Marc A. Shampo

Statistical Analysis in Clinical Laboratory Medicine: Fundamentals and Common Uses

Medical research studies may be classified into two broad categories: descriptive and inferential. Descriptive studies are intended to describe the characteristics of only the study group, using observations obtained from every member of the group. Inferential studies, on the other hand, are designed to enable the investigator to use observations from selected individuals (a sample) to make conclusions about the larger group (population) from which they were drawn. The first part of this paper deals with descriptive studies, focusing on summary statistics and graphic techniques. The remainder deals with inferential studies. We start by describing how characteristics of the population may be estimated from characteristics of a small number of its members, which are randomly selected. These principles are then applied to the problem of testing hypotheses about the population by use of some of the more common testing procedures. These include t tests, regression, chi-square tests, evaluating a new diagnostic procedure, estimating normal values, survivorship studies, and sequential methods.

Adolf Butenandt--Nobel Prize for chemistry.

M e t i a a B G F e a Institute, Boston, MA (D.P.S.). T he 1939 Nobel Prize in chemistry was shared by the German biochemist Adolf Friedrich J. Butenandt and the Croatian-Swiss chemist Leopold Ruzicka (1887-1976) for work on sex hormones, although these scientists worked independently of each other. Butenandt, the son of a German businessman, was born on March 24, 1903, in Bremerhaven-Lehe in northwestern Germany (about 35 miles north of Bremen) on the Weser River. He received his early education in the Oberrealschule in Bremerhaven, after which he entered the University of Marburg (in central Germany north of Frankfurt), where he began to study chemistry and biology. After receiving his baccalaureate degree, he did postgraduate work at the University of Göttingen in western Germany, receiving a PhD degree in 1927 with a thesis on the chemistry of rotenone, a compound used in insecticides. After receiving his doctoral degree, Butenandt remained at the University of Göttingen, during which time he was hired as a researcher by the Schering Corporation of Germany. From 1927 to 1930, he was Scientific Assistant at the Institute of Chemistry in Göttingen, and from 1931 to 1933, was Privatdozent in the university’s Department of Biological Chemistry. In 1929, Butenandt isolated estrone from the urine of pregnant women. Estrone is one of the hormones responsible for sexual development and function in females. Similar research was performed almost simultaneously by the American biochemist Edward A. Doisy (1893-1986). Butenandt continued his research on sex hormones, and in 1931, he isolated and identified androsterone, a male sex hormone. He remained in Göttingen until 1933, when he became professor and director at the Technische Hochschulen in Danzig, Germany (now Gdañsk, Poland). There, he isolated and identified the hors

Walter Clement Noel—First Patient Described With Sickle Cell Disease

Historical reviews of medical discoveries usually focus on the physician or scientist who first reported the novel finding or the scientific and intellectual climate at the time of the development. The individual study patient is rarely considered—sometimes because of privacy concerns but perhaps more often because the specific afflicted person is not considered of universal interest. However, for sickle cell disease, first described in 1910 by noted Chicago-based internist James Bryan Herrick (1861-1954), enough details are available about the first patient for a compelling story because of the determined research efforts of a medical historian and a physician (Savitt TL and Goldberg MF, JAMA 1989;261(2): 266-271). Walter Clement Noel (1884-1916), the sickle cell proband, was born on a large estate in the hilly bush country at the north end of Grenada, a small Carribean island that at that time was a British colony. His family was wealthy landholders. Despite chronic health problems, Noel received a quality education and attended Harrison College in Barbados, completing his undergraduate studies in the summer of 1904. In September of that year, Noel sailed from Barbados to New York on the SS Cearense; he developed a leg ulcer (a common complication of sickle cell disease) during the week-long journey. After customs and immigration processing in New York, he sought medical attention and was treated with topical iodine, and the leg wound quickly healed. Noel then traveled by train to Chicago, where he had been accepted as a dental student at the Chicago College of Dental Surgery on the city’s West Side, and he took a room on West Congress Street in the heart of the Chicago medical district. It was unusual in 1904 for a student of African descent such as Noel to be permitted to study in the United States outside of a traditionally “blacksonly” secondary school; however, Noel was well-to-do and was also a foreigner, and educational opportunities for black students were slowly beginning to expand. In late November 1904, Noel developed respiratory problems, now recognized as the leading acute cause of death in patients with sickle cell disease, that persisted for more than a month. He finally sought medical attention at the Presbyterian Hospital in Chicago (a private facility) and was evaluated by an intern, Ernest Irons (1877-1959). Irons performed a peripheral blood smear, which was a relatively recent addition to the clinical testing battery, and noted that Noel’s blood smear contained “many pear-shaped and elongated forms—some small.” Irons discussed the case at length with Herrick, his supervising physician. A thorough search for potential causes of these oddly shaped cells was unrevealing; malaria or a parasitic infection was suspected but not substantiated. With supportive care, Noel eventually recovered and returned to school. Throughout the next 2/2 years, as Noel progressed through his dental studies, he experienced several illnesses: he was hospitalized briefly for bronchitis and later confined for 2 months at the Frances Willard Hospital for “a bilious and muscular attack” (patients with sickle cell syndromes frequently have musculoskeletal painful crises and pigment gallstones). Irons also made house calls for Noel’s knee pain and for bronchitis. Irons kept dutiful case notes (Noel is described as “bright and intelligent”) and gave all these to Herrick at the end of his training. Irons later achieved distinction as a rheumatologist, and he was the president of the American College of Physicians and the American Medical Association in the 1940s. Herrick presented Noel’s case (without giving any credit to Irons) at a national meeting in 1910 and published a detailed report later that same year, but then he turned his attention to other matters; he is customarily given credit for being the first to describe myocardial infarction in 1912. A few months after the Noel case was published, a second, similar case was described in rural Virginia; the patient was a cook and housemaid named Ellen Anthony. The next clear instance was not published for 5 more years; however, by the early 1920s, enough experience had accumulated that Vernon Mason was able to name the illness sickle cell anemia. By the 1940s, the inheritance pattern and physical chemistry of hemoglobin S were well enough understood that renowned scientist Linus Pauling (1901-1994) could call sickle cell anemia “the first molecular disease.” Despite his illnesses, Noel graduated from dental school with his entering class in 1907 and then returned to Grenada to set up a private general dentistry practice in the capital city of St George’s. His mother owned the building where the practice was located; Noel lived upstairs, and his offices were at street level. Few details are available

Howard Walter Florey—Production of Penicillin

From Mayo Clinic, Rochester, MN (R.A.K., M.A.S.); and Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA (D.P.S.). W hile Alexander Fleming is often credited with discovering penicillin in 1928, Howard Walter Florey oversaw initial clinical trials and led the team that first produced large quantities of this antibiotic, which played an important role in the Allied victory in World War II. But the antibacterial activity of penicillin was first discovered decades before Fleming’s or Florey’s work. Ernest A.C. Duchesne, a young military physician in Lyons, France, discovered penicillin in 1897 during a thesis project investigating antagonism between bacteria and fungi. On the advice of his mentor, Professor Gabriel Roux, Duchesne inoculated guinea pigs with various bacteria and then injected them with either the broth of a Penicillium glaucum culture or saline. Most of the animals injected with the culture broth survived, while those given saline died. Duchesne’s thesis was not noticed by the Institut Pasteur, and he entered the French Army and was unable to continue his research. Penicillin remained unknown until Fleming left several bacterial culture plates on a laboratory bench, allegedly before leaving for his 1928 summer holiday. Fleming claimed that upon his return, he examined the culture plates and noted, “Around a large colony of a contaminatingmold, the staphylococcus coloniesbecame transparent and had obviously undergone lysis.” He subsequently carriedout experimentswith animals and then human conjunctivae, but had difficulty extracting adequate amounts of penicillin from culture and abandoned this work. Largescale production of penicillin was developed by Florey andGerman-born British biochemist Ernst B. Chain more than a decade later. Floreywas born inAdelaide, SouthAustralia, on September 26, 1898, the youngest of five children. His father was a manager for a shoe manufacturing company. Following education at St. Peters Collegiate School and the University of Adelaide, where he obtained his MBBS degree in 1921, Florey was selected as a Rhodes Scholar to Magdalen College at Oxford in 1922. In

Bruce Beutler: Innate Immunity and Toll-Like Receptors

From the Dana-Farber Cancer Institute, Boston, MA (D.P.S.), and Mayo Clinic, Rochester, MN (M.A.S., R.A.K.). B ruce Alan Beutler was born in Chicago, Illinois, in 1957 but spent most of his childhood in southern California, where his father, noted hematologist Ernest Beutler (1928-2008), became the Chairman of Medicine at the City of Hope National Medical Center in Duarte, California, in 1959. Young Bruce Beutler’s first intensive exposure to biology was in his father’s laboratory on weekends and school holidays, where he learned how to purify proteins and to assay red cell enzymes such as glucose 6-phosphate dehydrogenase. In high school, he also worked with Susumu Ohno, an immunologist and geneticist at City of Hope who collaborated with his father. Beutler distinguished himself early academically, graduating from the University of California at San Diego at age 18 and from the University of Chicago’s medical school at age 23. (Ernest Beutler had graduated from the same medical school at age 21, and he suggested to his son that medical training would be a good skill “to fall back on” in case his planned scientific career foundered.) Bruce Beutler’s later scientific focus on innate immunity was in part a result of medical school clinical experience with patients with inflammatory diseases and serious infections. Beutler’s medical internship was at the University of Texas Southwestern (UT Southwestern) Medical Center at Dallas, followed by a year as a neurology resident at the same institution. In 1983, he moved to Rockefeller University in New York and to the laboratory of Anthony Cerami, where Beutler was the first to isolate murine tumor necrosis factor (TNF). He discovered that the TNF pathwaymediated some of the potent inflammatory effects of lipopolysaccharide (LPS), a bacterial macromolecule important in the clinical signs of sepsis. In 1986, he joined the faculty of UT Southwestern and also became an assistant

Tadeus Reichstein—Work on Hormones of the Adrenal Glands

From the Mayo Clinic, Rochester, MN (M.A.S., R.A.K.); and Dana-Farber Cancer Institute, Boston, MA (D.P.S.). T he Polish-Swiss biochemist Tadeus Reichstein shared the 1950 Nobel Prize for Physiology or Medicine with 2 American medical scientists, Philip S. Hench (1896-1965) and Edward C. Kendall (18861972), for discoveries concerning the structure and effects of hormones of the adrenal cortex. Reichstein’s research on steroids, particularly on hormones of the adrenal cortex, paralleled that of Kendall in the United States. Reichstein’s “substance F,” described and named by him in 1936, proved to be identical to Kendall’s “compound E,” or cortisone. Hench was credited with the clinical application of Kendall’s and Reichstein’s discoveries. The work of the 3 laureates proved useful in the treatment of rheumatoid arthritis and many varieties of inflammatory disease. Reichstein, the eldest of 5 sons, was born on July 20, 1897, in Wloclawek, Poland (about 80 miles northwest of Warsaw). Reichstein spent the first 8 years of his life in Kiev, Ukraine (where his father worked as an engineer), and then attended a private boarding school in Jena, Germany. In early 1905, the familymoved to Berlin (Germany) and then later in 1905 to Zurich (Switzerland), where they settled permanently and acquired Swiss citizenship in 1914. In 1914, Reichstein, who had been tutored privately, entered the Oberrealschule (a junior technical school) in Zurich and graduated in 1916, after which he enrolled in the Eidgenossische Technische Hochschule (State Technical College [ETH]) and received a bachelor’s degree in chemical engineering in 1920. After receiving his bachelor’s degree, Reichstein spent 1 year as an industrial chemist in a flashlight factory, after which he returned to the ETH for graduate study. In 1922, he was awarded a PhD degree in organic chemistry. After receiving his doctorate, Reichstein worked as an assistant to Herman Staudinger (18811965), who won the Nobel Prize in chemistry in 1953. Their work involved the isolation

Hans Geiger—German Physicist and the Geiger Counter

The German physicist Hans Wilhelm Geiger is best known as the inventor of the Geiger counter to measure radiation. In 1908, Geiger introduced the first successful detector of individual alpha particles. Later versions of this counter were able to count beta particles and other ionizing radiation. The introduction in July 1928 of the Geiger-Muller counter marked the introduction of modern electrical devices into radiation research. Geiger, the eldest of 5 children of a professor of philology, was born on September 30, 1882, in Neustadt an der Hardt, Rhineland-Palatinate state in western Germany (about 20 miles southwest of Mannheim). He studied physics at the universities of Munich and Erlangen in Bavaria, Germany, and received the PhD degree from the latter university in 1906. At the University of Erlangen, he worked with Eilhard Wiedemann (1852-1928) and wrote a thesis on electrical discharges through gases. Geiger received a fellowship that enabled him to work as an assistant to physicist Arthur Schuster (1851-1934) at the University of Manchester (England). After Schuster’s retirement in 1907, Geiger continued to work with Schuster’s successor, Ernest Rutherford (1871-1937). In 1908, Rutherford and Geiger devised a counter for alpha particles, work that led to Rutherford’s nuclear theory of the atom, for which he won the 1908 Nobel Prize in chemistry. They used the counter and other radiation detectors in experiments that led to the identification of the alpha particle as the nucleus of the helium atom and to Rutherford’s correct proposal (1912) that, in any atom, the nucleus occupies a very small volume at the center. Geiger remained in England until 1912, when he was named head of the German National Institute of Science and Technology in Berlin, where he continued his studies on atomic structure and radiation counting. In 1913, Geiger was joined by two physicists, Walther Bothe (1891-1957), later the 1954 Nobel Prize winner in physics, and James Chadwick (1891-1974), later Sir James Chadwick and winner of the 1935 Nobel Prize in physics. Bothe investigated alpha scattering, and Chadwick counted beta particles. The work was interrupted in 1914, with the beginning of World War I (1914-1918). Geiger served in the German army in the field artillery. After the war, Geiger returned to his work, and in 1924, he used his device to confirm the Compton effect, namely, the increase in wavelength of electromagnetic radiation, especially of an X-ray or gamma-ray photon, scattered by an electron. The Compton effect was discovered by the American physicist Arthur Holly Compton (1892-1962), for which he was awarded the 1927 Nobel Prize in physics. In 1925, Geiger accepted his first teaching position, which was at the University of Kiel, Germany. Here, he and Walther Muller improved the sensitivity, performance, and durability of the counter, and it became known as the “Geiger-Muller counter.” It could detect not only alpha particles but also beta particles (electrons) and ionizing photons. The counter was essentially in the same form as the modern counter. In 1929, Geiger moved to the University of Tubingen (Germany), where he was named professor of physics and director of research at the Institute of Physics. In 1929, while at the Institute, Geiger made his first observations of a cosmic-ray shower. Geiger continued to investigate cosmic rays, artificial radioactivity, and nuclear fission after accepting a position in 1936 at the Technische Hochschule in Berlin, a position he held until his death. In 1937, with Otto Zeiller, Geiger used the counter to measure a cosmic-ray shower. During World War II (1939-1945), Geiger participated briefly in Germany’s abortive attempt to develop an atomic bomb. In June 1945, Geiger fled the Russian occupation of Berlin and went to nearby Potsdam, where he died on September 24, 1945, at the age of 62 years, less than 2 months after the American atomic bomb was dropped on Hiroshima, Japan. Geiger was honored on a stamp (Scott No. 2182) issued in 1998 by Antigua and Barbuda.

Vincent du Vigneaud—Nobel Prize in Chemistry

From the Mayo Clinic, Rochester, MN (M.A.S., R.A.K.); and Dana-Farber Cancer Institute, Boston, MA (D.P.S.). I n 1955, the Nobel Prize in chemistry was awarded to Vincent du Vigneaud for the isolation and synthesis of two pituitary hormones: vasopressin, which acts on the muscles of the blood vessels, causing an increase in blood pressure, and oxytocin, the principal agent causing contractions of the uterus and secretion of milk. du Vigneaud also contributed to the understanding of vitamins, insulin, and metabolic reactions. du Vigneaud was born on May 18, 1901, in Chicago, Illinois. His father was an inventor and designer of machines. Vincent attended public schools of Chicago and, in 1918, entered the University of Illinois at Champaign-Urbana. He received a BS degree in 1923 and an MS degree in 1924, both in chemistry. In 1924, du Vigneaud worked briefly at the Jackson Laboratories of E.I. Du Pont de Nemours and Company in Wilmington, Delaware. He left that company in 1924 to become an assistant biochemist at the Graduate School of Medicine at the University of Pennsylvania (Philadelphia) and at the Philadelphia General Hospital, where he worked in the chemistry laboratory. In 1925, du Vigneaud joined the faculty of the new School of Medicine at the University of Rochester in Rochester, New York. At the University of Rochester, he conducted research on the chemistry of insulin while working on his doctorate. In 1927, he was awarded a PhD degree in chemistry. After graduation, he became a National Research Council Fellow. This fellowship allowed him to study at the Johns Hopkins School of Medicine (Baltimore, Maryland) in the Department of Pharmacology, where he isolated the amino acid cystine from crystals of insulin.

Michael Servetus: unorthodox theologian, meticulous cartographer, and innovative anatomist.

From Dana-Farber Cancer Institute, Boston, MA (D.P.S.), and the Mayo Clinic, Rochester, MN (M.A.S., R.A.K.). M iguel (Michael) Servetus (15111553) was a gifted anatomist who had the misfortune to be an independent thinker and theological gadfly during a polarized and religiously intolerant age. His heterodox belief that the Christian doctrine of the Trinity lacked a basis in the Holye Scripturesda doctrine that had been considered orthodox dogma by the Church since the Council of Nicea in 325eADdinfuriated many of the chief religious figures of the 16th century and ultimately proved to be Servetus’ undoing. Servetus was one of a few Renaissance thinkers who managed to draw the ire of both the Catholic Inquisition and Protestant Reformers. Servetuswas born near Villanueva, Spain, in either 1509 or 1511. Little is known of his early life, but by the age of 15, he had attached himself to a prominent Franciscan monk and, in 1528, attended the University of Toulouse under the monk’s patronage. At the University, he read the Bible for the first time and was apparently surprised to find no direct mention of the Trinity. He grew increasingly critical of the establishedChurch, rejected hismaster during a sojourn to Italy in 1530, and joined Protestant groups in Basel and Strasbourg. Servetus published De Trinitatis erroribus (On the Errors of the Trinity) in 1531. It is not clear what sort of reception he expected for his views. In any event, not only were Reformation leaders critical, he was summoned by the Inquisition in 1532. He fled to Paris to escape the Inquisition, where he studied mathematics and also briefly read medicine. Working under an assumed name, he next moved to Lyon, where he made a living correcting contemporary editions of ptolemaic maps for publication by the Treschel firm; these cartographic works are now rare and highly valued by collectors. From 1536 to 1538, Servetus returned to the University of Paris to study medicine again,

Sir Brian Gerald Barratt-Boyes—Pioneer Cardiac Surgeon

N t h F t Dana-Farber Cancer Institute, Boston, MA (D.P.S.). B orn on January 13, 1924, in Wellington, New Zealand, Brian Barratt-Boyes attended Wellington College and then Victoria University, both in Wellington. He graduated in medicine from Otago Medical School (Dunedin, New Zealand) in 1946. He received initial surgical training in New Zealand and then trained with pioneering cardiac surgeon John Kirklin at Mayo Clinic in Rochester, Minnesota, from 1953 to 1955. Following a 1-year Nuffield Fellowship in England—a program endowed in 1940 by Viscount Nuffield of Oxford (William Morris, founder of Morris Motors) to support research in health care policy—BarrattBoyes was recruited by Sir Douglas Robb, New Zealand’s pioneer of heart surgery, to Green Lane Hospital in Auckland, New Zealand. At Green Lane, Barratt-Boyes pioneered the development of cardiopulmonary bypass surgery, using this technique for the first time on a patient in New Zealand in 1958. He was also one of the first surgeons to implant a pacemaker (a prototype built in a university laboratory) in the human heart before these devices became commercially available in 1961. He credited these early successes in part to the remoteness of New Zealand with its accompanying freedom from the distractions of large urban centers, and he compared this to the single-minded devotion that the small city of Rochester had allowed Kirklin. In 1962, independently and almost simultaneously with Donald Ross of London, Barratt-Boyes introduced human cadaveric aortic homografts for aortic valve replacement. He championed the physiologic advantages of such grafts and simplified their surgical implantation technique. He also introduced the use of hypothermia and cardiac arrest for major surgery in neonates and infants. He was coauthor t