Erythrocyte Sedimentation Rate

Published in Microbiology
Monday, 09 May 2016 22:12
The Erythrocyte Sedimentation Rate (ESR) measures the rate of settlement of erythrocytes in anticoagulated  blood. Anticoagulated blood is allowed to stand in a glass tube for an hour and the plasma above the red cells is measured in millimeters; this is called ESR.
  • Stage 1: Lag pahse or rouleaux formation: The RBCs stack together and form a structure like package of coins in a shape of canned. (10 Minutes)
  • Stage 2: Submerged of rouleaux. (40 Minutes)
  • Stage 3: Slow sedimentation. (10 Minutes)

  1. Red Blood Cells: In polycythemia, the mass of the red blood cells increases which cause the decrease of ESR. In Anemia the mass of the red cells decreases which cause the increase of ESR. In other words, Erythrocyte Sedimentation Rate is indirectly proportional to ratio between mass of the red cells and plasma.
  2. Plasma: The most important component influencing on Erythrocyte Sedimentation Rate is the composition of plasma. High level of C-Reactive Protein, fibrinogen, heptoglobin, α1-antitrypsin, ceruloplasmin and immunoglobulins causes the elevation of Erythrocyte Sedimentation Rate. When the level of proteins increases in plasma, it reduce the negative charge from the surface of red cells and depreciate the zeta potential; this facilitate the attraction between red blood cells, and form rouleaux.
  3. Technical Issues: Elevation in room temperature also affects the Erythrocyte Sedimentation Rate. Moving tube in sloping position, length and calibre of the tube also affect Erythrocyte Sedimentation Rate.
Erythrocyte Sedimentation Rate is not a specific and diagnostic test for any particular disease. However, Erythrocyte Sedimentation Rate is elevated in a wide range of infectious diseases.
There are four methods for the estimation of Erythrocyte Sedimentation Rate.
  1. Wintrobe method
  2. Westergren method
  3. Micro-ESR
  4. Zeta Sedimentation Ratio
Wintrobe tube is used for both packed cell volume (PCV) and Erythrocyte Sedimentation Rate. Wintrobe’s method is more trustworthy when Erythrocyte Sedimentation Rate is low, while Westergren’s method is more impervious for elevated Erythrocyte Sedimentation Rate. Ethylenediaminetetraacetic acid (C10H16N2O8) is used as an anticoagulant. The internal diameter of Wintrobe tube is about 3 mm and the length is about 110 mm.
After getting the result of Erythrocyte Sedimentation Rate in the first hour, the tube can be whirl in a centrifuge to get the Packed Cell Volume (PCV).

Westergren ESR tube and Westergren stand.
The composition of Trisodium citrate dihydrate (C6H5Na3O7.2H2O or C6H9Na3O9) is as follows:
  • Trisodium citrate dihydrate 32.08 gm
  • Distilled water upto 1000 ml
After making this composition, the mixture is filtered through a sterile membrane (0.22 μm) and stored in a refrigerator at 4°C. The shelf life of this solution is of few months. When the solution becomes turbid (due to the growth of moulds), it should be disposed.
Venous blood is collected in trisodium citrate solution in 4:1 (blood:citrate) proportion. The test should be performed within 4 hours of blood collection.
  1. Mix the anticoagulated  blood smaple thoroughly. Fill the Westergren tube with blood upto “ZERO” mark. Note that there is no air bubbles in the blood.
  2. Place the tube is vertical position in the ESR stand and left for an hour.
  3. Just exactly after an hour, read the height of the column of plasma above the red cells column in mm.
  4. Result is express in the following manner:
    Erythrocyte Sedimentation Rate = ________ mm in an hour.
  1. Always use the correct ratio of anticoagulant and blood. Blood should be check for clots and air bubbles before going to the further process. Blood and anticoagulant should be mix thoroughly.
  2. Make sure that the temperature of the room is between 18-25°C. If the room temperature is elevated than  25°C, Erythrocyte Sedimentation Rate will increase and different reference range will acquire.
  3. ESR tube must be in strict vertical position. Even a slight tilting will cause elevation in Erythrocyte Sedimentation Rate.
Capillary blood is uses for the estimation of Micro-ESR. This method is recommended for the estimation of Erythrocyte Sedimentation Rate in small children.
In this method a special device is uses named zetafuge. Zeta Sedimentation Ratio is not pretended due to anemia, unlike Westergren method.
Erythrocyte Sedimentation Rate by Wintrobe Method
  • Male: 0-9 mm in an hour
  • Females: 0-20 mm in an hour
  • Children: 0-13 mm in an hour

Erythrocyte Sedimentation Rate by Westergren Method
  • Males < 50 years: 0-15 mm in an hour.
  • Females < 50 years: 0-20 mm in an hour.
  • Children: 0-10 mm in an hour.
  • Elderly males > 50 years: 0-20 mm in an hour.
  • Elderly females > 50 years: 0-30 mm in an hour

Lucy (Australopithecus)

Published in Biodiversity
Wednesday, 04 May 2016 01:43
This article uses material from the Wikipedia article Lucy (Australopithecus), which is released under the Creative Commons Attribution-ShareAlike 3.0 Unported License (view authors).

Lucy is the common name of AL 288-1, several hundred pieces of bone fossils representing 40 percent of the skeleton of a female of the hominin species Australopithecus afarensis. In Ethiopia, the assembly is also known as Dinkinesh, which means "you are marvelous" in the Amharic language. Lucy was discovered in 1974 near the village Hadar in the Awash Valley of the Afar Triangle in Ethiopia by paleoanthropologist Donald Johanson.

The Lucy specimen is an early australopithecine and is dated to about 3.2 million years ago. The skeleton presents a small skull akin to that of non-hominin apes, plus evidence of a walking-gait that was bipedal and upright, akin to that of humans (and other hominins); this combination supports the view of human evolution that bipedalism preceded increase in brain size.

"Lucy" acquired her name from the song "Lucy in the Sky with Diamonds" by the Beatles, which was played loudly and repeatedly in the expedition camp all evening after the excavation team's first day of work on the recovery site. After public announcement of the discovery, Lucy captured much public interest, becoming almost a household name at the time.

Lucy became famous worldwide, and the story of her discovery and reconstruction was published in a book by Johanson. Beginning in 2007, the fossil assembly and associated artifacts were exhibited publicly in an extended six-year tour of the United States; the exhibition was called Lucy’s Legacy: The Hidden Treasures of Ethiopia. There was discussion of the risks of damage to the unique fossils, and other museums preferred to display casts of the fossil assembly.[5] The original fossils were returned to Ethiopia in 2013, and subsequent exhibitions have used casts.

WHO Hemoglobin Color Scale for the Estimation of Hemoglobin

Published in Microbiology
Sunday, 01 May 2016 11:30
This method conceived and formulated by Stott and Lewis in 1995. This method is much similar in principle to the now outdated Tallqvist method. Positive technical changes have been made to improve the validity, accuracy and reliability. This method is simple, swift, reliable and inexpensive. This method is reliable and trustworthy within 1 gram/dl for diagnosis of anemia. The World Health Organization (WHO) Hemoglobin Color Scale consists of a printed set of colors corresponding to the hemoglobin value from 4 to 14 grams/dl. On a strip of chromatography paper, a drop of blood is placed and then the developed color is matched visually against the printed color scale. Research has proven that performance is greater than 90% in detecting anemia and 86% in classifying the grade of anemia. The World Health Organization (WHO) has developed hemoglobin color scale after extensive and vast field trails. It is mainly planned for the detection, treatment and control of anemia in under-resourced countries. It is especially use for the screening of blood donors, for screening women and children in health scheme, examine iron therapy, selection-making concerning referral to a hospital, and as a point of care tool.

Difference Between Total Leukocyte Count and Differential Leukocyte Count!

Published in Microbiology
Friday, 29 April 2016 23:53
Difference Between Total Leukocyte Count and Differential Leukocyte Count!

Variations of leukocyte count occur in many infectious, hematologic, inflammatory, and neoplastic diseases.
Therefore, laboratory evaluation of almost all patients begins with the examination of the patient’s blood for total leukocyte count and examination of the peripheral blood smear for the differential leukocyte count as well as blood cells picture. Usually, some clinical interpretations may be made from the total leukocyte count and differential leukocyte count. 
From total leukocyte count and differential leukocyte count, the absolute count of each leukocyte type can be calculated. The absolute leukocyte count provides a more accurate picture than the differential leukocyte count. (For example, a chronic lymphocytic leukemia patient has a total leukocyte count of 100 x 103 cells µI and a differential leukocyte count of 7 percent neutrophils and 90 percent lymphocytes. By looking at the differential leukocyte count of 7 percent neutrophils alone one get the impression of very low neutrophils in this patient But if the absolute count of neutrophils is calculated, one will be surprised to see a normal neutrophil number in this patient.
Absolute neutrophil count = Total leukocyte count x neutrophil percent
=100 x 103 x 7/100 = 7 x 103 = 7000 / µl
At times of acute bacterial infection enormous numbers of neutrophils are required. Therefore, large number of neutrophils is released from bone marrow to cope with this requirement. Consequently, the number as well as the percentage of neutrophils in the blood increases several fold. Hence, an increase in total leukocyte count with increase in percentage of neutrophil is taken as an important indication of acute bacterial infection.
The terms leukocytosis and leukopenia indicate increase or decrease in the total number of leukocytes, respectively. Increase in numbers of neutrophils, eosinophils, lymphocytes, and monocytes are known as neutrophilia, eosinophilia, lymphocytosis, and monocytosis respectively.
i. Neutrophilia is the increase in peripheral blood absolute neutrophil count, above the upper limit of normal of 7.5 x 109/L (in adults). Bacterial infection is one of the common causes of neutrophilia. Neutrophilia is not a feature of viral infections (However, the development of neutrophila late in the course of a viral infection may indicate the emergence of secondary bacterial infection). There is a storage pool of mature neutrophils in the bone marrow (Such storage pool does not occur for other leukocytes).
In response to stress, such as infections the neutrophils from storage pool are released into circulation, resulting in a rise in total leukocyte count and neutrophil percentage. Moreover, there is increased neutrophil production in the bone marrow. Increased neutrophil production during bacterial infection is usually associated with the entry of less mature neutrophils from bone marrow into blood. This is indicated by the appearance of neutrophils with lesser number of nuclear segmentation in the peripheral blood picture. Band cells may also be seen in the peripheral blood smear.
This is referred to as a ‘shift to the left’. The leukemoid reaction is a reactive and excessive leukocytosis, wherein the peripheral blood smear shows the presence of immature cells (e.g. myeloblasts, promyelocytes, and myelocytes). Leukemoid picture occurs in severe or chronic infections and hemolysis. Another most common change that occurs in neutrophils during infection is the presence of toxic basophilic inclusions in the cytoplasm.
Eosinophilia is an increase in peripheral blood absolute eosinophil count beyond 0.4 x 109/L (in adults). Eosinophilia is usually associated with allergic conditions such as asthma and hay fever and parasitic infections. Eosinophilia may also occur in reactions to drugs.
Decrease in total leukocyte count is known as leukopenia. Reduction in the number of neutrophils is the most frequent cause of leukopenia.
i. Neutropenia is the decrease in peripheral blood absolute neutrophil count below the lower limit of normal of 2 x 109/L (in adults). Neutropenic patients are more vulnerable to infection.
ii. Lymphopenia in adults is the decrease in peripheral blood absolute lymphocyte count below the lower limit of normal of 1.5 x 109/L. Lymphopenia is common in the leukopenic prodromal phase of many viral infections. A selective depletion of helper T lymphocytes (CD4+) with or without absolute lymphopenia occurs in acquired immunodeficiency disease (AIDS).

15 Main Theories of Biological Evolution of Man (with Statistics)

Published in Zoology
Wednesday, 29 April 2009 03:38

15 Main Theories of Biological Evolution of Man (with Statistics)

Read this essay to learn about the 15 main Theories of Biological Evolution of Man !


1. Theory of Eternity:

This is an orthodox theory. It believes that some organisms were there from the very beginning of the Universe. Those organisms still exist and will be continued in future in addition to some new forms. According to this theory, the original forms are eternal, and they have been preserved automatically. But this view is not at all popular; it is held by a few people only.

2. Theory of Divine Creation:

A Spanish Monk, Father Sudrez (1548 – 1617) proposed this theory. It was based on the Biblical book of Genesis. According to Genesis, of Old Testament of Bible, the world was created by the supernatural power (God) in six natural days.
The theory specifies that all creations, including plants, animals and man on earth were created during those six days. Since all species were made individually by god, the theory does not accept the idea of origin of new species from ancestral forms. Life is considered as a vital spirit according to this theory.
The Hebrew and the Christian Church authorities had supported this view for many Centuries. To them, god created Adam and Eve, the two companions of opposite sex about 6,000 years ago, from whom the human beings have descended.
Archbishop Ussher (1581 – 1656) pointed out 4004 BC as the exact year for the creation of man. Each and every followers of this theory believed that all creations of god are arranged in a chain where human is posited at the top.

3. Theory of Spontaneous Origin:

The theory contends that life had originated repeatedly from inanimate materials or non-living things in a spontaneous manner. The concept was held by early Greek philosophers like Thales (624 – 547BC), Empedocles (485 – 425BC), Democritus (460 – 370BC), Aristotle (384 – 322BC) and others.
Aristotle thought that fireflies originated from morning dew and mice from the moist soil spontaneously. All succeeding Greek philosophers and many scientists shared Aristotle’s view till the middle of the seventeenth Century. Louis Pasteur partially accepted this theory.

4. Theory of Catachysm or Catastrophism:

French geologist Georges Cuvier (1769 – 1832) proposed this theory. His observation was based on the fossil remains of varied organisms. According to him, the earth had to face severe natural calamities at different times for which many animal species have been destroyed. But each time when the earth settled after a great Catastrophe, relatively higher forms of animals appeared to replace the situation.
Cuvier did not believe in continuous evolution. To him the species never evolved by modification and re-modification; a series of Catastrophes were responsible behind changes where previous sets of living creatures get replaced by new creatures of complex structure.
As per his scheme, corals, molluscs and crustaceous appeared in the first phase. Then came the first plants being followed by the fish and reptiles. The birds and mammals appeared thereafter and in the last phase man emerged about five to six thousand years ago.

5. Theory of Uniformitarianism:

This theory was presented by Charles Lyell (1797 – 1837) in his work ‘Principles of Geology’. Being a geologist, he could not accept the concept of an unchanging earth. By studying the rocks and geological processes, he came to the conclusion that, at the beginning, some forces were in operation to shape and reshape the earth. Animal forms gradually evolved along with this change. Fossils were the main support for his evidence. This theory on one hand discarded the “theory of Catastrophism” and on the other hand nullified the “theory of divine Creation”.

6. Theory of Cosmic Origin of life:

This theory advocated that the first life seed had been transported through the cosmic particles from other planet. Richter (1865) developed this theory and he was supported by Thomson, Helmholtz (1884), Von Tieghem (1891) and others.
According to them the meteorites that travelled through the earth’s atmosphere, contained embryos and spores in them; those gradually grew and evolved into different types of organisms. But the concept lacked evidences and interplanetary exchange of viable spores and embryos could hardly be possible in the light of current understandings.

7. Theory of Cynogen:

German scientist Fluger proposed this theory. According to him, the cynogen, a complex chemical compound was developed by sudden reaction between the atmospheric nitrogen and carbon. This cynogen later gave rise to first protein substance, which ultimately created life through various types of chemical synthesis.

8. Theory of Chemo-synthesis:

This theory also recognized a complex type of chemical synthesis. It pointed out different kinds of materials, which in varied natural environment produced a large number of actions and interactions. As a consequence, life developed in a peculiar set up following a complicated situation.

9. Theory of Virus

Some scientists believed that virus was initially responsible for the emergence of life. The viruses hold a transitional stage between living and non-living. By nature a virus is non-living, but when it reaches to the body cell of the living host, it behaves as living. Therefore, it was thought that such a creature might possess a role in the emergence of life.

10. Theory of Organic evolution

According to this theory, origin of life must have taken place in this world. First living existence was very minute and in the form of unicellular structure. As the time passed on, most of the unicellular forms were transformed to multicellular forms under the various environmental oscillations. Gradually and gradually simple form of animals was converted to very complex type of animals.
As a matter of fact, the geo-environment of the earth underwent a process of continuous change and influenced the animal forms. Complex forms of animals evolved out of the simple forms in a slow and steady way. This process of change has been designated as organic evolution. The conception of organic evolution maintains its conformity with ancient Hindu religious thought. B.M. Das (1961) wanted to prove this with the example often incarnations of Lord Krishna (Dasha avatar).
He mentioned that the first incarnation was a fish (Matsya avatar). He justified his remark by comparing it with the western belief where the life was thought to be originated in water. The second incarnation according to Das was a turtle (Kurma avatar), an amphibian. The next incarnation was a wild pig (Baraha avatar) which represents land-living animals. The fourth incarnation was a mixed form with half man and half animal (Nrisingha avatar). This idea complies with anthropological outlook.
All of the anthropologists now agree that the stage before true man was a combination of man and ape. However, the fifth one was a short-statured incarnation (Baman avatar). It indicates the fact that early men were short stature.
In this way Prof Das described not only the biological evolution, but the cultural revolution too. He also mentioned that Parasurama was defeated by Rama, as Rama possessed bow and arrow, a superior weapon than the axe. The stage corresponded to the food-gathering stage of prehistory and it was followed by a food-producing stage as depicted in the story of Lord Krishna who used to look after the cattle in his childhood and his elder brother Balaram carried a plough most of the time.
In the Christian era, before Darwin, several scientists and philosophers expressed their views regarding the evolution. In this context, Carl Linnaeus (1707 – 1778) made a classic work “Systema Natural” where he described a system of classification involving the plants and animals, known as taxonomy. 
He placed man in the order Primate along with apes and monkeys, but he did not suggest any common ancestry for them. Further, he believed that each species was created specially and separately; their position remains unchangeable. In this way, the proposition of Linnaeus was a combination of the Old belief and the new thought.
Men boddo (1714-1790) by observing the origin of species traced the evolution of man from the monkeys. Bonnet (1720 – 1793) also worked on the process of evolution and proposed a ‘scale of beings’. His proposition went on an ascending order from the mineral to man. Many more scientists worked with the origin of man. Among them, the contributions of Erasmus, Darwin (1731- 1802), Karl von Baer (1792-1876), Schopenauer (1788 -1860) and Charles Lyell (1797 – 1875) seem to be indispensable for proper understanding of the facts of evolution. Imanuel Kant (1724 – 1804) proposed that the man be descended from the monkey.
According to a group of scholars, the expression of Goethe (1749 – 1832) was so meaningful in respect of evolution that he may be regarded as a predecessor of Charles Darwin. Again, another scientist, Malthus (1766 -1834) kept valuable contribution towards formulating the theory of natural selection. It is justified to trace the history of evolutionary thought from the beginning of nineteenth Century. The first systematic attempt was made by Jean Baptiste Lamarck (1744 – 1829), a French biologist who was an eminent pre-Darwian student of evolution.
His theory was published in 1802 in which he proposed the ‘inheritance of acquired characters’ during the lifetime of the individual. Following Lamarck’s proposition, Charles Darwin and Alfred Russell Wallace jointly proposed the theory of the ‘Origin of Species’ by Natural Selection.
Charles Darwin’s evolutionary theory had its base on the accumulation of small fluctuating variations. He had realized that heredity was an essential factor in the study of evolution, though he did not put much importance to it. August Weismann realized the importance of heredity better than Darwin did.
He emphasized on the ‘continuity- of the germ plasm’ and tried to project the transmission of inherited qualities from generation to generation by the germ cells. Hugo de Vries, one of the re-discoverers of Mendel’s laws of heredity, announced mutation theory of evolution in 1901. He considered mutation (i.e. sudden hereditary changes) as a factor behind evolution.
Natural selection found very little or no place in his mutation theory. But, later the geneticists, biometricians, and palaeontologists revived the faith in natural selection. Of these, the most important development took place in the field of genetics; the natural selection was started to be restudied and reinterpreted by the geneticists. Mention may be made of Theodore Dobzhansky and R.B. Goldschmidt, who laid the foundation for the Neo-Darwinian theory.
The genetic theory of Natural Selection is therefore referred as Neo-Darwinism. R.S. Fisher, J.B.S. Haldane and Sewall Wright made valuable contribution to the statistical analysis of population and secured own position among the principal proponents of Neo-Darwinism. However, the important theories have been discussed in the following pages.

11. Theory of Lamarck (Lamarckism):

The French biologist, Jean Baptiste Lamarck (1744 – 1829) spent his early years in military service but when he was stationed at Monaco, he acquired interest in Botany. He also established himself as a distinguished zoologist. His extensive studies on invertebrates formed a base in zoological classification.
He was the first scholar to recognize the distinction between invertebrates and vertebrates. But Lamarck’s name is usually associated with the ‘theory of inheritance of acquired characters’. Of his several writings, mention must be made about three publications relating to the theory of evolution: Recherches Sur L ‘Organization des Corps Vivant (1802), Philosophic Zoologique (1809) and Historie Naturelle des Animaux sans Vertebrates (1815 – 1822).
Lamarck expressed the fact that the acquired characters could be inherited. His theory, known as Lamarckism was based on two laws:
i. The law of use and disuse of organs, and
ii. The inheritance of acquired characters.
According to Lamarck, a living body is always influenced by the environmental factors and ultimately this phenomenon initiates an adaptation of organism to its surroundings. As per necessity, some parts of the body may be used more and more.
Therefore, those parts tend to show more development or changes in course of time. On the contrary, other parts of the body, which may not be required much, will be weak or demolished due to constant disuse. This change in body structure is reflected in future generations. This means, the characters that are acquired by the use or disuse of different organs can be transmitted to the succeeding generations.
To support his theory Lamarck presented several examples. The most remarkable one is associated with the long neck and high front legs of giraffes. He stated that this animal originally possessed short neck and small front legs.
As an herbivorous animal, the forerunners of modern giraffe were acquainted with grass and the leaves of dwarf trees. But following a sudden scarcity of these plants, giraffes had to stretch out their necks to reach the leaves of the tall trees. This stretching affected the muscles and bones of the neck, which started to be modified with time. Not only had the neck become longer the front legs also increased in size. This phenomenon is nothing but an adaptation to the environment, in the way to survival.
The modified traits were continued in subsequent generations and eventually all the giraffes got very long necks and well-built long front legs. In another example, he mentioned that the ducks are unable to fly because their wings became weak when they stopped flying.
Again, the birds that started to live in an aquatic environment gradually acquired webbed feet through the conquest of survival. Lamarck also cited other examples like limblessness in snake, blindness of moles and certain cave-dwelling forms, aquatic plants with dimorphic leaves (having submerged and aerial leaves), etc. All these changes were held to be cumulative from generation to generation, and also hereditary.
Lamarck’s theory had met criticisms from several angles. Although some of his views were admitted by a few scholars, most of the scholars did not accept his theory. The German scientist August Weismann ridiculed the essence of Lamarckism (inheritance or acquired characters) by his experiments, which involved cutting of tails of mice for over twenty generations.
All tailless mice in all generations (even in the last generation) produced their offspring’s with tails. Therefore he reached to the conclusion that the environmental factors might have an influence on the body cells, but it is not enough to profess a change of reproductive cells.
Characters of an organism would not be inherited unless the change could occur in the reproductive cells. However, the proposition of Weismann is popularly known as ‘Germ-Plasm theory’ as contrary to the theory of Lamarck. According to Weismann the body of an animal is composed of two parts viz. Germplasm (germ cells) and somatoplasm (body cells); only those characters which are located in the germplasm will be inherited by the offspring.
The evidence against Lamarckism was also criticized by others, on the ground that cutting of tail is rather mutilation, in which the animal did not participate actively so some specific cases were required where organisms can actively participate in the activity. In this respect, McDougall (1938) conducted a series of experiments on learning, using white rats. He designed a water tank having two exits, one lighted and the other dark.
The lighted exit received electric shock, while the dark exit did not have any arrangement to receive the electric shock. The white rats were dropped into such an experimental tank, and then trained to escape through the dark exit. A number of trials were required for the rat to learn the way to escape from the dark exit. These trials constituted a measure of the speed of learning.
The trained rats were bred, and their offspring’s were taught the same problem. In this manner, he subjected the rats for experimentation, for forty-five generations. McDougall observed that the number of errors made in learning, the problem decreased progressively from generation after generation. On the basis of this experiment, he concluded that an acquired character (learning or training) is inherited.
Unfortunately, McDougall’s experiments met with severe criticism, mainly because the repetition of similar experiments in other laboratories had failed to produce similar results. They could not control the genetic constitution of the experimental rats.Limitation of various other experiments probably initiated the scholars for seeking evidence in favour of Lamarck. A new school of thought in the name of Neo-Lamarckism soon appeared in the scene, which tried to modify the principles of Lamarck in order to make it acceptable to the students of evolution.
The foremost position was occupied by Giard (1846 – 1908) of France and Cope (1840 – 1897) of America. However, Neo- Lamarckism was based on the idea of adaptation, integrated with direct and casual relationship between structure-function and environment. The difference between the Lamarckism and Neo- Lamarckism was that, Lamarck believed in direct action of the environment, which, he thought was responsible to achieve final perfection of the individual. But Neo-Lamarckism omitted the very idea.
The Neo-Lamarckians argued that a considerable period of time was required for getting the effect of external factors. They also pointed out that if the external factors failed to influence the reproductive cells of the parents, their offspring’s would never inherit any of the modifications.
Rapid progress of science in twentieth Century favoured the growth of ‘genetics’, which supported none of the theories – Lamarckism and Neo-Lamarckism. Still Lamarck deserves appreciation as his proposition helped to open new avenues of thought in the science of evolution.

12. Theory of Darwin (Darwinism):

Charles Robert Darwin (1809 -1882) was born as the fifth son of his parents. He had an elementary schooling in Shrewsbury, England. In childhood he took little interest in studies, but showed great interest in hunting birds and shooting dogs. His father and teacher considered him as ‘a little below average in intelligence’. Although in school, he showed some interest in mathematics and chemistry, but most of his time was spent in watching the habits of birds, collecting insects and minerals.
In 1825, Darwin was sent to Edinburgh to study medicine, but soon he discontinued the course. After this his father wanted him to be prepared for the post of a clergyman, in the Church of England. So Darwin was sent to Cambridge. While studying at Cambridge, he gained friendship with some distinguished men of science, such as, the botanist Dr. Henslow and the geologist Sedgwick. Dr. Henslow’s friendship entirely changed the course of Darwin’s life; he nominated Darwin in the position of a young naturalist for the voyage on H.M.S. Beagle (a ship, in which Charles Darwm sailed around the world).
The voyage on the Beagle started on 27th. Dec. 1831 and Darwin visited many Islands in Atlantic ocean, some of the islands in the Pacific ocean including Galapagos islands, many places on the coasts of South America and finally returned after five years on 2nd. Oct. 1936. While on the Beagle, Darwin took notes on the flora, fauna, and the geology of the places visited; and also made extensive collections of living and fossil minerals. All these constituted the basis for his future publications.
Darwin’s first publication, Journal of Researches (1839) met with immediate success. In October 1838 he accidentally came across Robert Malthus’ essay on population. This essay provided a clue for which Darwin was able to think of the ‘struggle for existence’ among the animals and plant kingdom.
In this respect, he started to collect the data from 1842. The famous geologist of that period, Sir Charles Lyell suggested him to write about the origin of species. In 1858, when Darwin was halfway in his writing, he received a manuscript entitled, “On the tendency of varieties to Depart Indefinitely from the Original type” from Alfred Russell Wallace (1823 – 1913).
Wallace requested Darwin to read his essay and to make comments on it. Darwin found that the essay was complete in all respects and contained the essence of his theory of natural selection. Being generous, he decided to withhold his half-completed work, in favour of Wallace. So he wrote to Lyell with a recommendation to publish Wallace’s paper at once.
But Lyell, being aware of Darwin’s strenuous effort since 1842, urged Darwin to write a short abstract of his theory. He wished that Wallace’s paper would be published simultaneously with Darwin’s abstract. Reluctance of Darwin could not stand against the insistence of Lyell.
Thus, in 1859, Wallace’s paper and an abstract of Darwin’s manuscript together appeared in the Journal of the Proceedings of the Linnean society. To start with, Darwin intended to complete his work in four volumes but subsequently he condensed the work into a single volume, entitled ‘Origin of Species’ which was published in November 1859.
The work of Darwin was submitted fifty years after Lamarck and his theory is commonly known as Darwinism. But, the credit went to both the scholars – Darwin and Wallace; the first systematic as well as comprehensive approach in the perspective of evolutionary development was made by them.
Darwin’s theory of evolution is based upon four main, rather easily understandable postulates, which may be summarized as follows:
1. Prodigality of Nature:
All species have a tendency to produce more and more offspring’s in order to increase the number of population. For example, a salmon produces 28,000,000 eggs in a single season; a single spawning of an Oyster may yield as many as 114,000,000 eggs; a common roundworm (Ascaris lumbricoides) lays about 70,000,000 eggs in a day.
Darwin has even cited examples from slow breeding animals. Elephants appear to be one of the slowest breeders, having a life span of about hundred years. The active breeding age continues from thirty to ninety years, during which a single female may produce six young ones.
Taking this estimation into consideration, Darwin calculated that a single pair of elephants, at this rate of reproduction (provided all the descendants survived and reproduced at the same rate) would produce 19,000,000 elephants after 750 years.
All these examples furnish instances of tremendous reproductive potential among all species of organisms. The basic reason behind this huge production is to ensure the survival. Because, in reality we find that, in spite of the rapid reproductive potential, the size of a given species, in a given area, remains relatively constant.
2. Struggle for Existence:
Above observation led to the conclusion that all the progeny produced by any generation do not complete their life cycle, many of them die during juvenile stages. Darwin therefore proposed his of Struggle for Existence’; the struggle is often generated for the want of enough resource All individuals cannot survive under struggle.
According to Darwin, the Struggle for existence may be of different types. It may be a Struggle to overcome adverse environmental conditions (like cold or drought), or to obtain food from a limited source of supply. It may be a fight for occupying a living pace, or even to escape from the enemies. However, any of these said situations, evidently leads the members of a group towards competition, in order to meet their requirements.
Thus the nature of struggle may be of three types according to the situations:
(i) Intra-specific struggle:
When the members of a same species struggle among themselves, the situation is considered as intra-specific struggle. Such a struggle is usually centered round the consumption.
(ii) Inter-specific struggle:
The individuals from different species also may go on fighting for survival. An individual from one species may hunt another individual of other species as food. For example, tiger hunt goat and deer; cat hunt rat; lizard hunt cockroach and different small insects; and so on. According to Darwin in the animal kingdom, a species often stand as prey to other species, which clearly indicates a struggle for existence. Such happenings have been referred as Inter-specific struggle.
(iii) Environmental struggle:
The environmental struggle is different from the inter-specific or intra-specific struggle. Here individuals irrespective of their species-identity struggle against the environmental hazards like earthquake, flood, drought etc. Those who have greater potentiality for resistance, only they survive.
Darwin believed that the struggle is a continuous phenomenon in the way to survival It is severe among the members of the same species (intra-specific competition), as they depend on identical requirements of life. The inter-specific competition is though very common, but its frequency is lesser than the intra-specific competition.
3. Organic variation:
Darwin observed that variation is a universal phenomenon. Except the identical twins no two organisms are exactly alike. Even the two leaves of a plant or two peas in a pod often show easily recognizable differences. Therefore individuals of a single species must vary from each other.
At times, an entire population may exhibit a definite pattern of variation for which it is distinguished from the rest of the species. Such a population showing definite pattern of variation is often referred to as subspecies. Darwin considered these subspecies as incipient species, and he believed that in course of time, these subspecies would be subjected to further variation to give rise a new species.
Although natural variations are neither advantageous nor disadvantageous to the species concerned but some variations are considered as favourable and others are unfavourable. In fact, the variations in terms of physiological, structural and behavioural traits play very important role for adaptation in the environment. The new variants are produced continuously but when those variants cannot cope up the environment, it is termed as un-favourable variation.
Organisms with un-favourable variation easily get defeated in the struggle for survival and in course of time they become eliminated from the world. On the other hand, the new variants that are capable to adopt the pressure of the environment survive long. The new traits of advantageous characteristics pass on to the future generation.
Darwin recognized two main types of variation in nature, viz. Continuous variation and discontinuous variation. By the term continuous variation he wanted to mean small fluctuations of evolutionary significance. It was held as a force for attaining perfection being selected by nature For example, the long neck of giraffe was evolved out of continuous evolution.
Contrary to this discontinuous variation is mostly large and rare in occurrence. However, they appear suddenly and do not show any graded series. Such discontinuous variations have been regarded as ‘sports’ by Darwin; to which, Hugo de Vries has given the name ‘mutation’, at a later period. In the eye of Darwini discontinuous variation had no evolutionary importance.
Darwin draws the example of Dinosaurs. The enormous size and giant stature of Dinosaurs were the result of discontinuous variation. He found negative mode of natural selection behind the extinction of those Dinosaurs.
4. Natural Selection:
Natural selection is the final outcome of Darwin’s evolutionary thought. Individuals differ from each other because of organic variation, which evidently means that some individuals are better adapted to survive under the existing environmental conditions than others.
In the struggle for existence, the better-adapted individuals possess a better chance of survival than those who are less adapted. The less adapted individuals therefore get eliminated before reaching maturity and thus a large number of individuals die in the struggle for existence.
However, the traits having greater survival value are preserved in the individuals and transmitted to the offspring’s, who are supposed to be the progenitors of the next generation. Darwin called this principle, by which preservation of useful variation is brought about, as natural selection. The same principle (natural selection) has been designated by Herbert Spencer as ‘survival of the fittest’. In the words of Darwin “the expression often used by Spencer, of the survival of the fittest, is more accurate, and is sometimes equally convenient”.
The theme of Darwin’s theory may finally be summed up in the following words: The organisms always struggle to maintain their existence as nature decides the survival of the fittest one. Adaptive traits preserved through natural selection gradually change the characteristics of species and thus evolution occurs.
The theory of the origin of species by natural selection, though is regarded as a major advancement in evolutionary thought, it lacked the knowledge of heredity, which was essential for the understanding of evolutionary studies. It was really unfortunate that Darwin never came across Mendel’s work, who by then invented the basic principles of heredity. Had Darwin come across Mendel and his work, he would not have to write in the last edition of his ‘origin of species’ that “the fundamental principles of heredity are still unknown”.
The human ancestry was discussed by Darwin in his book, ‘The Descent of Man’ which was published in 1871. He said that life ascended from simplest form of minute organisms to the complex forms through different stages of evolution where man is found at the summit.
But, at the time of Darwin very few fossil evidences were discovered; those were insufficient to establish the proposition. This was the first weakness of Darwinism. The second weakness was hidden in the process itself. Darwin wanted to explain heredity by the ‘theory of Pangenesis’, which declared that all parts of the body produce minute particles called pangenes that ultimately get deposited to the sex-cells being carried by blood.
Those particles are further carried to the next generation when fertilization takes place and same kinds of organ, cell, tissue etc. are reproduced. However, the theory of Pangenesis, like the Lamarck’s principle, accounts for the inheritance of acquired characters. But it too was universally discarded for the lack of evidence. The flaws of Darwin were rectified later, after the development of the science of genetics and the rectified theory was known as Neo-Darwinism.

13. Mutation Theory of Hugo De Vries:

Hugo de Vries (1840 – 1935) was a Dutch Botanist, who proposed the third theory of evolution. His ‘mutation theory’ which appeared in 1901, focused attention upon the importance of mutation in evolution. In this theory, de Vries declared that evolution is not a slow and gradual process involving accumulation of numerous small changes by natural selection. Conversely, the evolutionary changes appear suddenly and are a result of large jumps, which he designated as mutation.
The publication of de Vries’ work raised much controversy among the adherents of Darwinism and the mutationists. The early geneticists extended their wide support to de Vries’ theory, mainly because the variations, which they noted in their experiments, conformed to de Vries’ observations but hardly with Darwin’s concept.
Even eminent geneticists like William Bateson, Thomas Hunt Morgan and others were attracted by this mutation theory Mutation theory distinguished heritable variations from environmental variations, which Darwin failed to understand in his ‘Natural Selection’. As a consequence, in the early years of twentieth Century Darwin’s natural selection was totally rejected in explaining the process of evolution.

14. Theory of Gregor Mendel:

The work of Gregor Mendel virtually remained unknown from 1865 to 1900 until it was rediscovered by three geneticists in 1900, Carl Correns, Hugo de Vries and Eric Von Tschermak. The real mechanism of mutation was properly understood through the work of Gregor Mendel and the recent discoveries in the field of molecular biology.
De Vries’ hypothesis on mutation highlighted chromosomal changes, rather than the changes in the gene themselves. So his mutation theory is considered as out modded on the ground that it did not indicate true mutation.
The mutations as understood today are concerned with genes, the discrete units of heredity, which occupy particular loci on the chromosomes. It tells that each gene controls a specific developmental process and responsible for the appearance of specific traits in an organism.
Mendel used the term ‘factor’, when he described his ‘Law of Inheritance’. But in 1900 the term was replaced by the new term ‘gene’ and a new science gradually developed with the name ‘Genetics’ Now It IS known that a gene represents a specific segment of the DNA molecule.
The product of a gene action in many cases, is a protein; and the developmental process in a given organism depends on specific kind of proteins produced under the instruction of a particular set of genes. A mutation in a gene often causes corresponding changes in the protein concerned. If mutation occurs in the gem cells of an organism, the change will be inherited by its offspring.
Therefore, only those mutations that cause changes in the reproductive cells of the organism are of evolutionary significance But the structural changes of chromosomes cannot be undermined because they often bring considerable effects in the evolution as found in many plants and a few animals like Drosophila, crepis etc.
Although the knowledge of genetics brought a revolution in the field of evolution Mendel’s Law of Inheritance’ is fundamental in identifying the nature of the offspring’s. It explained the basic process of heredity.

15. Synthetic Theory of Evolution (Neo-Darwinism):

Darwinism in its original form failed to explain satisfactorily the mechanism of evolution and the origin of new species. The inherent drawbacks in the Darwinian ideas were the lack of clarity as to the sources of variation and the nature of heredity.
In the middle of twentieth Century, Scientists had come to a consensus to employ all sorts of knowledge – genetic, ecological, geographical morphological, palaeontological etc. in order to understand the actual mechanism of evolution. Due importance was given to both mutation and natural selection, among other forces of evolution This led o the emergence of a synthetic theory of evolution, which we also call as Genetical Theory of evolution, or ‘Biological theory of Evolution’.
Some authors namely David J. Merrell (author of ‘Evolution and Genetics’) and Edward O Dodson (author of ‘Evolution: Process and Product’) have called this new theory as Neo-Darwinism. But, George Gaylord Simpson and his followers strongly warned against equating the synthetic theory of evolution with ‘Neo-Darwinism’. Simpson argued that the synthetic theory had no Darwin. It was not only different from Darwin’s; it had drawn its material from a variety of non-Darwinian sources.
After the development of the science of genetics, it has been known mat a population snares a common gene pool. Accordingly, the evolution denotes a change of gene -frequency in the gene pool of a population over certain span of time.
The synthetic theory of evolution does not discard all previous propositions, rather considers them as partially important. Therefore, we find amalgamation of various concepts viz. Natural selection, Mendelian principles, Mutation, population genetics in this theory of evolution. But it is interesting to note that modem genetics does not acknowledge to mutation theory in its original form, as proposed by de Vries. Because that original theory had out- rightly rejected the basic concept, ‘natural selection’ as delivered by Darwin and advocated ‘mutation’ as the sole force of evolution. However, at present evolution appears to be a complex process involving several complex forces.

Red Blood Cells (RBC's)

Published in Microbiology
Friday, 29 April 2016 03:16
Size: 6.7 to 7.7 μ in diameter.
Cytoplasm: Pink in color.
The mature red blood cell is a nonnucleated, round, biconcave cell.
Erythropoiesis: The main function of the red blood cell is to transport oxygen to the tissues. Production of red blood cells (erythropoiesis) is initiated by a hormone produced by the kidney called erythropoietin. When a person’s hemoglobin level is below normal, his tissues will not receive an adequate supply of oxygen, and this will stimulate the kidneys to increase their production of erythropoietin. The increased erythropoietin will then stimulate the stem cells of the bone marrow to differentiate into the pronormoblast, and there will be an increased number of red blood cells produced. As the red cells are maturing they undergo several cellular divisions. Once the orthochromic normoblast stage is reached, however, the cell is no longer capable of mitosis but will continue to mature in the bone marrow. The reticulocyte remains in the marrow for approximately two days and is then released into the peripheral blood. The red cells of the circulating blood have a lifespan of approximately 120 days, ±20 days.
Hemoglobin structure and synthesis: Hemoglobin is made up of the protein, globin, and heme. In normal adult hemoglobin, the globin portion of each molecule consists of four polypeptide chains: two α and two β chains. These chains, in turn, are composed of 141 and 146 amino acids (arranged in a specific sequence), respectively. Each chain is bent and coiled. The heme group is composed of four pyrrole rings connected by methene bridges. In the center of this structure is an atom of iron to which oxygen is attached, when the iron is in the ferrous state (Feˉˉ).
One heme molecule will be attached to each of the α and β chains. Two α and two β chains come together to form a tetramer. The single hemoglobin molecule, therefore, consists of two α chains, two β chains, and four heme groups (thus, four atom of iron). Mature red blood cells are incapable of hemoglobin synthesis. The production of heme and globin takes place independently of each other, beginning in the polychromatic normoblast, and ending in the reticulocyte stage.

Urine Albumin and Albumin/Creatinine Ratio

Published in Biochemistry
Friday, 29 April 2016 03:04
The urine albumin test or albumin/creatinine ratio (ACR) is used to screen people with chronic conditions, such as diabetes and high blood pressure (hypertension) that put them at an increased risk of developing kidney disease. Studies have shown that identifying individuals in the very early stages of kidney disease helps people and healthcare providers adjust treatment. Controlling diabetes and hypertension by maintaining tight glycemic control and reducing blood pressure delay or prevent the progression of kidney disease.
Albumin is a protein that is present in high concentrations in the blood. Virtually no albumin is present in the urine when the kidneys are functioning properly. However, albumin may be detected in the urine even in the early stages of kidney disease. (See the "What is being tested?" section for more.)
If albumin is detected in a urine sample collected at random, over 4 hours, or overnight, the test may be repeated and/or confirmed with urine that is collected over a 24-hour period (24-hour urine).
Most of the time, both albumin and creatinine are measured in a random urine sample and an albumin/creatinine ratio (ACR) is calculated. This may be done to more accurately determine how much albumin is escaping from the kidneys into the urine. The concentration (or dilution) of urine varies throughout the day with more or less liquid being released in addition to the body's waste products. Thus, the concentration of albumin in the urine may also vary.
Creatinine, a byproduct of muscle metabolism, is normally released into the urine at a constant rate and its level in the urine is an indication of the urine concentration. This property of creatinine allows its measurement to be used to correct for urine concentration in a random urine sample. The American Diabetes Association has stated a preference for the ACR for screening for albuminuria indicating early kidney disease. Since the amount of albumin in the urine can vary considerably, an elevated ACR should be repeated twice within 3 to 6 months to confirm the diagnosis.
When is it ordered?
According to the American Diabetes Association and National Kidney Foundation, everyone with type 1 diabetes should get tested annually, starting 5 years after onset of the disease, and all those with type 2 diabetes should get tested annually, starting at the time of diagnosis. If albumin in the urine (albuminuria) is detected, it should be confirmed by retesting twice within a 3-6 month period. People with hypertension may be tested at regular intervals, with the frequency determined by their healthcare provider.
What does the test result mean?
Moderately increased albumin levels found in both initial and repeat urine tests indicate that a person is likely to have early kidney disease. Very high levels are an indication that kidney disease is present in a more severe form. Undetectable levels are an indication that kidney function is normal.
The presence of blood in the urine, a urinary tract infection, vigorous exercise, and other acute illnesses may cause a positive test result that is not related to kidney disease. Testing should be repeated after these conditions have resolved.
Is there anything else I should know?
Studies have shown that elevated levels of urinary albumin in people with diabetes or hypertension are associated with increased risk of developing cardiovascular disease (CVD). More recently, research has been focused on trying to determine if increased levels of albumin in the urine are also indicative of CVD risk in those who do not have diabetes or high blood pressure.

Cholinesterase Tests

Published in Immunology
Friday, 29 April 2016 02:55
Cholinesterase testing has two main uses:
  • It can be used to detect and diagnose organophosphate pesticide exposure and/or poisoning. It may also be used to monitor those who may be at increased risk of exposure to organophosphate compounds, such as those who work in agricultural and chemical industries, and to monitor those who are being treated for exposure. Typically, tests for red blood cell acetylcholinesterase (AChE) and serum pseudocholinesterase (PChE) are used for this purpose.
  • It can be used several days prior to a surgical procedure to determine if someone with a history of or family history of post-operative paralysis following the use of succinylcholine, a common muscle relaxant used for anesthesia, is at risk of having this reaction. In these cases, the test for pseudocholinesterase is usually used. A second test, referred to as a dibucaine inhibition test, may be done to help determine the extent to which the activity of the enzyme is decreased.
When is it ordered?
People who work with organophosphate compounds in the farming or chemical industries may be routinely monitored to assess any adverse exposure, once baseline levels have been established. Cholinesterase testing can also be used to assess any acute exposure to these compounds, which can cause neuromuscular damage. Toxicity can follow a rapid absorption of the compound in the lungs, skin, or gastrointestinal tract. The symptoms of toxicity are varied depending on the compound, quantity, and the site of exposure. Early symptoms may include:
  • Headache, dizziness
  • Nausea
  • Excessive tearing in the eyes, sweating and/or salivation
As the effects of the poisoning worsen, some additional symptoms may appear:
  • Vomiting, diarrhea
  • Dark or blurred vision due to constricted pupils
  • Muscle weakness, twitching, lack of coordination
  • Slowed breathing leading to respiratory failure, requiring lifesaving ventilation
  • In serious cases, seizures, coma, and death
Pre-operative screening for pseudocholinesterase activity is advised if a person or a close relative has experienced prolonged paralysis and apnea after the use of succinylcholine for anesthesia during an operation.
What does the test result mean?
In monitoring for occupational pesticide exposure
Following exposure to organophosphate compounds, AChE and PChE activity can fall to about 80% of normal before any symptoms occur and drop to 40% of normal before the symptoms become severe. Those who are regularly exposed to these compounds may be monitored for toxic exposure by establishing a baseline activity level and then testing on a regular basis to watch for a significant reduction on activity of acetylcholinesterase or pseudocholinesterase.
In testing for acute pesticide exposure/poisoning
Significantly decreased cholinesterase activity levels usually indicate excessive absorption of organophosphate compounds. Pseudocholinesterase and RBC acetylcholinesterase activity are usually decreased within a few minutes to hours after exposure. Pseudocholinesterase activity may regenerate in a few days to weeks, while acetylcholinesterase activity will remain low for as long as one to three months. Both plasma and RBC activities are immediately affected by pesticide exposure but, upon removal from exposure, AChE and PChE regenerate at different rates since AChE is produced in blood cells, which have a lifespan of 120 days, whereas PChE is produced in the liver, with a half-life of about two weeks.
In testing for succinylcholine sensitivity
About 3% of people have low activity levels of pseudocholinesterase due to an inherited deficiency and will have prolonged effects from the muscle relaxant succinylcholine. Total quantitative pseudocholinesterase levels will be evaluated prior to surgery for patients with a history or family history of prolonged apnea after use of this drug. Low activity levels of pseudocholinesterase levels indicate that these people may be at increased risk of experiencing prolonged effects of the muscle relaxant. A second test, the dibucaine inhibition test, may also be performed to help characterize the degree of a person's sensitivity to the drug. The lower the result from a dibucaine inhibition test, the greater the risk of drug sensitivity.
Reduced cholinesterase levels can also be caused by chronic liver disease and malnutrition. Total cholinesterase activity can be lowered in a number of other conditions, including pregnancy, renal disease, shock, and some cancers.
Is there anything else I should know?
If someone unexpectedly has prolonged apnea after surgery, testing for succinylcholine sensitivity may be performed; however, the sample should be obtained after 24 to 48 hours have elapsed following the surgery to avoid interference by any drugs given during the surgery that could affect the results. Drugs called cholinesterase inhibitors may have a moderate benefit in those with early diagnosed Alzheimer's disease.

Platelets Count

Published in Microbiology
Friday, 29 April 2016 02:26

Platelets, also called "thrombocytes", are blood cells whose function (along with the coagulation factors) is to stop bleeding. Platelets have no nucleus: they are fragments of cytoplasm which are derived from the megakaryocytes of the bone marrow, and then enter the circulation. These unactivated platelets are biconvex discoid (lens-shaped) structures, 2–3 µm in greatest diameter. Platelets are found only in mammals, whereas in other animals (e.g. birds, amphibians) thrombocytes circulate as intact mononuclear cells. There are two methods for estimation of erythrocyte count:
•    Manual or microscopic method
•    Automated method
Free-flowing capillary or well-mixed anticoagulated venous blood is added to a diluent at a specific volume in the Unopette reservoir.  The diluents (1% ammonium oxalate) lyses the erythrocytes but preserves leukocytes and platelets.  A 20 µL pipette is used with 1.98 ml of diluents to make a 1:100 dilution. The diluted blood is added to the hemacytometer chamber.  Cells are allowed to settle for 10 minutes before leukocytes and platelets are counted. (Always refer to the manufacturer’s instructions for the procedure.)
Hemocytometer with cover glass, compound microscope. Unopette capillary pipette, lint-free wipe, alcohol pads,  hand counter, petri dish with moist filter paper.
Ammonium oxalate: 11.45 gm
Sorensen’s phosphate buffer: 1.0 gm
Thimerosal: 0.1 gm
Distilled water: 1000 ml
EDTA-anticoagulated blood or capillary blood is preferred.
(1) Using the protective shield on the capillary pipette, puncture diaphragm of  Unopette reservoir.    
(2) Remove shield from pipette assembly by twisting. Holding pipette almost horizontally, touch tip of pipette to blood.  Pipette will fill by capillary action. Filling will cease automatically when the blood reaches the end of the capillary bore in the neck of the pipette.
(3) Wipe the outside of the capillary pipette to remove excess blood that would interfere with the dilution factor.
(4) Squeeze reservoir slightly to force out some air while simultaneously maintaining pressure on reservoir.
(5) Cover opening of overflow chamber of pipette with index finger and seat pipet securely in reservoir neck.
(6) Release pressure on reservoir. Then remove finger from pipette opening. At this  time negative pressure will draw blood into reservoir.
(7) Squeeze reservoir gently two or three times to rinse capillary bore forcing diluent up int, but not out of, overflow chamber, releasing pressure each time to return mixture to reservoir.
(8) Place index finger over upper opening and gently invert several times to thoroughly mix blood with diuent.
(9) Cover overflow chamber with pipette shield and incubate at room temperature for 10 minutes before charging the hemacytometer.
(10) Meticulously clean the hemacytometer with alcohol or other cleaning solution. This is important because dust particles and other debris can be mistaken for platelets especially on a light microscope. Allow to dry completely before charging with diluted specimen.
(11) To charge the hemacyto-meter, convert to dropper assembly by withdrawing pipette from reservoir and reseating securely in reverse position.
(12) Invert reservoir and discard the first 3 or 4 drops of mixture.
(13) Carefully charge hemacyto-meter with diluted blood by gently squeezing sides of reservoir to expel contents until chamber is properly filled.
(14) Place hemacytometer in moist Petri dish for 10 minutes to allow platelets to settle.  (Moistened filter paper retains evaporation of diluted specimen while standing.)
(15) Mount the hemacytometer on the microscope and lower its condenser.
(16) Procedure for counting platelets:

• Under 40x magnification, scan to ensure even distribution.  Platelets are counted in all twenty-five small squares within the large center square. Platelets appear greenish, not refractile.
• Count cells starting in the upper left of the large middle square.  Continue counting to the right hand square, drop down to the next row; continue counting in this fashion until the total area in that middle square (all 25 squares) have been counted.
• Count all cells that touch any of the upper and left lines, do not count any  cell that touches a lower or right line.
• Count both sides of the hemocyt-ometer and take the average.
cells/mm3 =      Tc x Rd     
                    Ns x As x Ds
     Where Tc is the number of cells counted, Rd is the reciprocal of dilution, Ns is the number of squares counted, As area of each square and Ds is the depth of the solution.
Total number of cells= 230
Dilution 1:100
Number of squares counted: 1
Area of each square: 1 mm3
Depth of solution: 0.1mm

cells/mm3 =         230 x 100        
                  1 x 1 mm2 x 0.01 mm
               = 230,000/mm3 (µL)
               = 230 x 103/L
• 150,000 - 450,000/µL
• 150 - 450 x 109/L
1. Brown, B.A., Haemotology, Principles and Procedures, Lea & Febiger, U.S.A., 1976.
2. Hoffbrand, A. V. and Pettit, 1. E., Essential Haemotology, Blackwell Scientific Publication, U.S.A., 1980.
3. Kassirsky, I. and Alexeev, G., Clinical Haemotology, Mir Publishers, U.S.S.R., 1972.
4. Widmann, F.K., Clinical interpretation of Laboratory tests, F.A. Davis Company, U.S.A., 1985.
5. Kirk, C.J.C. et al, Basic Medical Laboratory Technology, Pitman Book Ltd., U.K. 1982.
6. Green, J.H., An Introduction to human Physiology, Oxford University Press, U.K., 1980.

Serum Angiotensin Converting Enzyme

Published in Immunology
Friday, 29 April 2016 02:18
Angiotensin-converting enzyme (ACE) is an enzyme that helps regulate blood pressure. An increased blood level of ACE is sometimes found in sarcoidosis, a systemic disorder of unknown cause that often affects the lungs but may also affect many other body organs, including the eyes, skin, nerves, liver, and heart., This test measures the amount of ACE in the blood.
A classic feature of sarcoidosis is the development of granulomas, small tumor-like masses of immune and inflammatory cells and fibrous tissue that form nodules under the skin and in organs throughout the body. Granulomas change the structure of the tissues around them and, in sufficient numbers, they can cause damage and inflammation and may interfere with normal functions. The cells found at the outside borders of granulomas can produce increased amounts of ACE. The level of ACE in the blood may increase when sarcoidosis-related granulomas develop.
The angiotensin-converting enzyme (ACE) test is primarily ordered to help diagnose and monitor sarcoidosis. It is often ordered as part of an investigation into the cause of a group of troubling chronic symptoms that are possibly due to sarcoidosis.
Sarcoidosis is a disorder in which small nodules called granulomas may form under the skin and in organs throughout the body. The cells surrounding granulomas can produce increased amounts of ACE and the blood level of ACE may increase when sarcoidosis is present.
The blood level of ACE tends to rise and fall with disease activity. If ACE is initially elevated in someone with sarcoidosis, the ACE test can be used to monitor the course of the disease and the effectiveness of corticosteroid treatment.
A health practitioner may order ACE along with other tests, such as AFB tests that detect mycobacterial infections or fungal tests. This may help to differentiate between sarcoidosis and another condition causing granuloma formation.
When is it ordered?
An ACE test is ordered when someone has signs or symptoms that may be due to sarcoidosis, such as:
  • Granulomas
  • A chronic cough or shortness of breath
  • Red, watery eyes
  • Joint pain
This is especially true if the person is between 20 and 40 years of age, when sarcoidosis is most frequently seen.
When someone has been diagnosed with sarcoidosis and initial ACE levels were elevated, a health practitioner may order ACE testing at regular intervals to monitor the change in ACE over time as a reflection of disease activity.
What does the test result mean?
An increased ACE level in a person who has clinical findings consistent with sarcoidosis means that it is likely that the person has an active case of sarcoidosis, if other diseases have been ruled out. ACE will be elevated in 50% to 80% of those with active sarcoidosis. The finding of a high ACE level helps to confirm the diagnosis.
A normal ACE level cannot be used to rule out sarcoidosis because sarcoidosis can be present without an elevated ACE level. Findings of normal ACE levels in sarcoidosis may occur if the disease is in an inactive state, may reflect early detection of sarcoidosis, or may be a case where the cells do not produce increased amounts of ACE. ACE levels are also less likely to be elevated in cases of chronic sarcoidosis.
When monitoring the course of the disease, an ACE level that is initially high and then decreases over time usually indicates spontaneous or therapy-induced remission and a favorable prognosis. A rising level of ACE, on the other hand, may indicate either an early disease process that is progressing or disease activity that is not responding to therapy.




CHOLERA is a specific infectious disease that affects the lower portion of the intestine and is char...

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