Introduction to HIV types, groups and subtypes

Introduction to HIV types, groups and subtypes

HIV is a highly variable virus which mutates very readily. This means there are many different strains of HIV, even within the body of a single infected person.
Based on genetic similarities, the numerous virus strains may be classified into types, groups and subtypes.
There are two types of HIV: HIV-1 and HIV-2. Both types are transmitted by sexual contact, through blood, and from mother to child, and they appear to cause clinically indistinguishable AIDS. However, it seems that HIV-2 is less easily transmitted, and the period between initial infection and illness is longer in the case of HIV-2.
Worldwide, the predominant virus is HIV-1, and generally when people refer to HIV without specifying the type of virus they will be referring to HIV-1. The relatively uncommon HIV-2 type is concentrated in West Africa and is rarely found elsewhere.
Subtypes of HIV-1
The strains of HIV-1 can be classified into three groups : the "major" group M, the "outlier" group O and the "new" group N. These three groups may represent three separate introductions of simian immunodeficiency virus into humans.
Group O appears to be restricted to west-central Africa and group N - discovered in 1998 in Cameroon - is extremely rare. More than 90% of HIV-1 infections belong to HIV-1 group M and, unless specified, the rest of this page will relate to HIV-1 group M only.
Within group M there are known to be at least nine genetically distinct subtypes (or clades) of HIV-1. These are subtypes A, B, C, D, F, G, H, J and K.
Occasionally, two viruses of different subtypes can meet in the cell of an infected person and mix together their genetic material to create a new hybrid virus (a process similar to sexual reproduction, and sometimes called "viral sex").1 Many of these new strains do not survive for long, but those that infect more than one person are known as "circulating recombinant forms" or CRFs. For example, the CRF A/B is a mixture of subtypes A and B.
The classification of HIV strains into subtypes and CRFs is a complex issue and the definitions are subject to change as new discoveries are made. Some scientists talk about subtypes A1, A2, A3, F1 and F2 instead of A and F, though others regard the former as sub-subtypes.
Subtypes E and I? : One of the CRFs is called A/E because it is thought to have resulted from hybridization between subtype A and some other "parent" subtype E. However, no one has ever found a pure form of subtype E. Confusingly, many people still refer to the CRF A/E as "subtype E" (in fact it is most correctly called CRF01_AE).2
A virus isolated in Cyprus was originally placed in a new subtype I, before being reclassified as a recombinant form A/G/I. It is now thought that this virus represents an even more complex CRF comprised of subtypes A, G, H, K and unclassified regions. The designation "I" is no longer used.
Different subtypes and CRFs
The HIV-1 subtypes and CRFs are very unevenly distributed throughout the world, with the most widespread being subtypes A and C.
Subtype A and CRF A/G predominate in West and Central Africa, with subtype A possibly also causing much of the Russian epidemic.4
Historically, subtype B has been the most common subtype/CRF in Europe, the Americas, Japan and Australia. Although this remains the case, other subtypes are becoming more frequent and now account for at least 25% of new infections in Europe.
Subtype C is predominant in Southern and East Africa, India and Nepal. It has caused the world's worst HIV epidemics and is responsible for around half of all infections.
Subtype D is generally limited to East and Central Africa. CRF A/E is prevalent in South-East Asia, but originated in Central Africa. Subtype F has been found in Central Africa, South America and Eastern Europe. Subtype G and CRF A/G have been observed in West and East Africa and Central Europe.
Subtype H has only been found in Central Africa; J only in Central America; and K only in the Democratic Republic of Congo and Cameroon.
A study presented in 2006 found that Ugandans infected with subtype D or recombinant strains incorporating subtype D developed AIDS sooner than those infected with subtype A, and also died sooner. The study's authors suggested that subtype D is more virulent because it is more effective at binding to immune cells.5 This result was supported by another study presented in 2007, which found that Kenyan women infected with subtype D had more than twice the risk of death over six years compared with those infected with subtype A.6 An earlier study of sex workers in Senegal, published in 1999, found that women infected with subtype C, D or G were more likely to develop AIDS within five years of infection than those infected with subtype A.
Differences in transmission
It has been observed that certain subtypes/CRFs are predominantly associated with specific modes of transmission. In particular, subtype B is spread mostly by homosexual contact and intravenous drug use (essentially via blood), while subtype C and CRF A/E tend to fuel heterosexual epidemics (via a mucosal route).
Whether there are biological causes for the observed differences in transmission routes remains the subject of debate. Some scientists, such as Dr Max Essex of Harvard, believe such causes do exist. Among their claims are that subtype C and CRF A/E are transmitted much more efficiently during heterosexual sex than subtype B.8 9 However, this theory has not been conclusively proven.
More recent studies have looked for variation between subtypes in rates of mother-to-child transmission. One of these found that such transmission is more common with subtype D than subtype A.12 Another reached the opposite conclusion (A worse than D), and also found that subtype C was more often transmitted that subtype D. A third study concluded that subtype C is more transmissible than either D or A. Other researchers have found no association between subtype and rates of mother-to-child transmission.
Until about 1994, it was generally thought that individuals do not become infected with multiple distinct HIV-1 strains. Since then, many cases of people coinfected with two or more strains have been documented.
All cases of coinfection were once assumed to be the result of people being exposed to the different strains more or less simultaneously, before their immune systems had had a chance to react. However, it is now thought that "superinfection" is also occurring. In these cases, the second infection occurred several months after the first. It would appear that the body's immune response to the first virus is sometimes not enough to prevent infection with a second strain, especially with a virus belonging to a different subtype. It is not yet known how commonly superinfection occurs, or whether it can take place only in special circumstances.
Tests for HIV
Initial tests for HIV are usually conducted using the EIA (or ELISA) antibody test or a rapid antibody test.
EIA tests which can detect either one or both types of HIV have been available for a number of years. According to the US Centers for Disease Control and Prevention, current HIV-1 EIAs "can accurately identify infections with nearly all non-B subtypes and many infections with group O HIV subtypes."21 However, because HIV-2 and group O infections are extremely rare in most countries, routine screening programs might not be designed to test for them. Anyone who believes they may have contracted HIV-2, HIV-1 group O or one of the rarer subtypes of group M should seek expert advice.
Rapid tests - which can produce a result in less than an hour - are becoming increasingly popular. Most modern rapid HIV-1 tests are capable of detecting all the major subtypes of group M.22 Rapid tests which can detect HIV-2 are also now available.
Treatment implications
Most current HIV-1 antiretroviral drug regimens were designed for use against subtype B, and so hypothetically might not be equally effective in Africa or Asia where other strains are more common. At present, there is no compelling evidence that subtypes differ in their sensitivity to antiretroviral drugs. However, some subtypes may occasionally be more likely to develop resistance to certain drugs. In some situations, the types of mutations associated with resistance may vary. This is an important subject for future research.
The effectiveness of HIV-1 treatment is monitored using viral load tests. It has been demonstrated that some such tests are sensitive only to subtype B and can produce a significant underestimate of viral load if used to process other strains. The latest tests do claim to produce accurate results for most Group M subtypes, though not necessarily for Group O. It is important that health workers and patients are aware of the subtype/CRF they are testing for and of the limitations of the test they are applying.
Not all of the drugs used to treat HIV-1 infection are as effective against HIV-2. In particular, HIV-2 has a natural resistance to NNRTI antiretroviral drugs and they are therefore not recommended. As yet there is no FDA-licensed viral load test for HIV-2 and those designed for HIV-1 are not reliable for monitoring the other type. Instead, response to treatment may be monitored by following CD4+ T-cell counts and indicators of immune system deterioration. More research and clinical experience is needed to determine the most effective treatment for HIV-2
The development of an AIDS vaccine is affected by the range of virus subtypes as well as by the wide variety of human populations who need protection and who differ, for example, in their genetic make-up and their routes of exposure to HIV. In particular, the occurrence of superinfection indicates that an immune response triggered by a vaccine to prevent infection by one strain of HIV may not protect against all other strains. The effectiveness of a vaccine is likely to vary in different populations unless some innovative method is developed which guards against many virus strains.
Inevitably, different types of candidate vaccines will have to be tested against various viral strains in multiple vaccine trials, conducted in both high-income and developing countries.
Types of HIV Infections (AIDS)
For the first few years of the AIDS epidemic, it appeared that some HIV-infected people sickened and died quickly while the others stayed healthy indefinitely or slowly progressed into sickness. Now it appears the greater majority of HIV-infected will gradually become very sick and eventually die. There are reports of long-term survivors of HIV infection; but, they seem to be the exception rather than the rule.
The average (the mean) incubation time for HIV infection is 10 years. Incubation time means the time from initial infection until the development of "full-blown AIDS," discussed below. The average is a bell curve, with 10 years at the center. Some individuals develop illness sooner than 10 years and some later than 10 years.
Most symptoms and diseases common to HIV infection are listed in Figures 4, 5 and 6. The presence of these symptoms and diseases varies from one patient to another. These diseases may occur in sequence or simultaneously.
Obviously, many of these symptoms can be caused by a number of common illnesses. These diseases are listed here for the sake of education, not for the purpose of self-diagnosis. In case of any persistent illness, consult your health care provider.
The popular classification system of HIV infections, used here, is a collection of haphazard definitions that evolved as the AIDS epidemic unfolded. These labels are ones of convenience, not ones of scientific or medical accuracy. Medical authorities use different, more complex, classification systems.
Basically, four loosely defined different stages of HIV infection exist: I ) the healthy carrier state, 2) the lymphadenopathy syndrome (LAS), 3) AIDS-related complex (ARC), and 4) AIDS or "frank AIDS," or "full-blown AIDS." These forms or the symptoms of each may overlap the other.
Healthy Carrier State
A carrier is someone who is infected with a disease and shows no clinical symptoms, but who is capable of infecting other people with the disease. ("Clinical" means "seen in the doctors office.")
HIV has been isolated (removed) and cultured ("grown" in a laboratory dish) from healthy people who show no clinical signs of HIV infection.
It is not yet clear when an HIV-infected person becomes infectious. At this time, the only safe practice is to assume that anyone carrying the virus is capable of transmitting it to others.
Lymphadenopathy Syndrome (LAS)
Lymphadenopathy Syndrome (LAS) is a mild form of HIV infection, generally characterized by some of the symptoms in Figure 4.
Lymphadenopathy means "disease of the lymphatic system." The lymphatic system is the human body's second fluid system which contains a clear fluid called lymph (see Figure 3). The lymphatic system aids the blood system by draining fluid out of the body' s tissues. The lymphatic system is not a closed loop like the bloodstream, meaning it does not flow in a circle, and it has no pump like the heart. Nevertheless, lymph flows from smaller vessels into larger lymph ducts in the upper chest. In doing so, lymphatic fluid passes through a series of filtering stations called lymph nodes, or lymph glands. Lymph nodes filter bacteria (one-celled organisms), foreign substances, and dead white blood cells out of the fluid.
The lymphatic system is a vital part of the body's immune system. Lymph nodes store and mature lymphocytes and other white blood cells and also manufacture antibodies. T-cells and macrophages can migrate back and forth between the blood system and the lymphatic system, perhaps exposing newly generating cells to HIV during their formative stages.
One of the key signs of lymphadenopathy is swollen lymph glands. Of course, any infection, such as the flu, causes the lymph nodes to swell; but, nodal swelling due to normal infections passes quickly. With HIV infection, this nodal swelling may persist for months, with no other signs of a temporary infectious disease. Consequently, lymphadenopathy is sometimes called persistent generalized lymphadenopathy (PGL).

Symptoms of Lymphadenopathy Syndrome (LAS)
• Unexplained fever
• Difficulty in swallowing
• Swollen glands
• Fatigue/Lethargy
• Night sweats and chills
• Apathy
• Gradual loss of weight
• Diarrhea
• Sore throat
• Impotence
AIDS-related Complex (ARC)
AIDS-related Complex is a more advanced level of HIV infection. Symptoms generally include the symptoms of lymphadenopathy, plus abnormal body conditions revealed by laboratory tests, and/or the presence of one or more opportunistic infections.
A person with ARC has a discomforting illness. His or her everyday activity may be restricted and he or she is probably manifesting bouts of illness that require short-term or long-term medical treatment in and out of the hospital.
Acquired Immune Deficiency Syndrome (AIDS)
AIDS is the "full-blown" syndrome, also called "frank" AIDS. Patients suffering from AIDS often have any number of the opportunistic diseases listed in Figure 6. These diseases develop because of the widespread failure of the immune system. Drug treatments are available for many of these infections; but, without the support of the immune system, the drugs fail to cure the disease fully or are unable to keep the disease from returning. These opportunistic infections, curable under other circumstances, cause the death of most AIDS patients.
Symptoms and Conditions of ARC and AIDS
• Anergy: lack of skin allergic response
• Anemia: lack of red blood cells
• Autoimmune Disorders: immune system attacks own body
• Candidiasis/Oral Thrush: See Figure 6
• Hyperplasia: excessive growth of normal cells in organ
• Kidney Dysfunction: kidneys fail or function poorly
• Leukopenia: decreased number of leukocytes (white blood cells that engulf germs)
• Lymphomas: lymphatic system cancers
• Lymphopenia: decreased number of lymphocytes
• Nerve Damage: possible blindness, deafness, paralysis
• Oral Thrush: caused by Epstein-Barr Virus (Figure 6)
• Wasting: severe weight loss, perhaps death, from diarrhea and malnutrition
Diseases Common to AIDS
Pneumocystis carinii pneumonia (PCP).
Caused by fungus-like single-celled parasite, Pneumocystis carinii, common world-wide. Infects lungs. Previous to AIDS, found in kidney transplant patients whose immune system had been chemically suppressed. Occurs in 60% to 80% of AIDS patients. Initially responsible for 30% to 50% of deaths among AIDS patients, now brought under better control due to chemical prophylaxis, that is, chemically treating the patient before symptoms occur.
Kaposi's sarcoma (KS).
Malignant skin cancer. Appear first as pink, purple or brown lesions (wounds), usually on arms and/or legs; then spreading around body. In AIDS patients, may spread to gastrointestinal tract, lungs, other internal organs. Initially occurred in 46% of homosexual AIDS patients, in only 3.8% of heterosexual IV drug abuser AIDS patients. Onset is statistically associated, in homosexual males, with oral-anal sex and fecal (feces) contact-possible infectious agent involved.
Toxoplasmosis.
Caused by Toxoplasmosa gondii. Infects blood and many tissues. Common to humans, many domestic and wild animals. Humans may catch from droppings of cats and undercooked meat, especially mutton. In AIDS patients, tendency to infect tissues of central nervous system (brain and nerves). Also causes pneumonia and hepatitis inflammation/dysfunction of the liver). Many minor, non-life-threatening outbreaks occur in day-care centers. In AIDS patients, can be a major cause of mortality.


Candidiasis.
Caused by species of Candida, a fungus common to skin, mouth, vagina, gastrointestinal tract of humans. In AIDS patients, usually takes oral form: white spots or patches on lateral sides of tongue, perhaps inside mouth on mucous membranes of cheeks; commonly lodges under nailbeds and skin around armpits, groin, and rectum. Sometimes affects lungs. Frequently, first clinical (as seen in doctor's office) sign of HIV infection .
Cryptococcosis.
Caused by Cryptococcus neoformans, a fungus found in pigeon manure. Common among humans and other mammals, especially cats. Causes pneumonia in rare instances, most often causes meningitis (inflammation of the spinal cord and brain membranes). Also causes endocarditis (inflammation of lining of heart); and skin ulcers. Some increasing success with drug therapy.
Herpes infections.
Caused by herpes simplex viruses 1 (cold sores on lips) and 2 (sores on genitals). In HIV-infected patients, herpes simplex infections form chronic ulcers, often affecting face and sometimes the eyes; anal area often affected in homosexual males. Herpes infections are commonly found in people who are not infected with HIV; forming a cluster(s) of small, painful blisters, often, but not necessarily, on face.
Herpes zoster infection.
Caused by another herpes virus. Also known as shingles or chickenpox. Herpes zoster viruses may remain latent (inactive) for years (perhaps left over from childhood), but may be reactivated by HIV infection, causing inflammation of the spinal and cranial ganglia (nerve roots). In AIDS patients, can be disseminated (widespread) throughout the body. Often an initial clinical symptom in HIV-infected individuals. Herpes zoster is common among people not infected with HIV.
Mycobacterium infection.
Caused by Mycobacterium avium intracellulare, a bacterium commonly found in human saliva. Causes type of tuberculosis in humans, producing lesions in lungs. Disseminated, it cause problems in the intestines, blood, liver, and spleen.
Epstein-Barr infection.
Caused by Epstein-Barr virus (EBV), suspected cause of mononucleosis and some lymphomas (cancers of the lymph tissue). Implicated in number of auto-immune conditions (body's immune system attacking itself, as sometimes occurs in advanced HIV infection.) Thought to disrupt T-cell function. In HIV-infected, causes oral hairy leukoplakia, fuzzy white spots on the tongue which do not rub off as does "hairy tongue" caused by smoking. Possibly remains dormant until HIV infection occurs.
Cytomegalovirus (CMV) infection.
Normally present in salivary glands of humans. Often widely scattered throughout the body in patients with advanced HIV infection. Causes problems in eyes, colon, lungs, liver, and adrenal glands. Suspected in promoting appearance of Kaposi's sarcoma. After PCP prophylaxis became effective, CMV infection became the major cause of mortality among AIDS patients. Cytomegalovirus is frequently spread in day-care centers, where it has been shown to survive on toys and plexiglass for 30 minutes.
Cryptosporidiosis.
An enteritis (inflammation/swelling of intestines) caused by Cryptosporidia muris and/or C. difficile; a one-celled parasite common to domestic and wild animals. Many minor, non-life-threatening outbreaks occur in day-care centers. In AIDS patients, may be major cause of mortality.
Tuberculosis (TB).
Caused by Mycobacterium tuberculosis, a bacterium and a non-opportunistic infection found in non-HIV-infected people. Infects lungs, disseminated in some AIDS patients. A major killer in the past, social hygiene education and effective medical treatment eliminated TB from most of the Western world, except among populations lacking adequate access to medical care. Statistically associated with AIDS (found in some AIDS patients), it may reflect socio-economic status rather than being an opportunistic infection due to AIDS. Infection may occur prior to HIV infection as a damaged immune system is not required to catch TB.

Diabetes

Diabetes mellitus

Diabetes mellitus is a disorder of carbohydrate metabolism. It is a disease characterized by persistent hyperglycemia (high blood sugar levels). It is a metabolic disease that requires medical diagnosis, treatment and lifestyle changes. The World Health Organization recognizes three main forms of diabetes: type 1, type 2 and gestational diabetes (or type 3, occurring during pregnancy)[1], although these three "types" of diabetes are more accurately considered patterns of pancreatic failure rather than single diseases. Type 1 is due to autoimmune destruction of the insulin-producing cells, while type 2 and gestational diabetes are due to insulin resistance by tissues. Type 2 may progress to destruction of the insulin producing cells of the pancreas, but is still considered Type 2, even though insulin administration may be required..
Since the first therapeutic use of insulin (1921) diabetes has been a treatable but chronic condition, and the main risks to health are its characteristic long-term complications. These include cardiovascular disease (doubled risk), chronic renal failure (it is the main cause for dialysis in developed world adults), retinal damage which can lead to blindness and is the most significant cause of adult blindness in the non-elderly in the developed world, nerve damage, erectile dysfunction (impotence), to gangrene with risk of amputation of toes, feet, and even legs.
The term diabetes was coined by Aretaeus of Cappadocia. It is derived from the Greek diabaínein that literally means "passing through," or "siphon," a reference to one of diabetes' major symptoms—excessive urine production. In 1675 Thomas Willis added mellitus from the Latin word for honey (mel in the sense of "honey sweet") when he noted that the blood and urine of a diabetic has a sweet taste. This had been noticed long before in ancient times by the Greeks, Chinese, Egyptians, and Indians. In 1776 it was confirmed the sweet taste was because of an excess of a kind of sugar in the urine and blood of people with diabetes.[citation needed]
The ancient Indians tested for diabetes by observing whether ants were attracted to a person's urine, and called the ailment "sweet urine disease" (Madhumehalai); medieval European doctors tested for it by tasting the urine themselves, a scene which was occasionally depicted in Gothic reliefs.
While the term diabetes without a modifier usually refers to diabetes mellitus, there is another, rarer condition named diabetes insipidus (unquenchable diabetes) in which the urine is not sweet; it can be caused by either kidney (nephrogenic DI) or pituitary gland (central DI) damage.
History
Although diabetes has been recognized since antiquity, and treatments of various efficacy have been known in various regions since the Middle Ages, and in legend for much longer, the elucidation of the pathogenesis of diabetes occurred mainly in the 20th century.
The discovery of the role of the pancreas in diabetes is generally ascribed to Joseph von Mering and Oskar Minkowski, European researchers who in 1889 found that when they completely removed the pancreas of dogs, the dogs developed all the signs and symptoms of diabetes and died shortly afterward. In 1910, Sir Edward Albert Sharpey-Schafer of Edinburgh suggested that people with diabetes were deficient in a single chemical that was normally produced by the pancreas—he proposed calling this substance insulin. The term is derived from the Latin insula, meaning island, in reference to the islets of Langerhans in the pancreas that produce insulin.
The endocrine role of the pancreas in metabolism, and indeed the existence of insulin, was not fully clarified until 1921, when Sir Frederick Grant Banting and Charles Herbert Best repeated the work of Von Mering and Minkowski, but went further and demonstrated that they could reverse induced diabetes in dogs by giving them an extract from the pancreatic islets of Langerhans of healthy dogs.Banting, Best, and colleagues (particularly the chemist Collip) went on to isolate the hormone insulin from bovine pancreases at the University of Toronto in Canada. This led to the availability of an effective treatment—insulin injections—and the first clinical patient was treated in 1922. For this, Banting and MacLeod received the Nobel Prize in Physiology or Medicine in 1923; both shared their Prize money with others in the team who were not recognized, in particular Best and Collip. Banting and Best made the patent available without charge and did not attempt to control commercial production. Insulin production and therapy rapidly spread around the world, largely as a result of this decision.Despite the availability of treatment, diabetes remained a major cause of death. For instance, statistics reveal that the cause-specific mortality rate during 1927 amounted to about 47.7 per 100,000 population in Malta.

The distinction between what is now known as type 1 diabetes and type 2 diabetes was first clearly made by Sir Harold Percival (Harry) Himsworth in 1935 and was published in January 1936.
Glucose metabolism
Mechanism of insulin release in normal pancreatic beta cells (that is, glucose dependence). Insulin secretion does not depend on blood glucose levels; it is stored pending release which does depend on blood glucose levels. Since insulin is the principal hormone that regulates uptake of glucose into most cells from the blood (primarily muscle and fat cells, but not central nervous system cells), deficiency of insulin or the insensitivity of its receptors plays a central role in all forms of diabetes mellitus.
Much of the carbohydrate in food is converted within a few hours to the monosaccharide glucose, the principal carbohydrate in blood. Some carbohydrates are not; fruit sugar (fructose) is usable as cellular fuel but is not converted to glucose and does not participate in the insulin / glucose metabolic regulatory mechanism, nor does the carbohydrate cellulose (though it is actually many glucoses in long chains) as humans and many animals have no digestive pathway capable of handling it. Insulin is released into the blood by beta cells (β-cells) in the pancreas in response to rising levels of blood glucose (e.g., after a meal). Insulin enables most body cells (about 2/3 is the usual estimate, including muscle cells and adipose tissue) to absorb glucose from the blood for use as fuel, for conversion to other needed molecules, or for storage. Insulin is also the principal control signal for conversion of glucose (the basic sugar used for fuel) to glycogen for internal storage in liver and muscle cells. Reduced insulin levels result both in the reduced release of insulin from the beta cells and in the reverse conversion of glycogen to glucose when glucose levels fall, although only glucose thus recovered by the liver re-enters the bloodstream as muscle cells lack the necessary export mechanism.
Higher insulin levels increase many anabolic ("building up") processes such as cell growth and duplication, protein synthesis, and fat storage. Insulin is the principal signal in converting many of the bidirectional processes of metabolism from a catabolic to an anabolic direction, and vice versa. In particular, it is the trigger for entering or leaving ketosis (ie, the fat burning metabolic phase).
If the amount of insulin available is insufficient, if cells respond poorly to the effects of insulin (insulin insensitivity or resistance), or if the insulin itself is defective, glucose will not be handled properly by body cells (about ⅔ require it) or stored appropriately in the liver and muscles. The net effect is persistent high levels of blood glucose, poor protein synthesis, and other metabolic derangements, such as acidosis.
Type 1 diabetes mellitus
Type 1 diabetes mellitus - formerly known as insulin-dependent diabetes (IDDM), childhood diabetes, or juvenile-onset diabetes - is characterized by loss of the insulin-producing beta cells of the islets of Langerhans of the pancreas leading to a deficiency of insulin. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages. This type comprises up to 10% of total cases in North America and Europe, though this varies by geographical location. This type of diabetes can affect children or adults, but has traditionally been termed "juvenile diabetes" because it represents a majority of cases of diabetes affecting children. The most common cause of beta cell loss leading to type 1 diabetes is autoimmune destruction, accompanied by antibodies directed against insulin and islet cell proteins. The principal treatment of type 1 diabetes, even from the earliest stages, is replacement of insulin. Without insulin, ketosis and diabetic ketoacidosis can develop and coma or death will result.
Currently, type 1 diabetes can be treated only with insulin, with careful monitoring of blood glucose levels using blood testing monitors. Emphasis is also placed on lifestyle adjustments (diet and exercise). Apart from the common subcutaneous injections, it is also possible to deliver insulin via a pump, which allows infusion of insulin 24 hours a day at preset levels, and the ability to program a push dose (a bolus) of insulin as needed at meal times. This is at the expense of an indwelling subcutaneous catheter. It is also possible to deliver insulin via an inhaled powder.
Type 1 treatment must be continued indefinitely at present. Treatment does not impair normal activities, if sufficient awareness, appropriate care, and discipline in testing and medication. The average glucose level for the type 1 patient should be as close to normal (80–120 mg/dl, 4–6 mmol/l) as possible. Some physicians suggest up to 140–150 mg/dl (7-7.5 mmol/l) for those having trouble with lower values, such as frequent hypoglycemic events. Values above 200 mg/dl (10 mmol/l) are often accompanied by discomfort and frequent urination leading to dehydration. Values above 300 mg/dl (15 mmol/l) usually require immediate treatment and may lead to ketoacidosis. Low levels of blood glucose, called hypoglycemia, may lead to seizures or episodes of unconsciousness.
Type 2 diabetes mellitus: Type 2 diabetes mellitus - previously known as adult-onset diabetes, maturity-onset diabetes, or non-insulin dependent diabetes mellitus (NIDDM) - is due to a combination of defective insulin secretion and defective responsiveness to insulin (often termed insulin resistance or reduced insulin sensitivity), almost certainly involving the insulin receptor in cell membranes. In early stages, the predominant abnormality is reduced insulin sensitivity, characterized by elevated levels of insulin in the blood. In the early stages, hyperglycemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver, but as the disease progresses the impairment of insulin secretion worsens, and therapeutic replacement of insulin often becomes necessary. There are numerous theories as to the exact cause and mechanism for this resistance, but central obesity (fat concentrated around the waist in relation to abdominal organs, not it seems, subcutaneous fat) is known to predispose for insulin resistance, possibly due to its secretion of adipokines (a group of hormones) that impair glucose tolerance. Abdominal fat is especially active hormonally. Obesity is found in approximately 90% of Developed world patients diagnosed with type 2 diabetes. Other factors may include aging and family history, although in the last decade it has increasingly begun to affect children and adolescents.
Type 2 diabetes may go unnoticed for years in a patient before diagnosis, since the symptoms are typically milder (eg, lack of ketoacidotic episodes) and can be sporadic. However, severe complications can result from unnoticed type 2 diabetes, including renal failure, vascular disease (including coronary artery disease), vision damage, etc.
Type 2 diabetes is usually first treated by changes in physical activity (usually increase), diet (generally decrease carbohydrate intake, especially glucose generating carbohydrates), and through weight loss. These can restore insulin sensitivity, even when the weight loss is modest, for example, around 5 kg (10 to 15 lb), most especially when it is in abdominal fat deposits. The next step, if necessary, is treatment with oral antidiabetic drugs. As insulin production is initially unimpaired, oral medication (often used in combination) can still be used that improves insulin production (eg, sulfonylureas) and regulate inappropriate release of glucose by the liver (and attenuate insulin resistance to some extent (eg, metformin), and substantially attenuate insulin resistance (eg, thiazolidinediones). If these fail, insulin therapy will be necessary to maintain normal or near normal glucose levels. A disciplined regimen of blood glucose checks is recommended in most cases, most particularly and necessarily when taking most of these medications.
Gestational diabetes
Gestational diabetes, Type 3, also involves a combination of inadequate insulin secretion and responsiveness, resembling type 2 diabetes in several respects. It develops during pregnancy and may improve or disappear after delivery. Even though it may be transient, gestational diabetes may damage the health of the fetus or mother, and about 20%–50% of women with gestational diabetes develop type 2 diabetes later in life.
Gestational diabetes mellitus occurs in about 2%–5% of all pregnancies. It is temporary, and fully treatable, but, if untreated, may cause problems with the pregnancy, including macrosomia (high birth weight) of the child. It requires careful medical supervision during the pregnancy.
Other types
There are several rare causes of diabetes mellitus that do not fit into type 1, type 2, or gestational diabetes:
Genetic defects in beta cells (autosomal or mitochondrial)
Genetically-related insulin resistance, with or without lipodystrophy (abnormal body fat deposition)
Diseases of the pancreas (e.g. chronic pancreatitis, cystic fibrosis)
Hormonal defects
Chemicals or drugs.
The tenth version of the International Statistical Classification of Diseases (ICD-10) contained a diagnostic entity named "malnutrition-related diabetes mellitus" (MRDM or MMDM, ICD-10 code E12). A subsequent WHO 1999 working group recommended that MRDM be deprecated, and proposed a new taxonomy for alternative forms of diabetes.Classifications of non-type 1, non-type 2, non-gestational diabetes remains controversial.[citation needed]
Genetics
Both type 1 and type 2 diabetes are at least partly inherited. Type 1 diabetes appears to be triggered by some (mainly viral) infections, or in a less common group, by stress or environmental factors (such as exposure to certain chemicals or drugs). There is a genetic element in individual susceptibility to some of these triggers which has been traced to particular HLA genotypes (i.e. genetic "self" identifiers used by the immune system). However, even in those who have inherited the susceptibility, type 1 diabetes mellitus seems to require an environmental trigger. A small proportion of people with type 1 diabetes carry a mutated gene that causes maturity onset diabetes of the young (MODY).
There is a rather stronger inheritance pattern for type 2 diabetes. Those with first-degree relatives with type 2 have a much higher risk of developing type 2. Concordance among monozygotic twins is close to 100%, and 25% of those with the disease have a family history of diabetes. It is also often connected to obesity, which is found in approximately 85% of (North American) patients diagnosed with this type, so some experts believe that inheriting a tendency toward obesity also contributes.
Diagnosis
Signs and symptoms
The classical triad of diabetes symptoms is polyuria (frequent urination), polydipsia (increased thirst, and consequent increased fluid intake) and polyphagia (increased appetite). These symptoms may develop quite fast in type 1, particularly in children (weeks or months), but may be subtle or completely absent - as well as developing much more slowly - in type 2. In type 1 there may also be weight loss (despite normal or increased eating), increased appetite, and irreducible fatigue. These symptoms may also manifest in type 2 diabetes in patients whose diabetes is poorly controlled.
Thirst develops because of osmotic effects—sufficiently high glucose (above the "renal threshold") in the blood is excreted by the kidneys, but this requires water to carry it and causes increased fluid loss, which must be replaced. The lost blood volume will be replaced from water held inside body cells, causing dehydration. Prolonged high blood glucose causes changes in the shape of the lens in the eye, leading to vision changes. Blurred vision is a common complaint leading to a diagnosis of type 1; it should always be suspected in such cases.
Patients (usually with type 1 diabetes) may also present with diabetic ketoacidosis (DKA), an extreme state of dysregulation characterized by the smell of acetone on the patient's breath, Kussmaul breathing (a rapid, deep breathing), polyuria, nausea, vomiting and abdominal pain and any of many altered state of consciousness or arousal (eg, hostility and mania or, equally, confusion and lethargy). In severe DKA, coma (unconsciousness) may follow, progressing to death if untreated. In any form, DKA is a medical emergency and requires expert attention.
A rarer but equally severe presentation is hyperosmolar nonketotic state, which is more common in type 2 diabetes, and is mainly the result of dehydration due to the polyuria. Often, the patient has been drinking extreme amounts of sugar-containing drinks, leading to a vicious circle in regard to water loss.
Diagnostic approach
The diagnosis of type 1 diabetes and many cases of type 2 is usually prompted by recent-onset symptoms of excessive urination (polyuria) and excessive thirst (polydipsia), often accompanied by weight loss. These symptoms typically worsen over days to weeks; about 25% of people with new type 1 diabetes have developed a degree of diabetic ketoacidosis by the time the diabetes is recognized. The diagnosis of other types of diabetes is usually made in many other ways. The most common are
(1) health screening,
(2) detection of hyperglycemia when a doctor is investigating a complication of
longstanding, unrecognized diabetes.
(3) new signs and symptoms attributable to the diabetes.
Diabetes screening is recommended for many types of people at various stages of life or with several different risk factors. The screening test varies according to circumstances and local policy and may be a random glucose, a fasting glucose and insulin, a glucose two hours after 75 g of glucose, or a formal glucose tolerance test. Many healthcare providers recommend universal screening for adults at age 40 or 50, and sometimes occasionally thereafter. Earlier screening is recommended for those with risk factors such as obesity, family history of diabetes, high-risk ethnicity (Hispanic/Latin American, American Indian, African American, Pacific Island, and South Asian ancestry).
Many medical conditions are associated with a higher risk of various types of diabetes and warrant screening. A partial list includes: high blood pressure, elevated cholesterol levels, coronary artery disease, past gestational diabetes, polycystic ovary syndrome, chronic pancreatitis, fatty liver, hemochromatosis, cystic fibrosis, several mitochondrial neuropathies and myopathies, myotonic dystrophy, Friedreich's ataxia, some of the inherited forms of neonatal hyperinsulinism, and many others. Risk of diabetes is higher with chronic use of several medications, including high-dose glucocorticoids, some chemotherapy agents (especially L-asparaginase), and some of the antipsychotics and mood stabilizers (especially phenothiazines and some atypical antipsychotics). Diabetes is often detected when a person suffers a problem frequently caused by diabetes, such as a heart attack, stroke, neuropathy, poor wound healing or a foot ulcer, certain eye problems, certain fungal infections, or delivering a baby with macrosomia or hypoglycemia.
Diagnostic criteria
Diabetes mellitus is characterized by recurrent or persistent hyperglycemia, and is diagnosed by demonstrating any one of the following:
Fasting plasma glucose level at or above 126 mg/dL or 7.0 mmol/l.
Plasma glucose at or above 200 mg/dL or 11.1 mmol/l two hours after a 75 g oral glucose load in a glucose tolerance test.
Random plasma glucose at or above 200 mg/dL or 11.1 mmol/l.
A positive result should be confirmed by any of the above-listed methods on a different day, unless there is no doubt as to the presence of significantly-elevated glucose levels. Most physicians prefer measuring a fasting glucose level because of the ease of measurement and time commitment of formal glucose tolerance testing, which can take two hours to complete. By definition, two fasting glucose measurements above 126 mg/dL or 7.0 mmol/l is considered diagnostic for diabetes mellitus.
Patients with fasting sugars between 6.1 and 7.0 mmol/l (110 and 125 mg/dL) are considered to have "impaired fasting glucose" and patients with plasma glucose at or above 140mg/dL or 7.8 mmol/l two hours after a 75 g oral glucose load are considered to have "impaired glucose tolerance". "Prediabetes" is either impaired fasting glucose or impaired glucose tolerance; the latter in particular is a major risk factor for progression to full-blown diabetes mellitus as well as cardiovascular disease.
While not used for diagnosis, an elevated level of glucose bound to hemoglobin (termed glycosylated hemoglobin or HbA1c) of 6.0% or higher (2003 revised U.S. standard) is considered abnormal by most labs; HbA1c is primarily a treatment-tracking test reflecting average blood glucose levels over the preceding 90 days (approximately). However, some physicians may order this test at the time of diagnosis to track changes over time. The current recommended goal for HbA1c in patients with diabetes is <7.0%, as defined as "good glycemic control", although some guidelines are stricter (<6.5%). People with diabetes that have HbA1c levels within this goal have a significantly lower incidence of complications from diabetes, including retinopathy and diabetic nephropathy.
Complications
The complications are far less common and less severe in people who have well-controlled blood sugar levels.In fact, the better the control, the lower the risk of complications. Hence patient education, understanding and participation is vital. Healthcare professionals who treat diabetes also address other health problems that may accelerate the deleterious effects of diabetes. These include smoking (abstain), elevated cholesterol levels (control with diet, exercise or medication), obesity (even modest weight loss can be beneficial), high blood pressure, and lack of regular exercise.
Acute Problems.
Diabetic ketoacidosis , Nonketotic hyperosmolar coma , Hypoglycemia , and Diabetic coma.
Diabetic ketoacidosis
Diabetic ketoacidosis (DKA) is an acute, dangerous complication and is always a medical emergency. On presentation at hospital, the patient in DKA is typically dehydrated and breathing both fast and deeply. Abdominal pain is common and may be severe. The level of consciousness is normal until late in the process, when lethargy (dulled or reduced level of alertness or consciousness) may progress to coma. The ketoacidosis can become severe enough to cause hypotension and shock. Prompt proper treatment usually results in full recovery, though death can result from inadequate treatment, delayed treatment or from a variety of complications. It is much more common in type 1 diabetes than type 2, but can still occur in patients with type 2 diabetes.
Nonketotic hyperosmolar coma
While not always progressing to coma, this hyperosmolar nonketotic state (HNS) is another acute problem associated with diabetes mellitus. It has many symptoms in common with DKA, but a different cause, and requires different treatment. In anyone with very high blood glucose levels (usually considered to be above 300 mg/dl or 16 mmol/l), water will be osmotically driven out of cells into the blood. The kidneys will also be "dumping" glucose into the urine, resulting in concomitant loss of water, causing an increase in blood osmolality. If the fluid is not replaced (by mouth or intravenously), the osmotic effect of high glucose levels combined with the loss of water will eventually result in such a high serum osmolality (dehydration).The body's cells may become progressively dehydrated as water is drawn out from them and excreted. Electrolyte imbalances are also common. This combination of changes, especially if prolonged, will result in symptoms of lethargy (dulled or reduced level of alertness or consciousness) and may progress to coma. As with DKA urgent medical treatment is necessary, especially volume replacement. This is the diabetic coma which more commonly occurs in type 2 diabetics.
Hypoglycemia
Hypoglycemia, or abnormally low blood glucose, is a complication of several diabetes treatments. It may develop if the glucose intake does not match the treatment. The patient may become agitated, sweaty, and have many symptoms of sympathetic activation of the autonomic nervous system resulting in feelings similar to dread and immobilized panic. Consciousness can be altered, or even lost, in extreme cases, leading to coma and/or seizures or even brain damage and death. In patients with diabetes this can be caused by several factors, such as too much or incorrectly timed insulin, too much exercise or incorrectly timed exercise (which decreases insulin requirements) or not enough food or insufficient amount of carbohydrates in food. In most cases, hypoglycemia is treated with sweet drinks or food. In severe cases, an injection of glucagon (a hormone with the opposite effects of insulin) or an intravenous infusion of glucose is used for treatment, but usually only if the diabetic is unconscious.
Chronic problems.
Microvascular disease
Chronic elevation of blood glucose level leads to damage of blood vessels. In diabetes, the resultant problems are grouped under "microvascular disease" (due to damage to small blood vessels) and "macrovascular disease" (due to damage to the arteries).
The damage to small blood vessels leads to a microangiopathy, which causes the following organ-related problems:
Diabetic retinopathy, growth of friable and poor-quality new blood vessels in the retina as well as macular edema (swelling of the macula), which can lead to severe vision loss or blindness. Retinal damage (from microangiopathy) makes it the most common cause of blindness among non-elderly adults in the US.
Diabetic neuropathy, abnormal and decreased sensation, usually in a stocking distribution starting at the feet but potentially in other nerves. When combined with damaged blood vessels this can lead to diabetic foot (see below). Other forms of diabetic neuropathy may present as mononeuritis or autonomic neuropathy.
Diabetic nephropathy, damage to the kidney which can lead to chronic renal failure, eventually requiring dialysis. Diabetes mellitus is the most common cause of adult kidney failure worldwide.
Macrovascular disease
Macrovascular disease leads to cardiovascular disease, mainly by accelerating atherosclerosis:
Coronary artery disease, leading to myocardial infarction ("heart attack") or angina
Stroke (mainly ischemic type)
Peripheral vascular disease, which contributes to intermittent claudication (exertion-related foot pain) as well as diabetic foot.
Diabetic myonecrosis
Diabetic foot, often due to a combination of neuropathy and arterial disease, may cause skin ulcer and infection and, in serious cases, necrosis and gangrene. It is the most common cause of adult amputation, usually of toes and or feet, in the US and other Western countries.
Carotid artery stenosis does not occur more often in diabetes, and there appears to be a lower prevalence of abdominal aortic aneurysm. However, diabetes does cause higher morbidity, mortality and operative risks with these conditions.
Treatment and management.
Diabetes management
Diabetes is a chronic disease, and emphasis is on managing short-term as well as long-term diabetes-related problems. There is an important role for patient education, nutritional support, self glucose monitoring, as well as long-term glycemic control. A scrupulous control is needed to help reduce the risk of long term complications. In addition, given the associated higher risks of cardiovascular disease, lifestyle modifications must be implemented to control blood pressure and cholesterol by exercising more, smoking cessation, and consuming an appropriate diet.

The Nobel Prize in Physiology or Medicine 2007

The Nobel Prize in Physiology or Medicine 2007 is won by Mario R. Capecchi, Sir Martin J. Evans, Oliver Smithies "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells"

HEALTHY JUICES

HEALTHY JUICES

Carrot + Ginger + Apple - Boost and cleanse our system.

Apple + Cucumber + Celery - Prevent cancer, reduce cholesterol, and improve stomach upset and headache.

Tomato + Carrot + Apple - Improve skin complexion and bad breath.

Bitter gourd + Apple + Milk - Avoid bad breath and reduce internal body heat.

Orange + Ginger + Cucumber - Improve Skin texture and moisture and reduce body heat.

Pineapple + Apple + Watermelon - To dispel excess salts, nourishes the bladder and kidney.

Apple + Cucumber + Kiwi - To improve skin complexion.

Pear & Banana - regulates sugar content.

Carrot + Apple + Pear + Mango - Clear body heat, counteracts toxicity, decreased blood pressure and fight oxidization .

Honeydew + Grape + Watermelon + Milk - Rich in vitamin C + Vitamin B2 that increase cell activity and strengthen body immunity.

Papaya + Pineapple + Milk - Rich in vitamin C, E, Iron. Improve skin complexion and metabolism.

Banana + Pineapple + Milk - Rich in vitamin with nutritious and prevent constipation.

IMMUNOGLOBULINS - STRUCTURE AND FUNCTION

IMMUNOGLOBULINS - STRUCTURE AND FUNCTION

Kannan.S.Das
M.Sc.Biochemistry

I. DEFINITION
Immunoglobulins (Ig) Glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that they migrate with globular proteins when antibody-containing serum is placed in an electrical field

II. GENERAL FUNCTIONS OF IMMUNOGLOBULINS
A. Antigen binding Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host. The valency of antibody refers to the number of antigenic determinants that an individual antibody molecule can bind. The valency of all antibodies is at least two and in some instances more.
B. Effector Functions Frequently the binding of an antibody to an antigen has no direct biological effect. Rather, the significant biological effects are a consequence of secondary "effector functions" of antibodies. The immunoglobulins mediate a variety of these effector functions. Usually the ability to carry out a particular effector function requires that the antibody bind to its antigen. Not every immunoglobulin will mediate all effector functions. Such effector functions include:
1. Fixation of complement - This results in lysis of cells and release of biologically active molecules
2. Binding to various cell types - Phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins. This binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn
III. BASIC STRUCTURE OF IMMUNOGLOBULINS
The basic structure of the immunoglobulins is illustrated in the Figure 2. Although different immunoglobulins can differ structurally they all are built from the same basic units.
A. Heavy and Light Chains All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (23kD) and two identical heavy chains (50-70kD)
B. Disulfide bonds
1. Inter-chain disulfide bonds - The heavy and light chains and the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions The number of inter-chain disulfide bonds varies among different immunoglobulin molecules.
2. Intra-chain disulfide binds - Within each of the polypeptide chains there are also intra-chain disulfide bonds.
C. Variable (V) and Constant (C) Regions After the amino acid sequences of many different heavy chains and light chains were compared, it became clear that both the heavy and light chain could be divided into two regions based on variability in the amino acid sequences. These are the:
1. Light Chain - VL (110 amino acids) and CL (110 amino acids)
2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)
D. Hinge Region This is the region at which the arms of the antibody molecule forms a Y. It is called the hinge region because there is some flexibility in the molecule at this point.
E. Domains Three dimensional images of the immunoglobulin molecule show that it is not straight as depicted in Figure 2A. Rather, it is folded into globular regions each of which contains an intra-chain disulfide bond (figure 2B-D). These regions are called domains.
1. Light Chain Domains - VL and CL
2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)
F. Oligosaccharides Carbohydrates are attached to the CH2 domain in most immunoglobulins. However, in some cases carbohydrates may also be attached at other locations.
IV. STRUCTURE OF THE VARIABLE REGION
A. Hypervariable (HVR) or complementarity determining regions (CDR)
Comparisons of the amino acid sequences of the variable regions of immunoglobulins show that most of the variability resides in three regions called the hypervariable regions or the complementarity determining regions as illustrated in Figure 3. Antibodies with different specificities (i.e. different combining sites) have different complementarity determining regions while antibodies of the exact same specificity have identical complementarity determining regions (i.e. CDR is the antibody combining site). Complementarity determining regions are found in both the H and the L chains.
B. Framework regions
The regions between the complementarity determining regions in the variable region are called the framework regions (Figure 3). Based on similarities and differences in the framework regions the immunoglobulin heavy and light chain variable regions can be divided into groups and subgroups. These represent the products of different variable region genes.
V. IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS
Immunoglobulin fragments produced by proteolytic digestion have proven very useful in elucidating structure/function relationships in immunoglobulins.
A. Fab Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond Figure 4. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain.
Antigen binding - These fragments were called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL. An antibody is able to bind a particular antigenic determinant because it has a particular combination of VH and VL. Different combinations of a VH and VL result in antibodies that can bind a different antigenic determinants.
B. Fc Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it was easily crystallized.
Effector functions - The effector functions of immunoglobulins are mediated by this part of the molecule. Different functions are mediated by the different domains in this fragment (Figure 5). Normally the ability of an antibody to carry out an effector function requires the prior binding of an antigen; however, there are exceptions to this rule.
C. F(ab')2 Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites (Figure 6). This fragment was called F(ab')2 because it was divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab')2 binds antigen but it does not mediate the effector functions of antibodies.
VI. HUMAN IMMUNOGLOBULIN CLASSES, SUBCLASSES, TYPES AND SUBTYPES
A. Immunoglobulin classes The immunoglobulins can be divided into five different classes, based on differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a given class will have very similar heavy chain constant regions. These differences can be detected by sequence studies or more commonly by serological means (i.e. by the use of antibodies directed to these differences).
1. IgG - Gamma heavy chains
2. IgM - Mu heavy chains
3. IgA - Alpha heavy chains
4. IgD - Delta heavy chains
5. IgE - Epsilon heavy chains
B. Immunoglobulin Subclasses The classes of immunoglobulins can de divided into subclasses based on small differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a subclass will have very similar heavy chain constant region amino acid sequences. Again these differences are most commonly detected by serological means.
1. IgG Subclasses
a) IgG1 - Gamma 1 heavy chains
b) IgG2 - Gamma 2 heavy chains
c) IgG3 - Gamma 3 heavy chains
d) IgG4 - Gamma 4 heavy chains
2. IgA Subclasses
a) IgA1 - Alpha 1 heavy chains
b) IgA2 - Alpha 2 heavy chains
C. Immunoglobulin Types Immunoglobulins can also be classified by the type of light chain that they have. Light chain types are based on differences in the amino acid sequence in the constant region of the light chain. These differences are detected by serological means.
1. Kappa light chains
2. Lambda light chains
D. Immunoglobulin SubtypesThe light chains can also be divided into subtypes based on differences in the amino acid sequences in the constant region of the light chain.
1. Lambda subtypes
a) Lambda 1
b) Lambda 2
c) Lambda 3
d) Lambda 4
E. NomenclatureImmunoglobulins are named based on the class, or subclass of the heavy chain and type or subtype of light chain. Unless it is stated precisely you are to assume that all subclass, types and subtypes are present. IgG means that all subclasses and types are present.
F. HeterogeneityImmunoglobulins considered as a population of molecules are normally very heterogeneous because they are composed of different classes and subclasses each of which has different types and subtypes of light chains. In addition, different immunoglobulin molecules can have different antigen binding properties because of different VH and VL regions.
VII. STRUCTURE AND SOME PROPERTIES OF IG CLASSES AND SUBCLASSES
A. IgG
1. StructureThe structures of the IgG subclasses are presented in Figure 7. All IgG's are monomers (7S immunoglobulin). The subclasses differ in the number of disulfide bonds and length of the hinge region.
2. PropertiesMost versatile immunoglobulin because it is capable of carrying out all of the functions of immunoglobulin molecules.
a) IgG is the major Ig in serum - 75% of serum Ig is IgG
b) IgG is the major Ig in extra vascular spaces
c) Placental transfer - IgG is the only class of Ig that crosses the placenta. Transfer is mediated by receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally; IgG2 does not cross well.
d) Fixes complement - Not all subclasses fix equally well; IgG4 does not fix complement
e) Binding to cells - Macrophages, monocytes, PMN's and some lymphocytes have Fc receptors for the Fc region of IgG. Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fc receptors. A consequence of binding to the Fc receptors on PMN's, monocytes and macrophages is that the cell can now internalize the antigen better. The antibody has prepared the antigen for eating by the phagocytic cells. The term opsonin is used to describe substances that enhance phagocytosis. IgG is a good opsonin. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions.
B. IgM
1. StructureThe structure of IgM is presented in Figure 8. IgM normally exists as a pentamer (19S immunoglobulin) but it can also exist as a monomer. In the pentameric form all heavy chains are identical and all light chains are identical. Thus, the valence is theoretically 10. IgM has an extra domain on the mu chain (CH4) and it has another protein covalently bound via a S-S bond called the J chain. This chain functions in polymerization of the molecule into a pentamer.
2. Properties
a) IgM is the third most common serum Ig.
b) IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when it is stimulated by antigen.
c) As a consequence of its pentameric structure, IgM is a good complement fixing Ig. Thus, IgM antibodies are very efficient in leading to the lysis of microorganisms.
d) As a consequence of its structure, IgM is also a good agglutinating Ig . Thus, IgM antibodies are very good in clumping microorganisms for eventual elimination from the body.
e) IgM binds to some cells via Fc receptors.
f) B cell surface Ig Surface IgM exists as a monomer and lacks J chain but it has an extra 20 amino acids at the C-terminus to anchor it into the membrane (Figure 9). Cell surface IgM functions as a receptor for antigen on B cells. Surface IgM is noncovalently associated with two additional proteins in the membrane of the B cell called Ig-alpha and Ig-beta as indicated in Figure 10. These additional proteins act as signal transducing molecules since the cytoplasmic tail of the Ig molecule itself is too short to transduce a signal. Contact between surface immunoglobulin and an antigen is required before a signal can be transduced by the Ig-alpha and Ig-beta chains. In the case of T-independent antigens, contact between the antigen and surface immunoglobulin is sufficient to activate B cells to differentiate into antibody secreting plasma cells. However, for T-dependent antigens, a second signal provided by helper T cells is required before B cells are activated.
C. IgA
1. StructureSerum IgA is a monomer but IgA found in secretions is a dimer as presented in Figure 11. When IgA exits as a dimer, a J chain is associated with it.
When IgA is found in secretions is also has another protein associated with it called the secretory piece or T piece; sIgA is sometimes referred to as 11S immunoglobulin. Unlike the remainder of the IgA which is made in the plasma cell, the secretory piece is made in epithelial cells and is added to the IgA as it passes into the secretions (Figure 12). The secretory piece helps IgA to be transported across mucosa and also protects it from degradation in the secretions.
2. Properties
a) IgA is the 2nd most common serum Ig.
b) IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus. Since it is found in secretions secretory IgA is important in local (mucosal) immunity.
c) Normally IgA does not fix complement, unless aggregated.
d) IgA can binding to some cells - PMN's and some lymphocytes.
D. IgD
1. StructureThe structure of IgD is presented in the Figure 13. IgD exists only as a monomer.
2. Properties
a) IgD is found in low levels in serum; its role in serum uncertain.
b) IgD is primarily found on B cell surfaces where it functions as a receptor for antigen. IgD on the surface of B cells has extra amino acids at C-terminal end for anchoring to the membrane. It also associates with the Ig-alpha and Ig-beta chains.
c) IgD does not bind complement.
E. IgE
1. StructureThe structure of IgE is presented in Figure 14. IgE exists as a monomer and has an extra domain in the constant region.
2. Properties
a) IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen.
b) Involved in allergic reactions - As a consequence of its binding to basophils an mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release of various pharmacological mediators that result in allergic symptoms.
c) IgE also plays a role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite.
d) IgE does not fix complement.
Clinical Implications of Human Immunoglobulin Classes
Adapted from:F.T. Fischbach in "A Manual of Laboratory Diagnostic Tests," 2nd Ed., J.B. Lippincott Co., Philadelphia, PA, 1984.

IgG
1. Increases in:
a) Chronic granulomatous infectionsb) Infections of all typesc) Hyperimmunizationd) Liver diseasee) Malnutrition (severe)f) Dysproteinemiag) Disease associated with hypersensitivity granulomas, dermatologic disorders, and IgG myelomah) Rheumatoid arthritis
2. Decreases in:
a) Agammaglobulinemiab) Lymphoid aplasiac) Selective IgG, IgA deficiencyd) IgA myelomae) Bence Jones proteinemiaf) Chronic lymphoblastic leukemia
IgM
1. Increases (in adults) in:
a) Waldenström's macroglobulinemiab) Trypanosomiasisc) Actinomycosisd) Carrión's disease (bartonellosis)e) Malariaf) Infectious mononucleosisg) Lupus erythematosush) Rheumatoid arthritisI) Dysgammaglobulinemia (certain cases)
Note: In the newborn, a level of IgM above 20 ng./dl is an indication of in utero stimulation of the immune system and stimulation by the rubella virus, the cytomegalovirus, syphilis, or toxoplasmosis.
2. Decreases in:
a) Agammaglobulinemiab) Lymphoproliferative disorders (certain cases)c) Lymphoid aplasiad) IgG and IgA myelomae) Dysgammaglobulinemiaf) Chronic lymphoblastic leukemia
IgA
1. Increases in:
a) Wiskott-Aldrich syndromeb) Cirrhosis of the liver (most cases)c) Certain stages of collagen and other autoimmune disorders such as rheumatoid arthritis and lupus erythematosusd) Chronic infections not based on immunologic deficienciese) IgA myeloma
2. Decreases in:
a) Hereditary ataxia telangiectasiab) Immunologic deficiency states (e.g., dysgammaglobulinemia, congenital and acquired agammaglobulinemia, and hypogammaglobulinemia)c) Malabsorption syndromesd) Lymphoid aplasiae) IgG myelomaf) Acute lymphoblastic leukemiag) Chronic lymphoblastic leukemia
IgD
1. Increases in:
a) Chronic infectionsb) IgD myelomas
IgE
1. Increases in:
a) Atopic skin diseases such as eczemab) Hay feverc) Asthmad) Anaphylactic shocke) IgE-myeloma
2. Decreases in:
a) Congenital agammaglobulinemiab) Hypogammaglobulinemia due to faulty metabolism or synthesis of immunoglobulins

MAJOR BIOCHEMICAL ESTIMATIONS

MAJOR BIOCHEMICAL ESTIMATIONS

Kannan.S.Das
M.Sc Biochemistry
Acid Phosphatase(AcP)
AcP is the name given to a group of Phosphatases with optimal activity below 7.0 and found in various tissues including Liver, Spleen, Prostate , Erythrocytes and Platelets.
Clinical interpretations.
Determination of prostatic AcP activity in serum is useful in monitoring carcinoma of the Prostate.
Normal values
Specimen used
Serum
Plasma – EDTA or Heparin.
Don’t use hemolysed specimens.
Albumin
Of all serum proteins albumin is present in the highest concentration. It maintains the plasma oncotic pressure and the transport of many substances.
Clinical interpretations.
Increased serum albumin may indicate dehydration or hyper fusion with albumin , a decrease is found in rapid hydration , over hydration , severe malnutrition , malabsorption , severe diffuse , liver necrosis , chronic active hepatitis and neoplasia.
Decreased serum albumin may indicate chronic alcoholism , pregnancy , renal protein loss, thyroid dysfunction ,peptic ulcer disease, chronic inflammatory diseases.
Specimen used.
Serum
Plasma –heparin
Alkaline Phosphatase(ALKP)
ALKP is present mainly in bone, liver, kidney, intestine, placenta and lung.
Clinical interpretations.
Serum ALKP may be elevated in increased bone metabolism (in adolescencets and during the healing of a fracture) primary and secondary hyperparathyroidism, Paget’s disease of bone, carcinoma metastatic to bone, Osteogenic Sarcoma, Hodgkin’s disease if bones are invaded. Hepatobiliary diseases involving Cholestasis, inflammation or cirrhosis increase alkaline phosphate activity, renal infarction and failure and in the complications of pregnancy.
Low ALKP activity may occasionally seen in hypothyroidism.
Specimen used.
Serum
Plasma –heparin
EDTA, Citrate fluoride oxalate will interfere with this enzyme, so don’t use this in plasma.
Alanine Aminotransferase (ALT)
ALT activity is present high in Liver, Skeletal muscle, heart and Kidney.
Clinical interpretations.
Serum ALT increase rapidly in liver cell necrosis, hepatitis, hepatic cirrhosis, liver tumors, obstructive jaundice, Reye’s syndrome, extensive trauma to skeletal muscle, myositis, myocarditis and myocardial infraction.
Specimen used
Serum
Plasma – EDTA or Heparin.
Ammonia
Ammonia is a waste product of protein catabolism. It is potentially toxic to the CNS.
Clinical interpretations.
Increased plasma ammonia may be indicative of hepatic encephalopathy , increased coma in terminal stages of liver cirrhosis , hepatic failure , acute and sub acute liver necrosis and Reye’s syndrome. Hyper ammonia may also be found with increasing dietary protein intake.
Specimen Collection. :Plasma – EDTA or Heparin.
Amylase
Amylase is an amylolytic digestive enzyme produced by the exocrine pancreas and salivary glands.
Clinical interpretations.
Amylase is increased in acute pancreatitis, pancreatic abscess or psedocyst, pancreatic trauma, amyloidosis, pancreatic neoplasm, common-bile duct obstruction, after thoracic surgery , mumps parotitis, renal insufficiency.
Specimen used.
Serum
Plasma –heparin
Urine
* plasma activities are approximately 20 U/L higher than serum activities.
Specimen not recommended if the plasma is mixed with Citrate, EDTA, and Fluoride oxalate. If the sample is urine, don’t add Boric acid/Sodium formate, Glacial acetic acid, concentrated hydrochloric acid.
Bilirubin
The term direct bilirubin means bilirubin conjugated to glucuronic acid (Bc) and the term Indirect bilirubin means Unconjugated bilirubin (Bu).The concentration of each of the different forms of serum bilirubin provides important additional diagnostic information when compared to the measurement of Total bilirubin (TBIL) alone.
Normal values
TBIL → 0.3 - 1.0 mg/dl
Bc → 0.1 – 0.3 mg/dl
Bu → 0.2 – 0.7 mg/dl
Direct bilirubin → 0.0 – 0.4 mg/dl
Neonatal bilirubin → 1.0 – 10.5 mg/dl
Clinical interpretations.
a) Unconjugated bilirubin fraction increased during
i) impaired conjugation due to physiological Jaundice Crigler-Najjar Syndrome.
ii) increased production due to Hemolytic jaundice ( Kernicterus in Neonates).
iii)decreased uptake is due to Gillbert’s disease.
iv) Ineffective erythropoiesis.
v) Presence of drugs competing for Glucuronide.
b)Conjugated bilirubin fraction increased during
i) Biliary obstruction due to biliary calculi.
ii) Impaired secretion due to Dubin-Johnson syndrome.
iii) Liver Cell damage.
iv) Rotor Syndrome.
Specimen used.
Serum
Plasma –heparin
Blood Urea Nitrogen.
The major pathway of nitrogen excretion is in the form of Urea that is synthesized in the liver , released into the blood and cleared by the kidneys.
Clinical interpretations.
A high serum urea nitrogen occurs in the Glomerulonephritis, Shock, Urinary tract obstruction, Pyelonephritis, acute or chronic renal failure, severe congestive heart failure, hyperalimentation, diabetic ketoacidosis, dehydration, bleeding from the gastrointestinal tract.
Low urea nitrogen occurs in normal pregnancy, with decreased protein intake, acute liver failure and with intravenous fluid administration.
Specimen Collection
Serum
Plasma – EDTA or Heparin.
Sodium fluoride inhibits the enzyme urease activity, so it is not used for specimen collection.
Note: If we want to convert the BUN value to Urea value, then multiply the BUN value with a factor 2.145.
Calcium.
Calcium is the major mineral component of bone , 99% of the body’s calcium is in bone. Calcium ions plays an important role in the transmission of nerve impulses and in maintaining the normal muscle contraction.
Clinical interpretations.
Abnormal concentrations of serum calcium may indicate malfunction of the parotid gland ,bone diseases, carcinoma, malabsorption syndrome and malnutrition, vitamin D deficiency, overdose with calcium containing antacids and renal diseases.
Specimen used
Serum
Plasma – Heparin.
Urine.
Caution : Protective gloves manufactured with calcium carbonate powders may cause an elevated test results because of the contamination of the sample handling supplies(pipette tips, transfer pipettes, sample cups, caps).Supplies that have come in contact with powdered gloves may subsequently contaminate the test specimen during sample metering. Don’t use blood from patients on EDTA therapy.
Cholinesterase
There are two types of Cholinesterase.
a) Acetylcholinesterase which is found in RBC and Nerve tissues.
b) Cholinesterase which is found in plasma, liver, heart and other tissues.
Clinical interpretations.
These measurements are useful in the diagnosis of pesticide poisoning, liver diseases and sensitivity to succinylcholine administration.
a)Pesticide Poisoning: Organophosphate and Carbamate pesticides are inhibitors of both cholinesterase and Acetylcholinesterase. Although the toxic effect is caused by the inhibition of Acetylcholinesterase in the nerve endings , cholinesterase is often used clinically because it is present in high activities and is easy to measure.
b)Liver Disease: Cirrhosis, hepatitis and carcinoma with metastasis to the liver are known to lower cholinesterase activity. A decrease in the cholinesterase activity is considered a sensitive measure of a drop in liver synthetic capacity , because high activities of cholinesterase are normally present in serum.
c)Sensitivity to succinylcholine administration: Succinylcholine is a short acting muscle relaxant administered during the surgery. It is a reversible inhibitor of acetylcholinesterase and is hydrolyzed by serum cholinesterase. Individuals without sufficient serum cholinesterase activity or with certain genetic variants may be unable to metabolize the drug quickly resulting in prolonged apnea. Low cholinesterase acclivities may be chronic for the individual or transient due to pesticide exposure, liver disorder, pregnancy or the use of oral contractives.
Specimen used
Serum
Plasma – Heparin.
Cholesterol
Cholesterol is present in tissues and in serum & plasma either as cholesterol or cholesterol esters bound to proteins. cholesterol is an essential structural component of cell membranes and the outer layers of the plasma lipoproteins and is the precursor of all steroid hormones, including sex and adrenal hormones ,bile acids and vitamin D.
Cholesterol measurements are used to evaluate the risk of developing coronary artery occlusion ,atherosclerosis ,myocardial infarction, cerebrovascular disease.
Clinical interpretations.
Cholesterol level is increased during coronary atherosclerosis, primary hyper cholestrolemia, secondary hyper lipoproteinemia ,nephritic syndrome, primary biliary cirrhosis, hypothyroidism, some cases of diabetes mellitus.
Low cholesterol level is found in malnutrition , malabsorption, advanced malignancy and hyper thyroidism.
Serum cholesterol level depends on many factors including age and sex.
Specimen used
Serum
Plasma – Heparin
Creatine kinase(CK) / Creatine phosphokinase.
CK is a cellular enzyme with a wide tissue distribution. CK is found mainly in skeletal and cardiac muscle. CK’s physiological role is associated with ATP generation for contractile or transport systems.
Clinical interpretations.
Serum Ck is almost always increased during myocardial infarction or skeletal muscle damage. The enzyme is commonly elevated in myocarditis of any cause, cerebrovascular accidents ,rhabdomyolysis, polymyositis and acute physical exertion, muscular dystrophies in Duchene’s muscular dystrophy.
Low CK is due to decrease muscle mass or muscle wasting. Low serum CK activities are common in elderly ,in the bedridden and in patients with advanced malignancy.
Specimen used
Serum
Plasma – Heparin
Creatine Kinase MB
The CK-MB isoenzyme is found primarily in cardiac muscle, trace amounts are present in skeletal muscle.
Clinical interpretations.
CK-MB is elevated in acute myocardial infarction. CK-MB usually peaks between 12 and 24 hours after myocardial infarction and returns to the normal in 48 to 72 hours in an uncomplicated case.
CK-MB is also increased in myocarditis ,Duchene’s muscular dystrophy, polymyositis, rhabdomyolysis and other myocardial or myopathic disorders.
Specimen used
Serum
Chloride( cl- )
Chloride is the major anion in the extra cellular water space, its physiological significance is in maintaining proper body water distribution, osmotic pressure, and normal anion-cation balance in the ECF compartment.
Clinical interpretations.
Chloride is increased in dehydration ,renal tubular acidosis( hyperchloremia metabolic acidosis) and in excessive infusion of isotonic saline.
Chloride is decreased in over hydration, chronic respiratory acidosis, salt losing nephritis, metabolic alkalosis and congestive heart failure.
Specimen used
Serum
Plasma – Heparin
Creatinine
Serum creatinine and urinary creatinine excretion is a function of lean body mass in normal persons and shows little or no response to dietary changes. Serum creatinine is higher in men than in women. Since urinary creatinine is excreted mainly by glomerular filtration, with only small amounts due to tubular secretion, serum creatinine and 24 hour urine creatinine excretion can be used to estimate the glomerular filtration rate.
Clinical interpretations.
Serum creatinine is increased in acute or chronic renal failure, urinary tract obstruction, reduced renal blood flow, shock, dehydration and rhabdomyolysis. Exercise may cause an increased creatinine clearance.
Low serum creatinine concentration include debilitation and decreased muscle mass.
The creatinine clearance rate is unreliable if the urine flow is low.
Creatinine Clearance Test (CCT) = Total Urine Volume X urine creatinine
1440 Serum creatinine It is useful to correct the clearance value with body surface area. This is important especially in children, persons with short or tall frame. Body surface area may be calculated from the formula
Log A = 0.425 log W + 0.725 log H – 2.144 { where A= Body surface in m2 , W= weight in Kg and H= height in cm. The standard body surface area is 1.73 m2
CCT= Total Urine Volume X urine creatinine X 1.73
1440 Serum creatinine Body surface in m2
Specimen used →Serum, Heparinized Plasma , Urine.
C-reactive protein
C-reactive protein is synthesized by the liver and is one of the acute phase proteins .In the acute phase response, increased concentrations of a number of plasma proteins including C-reactive protein are observed.
Clinical interpretations.
C-reactive protein concentration measurements are useful in the detection and elevation of inflammatory disorders, tissue injury and infections.
Specimen used
Serum
Plasma – Heparin /EDTA.
Direct Bilirubin.
Direct Bilirubin is used for evaluating liver and biliary diseases. An increased direct bilirubin occurs in both intrahepatic and extrahepatic biliary tract obstructions. Hepatocellular causes of elevation include hepatitis, cirrhosis and advanced neoplastic states. It is also increased in Dubin-Johnson syndrome and in Rotor Syndrome.
Direct Bilirubin = Total bilirubin – Bilirubin Unconjugated.
Direct HDL Cholesterol.
Clinical interpretations.
HDL cholesterol is used to evaluate the risk of developing coronary heart disease(CHD).
The risk of CHD increases with lower HDL cholesterol concentrations.
Specimen used
Serum
Plasma – Heparin /EDTA.
Carbon dioxide.
The Carbonic acid – Bicarbonate buffer system is one of the important buffer systems that maintains the pH of the blood. Total CO2 measurements are used for the evaluation of acid—base disorders.
Clinical interpretations.
Total CO2 is increased in respiratory acidosis, metabolic acidosis and excessive alkali intake.
CO2 decreased in compensated respiratory alkalosis, metabolic acidosis and in renal disorders where H+ ions cannot be excreted.
Specimen used
Serum
Plasma – Heparin
Iron (Fe)
Most body iron is found in Hemoglobin. The serum measurement of iron is useful in the differential diagnosis of anemia, iron deficiency anemia, thalassemia, possible sideroblastic anemia and iron poisoning.
Clinical interpretations.
hemosiderosis ,hemolytic anemia’s, Thalassemia, Sideroblastic anemia’s, hepatitis, acute hepatic necrosis, Hemochromatosis, inappropriate iron therapy and iron poisoning.
Serum iron is decreased in cases of insufficient dietary iron, chronic blood loss, inadequate absorption of iron, impaired release of iron stores, infection and chronic diseases.
Specimen used
Serum
Plasma – Heparin
Gamma Glutamyl Transferase.( GGT )
GGT plays a major role in glutamine metabolism and in resorption of amino acids from the glomerular filtrate. It is also important in the absorption of amino acids from the intestinal lumen. GGT is found mainly in the liver, pancreas and kidney, although lower activities can be demonstrated in most of organs.
Clinical interpretations.
Serum GGT is a sensitive indicator of Hepatobiliary disease and is useful in the diagnosis of obstructive jaundice and chronic alcoholic liver disease, in the follow up of chronic alcoholics undergoing treatment and in the detection of hepatotoxicity.
GGT is more responsive to biliary obstruction than AST, ALT, and ALKP. GGT is also increased in hepatoma, carcinoma of pancreas and carcinoma metastatic to the liver.
Specimen used
Serum
Plasma – Heparin /EDTA.
Glucose.
Glucose is the primary cellular energy source.
Clinical interpretations.
Fasting plasma glucose concentrations and tolerance to a dose of glucose are used to establish the diagnosis of diabetes mellitus and disorders of carbohydrate metabolism. Glucose measurements are used to monitor therapy in diabetics and in patients with dehydration, coma, hypoglycemia, insulinoma, acidosis and ketoacidosis.
Specimen used.
Serum
Plasma – Heparin /EDTA/Sodium fluoride/Potassium oxalate.
Urine
CSF
Potassium ( K+ )
Potassium is the major cation of the intracellular fluid.
Measurements of serum potassium are used for the electrolyte imbalance, cardiac arrhythmias, muscular weakness, hepatic encephalopathy, renal failure and for the monitoring of ketoacidosis in the diabetes mellitus and intravenous fluid replacement therapy.
Clinical interpretations.More than 90% of hypertensive patients with aldosteronism have a low K+, a low K+ is also common in vomiting, diarrhea, alcoholism and folic acid deficiency. High K+ values occur in rapid K+ infusions, end stage renal failure, hemolysis, trauma, Addison’s disease, metabolic acidosis, acute starvation, dehydration and acute medical emergency.
Normally K+ is freely filtered by the glomerulus’s but tends to be conserved if the serum K+ is low. Urinary potassium may be elevated with dietary increase, hyperaldosteronism, renal tubular acidosis and at the onset of alkalosis.
Specimen used.
Serum
Plasma – Heparin
Urine
Lactate
Lactate is the end product of the anaerobic metabolism of glucose. The concentration of lactate in the blood is dependent on the rate of production in muscle cells and erythrocytes and the metabolism in the liver. Lactic acidosis usually results from overproduction or underutilization of lactate.
Clinical interpretations
Elevated lactate levels can occur as a result of tissue hyoxia,diabetes mellitus, phenformin therapy,malignancies,glycogen storage disease,ethanol,methanol or salicylate ingestion and metabolic acidosis.
Specimens used
Plasma-Fluoride oxalate
Heparinized plasma is acceptable, but precautions must be taken to retard glycolyisis by keeping the whole blood on ice.
LDH
LDH is an enzyme present in the cytosol of all human cells. It catalyses the reversible reduction of pyruvate to lactate using NADH.
Clinical interpretations
Causes of high LDH include neoplastic states, hypoxic cardio respiratory diseases, myocardial infractions, hemolytic anemias,megaloblastic anemias,hepatic cirrhosis, renal infraction,trauma,muscle damage, muscular dystrophy, shock and hypotension. In myocardial infraction cases,LDH begins to rise within about 12 hrs after infraction and usually returns to normal after two to five days.
Specimens used.
Serum
Plasma – Heparin
Serum is the right specimen for estimation of the LDH.
Specimens not recommended
Do not use hemolysed specimens.
Lipase
Lipase is a digestive enzyme that is mainly produced by the acinar cells of the exocrine pancreas. its physiological role is to hydrolyses the long-chain triglycerides in the small intestine.
Clinical interpretations.
Serum lipase uses rapidly in patients with acute and recurrent pancreatitis, pancreatic cancer, common bile duct obstruction and ingestion of drugs hat are toxic to the pancreas. It is increased by most inflammatory conditions in the abdominal cavity, biliary tract disease, abdominal abscesses and renal failure. Lipase is more specific than total amylase in the digestion of acute pancreatitis.
Specimens used. :Serum
Magnesium (Mg)
Mg is predominantly an intracellular cation and is essential in enzymatic reactions.
Clinical interpretations.
Mg deficiency may cause weakness, tremors, tetany and convulsions. Hypomagnesaemia is associated with hypocalcaemia, alcoholism, some types of malnutrition, malabsorption, chronic hemodialysis and pregnancy.
Increased serum Mg concentrations occur in patients with renal failure, dehydration, Addison’s disease.
Specimens used.
Plasma-EDTA /fluoride oxalate/ citrate.
Urine-- Boric acid with Sodium formate as a preservative.
Sodium(Na)
Sodium is the major cation of ECF.The kidneys regulate sodium content of the body.
Clinical interpretations.
Low sodium concentrations may be due to excessive urine loss, diarrhea, Addison’s disease and renal tubular disease. High Na levels may occur in severe dehydration, some types of brain injury, diabetic coma, excessive intake of sodium salts.
Specimens used.
Serum
Plasma-Heparin
Urine.
Neonatal Bilirubin (NBIL).
NBIL is the sum total of Unconjugated bilirubin and Conjugated bilirubin is increased in the erythroblasosis fetalisis(Hemolytic disease)of the new born which causes Jaundice in
the first two days of life. Other causes of neonatal jaundice includes physiological jaundice, hematomal/hemorrhage, hypothyroidism, obstruction jaundice.
Specimens used.
Serum
Plasma-Heparin
Phosphorus.
Phosphorus, as phosphate is distributed throughout the body. Cause of high serum P include dehydration, hyperparathyroidism, hypervitaminosis D, metastasis to bone sarcoidisis, pulmonary embolism, renal failure, diabetes mellitus with ketosis.
Clinical interpretations.
Low serum phosphorus is found in primary hyperparathyroidism and other causes of serum calcium elevation, sepsis, vitamin D deficiency, renal tubular acidosis, chronic hemodialysis, vomiting and decreased dietary phosphate intake.
Specimens used.
Serum
Plasma-Heparin
Urine.
Specimens not recommended:
Plasma – EDTA / Fluoride oxalate / Citrate.
Urine – Preservatives.


Cerebro Spinal Fluid (CSF) protein.
CSF proteins are those that remain in CSF following the ultracentrifugation of plasma through the choroidal capillary wall. Some proteins that are unique to the CSF are synthesized in the CNS.
Clinical interpretations.
In general diseases that interrupt the integrity of the capillary endothelial barrier lead to an increase in total CSF protein.CSF protein is generally increased in all types of meningitis, cerebral infarction, brain abscess, meningovascular syphilis, subarachinoid hemorrhage, some brain tumors, trauma to the brain, multiple sclerosis, encephalomyelitis, degenerative neurological disease.
Decreased CSF protein may occur in water intoxication, CSF leak and hyperthyroidism.
Specimen used
CSF
Total Bilirubin(TBIL)
TBIL in serum and plasma is the sum of Unconjugated bilirubin ,mono and di-glucuronide conjugated bilirubin and delta bilirubin a fraction covalently bound to albumin.
With the exception of anicteric jaundice, total serum bilirubin is invariably increased in jaundice.
Causes of jaundice are prehepatic, resulting from various hemolytic disease, hepatic; resulting from Hepatocellular injury or obstruction and post hepatic resulting from obstruction of the hepatic or common bile duct.
Specimen used
Serum
Plasma- Heparin
Total Iron Binding Capacity.(TIBC)
Most body iron is found in Hb. The serum measurement of iron is useful in the differential diagnosis of anemia, iron deficiency anemia, thalassemia, possible sideroblastic anemia and iron poisoning. TIBC in serum representing transferring concentration in Iron binding capacity is a useful index of nutritional iron status. Iron deficiency anemia is characterized by a decreased serum iron, increased TIBC or transferrin and a decreased transferrin saturation.
Clinical interpretations
Serum TIBC is increased in iron deficiency. Serum TIBC is decreased in anemia of chronic disease.
Specimen used
Serum.
Total Protein.(TP)
Serum proteins transport drugs ,metabolites and maintain plasma osmotic pressure. Most of the serum proteins are synthesized in the liver, with the exception of gamma globulins. One of the most important serum proteins produced in the liver is albumin. Total serum protein concentration can be used for the evaluation of nutritional status.
Clinical interpretations
Causes of high TP concentration include dehydration, Walden Strom’s macroglobulinemia, multiple myeloma, hyperglobulinemia, Granulomatous disease and some tropical diseases. TP concentration is occasionally increased in collagen diseases, Lupus erythematosus and other instances of chronic infection or inflammation.
Causes of low TP concentration includes pregnancy, excessive intravenous fluid administration, cirrhosis, or other liver diseases, chronic alcoholism, heart failure, nephritic syndrome, Glomerulonephritis, neoplasia, protein-losing enteropathies, malabsorption and sever malnutrition.
Specimen used
Serum
Plasma- heparin.
Triglyceride(TG)
TG, fatty acid esters of glycerol, represent the major form of fat found in the body. Their primary function is to store and provide cellular energy. The concentration of TG in the plasma at any given time is a balance between the rates of entry and removal. TG concentration in the plasma varies with age and gender.
Moderate increases occurs during growth and development.
Clinical interpretations.
TG is used for the evaluation of hyperlipidemias. High concentration may occur with hypothyroidism, nephrotic syndrome, glycogen storage disease and diabetes mellitus. Extremely high TG is common in acute pancreatitis.
Specimen used
Serum(It is the good choice) {12 hour fasting}
Plasma- heparin.
Urine Protein
The filtration and resorption of plasma proteins in the formation of urine are important functions of the intact , healthy kidney. The presence of elevated concentration of protein in urine is a key finding in primary renal disease of glomerular, tubular or mixed origin.
Clinical interpretations.
Proteinuria is also seen in patients with synthetic disorders that affect the kidneys such as diabetes mellitus, hypertension, vascular disease , neoplasia, drug toxicity and certain infectious diseases. Protein may also exist as either a benign or transient condition.
Specimen used
Urine.
Uric Acid
Uric acid is the end product of purine metabolism.
Clinical interpretations
Elevation of uric acid occurs in renal failure, prerenal azotemia, gout, lead poisoning, excessive cell destruction (example: following Chemotherapy) , hemolytic anemia, congestive heart failure and after myocardial infarction. Uric acid is also increased in some endocrine disorders, acidosis , toxemia of pregnancy, hereditary gout, and Glycogen storage disease Type I.
A low uric acid concentration may be found following treatment by some drugs (example: low dose Aspirin ) with low dietary intake of purines, in the presence of renal tubular defects and in Xanthinuria.
Specimen used
Serum
Plasma- heparin.
Low Density Lipoprotein ( LDL ) = Total Cholesterol ─ TG + HDL.{ VLDL = TG}
5 5

Prothrombin Time (PT)
PT is a rapid sensitive screening test for coagulation disorders in the domain of the extrinsic system[ factors II, V,VII & X ].Due to its high sensitivity for these coagulation factors is especially well suited for;
The induction and monitoring of oral anticoagulant therapy.
Diagnosing the genetically caused deficiencies in the coagulation factors of extrinsic system.
Diagnosing the required deficiencies in the coagulation factors.
Checking the synthesis performance of the liver in hepatic diseases.
Principle: The coagulation process is triggered by incubation of plasma with the optimal reaction time of Normal plasma in seconds
Result : the result is reported in seconds, Prothrombin ratio(PR), International normalized Ratio(INR)
To obtain the Prothrombin ratio, the reaction time of the sample is divided by the reaction time of the normal plasma.
PR = Reaction time of the sample (seconds)
Reaction time of normal plasma (Seconds)
If the Prothrombin ratio is determined using a normal plasma which does not have a PR of 1.0, the PR of this plasma has to be taken into account in the calculation.
PR = Reaction time of sample in seconds X PR of Normal Plasma.
Reaction time of Normal Plasma in seconds.
Prothrombin Ratio can be converted into Internationally comparable values by means of International Sensitivity Index (ISI).Result obtained is in INR.
INR = PT X a log factor
Control
Reference Values.
Normal → 12 to 15 seconds.
[Deep Vein Thrombosis, Pulmonary embolism, Arterial diseases, Myocardial infarction] → 2.0 to 3.0 seconds.
Recurrent systemic embolism, artificial cardiac valves → 2.5 to 4.5 seconds.
Activated Partial Thromboplastin Time ( APTT ).
APTT is a global screening procedure used primarily to evaluate coagulation abnormalities in the Intrinsic pathway, will also detect severe functional deficiencies in the factors II,V,X or fibrinogen. It is also used to monitor the effectiveness of heparin therapy, where the clotting time is prolonged in proportion to the level of heparin.
In summary the APTT is applicable for diagnosing Coagulant disorders and therapeutic monitoring of both hemorrhagic and thrombolic disease.
Principle: Factors of intrinsic coagulation system are activated by incubating the plasma with the optimal amount of Phospholipids and two surface activators. The addition of Ca ions triggers the coagulation process and the clotting time is then measured.

Immunoglobulin Assays
The method described here is immuno turbid metric assay. The anti human Ig antibodies ( for A,G,M,D & E) when mixed with samples containing the corresponding Ig’s will form insoluble complexes. These complexes cause an absorbance change depending upon the corresponding Ig concentration of the patient sample , that can be quantified by comparison from the calibrator of the corresponding known Ig concentration.
Clinical Significance of Ig’s.
IgA
IgA represents approximately 10 to 15 % of the total serum immunoglobulins. Its structure is monomeric , similar to the IgG molecule , but 10 to 15 % of the IgA in serum is polymeric , particularly IgA2 which is more resistant to destruction by some pathogenic bacteria. Another more important form of IgA is called secretory IgA which is found in skin , pulmonary , kidney infections and hepatic cirrhosis.
Increased monoclonal IgA concentrations may be found in multiple myeloma and other disturbances of plasmatic cells.
Normal range = 70 to 400 mg/dl.
IgG
IgG is the most important immunoglobulin produced by the plasma cells and represents about 75% of the total immunoglobulins. Its main function is to neutralize toxins in tisular spaces.
IgG deficit may be due to a congenital primary disturbance and is a special risk in children.
Polyclonal hyperimmunoglobulinemia is the normal response to infections, especially in hepatitis and cirrhosis as well as autoimmune diseases.
Increases of monoclonal IgG are found in multiple myeloma , lymphocytic leukemia and Waldenstrom macroglobulinemia.
Normal range = 700 to 1600 mg/dl.
IgM
IgM is the only immunoglobulin that a neonate normally synthesis and in adults it represents the 5 to 10 % of the total immunoglobulins. Its structure is a pentamer of five IgG molecules and its high molecular weight prevents its passage into the extra vascular spaces.
IgM concentrations is decreased in diseases related with hereditary or acquired deficiencies of the immunoglobulin production. Polyclonal increases in serum immunoglobulins are the normal response to infections. The IgM generally increases as a primary response to virus infections and blood stream infections such as malaria and primary biliary cirrhosis. In multiple myeloma , if the paraprotein proves to be IgM, the diagnosis is probably Waldenstrom macroglobulinemia.
Normal range = 40 to 230 mg/dl.

Clinical significance of Some other important Assays.
Alpha-fetoprotein ( AFP ).
AFP is a glycoprotein with a high molecular weight (approx: 68,000 D) consisting of a single polypeptide chain. AFP ,which belongs to the group of oncofetal proteins which is produced by the yolk sac and in the fetal liver.
In oncology, AFP is determined in patients with liver cell carcinoma or germ-cell tumours( non-seminomatous tumors of the testes , endodermal sinus tumors of the ovaries).
AFP plays an important role in pregnancy monitoring. During pregnancy, AFP levels in the maternal blood continuously increased. Between weeks 28 to 32 a maximum is reached , after this period a decrease can be observed until 15th week of gestation. Elevated AFP levels in early pregnancy indicate neural tube defects ( spina bifida, anencephaly ). Lower AFP concentrations in the maternal serum are indicative of Down’s syndrome.
The determination of serum AFP during therapeutic monitoring provides valuable information about the success or failure of treatment as well as the occurrence of recidivation.
Normal Range= healthy men and non-pregnant women normally shows AFP values below 5.5 IU/ml.
Thyroid Stimulating Hormone ( TSH ).
TSH is a glycoproteohormone with a molecular weight in the range of 28,000 to 30,000 Dalton and is composed of the two non-covalently bound subunits hTSHα and hTSHβ.
A characteristic feature of the glycoproteins TSH , Luteinising hormone (LH), Follicle Stimulating Hormone (FSH) and Human Chorionic Gonadotropin (HCG) is their relative carbohydrate content as well as the nearly identical sequential homology of their α-subunits .On the other hand the β-subunit has a different amino acid sequence in all four hormones.
TSH releases and synthesized in the anterior pituitary is stimulated by the hypothalamic thyrotropin-releasing Hormone (TRH).The TSH released stimulates the thyroidal release of the hormones Thyroxin(T4) and Triiodothyronine(T3) whose binding proteins are physiologically active in the peripheral tissues and regulates the thyroidal function via a pituitary feedback mechanism.Determination of basal TSH is generally sufficient in the monitoring of suppression or substitution therapy.
CA125 antigen.
Determination of CA125 antigen is used in the follow-up of patients with primary invasive ovarian carcinoma.
The two antigen determinants defined by the monoclonal antibodies OC125 and M11 are fond on a heterogeneous group of high molecular weight 200,000 to 1,000,000 glycoproteins.
They can be detected in a high percentage of nonmucinous epithelial ovarian tumours. Furthermore they are found in some fetal tissues (amnion, periderm, derivatives of the coelomic epithelium) and in adult tissues in the epithelium of the Fallopian tubes , apocrine sweat glands, breast glands, endometrium and endocervix.
Elevated CA125 assay values in serum are found in most of the patient with active epithelial ovarian cancer in early stages of the disease already and can therefore be used for therapeutic monitoring of such patients.
Normal range = 35 IU/ml. this cut-off can vary depending upon the age and menstrual cycle.
Hepatitis C Virus ( HCV ).
HCV is formerly described as the parenterally transmitted form of non-A, non-B hepatitis (NANBH), causes chronic liver disease in 50% of the cases. After blood transfusion from donors testing positive for HCV antibodies , 88% of recipients develop NANBH and seroconvert to positive anti-HCV test.
HCV can also be transmitted through intravenous drug abuse , sexual contact and administration of contaminated blood or blood products.
HCV is a single stranded RNA virus with some structural relation to the Flavivirus family. The HCV has been linked to cases of Cryptogenic Cirrhosis, Hepatocellular Carcinoma, Auto immune liver diseases and with a variety of extrahepatic disorders such as Glomerulonephritis, Polyarteritis and Cryoglobulinemia.
By using recombinant DNA techniques, the HCV genome is encoded. The genome encodes for three structural proteins Capsid (core) protein, Envelop Glycoproteins E1,E2 and other non structural proteins NS2, NS3, NS4, NS5.
Common tests done for diagnosing a disease.
Diabetes.
Glucose
Amylase
HDL
Pancreas function.
Lipase
Triglycerides
Hyperlipidemia.
Cholesterol
Cholesterol
Triglycerides
HDL
LDL
Liver Function Test ( LFT).
Bilirubin
GGT
Alkaline phosphate
Glucose
Albumin
Cholinesterase
AST
ALT
Ammonia
Gout
Urea
Uric acid
Creatinine
Myocardial infraction
Creatine kinase
Creatine kinase-MB
AST(GOT)
LDH
ALT(GPT)
Tumor Diagnosis
LDH
Alkaline Phosphate
Cholinesterase
Calcium
GGT
Kidney Function Test.
Creatinine
Uric acid
Urea
Total protein
Albumin
Sodium
Potassium
Calcium
Magnesium
Hemoglobin

Courtesy : Manual of Ortho diagnostic instrument.