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In this session we shall be looking at the following  Study of Pathology,  - Health and Disease,  - Common Terms in Pathology,  Evolution of Pathology,  - Prehistoric Times to Medieval Period,  - Human Anatomy and Era of Gross Pathology, - Era of Technology Development and Cellular Pathology,  - Modern Pathology,  - Telepathology  and Virtual Microscopy.  Subdivisions of Pathology - Morphological Branches,  - Non-Morphological Branches

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Pathology’  is derived from two Greek words— pathos  (meaning suffering) and  logos  (meaning study).  Pathology is, thus, scientific study of changes in the structure and function of the body in disease.  In other words, pathology consists of the abnormalities in normal anatomy (including histology) and normal physiology owing to disease.  Another commonly used term with reference to study of diseases is ‘ pathophysiology ’  ( patho   = suffering,  physiology  = study of normal function).   Pathophysiology , thus, includes study of disordered function (i.e. physiological changes) and breakdown of homeostasis in diseases (i.e. biochemical changes).  Pathologists contribute in patient management by providing final diagnosis of disease.

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Therefore, knowledge and understanding of pathology is essential for all would-be: - Doctors,  Medical laboratory scientist,  Pharmacist,  Nurses, Physiotherapy, As as well as general medical practitioners and specialists because unless they have knowledge and understanding  of  the  language  in  the  form of pathology laboratory reports, they would not be able to institute appropriate treatment or suggest preventive measures to the patient.

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For the student of any system of medical program, the discipline of pathology forms a vital bridge between initial learning phase of preclinical sciences and the final phase of clinical subjects.  The role and significance of learning of pathology in clinical medicine is quite well summed up by  Sir William Osler  (1849- 1919),  acclaimed physician and teacher in medicine considered as ‘Father of Modern Medicine’ whose famous quote state that the practice of medicine depend the understanding of pathology

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Before there were humans on earth, there was disease, albeit in early animals.  Since pathology is the study of disease, then what is  disease ? In simple language, disease is opposite of health i.e. what is not healthy is disease.  Health  may be defined as a condition when the individual is in complete accord with the surroundings, while  disease  is loss of ease (or comfort) to the body (i.e.  dis+ease ).  However, it must be borne in mind that in health there is a wide range of ‘normality’ e.g. in height, weight, blood and tissue chemical composition etc.  It also needs to be appreciated that at cellular level, the cells display wide range of activities within the broad area of health similar to what is seen in diseased cells.  Thus, a disease or an illness means a condition   marked by pronounced deviation from the normal healthy   state.  The term s yndrome  (meaning running together) is used for a combination of several clinical features caused by altered physiologic processes.

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It is important for a beginner in pathology to be familiar with the language used in pathology   Patient   is the person affected by disease.   Lesions   are the characteristic changes in tissues and cells produced by disease in an individual or experimental animal. Pathologic changes  or  morphology  consist of examination of diseased tissues.  These can be recognized with the naked eye  (gross) or macroscopic changes (microscopic examination  of tissues). Causal factors responsible for the lesions are included in  etiology   of disease (i.e. ‘why’ of disease). Mechanism by which the lesions are produced is termed  pathogenesis   of disease (i.e. ‘how’ of disease). Functional implications of the lesion felt by the patient are  symptoms   and those discovered by the clinician are the  physical signs . Clinical significance of the morphologic and functional changes together with results of other investigations help to arrive at an answer to what is wrong  ( diagnosis ) , what is going to happen  (prognosis),  what can be done about it  ( treatment ) , and finally what should be done to avoid complications and spread  ( prevention )  (i.e. ‘what’ of disease).

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Pathology as the scientific study of disease processes has its deep roots in medical history.  Since the beginning of mankind, there has been desire as well as need to know more about the causes, mechanisms and nature of diseases.  The answers to these questions have evolved over the centuries—from supernatural beliefs to the present state of our knowledge of modern pathology.  However, pathology is not separable from other multiple disciplines of medicine and owes its development to interaction and interdependence on advances in diverse neighboring branches of science, in addition to the strides made in medical technology.  Pathology has evolved over the years as a distinct discipline from anatomy, medicine and surgery, in that sequence. Pathology magnificently contributed outstandingly to learning and understanding the form of diseases, processes known by their names and stimulate interest in solving the problem.

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Present-day knowledge of primitive culture which was prevalent in the world in prehistoric times reveals that religion, magic and medical treatment were quite linked to each other in those times.  The earliest concept of disease understood by the patient and the healer was the religious belief that disease was the outcome of ‘curse from God’ or the belief in magic that the affliction had supernatural origin from ‘evil eye of spirits.’  To ward them off, priests through prayers and sacrifices, and magicians by magic power used to act as faith-healers and invoke supernatural powers and please the gods.  Remnants of ancient superstitions still exist in some parts of the world. The link between medicine and religion became so firmly established throughout the world that different societies had their gods and goddesses of healing; for example:  1- mythological Greeks had  Aesculapius  and  Apollo  as the principal gods of healing,  2-  Dhanvantri   as the deity of medicine in India, and  3- Orthodox Indians’ belief in  Mata  Sheetala  Devi  as the pox goddess.

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The insignia of healing, the Caduceus, having snake and staff, is believed to represent the god Hermes or Mercury, which according to Greek mythology has power of healing since snake has regenerative powers expressed by its periodic sloughing of its skin.  God of Greek medicine,  Aesculapius , performed his functions with a staff having a single serpent wound around it.  Later (around AD1800), however, the Caduceus got replaced with twin-serpents wound around a staff topped by a round knob and flanked by two wings and now represents the symbol of medicine instead of cross The period of ancient religious and magical beliefs was followed by the philosophical and rational approach to disease by the methods of observations.  This happened at the time when great Greek philosophers— Socrates, Plato  and  Aristotle,  introduced philosophical concepts to all natural phenomena.

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The  real practice of medicine began with  Hippocrates  (460–370 BC), the great Greek clinical genius of all times and regarded as ‘the father of medicine’.  Hippocrates dissociated medicine from religion and magic.  He firmly believed in study of patient’s symptoms and described methods of diagnosis.  He recorded his observations on cases in the form of collections of writings called Hippocratic Corpus which remained the mainstay of learning of medicine for nearly two thousand years.  However, the prevailing concept at that time on mechanism of disease based on disequilibrium of four basic humors (water, air, fire, and earth) was propagated by  Hippocates  too but this concept was later abandoned. HippoHcrates  followed rational and ethical attitudes in practice and teaching of medicine and is revered by the medical profession by taking  ‘Hippocratic oath’  at the time of entry into practice of medicine.

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It was extended to Italy which controlled Greek world after 146 BC and dominated the field of development of medicine in ancient Europe then.  In fact, since old times, many tongue-twisting terminologies in medicine have their origin from Latin language which was the official language of countries included in ancient Roman empire (Spanish, Portuguese, Italian, French and Greek languages have their origin from Latin). In Rome, Hippocratic teaching was propagated by Roman physicians, notably by  Cornelius  Celsus   (53 BC-7 AD) and  Claudius Galen  (130–200 AD).  Celsus  first described four cardinal signs of inflammation— rubor  (redness), tumor (swelling),  calor  (heat), and dolor (pain).  Galen postulated  humoral  theory, later called  Galenic  theory.  This theory suggested that the illness resulted from imbalance  between  four humors  (or body fluids): blood, lymph, black bile (believed at that time to be from the spleen), and  biliary  secretion from the liver. Hypothesis of disequilibrium of four elements constituting the body  ( Dhatus )  similar to Hippocratic doctrine finds mention in ancient Indian medicine books compiled about 200 AD— Charaka   Samhita , a finest document by  Charaka   on medicine listing 500 remedies, and  Sushruta   Samhita ,  similar

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Similar book of surgical sciences by  Sushruta , and includes about 700 plant-derived medicines. The end of Medieval period was marked by backward steps in medicine.  There were widespread and devastating epidemics which reversed the process of rational thinking again to supernatural concepts and divine punishment for ‘sins.’  The dominant belief during this period was that life was due to influence of vital substance under the control of soul  (theory of  vitalism ) .  Thus, dissection of human body was strictly forbidden at that time as that would mean hurting the ‘soul.’

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The backwardness of Medieval period was followed by the Renaissance period i.e. revival of learning.  The Renaissance began from Italy in late 15th century and spread to whole of Europe.  During this period, there was quest for advances in art and science.  Since there was freedom of thought, there was emphasis on philosophical and rational attitudes again. Beginning of  the  development  of  human  anatomy took place during this period with the art works and drawings of human muscles and embryos by famous Italian painter  Leonardo  da  Vinci  (1452–1519).  Dissection of human body was started by  Vesalius  (1514–1564) on freshly executed criminals.  His pupils,  Gabriel  Fallopius   (1523–1562) who described human oviducts (Fallopian tubes) and  Fabricius   who discovered lymphoid tissue around the intestine of birds (bursa of  Fabricius ) further  popularised  the practice of human anatomic dissection for which special postmortem amphitheatres came in to existence in various parts of ancient Europe.

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Antony van Leeuwenhoek  (1632–1723), a cloth merchant by profession in Holland, during his spare time invented the first ever microscope by grinding the lenses himself through which he  recognised  male spermatozoa as tiny preformed men (or “homunculi”) and other single - celled organisms which he called animalcules.  He also introduced histological staining in 1714 using saffron to examine muscle  fibres .  Marcello Malpighi  (1624–1694) used microscope extensively and observed the presence of capillaries and described the  malpighian  layer of the skin, and lymphoid tissue in the spleen ( malpighian  corpuscles).  Malpighi is known as ‘the father of histology.’

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The credit for beginning of the study of morbid anatomy (pathologic anatomy), however, goes to Italian anatomist- pathologist,  Giovanni B.  Morgagni   (1682–1771).   Morgagni  was an  excellent  teacher  in  anatomy,  a  prolific  writer  and a practicing clinician.  By  his  work,   Morgagni   demolished the ancient  humoral  theory of disease and published his life-time experiences based on 700 postmortems and their corresponding clinical findings.  He laid the foundations of  clinicopathologic  methodology in the study of disease and introduced the concept of  clinicopathologic  correlation (CPC), establishing a coherent sequence of cause, lesions, symptoms, and outcome of disease

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Sir  Percivall   Pott   (1714–1788), famous surgeon in England, described arthritic tuberculosis of the spine ( Pott’s  disease) and identified the first ever occupational cancer (cancer of scrotal skin) in the chimney sweeps in 1775 and discovered chimney soot as the first carcinogenic agent.  The study of anatomy in England during the latter part of 18th Century was dominated by the two Hunter brothers.  These were  John Hunter  (1728–1793), a student of Sir  Percivall   Pott , who rose to become the greatest surgeon-anatomist of all times and his elder brother  William Hunter  (1718–1788) who was a reputed anatomist-obstetrician.

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These brothers together  started  the first ever museum by collection of surgical specimens from their flourishing practice, arranged them into separate organ systems. Made comparison of specimens from animals and plants with humans, and included many clinical pathology specimens as well. Developed  the  first  museum of comparative anatomy and pathology in the world which became the  Hunterian  Museum, now housed in Royal College of Surgeons of London.  Among many pupils of John Hunter was  Edward Jenner  (1749–1823) whose work on inoculation in smallpox is well known.  Another prominent English pathologist was  Matthew Baillie  (1760–1823), nephew of Hunter brothers, who published first-ever systematic textbook of morbid anatomy in 1793.  The era of gross pathology had three more illustrious and brilliant physician-pathologists in England who were colleagues at Guy’s Hospital in London:

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Richard Bright  (1789–1858) who described non- suppurative  nephritis, later termed  glomerulonephritis  or  Bright’s  disease; Thomas Addison  (1793–1860) who gave an account of chronic  adrenocortical  insufficiency termed Addison’s disease; and Thomas Hodgkin  (1798–1866), who observed the complex of chronic enlargement of lymph nodes, often with enlargement of the liver and spleen, later called Hodgkin’s disease. Towards the end of 18th century,  Xavier Bichat  (1771–1802) in France described that organs were composed of tissue and divided the study of morbid anatomy into General Pathology and Systemic Pathology.  R.T.H. Laennec  (1781–1826), another

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French physician, dominated the early part of 19th century by his numerous discoveries.  He described several lung diseases (tubercles,  caseous  lesions,  miliary  lesions, pleural effusion, and  bronchiectasis ), chronic sclerotic liver disease (later called Laennec’s cirrhosis) and invented stethoscope. Morbid anatomy attained its zenith with appearance of  Carl F. von  Rokitansky   (1804–1878), self-taught German pathologist who performed nearly 30,000 autopsies himself.  He described acute yellow atrophy of the liver, wrote an outstanding monograph on diseases of arteries and congenital heart defects.  Unlike most other surgeons of that time. Rokitansky  did not do clinical practice of surgery but instead introduced the concept that pathologists should confine themselves to making diagnosis which became the accepted role of pathologist later.

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ERA OF TECHNOLOGICAL DEVELOPMENT AND CELLULAR  PATHOLOGY

Till the middle of the 19th century, correlation of clinical manifestations of disease with gross pathological findings at autopsy became the major method of study of disease.  Sophistication in surgery led to advancement in pathology.  The anatomist-surgeons of earlier centuries got replaced largely with surgeon-pathologists in the 19th century. Pathology started developing as a diagnostic discipline in later half of the 19th century with the evolution of cellular pathology which was closely linked to technology advance-  ments  in machinery manufacture for cutting thin sections of tissue, improvement in microscope, and development of chemical industry and dyes for staining

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The discovery of existence of disease-causing micro- organisms was made by French chemist  Louis Pasteur  (1822– 1895), demolishing the prevailing theory of spontaneous generation of disease and firmly established germ theory of disease.  Subsequently,  G.H.A. Hansen  (1841–1912) in Germany identified Hansen’s bacillus in 1873 as the first microbe causative for leprosy (Hansen’s disease).  While the study of infectious diseases was being made, the concept of immune tolerance and allergy emerged which formed the basis of immunization initiated by Edward Jenner.   Metchnikoff  (1845-1916), a Russian zoologist, introduced the existence of phenomenon of  phagocytosis  by human defense cells against invading microbes.

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Developments in chemical industry helped in switch over from earlier dyes of plant and animal origin to synthetic dyes; aniline violet being the first such synthetic dye prepared by  Perkin  in 1856. This led to emergence of a viable dye industry for histological and bacteriological purposes.  The impetus for the flourishing and successful dye industry came from the works of numerous pioneers as under: - Paul Ehrlich  (1854–1915), German physician, conferred Nobel prize in 1908 for his work in immunology, described Ehrlich’s test for  urobilinogen  using Ehrlich’s  aldehyde  reagent, staining techniques of cells and bacteria, and laid the foundations of clinical pathology Christian Gram  (1853–1938), Danish physician, developed bacteriologic staining by crystal violet.   D.L.  Romanowsky   (1861–1921), Russian physician, developed stain for peripheral blood film using eosin and  methylene  blue derivatives. - Robert Koch  (1843–1910), German bacteriologist, besides Koch’s postulate and Koch’s phenomena, developed techniques

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-  The development of  exfoliative  cytology for early detection of cervical cancer began with  George N.  Papanicolaou   (1883–1962), a Greek-born, American pathologist, in 1930s and is known as ‘father of  exfoliative  cytology’  . - Another pioneering contribution in pathology in the 20th century was by  an  eminent  teacher-author,   William Boyd  (1885–1979), psychiatrist-turned pathologist, whose textbooks—‘Pathology for Surgeons’ (first edition 1925) and ‘Textbook of Pathology’ (first edition 1932), dominated and inspired the students of pathology all over the world due to his flowery language and lucid style for a few generations. M.M.  Wintrobe   (1901–1986), a pupil of Boyd who discovered  haematocrit  technique, regarded him as a very stimulating teacher.

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Due to the strides made in the latter half of 20th century until recent times in 21st century have made it possible to study diseases at genetic and molecular level, and provide an evidence- based and objective diagnosis that may enable the physician to institute targeted therapy.  The major impact of advances in molecular biology are in the field of diagnosis and treatment of genetic disorders, immunology and in cancer. Some of the revolutionary discoveries during this time are as under. 1- Description of  the structure of DNA  of the cell by Watson and Crick in 1953. 2- Identification of  chromosomes and their correct number  in humans (46) by  Tijo   and  Levan   in 1956.

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3-  Identification of  Philadelphia chromosome  t(9;22) in chronic myeloid  leukaemia  by  Nowell  and  Hagerford  in 1960 as the first chromosomal abnormality in any cancer. 4-   In  Situ  hybridization   (ISH)  introduced  in  1969  in  which a  labelled  probe is employed to detect and  localise  specific RNA or DNA sequences  ‘in situ’  (i.e. in the original place). Its later modification employs use of fluorescence microscopy (FISH) to detect specific  localisation  of the defect on chromosomes. 5- Recombinant DNA technique  developed in 1972 using restriction enzymes to cut and paste bits of DNA.

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6-  Introduction of  polymerase chain reaction  (PCR) i.e. “ xeroxing ” of DNA fragments by  Kary  Mullis in 1983 has revolutionized the diagnostic molecular genetics. PCR analysis is more rapid than ISH, can be automated by thermal cyclers and requires much lower amount of starting DNA. 7-  Invention of  flexibility and dynamism of DNA  by Barbara McClintock for which she was awarded Nobel prize in 1983. 8-   Mammalian cloning  started in 1997 by  Ian  Wilmut   and his colleagues at  Roslin  Institute in Edinburgh, by successfully using a technique of somatic cell nuclear transfer to create the clone of a sheep named Dolly. Reproductive cloning for human beings, however, is very risky besides being absolutely unethical.

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9- The era of  stem cell research  started in 2000s by harvesting these primitive cells isolated from embryos and maintaining their growth in the laboratory.  There are 2 types of sources of stem cells in humans:  ( i ) - Embryonic stem cells (more numerous) and  (ii) - Adult stem cells (less numerous)     Stem cells are seen by many researchers as having virtually unlimited applications in the treatment of many human diseases such as Alzheimer’s disease, diabetes, cancer, strokes, etc.  At some point of time, stem cell therapy may be able to replace whole organ transplant and instead stem cells ‘harvested’ from the embryo may be used.

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10- Human Genome Project (HGP)  consisting of a consortium of countries was completed in April 2003 coinciding with 50 years of description of DNA double helix by Watson and Crick in April 1953.  The sequencing of human genome reveals that human genome contains approximately 3 billion base pairs of amino acids, which are located in the 23 pairs of chromosomes within the nucleus of each human cell.  Each chromosome contains an estimated 30,000 genes in the human genome which carry the instructions for making proteins.  The HGP has given us the ability to read nature’s complete genetic blueprint used in making of each human being (i.e. gene mapping).  Clinical trials by gene therapy on treatment of some single gene defects have resulted in some success, especially in  haematological  and immunological diseases.  Future developments in genetic engineering may result in designing new and highly effective individualized treatment options for genetic diseases as well as suggest prevention against diseases.

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Nucleus
p telomere
Chromatids
q telomere
p arm
Centromere
q arm
Histone
CELL
CHROMOSOME
Base pairs
DNA DOUBLE HELIX

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Telepathology   is defined as the practice of diagnostic pathology by a remote pathologist  utilising  images of tissue specimens transmitted over a telecommunication network.  The main  components  of a  telepathology  system are as under: -  Conventional light microscope. - Method of image capture, commonly a camera mounted on light microscope. - Telecommunications link between sending and receiving side. - Workstation at receiving end with a high quality monitor.

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Depending upon need and budget,  telepathology  system is of two  types : Static (store-and-forward, passive  telepathology )  In this, selected images are captured, stored  and  then  transmitted over the internet via e-mail attachment, file transfer protocol, web page or CD-ROM.  It is quite inexpensive and is more common  Suffers from disadvantage of having sender’s bias in selection of transmitted images. Dynamic (Robotic interactive  telepathology )  Here, the images are transmitted in real-time from a remote microscope. Robotic movement of stage of microscope is controlled remotely and the desired images and fields are accessioned from a remote/local server.  It almost duplicates to perfection the examination of actual slides under the microscope, hence is referred to as  Virtual Microscopy.  However, image quality and speed of internet can be major hurdles.

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The era of “ digital pathology ” in 21st Century has reached its zenith with availability of technology for preparation of  virtual pathology slides (VPS)  by high speed scanners and then storing the scanned data in large memory output computers.  VPS stored in the memory of the computer can then be examined and reported at any place on computer, without having to use microscope.  However, the most question remains whether current pathologists are used to conventional microscopy will get the same perception on monitor.  At present, this technology holds potential for pathology education, storage for records, clinical meetings and quality control.

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Human pathology  is conventionally studied under two broad divisions:  1-  General Pathology  dealing with general principles of disease, and 2-  Systemic Pathology  that includes study of diseases pertaining to the specific organs and body systems.   In general, the study of pathology includes  (a)- Morphological and  (b)- Non- morphological disciplines.

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These branches essentially involve application of microscope as an essential tool for the study and include histopathology,  cytopathology  and  haematology . A. HISTOPATHOLOGY  it is used synonymously with anatomic pathology, pathologic anatomy, morbid anatomy, or tissue pathology, is the classic method of study and still the most useful one which has stood the test of time.  The study includes structural changes observed by naked eye examination referred to as gross or macroscopic changes, and the changes detected by microscopy, which may be further supported by numerous special staining methods such as  histochemistry  and  immunohistochemistry  to arrive at the most accurate diagnosis.  Modern time anatomic pathology includes sub- specialities  such as cardiac pathology,  pulmo - nary pathology, neuropathology, renal pathology,  gynaeco - logic pathology, breast pathology,  dermatopathology , gastrointestinal pathology, oral pathology, and so on.

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Anatomic pathology includes the following subdivisions: Surgical pathology  It deals with the study of tissues removed from the living body by biopsy or surgical resection.  -  Surgical pathology forms the bulk of tissue material for the pathologist and includes study of tissue by conventional  paraffin embedding  technique;  intraoperative   frozen section  may be employed for rapid diagnosis. Experimental pathology  This is defined as production of disease in the experimental animal and study of morphological changes in organs after sacrificing the animal.    - However, all the findings of experimental work in animals may not be applicable to human beings due to species differences. Forensic pathology and Autopsy work   This includes the study of organs and tissues removed at postmortem for  medicolegal  work and for determining the underlying sequence and cause of death.    - By this, the pathologist attempts to reconstruct the course of events how they may have happened in the patient during life which culminated in his death.    - Postmortem anatomical diagnosis is helpful to the clinician to enhance his knowledge about the disease and his judgment while forensic autopsy is helpful for  medicolegal  purposes.  -  The significance of a careful postmortem examination is appropriately summed up in the old saying ‘the dead teach the living’.

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B. CYTOPATHOLOGY  is    a branch of anatomic pathology,  cytopathology  has developed as a distinct  subspeciality  in recent times.     It includes study of cells shed off from the lesions ( exfoliative  cytology) and fine-needle aspiration cytology (FNAC) of superficial and deep-seated lesions for diagnosis. C. HAEMATOLOGY  Haematology  deals with the diseases of blood.     It includes laboratory  haematology  and clinical  haematology ; the latter covers the management of patient as well.

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These diagnostic branches of pathology include  clinical pathology,  clinical biochemistry,  microbiology,  immunology,  Genetics and  Molecular pathology.  In these diagnostic branches, qualitative, semi-quantitative or quantitative deter-  minations  are carried out in the laboratory.  Microscope may also be required for at least some of these lab tests. CLINICAL PATHOLOGY  Analysis of various fluids including blood, urine, semen, CSF and other body fluids is included in this branch of pathology. Such analysis may be qualitative, semi-quantitative or quantitative. CLINICAL BIOCHEMISTRY  Quantitative determination of various biochemical constituents in serum and plasma, and in other body fluids is included in clinical biochemistry.

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Obviously, there is likely to be overlapping between clinical pathology and clinical biochemistry. MICROBIOLOGY  This is study of disease-causing microbes implicated in human diseases.  -  Depending upon the type of  microorganims  studied, it has further developed into such as bacteriology,  parasitology , mycology, virology etc. IMMUNOLOGY  Detection of abnormalities in the immune system of the body comprises immunology and  immunopathology . MEDICAL GENETICS  This is the branch of human genetics that deals with the relationship between heredity and disease. -  There have been important developments in the field of medical genetics e.g. in blood groups, inborn errors of metabolism, chromosomal aberrations in congenital malformations and  neoplasms  etc.

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MOLECULAR PATHOLOGY  The detection and diagnosis of abnormalities at the level of DNA of the cell is included in molecular pathology such as in situ  hybridisation , PCR etc.   - These methods are now not only used for research purposes but are also being used as a part of diagnostic pathology reports.  - The above divisions of pathology into several  subspecialties  are quite artificial since overlapping of disciplines is likely, ultimate aim of pathologist being to establish the final diagnosis and learn the causes and mechanisms of disease.   - Towards this aim, the beginner as well as the teacher in pathology remain lifelong students of pathology, eager to learn more in their quest to become better with every passing day.

PATHOLOGY LECTURE NOTE

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