a total of 8 possible genotypes of the offspring. Genotype Phenotype Summary- Offspring phenotype 3 AB 3 AB Phenotype Number Probability 3 AA 3A AB 3 3/8 or 37.5 % 1 AO 1A A 4 4/8 or 50.0 % 1 BO 1B B 1 1/8 or 12.5 % There is a 37.5% chance that the child will have Blood Type AB. There is a 50% chance that the couple’s child will have Blood Type A. There is a 12.5% chance that the child will have Blood Type B. 33 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  3. Both the mother and the father has Blood Type A. What are the possible blood types of the couple’s offspring? Analysis: Mother’s genotype can either be AA or AO Father’s genotype can either be AA or AO Possible Scenario 1 Possible Scenario 2 Mother: AA Father: AO Mother: AA Father: AA A O A A A AA AO A AA AA A AA AO A AA AA Possible Scenario 3 Possible Scenario 3 Mother: AO Father: AA Mother: AO Father: AO A A A O A AA AA A AA AO O AO AO O AO OO There are 16 possible genotypes of the offspring Summary- Offspring phenotype Genotype Phenotype Phenotype Number Probability 9 AA 9A A 15 15/16 or 6 AO 6A 93.75% 1 OO 1O O 1 1/16 or 6.25% There is a 93.75% chance that the offspring will have Blood Type A. There is a 6.25% chance that the offspring will have Blood Type O. 34 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  ROUTINE TESTS IN BLOOD BANK ABO BLOOD TYPING: The Four Major Blood Types Blood Type ANTIGEN on RBCs ANTIBODY in Plasma Type A A antigen Anti-B Type B B antigen Anti-A Type AB A and B antigens NO anti-A and anti-B Type O NO A and B antigens Anti-A and Anti-B A. DIRECT TYPING: MOST ROUTINE Also known as forward or cell typing which determines what antigens are present on the surface of the patient’s red blood cells. The specimen to be used here is whole blood and reagents are commercially prepared antisera. Blood Type Reagent: ANTISERA Anti-A (BLUE color) Anti-B (YELLOW color) Type A Positive Negative Type B Negative Positive Type AB Positive Positive Type O Negative Negative B. REVERSE TYPING: Also known as indirect or serum typing which determines what antibodies are present in the plasma or serum of the patient’s sample. This method utilizes Red Cell Suspension (RCS) as the reagent. Blood Type Reagent: Red Cell Suspension (RCS) A cell suspension B cell suspension Type A Negative Positive Type B Positive Negative Type AB Negative Negative Type O Positive Positive REVERSE TYPING 35 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  Rh BLOOD TYPING: This method detects the presence or absence of the D antigen on the surface of the red blood cells. It also categorizes individuals as Rh-positive or Rh-negative. The sample is whole blood and the reagent is commercially prepared Anti-D which is colorless. Type Reaction with Anti-D Interpretation Rh positive Positive Presence of D antigen Rh negative Negative Absence of D antigen NOTE: Results of both ABO and Rh blood typing are routinely written together. Sample results: A+ A, Rh positive Blood Type A; Has the D antigen A- A, Rh negative Blood Type A; Absence of D antigen CROSSMATCHING: Testing the compatibility of the donor blood and the recipient blood. The testing involves two parts: 1. MAJOR CROSSMATCH: o RSDC (Recipient serum + Donor cells): Recipient’s serum is mixed with donor’s red blood cells. o This detects if the recipient has antibodies which can destroy the transfused red blood cells from the donor. 2. MINOR CROSSMATCH: o DSRC (Donor serum + Recipient cell): Donor’s serum is mixed with the recipient’s red blood cells. o This detects if there are antibodies in the donor’s serum that can destroy the patient’s red blood cells. ACTIVITY 08 – EVALUATE ***Instructions shall be provided by your respective faculty course facilitator 36 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.