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HEMOGLOBIN

Hemoglobin is composed of heme (iron + protoporphyrin) and globin polypeptide chains. It is present in the red blood cells of all vertebrates except Channichthyidae (the family of fish: white-blooded fish also called crocodile fish found in southern South America and the Southern Ocean around Antarctica). It carries oxygen from the lungs to the tissues and carbon dioxide from tissues to the lungs.

In humans, hemoglobin is not homogeneous and normally different variants and derivatives exist. Normal hemoglobin variants are fetal hemoglobin (Hb F), adult hemoglobin (Hb A), Hb A2 and embryonic hemoglobins (Gower I, Gower II and Portland). They differ from each other on the basis of the structure and the type of polypeptide chains.

INDICATION FOR HEMOGLOBIN ESTIMATION

  1. Screening for polycythemia: Polycythemia is a disease state in which the hemoglobin level and hematocrit (HCT) or packed cell volume (PCV) value is elevated. It may be primary, secondary or relative.
  2. To determine presence and severity of anemia: Anemia is a disease state in which the hemoglobin concentration or oxygen-carrying capacity of blood is low. Clinical signs and symptoms (conjunctival vessels, polar of skin, mucosal membranes) are unreliable for the diagnosis of anemia. Anemia is best determined by estimation of hemoglobin and hematocrit (HCT) or packed cell volume (PCV).
  3. To assess response to specific therapy in anemia.
  4. Estimation of red cell indices (along with hematocrit (HCT) or packed cell volume (PCV) and red cell count) i.e. mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC).
  5. Selection of blood donors in the blood bank.

METHOD FOR ESTIMATION OF HEMOGLOBIN

There are different methods for estimation of hemoglobin. These are:

(1) Colorimetric methods: In these methods, the color comparison is made between the standard and the test sample, either visually or by colorimetric methods.

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(2) Gasometric method: In this method, oxygen-carrying capacity of red blood cells (RBCs) is measured in a Van Slyke apparatus. The amount of hemoglobin is then derived from the formula that 1 gram of hemoglobin carries 1.34 ml of oxygen. However, this method measures only physiologically active hemoglobin, which can carry oxygen. It does not measure methemoglobin, sulfhemoglobin, and carboxyhemoglobin. Also, this method is expensive and time-consuming, and the result is about 2% less than other methods.

(3) Chemical method: In this method, iron-content of hemoglobin is first evaluated. The value of hemoglobin is then derived indirectly from the formula that 100 grams of hemoglobin contain 374 mg of iron. This method is tiresome and time-consuming.

(4) Specific gravity method: In this method, an approximate value of hemoglobin is estimated from the specific gravity of blood as determined from copper sulfate technique. This method is simple and rapid. This method is useful and most common in mass screening like the selection of blood donors. See procedure.

Tallqvist Hemoglobin Chart

Tallqvist hemoglobin chart consists of a series of lithographed colors said to correspond to hemoglobin values ranging from 10% to 100%. In this method, a drop of blood obtained by finger puncture is placed on a piece of absorbent paper. The color produced is matched against the color on the chart and the corresponding reading is taken. The room of error is 20-50%. Although this method is very cheap and simple.

Red Cell Indices

  • 19 Jul 2016
Red Cell Indices

Red cell indices are mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC). They are also called as “absolute values”. They are derived from the values of hemoglobin, packed cell volume (PCV or hematocrit), and red cell count. Red cell indices are accurately measured by automated hematology analyzers. Recently, a new parameter called red cell distribution width (RDW) has been introduced.
 
USES OF RED CELL INDICES
 
(1) Morphological classification of anemia: Based on values of red cell indices, anemia is classified into three main types: normocytic normochromic, microcytic hypo-chromic, and macrocytic normo-chromic. Calculation of red cell indices is especially helpful in mild or moderate anemia when red cell changes are subtle and often difficult to appreciate on stained blood smear.
(2) Differentiation of iron deficiency anemia from thalassemia trait: In iron deficiency, MCV, MCH, and MCHC are low, while in thalassemia trait, MCV and MCH are low and MCHC is normal.
 
MEAN CELL VOLUME
 
MCV is a measure of average size of the red cells. It is measured directly by automated instruments from the measurement of volume of each red cell. With semiautomated instruments and by manual method, it is obtained by dividing PCV by red cell count.
 
MCV =                PCV in%                  x 10
            RBC count in million/cmm
 
     MCV is expressed in femtoliters or fl (10⁻¹⁵ of a liter). It corresponds with red cell diameter on blood smear. Normal MCV is 80-100 fl.
 
Causes of Increased MCV
 
• Megaloblastic anemia
• Non-megaloblastic macrocytosis: Chronic alcoholism, liver disease, hypothyroidism, normal pregnancy, reticulocytosis
• Newborns.
 
Causes of Low MCV
 
• Microcytic hypochromic anemia
 
MCV is normal in normocytic normochromic anemia (acute blood loss, hemolysis, aplastic anemia).
     In the presence of large number of abnormal red cells like sickle cells, and in dimorphic anemia (e.g. mixed normocytic and microcytic), MCV may be normal (since it is an average value) and thus unreliable for morphological classification.
     Mentzer index is derived by dividing MCV with red cell count. Ratio of less than 13 is seen in thalassemia while ratio is more than 13 in iron deficiency anemia.
 
MEAN CELL HEMOGLOBIN (MCH)
 
MCH is the average amount of hemoglobin in a single red cell. It is obtained by dividing hemoglobin value by red cell count.
 
MCH =    Hemoglobin in grams/dl     x 10
           RBC count in millions/cmm
 
MCH is expressed in picograms or pg (10⁻¹² gram). Reference range is 27-32 pg.
     MCH is decreased in microcytic hypochromic anemia, and increased in macrocytic anemia and in newborns.
 
MEAN CELL HEMOGLOBIN CONCENTRATION (MCHC)
 
MCHC is obtained by dividing hemoglobin value by PCV and expressed in grams/dl or grams/liter. It refers to concentration of hemoglobin in 1 dl or 1 liter of packed red cells.
 
MCHC = Hemoglobin in grams/dl  x 100
                        PCV in %
 
Reference range is 30-35 grams/dl. MCHC is raised in hereditary spherocytosis, and is decreased in hypochromic anemia. MCHC corresponds with degree of hemoglobinization of red cells on a blood smear. If MCHC is normal, red cell is normochromic, and if low, red cell is hypochromic.
 
Red Cell Distribution Width (RDW)
 
Some automated analyzers measure red cell distribution width or RDW. It is a measure of degree of variation in red cell size (anisocytosis) in a blood sample. It is helpful in differential diagnosis of some anemias. Amongst microcytic anemias, RDW is low in ß thalassemia trait, high in iron deficiency anemia, and normal in anemia of chronic disease. Normal RDW is 9.0 to 14.5.
 
REFERENCE RANGES
 
• Mean cell volume: 80-100 fl
• Mean cell hemoglobin: 27-32 pg
• Mean cell hemoglobin concentra-tion: 30-35 g/dl
• Red cell distribution width: 9.0-14.5
 
REFERENCES
 
1. Henry JB. Clinical diagnosis and management by laboratory methods (20th Ed). Philadelphia: WB Saunders Company, 2001.
2. Wallach J. Interpretation of Diagnostic Tests (7th Ed). Philadelphia: Lippincott Williams and Wilkins, 2000¹⁵

Porphyrias

  • 03 Jul 2016
Porphyrias

Porphyrias (from Greek porphura meaning purple pigment; the name is probably derived from purple discoloration of some body fluids during the attack) are a heterogeneous group of rare disorders resulting from disturbance in the heme biosynthetic pathway leading to the abnormal accumulations of red and purple pigments called as porphyrins in the body. Heme, a component of hemoglobin, is synthesized through various steps as shown in figure. Each of the steps is catalyzed by a separate enzyme; if any of these steps fails (due to hereditary or acquired cause), precursors of heme (porphyrin intermediates) accumulate in blood, get deposited in skin and other organs, and excreted in urine and feces. Depending on the site of defect, different types of porphyrias are described with varying clinical features, severity, and the nature of accumulated porphyrin.
 
Porphyria has been offered as a possible explanation for the medieval tales of vampires and werewolves; this is because of the number of similarities between the behavior of persons suffering from porphyria and the folklore (avoiding sunlight, mutilation of skin on exposure to sunlight, red teeth, psychiatric disturbance, and drinking of blood to obtain heme).
 
Porphyrias are often missed or wrongly diagnosed as many of them are not associated with definite physical findings, screening tests may yield false-negative results, diagnostic criteria are poorly defined and mild disorders produce an enzyme assay result within ‘normal’ range.
 
Heme is mainly required in bone marrow (for hemoglobin synthesis) and in liver (for cytochromes). Therefore, porphyrias are divided into erythropoietic and hepatic types, depending on the site of expression of disease. Hepatic porphyrias mainly affect the nervous system, while erythropoietic porphyrias primarily affect the skin. Porphyrias are also classified into acute and nonacute (or cutaneous) types depending on clinical presentation.
 
Inheritance of porphyrias may be autosomal dominant or recessive. Most acute porphyrias are inherited in an autosomal dominant manner (i.e. inheritance of one abnormal copy of gene). Therefore, the activity of the deficient enzyme is 50%. When the level of heme falls in the liver due to some cause, activity of ALA synthase is stimulated leading to increase in the levels of heme precursors up to the point of enzyme defect. Increased levels of heme precursors cause symptoms of acute porphyria. When the heme level returns back to normal, symptoms subside.
 
Accumulation of porphyrin precursors can occur in lead poisoning due to inhibition of enzyme aminolevulinic acid dehydratase in heme biosynthetic pathway. This can mimick acute intermittent porphyria.

“I can focus my slide under 10×, but not under 40×.”

A common reason for this is that the slide is upside down. Double check which side the smear is on (may not be the same side as the label!) and try focusing again. Another cause could be dried immersion oil on the 40× objective that is obstructing your view. When switching from oil immersion (100×) to 40×, there is a good chance that the tip of the 40× objective could be dragged through some immersion oil. If it is not immediately cleaned off, it will dry, producing a thick haze. To fx: Use lens paper and lens cleaner to clean the end of the 40× objective. This may need to be repeated several times depending on how thick the dried oil is. After cleaning, use a dry piece of lens paper to polish the objective. To avoid the problem: Clean up oil immediately after use. Clean the end of the 100× objective and any heavy oil present on the slide before moving back down to 40× objective.

“In hematology, when I focus under 40×, my red blood cells appear shiny.”

This is most likely due to water artifact during the staining and drying process. To make visualization of the cells easier, add a small drop of immersion oil to your slide. Gently spread the drop of oil over the area you will be examining. Wipe of excess oil using the side of your finger. Be very gentle when doing this, and use a clean finger each time you wipe. Wiping too hard or rough will cause your smear to rub off. This technique will leave a very thin layer of oil on your smear. The film is thin enough that you can use the 40× objective without running the risk of the lens becoming contaminated with oil. Try focusing under 40× again, and the shininess should have been resolved.

“There’s no light coming from the illuminator.”

The first assumption is always that the bulb is burnt out, but it is a good idea to check a couple of other possibilities as well. If the iris diaphragm is closed and the brightness of the illuminator is at its lowest, the light may be so small that it appears as if there is no light present. Check to make sure the cord is fully plugged into the back of the microscope. This plug can become dislodged slightly during transport and microscope set up. If your microscope is the type that uses fuses, it may be the fuse—not the bulb—that needs replacing. When the microscope is not in use, be sure to turn it off. This will help prolong the life of the bulb.

Clean up

When the use of the microscope is complete, following proper clean up procedures will improve the quality of images that are viewed and extend the life of the microscope and its components:

  1. Remove the slide from the stage and dispose of it properly.
  2. Clean any oil residue or sample material that may have contaminated the stage surface.
  3. Lower the stage and move the smallest objective into place.
  4. Clean the objective lens and oculars after every use. The order in which they are cleaned is important. Cleaning the 100× objective first and then moving onto other parts will result in immersion oil being spread onto all other components. Using lens tissue and lens cleaner, begin with cleaning the oculars, then the 4× objective, the 10× objective, 40× objective, and finish with the 100× objective lens.

When the recipient’s ABO and Rh blood groups are determined, the donor blood unit that is ABO and Rh compatible is selected, and compatibility test is carried out. The purpose of compatibility test is to prevent the transfusion of incompatible red cell units and thus avoidance of hemolytic transfusion reaction in the recipient. Compatibility test detects (i) major ABO grouping error, and (ii) most clinically significant antibodies reactive against donor red cells.

There are two types of cross-match: major cross-match (testing recipient’s serum against donor’s red cells) and minor cross-match (testing donor’s serum against recipient’s red cells). However, minor cross-match is considered as less important since antibodies in donor blood unit get diluted or neutralized in recipient’s plasma. Also, if antibody screening and identification is being carried out, minor cross-matching is not essential. Therefore, only the red cells from the donor unit are tested against the recipient’s serum and the name compatibility test has replaced the term cross-matching. For transfusion of platelets or fresh frozen plasma, cross-matching is not required. However, fresh frozen plasma should be ABO-compatible.

A full cross-matching procedure consists of:

  • Immediate spin cross-match at room temperature, and
  • Indirect antiglobulin test at 37°C.

IMMEDIATE SPIN CROSS MATCH

The purpose of this test is to detect ABO incompatibility. Equal volumes of 2% saline suspension of red cells of donor and recipient’s serum are mixed, incubated at room temperature for 5 minutes, and centrifuged. Agglutination or hemolysis indicates incompatibility.

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Causes of False-negative Test

  1. A2B donor red cells and group B recipient serum.
  2. Rapid complement fixation of potent ABO antibodies with bound complement interfering with agglutination.

Causes of False-positive Test

  1. Rouleaux formation
  2. Cold-reactive antibodies: If agglutination disappears by keeping the tube at 37°C for 10 minutes, presence of cold agglutinins is confirmed.

INDIRECT ANTIGLOBULIN TEST

Saline-suspended red cells of the donor after being incubated in patient’s serum are washed in saline and antiglobulin reagent is added. Following re-centrifugation, examine for agglutination or hemolysis. This test detects most of the clinically significant IgG antibodies.

If agglutination or hemolysis is not observed in any of the above stages, donor unit is compatible with recipient’s serum. Agglutination or hemolysis at any stage is indicative of incompatibility.

EMERGENCY CROSS-MATCH

If blood is required urgently, ABO and Rh grouping are carried out by rapid slide test and immediate spin cross match (i.e. the first stage of cross match) is performed (to exclude ABO incompatibility). If the blood unit is compatible, then after issuing it, remaining stage of the cross-match is completed. If any incompatibility is detected, the concerned physician is immediately informed about the incompatibility detected.

ANTIBODY SCREENING AND IDENTIFICATION

Screening for unexpected or irregular antibodies is done during pre-transfusion testing in recipient’s serum and in donor’s blood. In this test, serum of the recipient is tested against a set of three group O screening cells of known antigenic type. If unexpected antibodies are detected, then they are identified and blood unit that lacks the corresponding antigen is selected for compatibility test.

APTT is a measure of coagulation factors in intrinsic pathway (F XII, F XI, high molecular weight kininogen, prekallikrein, F IX, and F VIII) and common pathway (F X, F V, prothrombin, and fibrinogen).
 
Principle
Plasma is incubated with an activator (which initiates intrinsic pathway of coagulation by contact activation). Phospholipid (also called as partial thromboplastin) and calcium are then added and clotting time is measured.
 
Equipment
This is same as for Prothrombin Time test. (Click here to see)
 
Reagents
(1) Kaolin 5 gm/liter: This is a contact activator.
(2) Phospholipid: Various APTT reagents are available commercially, which contain phospholipids.
(3) Calcium chloride 0.025 mol/liter.
 
Specimen
Method
(1) Mix equal volumes of phospholipid reagent and calcium chloride solution in a glass test tube and keep in a waterbath at 37°C.
(2) Deliver 0.1 ml of plasma in another test tube and add 0.1 ml of kaolin solution. Incubate at 37°C in the waterbath for 10 minutes.
(3) After exactly 10 minutes, add 0.2 ml of phospholipidcalcium chloride mixture, start the stopwatch, and note the clotting time.
 
Normal Range
30-40 seconds.
 
Causes of prolongation of APTT
(1) Hemophilia A or B.
(2) Deficiencies of other coagulation factors in intrinsic and common pathways.
(3) Presence of coagulation inhibitors
(4) Heparin therapy
(5) Disseminated intravascular coagulation
(6) Liver disease
 
Uses of APTT
(1) Screening for hereditary disorders of coagulation: Since deficiencies of F VIII (hemophilia A) and F IX (hemophilia B) are relatively common, APTT is the most important screening test for inherited coagulation disorders. APTT detects deficiencies of all coagulation factors except F VII and F XIII. PT is also performed along with APTT. Prolongation of both PT and APTT is indicative of deficiency of coagulation factors in common pathway. Normal PT with prolongation of APTT is indicative of intrinsic pathway deficiency (particularly of F VIII or IX).
(2) To monitor heparin therapy: Heparin potentiates the action of natural anticoagulant antithrombin III which is an inhibitor of thrombin and activated factors IX, X, and XI. Full dose heparin therapy needs monitoring by APTT to maintain the dose in the therapeutic range (1.5 to 2.5 times the upper reference limit of APTT).
(3) Screening for circulating inhibitors of coagulation: APTT is prolonged in the presence of specific inhibitors (which are directed against specific coagulation factors) and non-specific inhibitors (which interfere with certain coagulation reactions).
 
Mixing experiment for detection of inhibitors: Mixing studies are used to distinguish between factor deficiencies and factor inhibitors (specific coagulation factor inhibitor or non-specific inhibitor such as lupus anticoagulant). If APTT is prolonged, patient’s plasma is mixed with an equal volume of normal plasma (called as a 50:50 mix) and APTT is repeated. In coagulation factor deficiency, prolongation of APTT gets corrected by more than 50% of the difference between the clotting times of control and test plasma. In the presence of lupus anticoagulant, there is no such correction. With lupus anticoagulant, APTT remains prolonged after mixing and for 2 hours following incubation. With F VIII inhibitor (which is time- and temperature-dependent), prolong-ed APTT gets immediately corrected after mixing, but becomes prolonged after incubation.
PT assesses coagulation factors in extrinsic pathway (F VII) and common pathway (F X, F V, prothrombin, and fibrinogen).
 
Principle
Tissue thromboplastin and calcium are added to plasma and clotting time is determined. The test determines the overall efficiency of extrinsic and common pathways.
 
Equipment
(1) Water bath at 37°C
(2) Test tubes (75 × 12 mm)
(3) Stopwatch
 
Reagents
(1) Thromboplastin reagent: This contains tissue factor and phospholipids and is available commercially.
(2) Calcium chloride 0.025 mol/liter.
 
Specimen
Venous blood is collected from antecubital fossa with a plastic, siliconized glass, or polypropylene syringe and a large bore needle (20 or 21 G in adults, 22 or 23 G in infants). Blood should never be collected from indwelling intravenous lines, as these often contain heparin. Glass syringe should not be used for collection since it activates coagulation. The blood is drawn gently but quickly after a single, smooth venepuncture. The needle is detached from the syringe, and the sample is passed gently into the plastic container. After capping the container, the blood and the anticoagulant are mixed immediately by gentle inversion 5 times. The anticoagulant used for coagulation studies is trisodium citrate (3.2%), with anticoagulant to blood proportion being 1:9. Most coagulation studies require platelet poor plasma (PPP). To obtain PPP, blood sample is centrifuged at 3000-4000 revolutions per minute for 15-30 minutes. Coagulation studies are carried out within 2 hours of collection of sample.
 
Method
(1) Deliver 0.1 ml of plasma in a glass test tube kept in water bath at 37°C.
(2) Add 0.1 ml of thromboplastin reagent and mix.
(3) After 1 minute, add 0.1 ml of calcium chloride solution. Immediately start the stopwatch and record the time required for clot formation.
 
Normal Range
11-16 seconds.
 
Causes of prolongation of PT
(1) Treatment with oral anticoagulants
(2) Liver disease
(3) Vitamin K deficiency
(4) Disseminated intravascular coagulation
(5) Inherited deficiency of factors in extrinsic and common pathways.
 
Uses of PT
(1) To monitor patients who are on oral anticoagulant therapy: PT is the standard test for monitoring treatment with oral anticoagulants. Oral anticoagulants inhibit carboxylation of vitamin K-dependent factors (Factors II, VII, IX, and X) and make these factors inactive.
In patients receiving oral anticoagulants, PT should be reported as a ratio of PT of patient to PT of control; it should not be reported as percentage. Various types of thromboplastin reagents obtained from different sources (like ox brain, rabbit brain, or rabbit lung) are available for PT test. These differ in their responsiveness to deficiency of vit. K-dependent factors. Technique of PT is also different in different laboratories. For standardization and to obtain comparable results, it is recommended to report PT (in persons on oral anticoagulants) in the form of an International Normalized Ratio (INR).

INR =  PT of Patient ISI
          PT of Control
 
International Sensitivity Index (ISI) of a particular tissue thromboplastin is derived (by its manufacturer) by comparing it with a reference thromboplastin of known ISI.
INR should be maintained in the therapeutic range for the particular indication (INR of 2.0-3.0 for prophylaxis and treatment of deep venous thrombosis; INR of 2.5-3.5 for mechanical heart valves). Therapeutic range provides adequate anticoagulation for prevention of thrombosis and also checks excess dosage, which will cause bleeding.
(2) To assess liver function: Liver is the site of synthesis of various coagulation factors, including vitamin Kdependent proteins. Therefore PT is a sensitive test for assessment of liver function.
(3) Detection of vitamin K deficiency: PT measures three of the four vitamin K-dependent factors (i.e. II, VII, and X).
(4) To screen for hereditary deficiency of coagulation factors VII, X, V, prothrombin, and fibrinogen.

BLEEDING TIME

The bleeding time test is dependent on appropriate functioning of platelets blood vessels and platelets and evaluates earliest hemostasis (platelets components and vascular).

In this test, incision (a surgical cut made in skin) or a superficial skin puncture is made and the time is measured for bleeding to stop.

There are three methods most commonly used to measure bleeding time:

  1. Duke’s method
  2. Ivy’s method
  3. Template method

In Duke’s method, ear lobe is puncture, and the time is measured for bleeding to stop.  This method is not recommended and cannot be standardized because it can cause a large local hematoma. In Ivy’s method, on the volar surface of the forearm, three punctures are made with a lancet (cutting depth 2-2.5 mm) under normal pulse pressure (between 30-40 mm Hg). A disadvantage of Ivy’s method is closure of puncture wound before stoppage of bleeding. In Template method, a special surgical blade is uses to make a larger cut of about 1 mm deep and 5 mm long. Although Template method is better than other methods, it may produce large scar and even form a keloid (irregular fibrous tissue formed at the site of a scar) in predisposed individuals. Ivy’s method for the measurement of bleeding time is described below.

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Ivy’s Method

Principle: On the volar surface of forearm, three normal punctures are made with the help of a lancet under normal pulse pressure (between 30-40 mm Hg).  The average time is measured for bleeding to stop from the puncture sites.

Equipment

  1. Disposable sterile lancets
  2. Sphygmomanometer
  3. Filter paper
  4. Stopwatch

Method

  1. Blood pressure of the patient is measured with the help of sphygmomanometer. The blood pressure of the patient should be normal before going to the further process.
  2. The volar surface of the forearm is cleansed with ethanol 70% and allowed to dry.
  3. With the help of a lancet, in quick succession, three punctures are made about 5 cm apart. Note that scars and superficial veins should be avoided.
  4. Start the stopwatch as soon as puncture made on the volar surface of the forearm.
  5. With the help of the filter paper, blood oozing from the puncture wound is gently absorbed with intervals of 15 seconds.
  6. The timer is stopped when blood no more mark the filter paper.
  7. Time measured for bleeding to stop from all the three puncture wound is recorded. The average time is calculated and reported as the bleeding time.

Reference Ranges

  • Normal range: 2 -7 minutes.
  • The greater numbers of individuals have bleeding time less than 4 minutes. The bleeding time should be reported in minutes or nearest half minute. If the bleeding continues more than twenty minutes, the test is stopped and the bleeding time should be reported as >20 minutes (more than 20 minutes).

Cause of extend of duration of bleeding time

  1. Disorders of blood vessels
  2. Thrombocytopenia: This term is uses when the platelet count is less than its normal value. The bleeding time test should not be performed if the platelet count is less than 1,00,000/ml. It may be difficult to control the bleeding if the platelet count is very low.
  3. Von Willebrand disease
  4. Disorder of platelet function
  5. Afibrinogenemia

CLOTTING TIME

In this test, required time is measured for the blood to clot in a glass test tube, kept at 37° C. Extend of duration of clotting time occurs only if severe deficiency of a clotting factor exists and is normal in moderate or mild deficiency.

Thrombin time assesses the final step of coagulation i.e. conversion of fibrinogen to fibrin by thrombin.
 
Principle
Thrombin is added to patient’s plasma and time required for clot formation is noted.
 
Equipment
(1) Water bath at 37°C
(2) Test tubes (75 × 12 mm)
(3) Stopwatch
 
Reagent
Thrombin solution.
 
Specimen
Venous blood is collected from antecubital fossa with a plastic, siliconized glass, or polypropylene syringe and a large bore needle (20 or 21 G in adults, 22 or 23 G in infants). Blood should never be collected from indwelling intravenous lines, as these often contain heparin. Glass syringe should not be used for collection since it activates coagulation. The blood is drawn gently but quickly after a single, smooth venepuncture. The needle is detached from the syringe, and the sample is passed gently into the plastic container. After capping the container, the blood and the anticoagulant are mixed immediately by gentle inversion 5 times. The anticoagulant used for coagulation studies is trisodium citrate (3.2%), with anticoagulant to blood proportion being 1:9. Most coagulation studies require platelet poor plasma (PPP). To obtain PPP, blood sample is centrifuged at 3000-4000 revolutions per minute for 15-30 minutes. Coagulation studies are carried out within 2 hours of collection of sample.
 
Method
Take 0.1 ml of buffered saline in a test tube and add 0.1 ml of plasma. Note clotting time after addition of 0.1 ml of thrombin solution.
 
Normal Range
± 3 seconds of control.
 
Causes of Prolongation of TT
(1) Disorders of fibrinogen: Prolongation of TT occurs in afibrinogenemia (virtual absence of fibrinogen), hypofibrinogenemia (fibrinogen less than 100 mgs/dl), and dysfibrinogenemia (dysfunctional fibrinogen).
(2) Heparin therapy: Heparin inhibits action of thrombin.
(3) Presence of fibrin degradation products (FDPs): These interfere with fibrin monomer polymeri-zation. TT is repeated using a mixture of normal plasma and patient’s plasma. If TT remains prolonged, then FDPs are present (provided patient is not receiving heparin).
This is a newly introduced screening test for platelet function that assesses both platelet adhesion and aggregation. This method uses an instrument called as PFA-100 in which anticoagulated whole blood is passed at a high shear rate through small membranes that have been coated with either collagen and epinephrine or collagen and ADP. Platelets adhere to each membrane and gradually occlude an aperture at the centre of the membrane. The time required for complete occlusion of the aperture is called as closure time. Normal closure time is 1-3 minutes. The PFA-100 test is performed initially with the collagen/epinephrine membrane; if closure time is normal, there is no significant platelet function defect. If closure time with collagen/epinephrine is prolonged, test with collagen/ADP is carried out; if normal, aspirin-induced platelet dysfunction is the probable cause; if prolonged, other platelet function defect (acquired or inherited) is likely. This test is more sensitive than bleeding time to assess primary hemostasis, sensitive for detection of von Willebrand disease and easy to perform. However, in the presence of thrombocytopenia and anemia, closure time is prolonged. Also, in the presence of a strong clinical suspicion of a platelet function defect and normal PFA-100 result, further testing is still necessary.
 
REFERENCES
1. Evatt BL, Gibbs WN, Lewis SM, McArthur JR. Fundamental Diagnostic Hematology: The Bleeding and Clotting Disorders (2nd ed), 1992. US Dept. of health and Human Services, Atlanta, Georgia and World Health Organization, Geneva, Switzerland.
2. Shrikhande AV, Warhadpande MS, Kawthalkar SM. A laboratory manual of coagulation (1994). Dept. of Pathology. Govt. Medical College, Nagpur.
3. Wallach J. Interpretation of Diagnostic Tests (7th ed). Philadelphia: Lippincott Williams and Wilkins, 2000.
4.  David J. Kuter, MD, DPhil. Laboratory Tests for Blood Disorders. The Merck™ Manuals, (2014).
Hemostais and Bleeding Disorders

Hemostasis or haemostasis (from the Ancient Greek: αἱμόστασις haimóstasis "styptic (drug)") is a process which causes bleeding to stop, meaning to keep blood within a damaged blood vessel (the opposite of hemostasis is hemorrhage). It is the first stage of wound healing. This involves blood changing from a liquid to a gel. Intact blood vessels are central to moderating blood's tendency to clot. The endothelial cells of intact vessels prevent blood clotting with a heparin-like molecule and thrombomodulin and prevent platelet aggregation with nitric oxide and prostacyclin. When endothelial injury occurs, the endothelial cells stop secretion of coagulation and aggregation inhibitors and instead secrete von Willebrand factor which initiate the maintenance of hemostasis after injury. Hemostasis has three major steps: 1) vasoconstriction, 2) temporary blockage of a break by a platelet plug, and 3) blood coagulation, or formation of a fibrin clot. These processes seal the hole until tissues are repaired.
     Bleeding disorders are the result of a generalized defect in hemostasis due to abnormalities of blood vessels, platelets, or coagulation factors.
     Initial tests, which should be performed in a suspected bleeding disorder, are complete blood count including blood smear, platelet count, bleeding time, clotting time, prothrombin time, and activated partial thromboplastin time. Depending on the results of these screening tests, one or more specific tests are carried out for definitive diagnosis (e.g. platelet function studies, assays of
coagulation factors, and test for fibrin degradation products). Abnormalities of blood vessels are usually not detectable by laboratory tests for hemostasis, and their diagnosis requires correlation of clinical and other investigations.
 
SCRENNING TESTS FOR HEMOSTASIS
(1) Complete Blood Count including Blood Smear
A complete blood count and a blood smear can provide information in the form of:
• Presence of cytopenia (anemia, leukopenia, thrombocytopenia)
• Red cell abnormalities (especially fragmented red cells which may indicate disseminated intravascular
coagulation)
• White cell abnormalities (like abnormal cells in leukemias)
• Abnormalities of platelets: thrombocytopenia (normally there is 1 platelets per 500-1000 red cells), giant platelets (seen in myeloproliferative disorders and Bernard-Soulier syndrome), and isolated discrete platelets without clumping in finger-prick smear (seen in uremia, Glanzmann’s thrombasthenia).
(2) Platelet Count
(3) Bleeding Time (BT)
(4) Clotting Time (CT)
(5) Prothrombin Time (PT)
(6) Activated Partial Thromboplastin Time (APTT)
(7) Thrombin Time (TT)
(8) Platelet Function Analyzer-100
 
LICENSE: This article uses material from the Wikipedia article "HEMOSTASIS", which is released under the Creative Commons Attribution-Share-Alike License 3.0.

Life history of malaria parasite consists of two cycles of development: asexual cycle or schizogony that occurs in humans and sexual cycle or sporogony that occurs in mosquitoes.

Asexual cycle (human cycle, schizogony)

This occurs in the liver cells and red blood cells of infected humans, and therefore humans are the intermediate hosts of the malaria parasite (Schizogony refers to the process of reproduction in protozoa in which there is production of daughter cells by fission). The human cycle begins when infected female Anopheles mosquito bites a person and sporozoites are injected into the circulation. There are four stages of human cycle.

(a) Pre-erythrocytic schizogony (Hepatic schizogony):

Inoculated sporozoites rapidly leave the circulation to enter the liver cells where they develop into hepatic (pre-erythrocytic) schizonts (Schizonts are cells undergoing schizogony). One sporozoite produces one tissue form. Hepatic schizonts rupture to release numerous merozoites in circulation (Merozoites are daughter cells produced after schizogony). Up to 40,000 merozoites are produced in the hepatic schizont.

In P. falciparum infection, all of the hepatic schizonts mature and rupture simultaneously; dormant forms do not persist in hepatocytes. In contrast, some of the sporozoites of P. vivax and P. ovale remain dormant after entering liver cells and develop into schizonts after some delay. Such persistent forms are called as hypnozoites; they develop into schizonts at a later date and are a cause of relapse.

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(b) Erythrocytic schizogony:

Merozoites released from rupture of hepatic schizonts enter the red blood cells via specific surface receptors. These merozoites become trophozoites that utilize red cell contents for their metabolism. A brown-black granular pigment (malaria pigment or hemozoin) is produced due to breakdown of hemoglobin by malaria parasites. The fully formed trophozoite develops into a schizont by multiple nuclear and cytoplasmic divisions. Mature schizonts rupture to release merozoites, red cell contents, malarial toxins, and malarial pigment. (This pigment is taken up by monocytes in peripheral blood and by macrophages of reticulo-endothelial system. In severe cases, organs which are rich in macrophages like spleen, liver, lymph nodes, and bone marrow become slate-gray or black in color due to hemozoin pigment). Rupture of red cell schizonts corresponds with clinical attack of malaria. Released merozoites infect new red cells and enter another erythrocytic schizogony cycle. This leads to rapid amplification of plasmodia in the red cells of the human host. In P. falciparum, P. vivax, and P. ovale infections, cycle of schizogony lasts for 48 hours, while in P. malarie infection it lasts for 72 hours. Merozoites of P. vivax and P. ovale preferentially invade young red cells or reticulocytes while those of P. falciparum infect red cells of all ages. Senescent red cells are preferred by P. malariae.

P. vivax, P. ovale, and P. malariae complete the erythrocyte schizogony in general circulation. Schizonts of P. falciparum induce membrane changes in red cells, which causes them to adhere to the capillary endothelial cells (cytoadherence). Therefore, in P. falciparum infection, erythrocyte schizogony is completed in capillaries of internal organs and usually only ring forms are seen in circulation.

(c) Gametogony:

After several cycles of erythrocytic schizogony, some merozoites, instead of developing into trophozoites and schizonts, transform into male and female gametocytes. These sexual forms are infective to mosquito and the person harboring them is called as a “carrier”. Gametocytes are not pathogenic for humans.

(d) Exoerythrocytic schizogony:

In P. vivax and P. ovale infections, some of the sporozoites in liver cells persist and remain dormant. These dormant forms in liver cells are called as hypnozoites. They become active and develop into schizonts a few days, months, or even years later. These schizonts rupture, release merozoites, and cause relapse. Exoerythrocytic schizogony is absent in P. falciparum infection and therefore relapse does not occur. Hence, P. vivax and P. ovale are called as relapsing plasmodia while P. falciparum and P. malariae are known as non-relapsing plasmodia.

Sexual cycle (mosquito cycle, sporogony)

The sexual cycle begins when a female Anopheles mosquito ingests mature male and female gametocytes during a blood meal. First, 4-8 microgametes are produced from one male gametocyte (microgametocyte) in the stomach of the mosquito; this is called as exflagellation. The female gametocyte (macrogametocyte) undergoes maturation to produce one macrogamete. By chemotaxis, microgametes are attracted toward the macrogamete; one of the microgametes fertilizes the macrogamete to produce a zygote. The zygote becomes motile and is called as ookinete. Ookinete penetrates the lining of the stomach and comes to lie on the outer surface of the stomach where it develops into an oocyst. On further growth and maturation, multiple sporozoites are formed within the oocyst. After complete maturation, oocyst ruptures to release sporozoites into the body cavity of the mosquito. Most of the sporozoites migrate to the salivary glands. Infection is transmitted to the humans by the bite of the mosquito through saliva when it takes a blood meal.

Reticulocytes

Reticulocytes are young or juvenile red cells released from the bone marrow into the bloodstream and that contain remnants of ribonucleic acid (RNA) and ribosomes but no nucleus. After staining with a supravital dye such as new methylene blue, RNA appears as blue precipitating granules or filaments within the red cells. Following supravital staining, any nonnucleated red cell containing 2 or more granules of bluestained material is considered as a reticulocyte (The College of American Pathology). Supravital staining refers to staining of cells in a living state before they are killed by fixation or drying or with passage of time. Reticulocyte count is performed by manual method.

PRINCIPLE

A few drops of blood (collected in EDTA) are incubated with new methylene blue solution which stains granules of RNA in red cells. A thin smear is prepared on a glass slide from the mixture and reticulocytes are counted under the microscope. Number of reticulocytes is expressed as a percentage of red cells.

REAGENT

New methylene blue solution is prepared as follows:

  • New methylene blue: 1.0 gm
  • Sodium citrate: 0.6 gm
  • Sodium chloride: 0.7 gm
  • Distilled water: 100 ml

Reagent should be kept stored in a refrigerator at 2-6°C and filtered before use.
Suitable alternatives to new methylene blue are brilliant cresyl blue and azure B.

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SPECIMEN

Capillary blood or EDTA anticoagulated venous blood can be used.

METHOD

(1) Take 2-3 drops of filtered new methylene blue solution in a 12 × 75 mm test tube.

(2) Add equal amount of blood and mix well.

(3) Keep the mixture at room temperature or at 37°C for 15 minutes.

(4) After gentle mixing, place a small drop from the mixture on a glass slide, prepare a thin smear, and allow to dry in the air.

(5) Examine under the microscope using oil-immersion objective. Mature red cells stain pale green blue. Reticulocytes show deep blue precipitates of fine granules and filaments in the form of a network (reticulum). Most immature reticulocytes show a large amount of precipitated material in the form of a mass, while the most mature reticulocytes show only a few granules or strands. Any nonnucleated red cell is considered as a reticulocyte if it contains 2 or more blue-stained particles of ribosomal RNA.

(6) Count 1000 red cells and note the number of red cells that are reticulocytes. Counting error is minimized if size of the microscopic field is reduced. This is achieved by using a Miller ocular disk inserted in the eyepiece; it divides the field into two squares (one nine times larger in size than the other). Reticulocytes are counted in both the squares and the red cells are counted in the smaller square.

REPORTING THE RESULT

(1) Reticulocyte percentage: The most common method of reporting is reticulocyte percentage which is calculated from the following formula:

Reticulocyte% =  NR   x 100
                           NRBC

Where NR is the Number of reticulocyte counted and NRBC is number of red blood cell counted.

Reference range is 0.5%-2.5% in adults and children. Reticulocyte count is higher in newborns.

(2) Absolute reticulocyte count = Reticulocyte percentage × Red cell count
Normal: 50,000 to 85,000/cmm

(3) Corrected reticulocyte count (Reticulocyte index)

                    = Reticulocyte % x PCV of Patient
                                                 Normal PCV

Corrected reticulocyte count > 2% indicates reticulocyte release appropriate for the degree of anemia. If < 2%, reticulocyte release is inappropriate.

(4) Reticulocyte maturation production index

  =         Corrected reticulocyte count
         Estimated maturation time in days

REFERENCE RANGES

  • Reticulocyte percentage: 0.5 2.5%
  • Absolute reticulocyte count: 50,000-85,000/cmm

Reticulocyte

  • 24 May 2016
Reticulocytes are young or juvenile red cells released from the bone marrow into the bloodstream and that contain remnants of ribonucleic acid (RNA) and ribosomes but no nucleus. After staining with a supravital dye such as new methylene blue, RNA appears as blue precipitating granules or filaments within the red cells. Following supravital staining, any nonnucleated red cell containing 2 or more granules of bluestained material is considered as a reticulocyte (The College of American Pathology). Supravital staining refers to staining of cells in a living state before they are killed by fixation or drying or with passage of time. Reticulocyte count is performed by manual method.
 
USES
  • As one of the baseline studies in anemia with no obvious cause
  • To diagnose anemia due to ineffective erythropoiesis (premature destruction of red cell precursors in bone marrow seen in megaloblastic anemia and thalassemia) or due to decreased production of red cells: In hypoplastic anemia or in ineffective erythropoiesis, reticulocyte count is low as compared to the degree of anemia. Increased erythropoiesis (e.g. in hemolytic anemia, blood loss, or specific treatment of nutritional anemia) is associated with increased reticulocyte count. Thus reticulocyte count is used to differentiate hypoproliferative anemia from hyperproliferative anemia.
  • To assess response to specific therapy in iron deficiency and megaloblastic anemias.
  • To assess response to erythropoietin therapy in anemia of chronic renal failure.
  • To follow the course of bone marrow transplantation for engraftment
  • To assess recovery from myelosuppressive therapy
  • To assess anemia in neonate
 
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PREPARATION OF BLOOD SMEAR (WEDGE METHOD)

(1) A small drop of blood (2-3 mm in diameter) is placed in the center line about 1 cm away from one end of a glass slide (typical size of slide is 75 × 25 mm; thickness about 1mm) with a wooden stick or glass capillary. Slide should be clean, dry, and grease-free. Blood sample may be venous (anticoagulated with EDTA) or capillary (finger prick). Better blood cell morphology is obtained if smear is made directly from a skin puncture. If EDTA-anticoagulated venous blood is used, smear should be prepared and stained within 2 hours of blood collection. If venous blood collected in a syringe is used, the last drop of blood in the needle after withdrawing (or first drop while dispensing) should be used.
(2) A 'spreader' slide is placed at an angle of 30° in front of the drop and then drawn back to touch the drop of blood. Blood spreads across the line of contact of two slides.
(3) Smear is made by smooth, forward movement of the 'spreader' along the slide. The whole drop should be used up 1 cm before the end of the slide. The length of the smear should be about 3 cm. The 'spreader' should not be raised above the slide surface till whole drop of blood is spread out.
(4) Smear is rapidly dried by waving it in the air or keeping it under an electric fan. Slow drying causes shrinkage artifact of red cells.
(5) Patient's name or laboratory number and date are written (with a lead pencil, a permanent marker pen, or a diamond pencil) on the thicker end of the smear.
(6) The smear is fixed immediately with absolute methyl alcohol (which should be moisture- and acetone-free) for 2-3 minutes in a covered jar (Absolute ethyl alcohol can also be used, but not methylated spirit as it contains water). Aim of fixation is to prevent washing off of the smear from the slide. Following this, color of the smear becomes light brown. This fixation is desirable even when Leishman stain is used which contains methyl alcohol. This is because Leishman stain may have absorbed moisture leading to poor fixation. If methanol is contaminated with water, sharpness of cell morphology is lost and there is vacuolation of red cells. Methanol should be acetone-free since acetone washes out nuclear stain. (In many laboratories, slide is stained immediately after air-drying without prior fixation, and the results are satisfactory; however, if delay of >4 hours is anticipated between air-drying and staining, the slide should be fixed. If not, a background gray-blue staining of plasma occurs).
 
Notes
(1) Making a 'spreader' slide—a glass slide with absolutely smooth edges should be selected and one or both corners at one end of the slide should be broken off. The 'spreader' slide should be narrower (width of about 15 mm) so that edges of the smear can be examined microscopically. The 'spreader' slide should be discarded after use. If the same is to be reused, its edge should be thoroughly cleaned and dried (otherwise carryover of cells or parasites can occur).
(2) A well-spread blood smear (a) is tongue-shaped with a smooth tail, (b) does not cover the entire area of the slide, (c) has both thick and thin areas with gradual transition, and (d) does not contain any lines or holes.
(3) By changing the angle of the 'spreader' and its speed, thickness of the blood smear can be controlled. In patients with anemia, a thicker smear can be obtained by increasing the angle and the speed of spreading. In patients with polycythemia, a thinner smear is obtained by decreasing the 'spreader' angle and the speed of spreading.
(4) Anticoagulant used may be EDTA (dipotassium salt) or sodium citrate. Heparin should not be used as an anticoagulant for making blood films since it causes platelet clumping and imparts a blue background to the film.
(5) It is recommended to stain blood films in reagent filled Coplin jars (rather than covering them with the staining solution) to avoid formation of stain precipitates due to evaporation.
(6) A drawback of this method is uneven distribution of leukocytes (i.e. monocytes, neutrophils, and abnormal cells are pushed towards the extreme tail end of the smear) and distortion of red cell morphology at the edges.
(7) Blood smear is covered with a coverslip and mounted in a mounting medium (e.g. DPX) for protection against mechanical damage and deterioration of staining with time on exposure to air.
(8) Cleaning of slides: (A) New slides: If new slides are not clean and grease-free, they are left overnight in a detergent solution, washed in running tap water, rinsed in distilled water, and wiped dry with a clean cloth. Before use, they are wiped with 95% methyl alcohol, dried, and then kept covered to protect from dust. (B) Used slides: The used slides are soaked in a detergent solution at 60°C for 20 minutes, washed in running tap water, rinsed in distilled water, and then wiped dry. Before use, they are wiped with 95% methyl alcohol, dried, and then kept covered to protect from dust.

STAINING OF BLOOD SMEAR

Blood smears are routinely stained by one of the Romanowsky stains. Romanowsky stains consist of a combination of acidic and basic dyes and after staining various intermediate shades are obtained between the two polar (red and blue) stains. Romanowsky stains include May-Grunwald-Giemsa, Jenner, Wright's, Leishman's, and Field's stains. Staining properties of the Romanowsky stains are dependent on two synthetic dyes: methylene blue and eosin. International Committee for Standardization in Haematology has recommended a highly purified standardized stain, which contains azure B and eosin Y; it, however, is very expensive. Romanowsky stains are insoluble in water but soluble in methyl alcohol. Methyl alcohol acts as a solvent as well as a fixative. Staining reaction is pH-dependent. These stains have a tendency towards precipitation and should be filtered before use.
Methylene blue and azure B are basic (cationic) dyes and have affinity for acidic components of the cells (like nucleic acids or basophil granules) and impart purpleviolet color to the nuclear chromatin, dark blue-violet color to the basophil granules, and deep blue color to the cytoplasm of lymphocytes. Eosin is an acidic (anionic) dye and has affinity for basic components like hemoglobin (stained pink-red), and granules in eosinophils (stained orange-red). Neutrophil granules are slightly basic and stain violet-pink or lilac.
Romanowsky stains impart more colours than just blue (from methylene blue or azure B) and red-orange (from eosin Y). Usefulness of the Romanowsky stains lies in their ability to differentially stain leucocyte granules.
A well-stained smear is pink in color in thinner portion and purple-blue in thicker portion. Excess blue coloration can be due to: (i) excessively thick smear, (ii) low concentration of eosin, (iii) impure dyes, (iv) too long staining time, (v) inadequate washing, or (vi) excessive alkaline pH of stain, buffer, or water. Excess red coloration can be due to: (i) impure dyes or incorrect proportion of dyes, (ii) excessive acid pH of stain, buffer, or water (as the red cells take up more acid dye i.e. eosin), (iii) too short staining time, or (iv) excessive washing. If there are granules of stain precipitate (masses of small black dots) on smear, stain needs to be filtered.
Method of Leishman staining is given below:

Reagents
(1) Leishman stain: William Boog Leishman, a British pathologist, modified the original Romanowsky method and devised a stain which is widely known as Leishman's stain. This consists of methylene blue and eosin dissolved in absolute methyl alcohol. Commercially available Leishman stain powder (0.6 gram) is mixed with water-free absolute methyl alcohol (400 ml). Prepared stain should be kept tightly stoppered in a brown bottle and stored in a cool, dark place at room temperature. Exposure to direct sunlight causes deterioration of the stain. After preparation, stain should be kept for 3-5 days before using since it improves the quality of the stain.
(2) Buffered water (pH 6.8).

Method
(1) Air-dry the smear and fix with methanol for 2-3 minutes.
(2) Cover the smear with Leishman stain for 2 minutes.
(3) After 2 minutes, add twice the volume of buffered water and leave for 5-7 minutes. A scum of metallic sheen forms on the surface.
(4) Wash the stain away in a stream of buffered water. Tap water can also be used for washing if it is not highly alkaline or highly acid.
(5) Wipe the back of the slide clean and set it upright in the draining rack to dry.
(6) Mount the slide in a suitable mounting medium (e.g. DPX) with a clean and dry 25 × 25 mm coverslip.
• Red cells: pink-red or deep pink
• Polychromatic cells (Reticulocyt-es): Gray-blue
• Neutrophils: Pale pink cytopla-sm; mauve-purple granules
• Eosinophils: Pale-pink cytoplasm; orange-red granules
• Basophils: Blue cytoplasm; dark blue-violet granules
• Monocytes: Gray-blue cytoplasm; fine reddish (azurophil) granules
• Small lymphocytes: Dark blue cy-toplasm
• Platelets: Purple
• Nuclei of all cells: Purple-violet

Blood Smear

  • 13 May 2016
Blood Smear

A blood film or peripheral blood smear is a thin layer of blood smeared on a microscope slide and then stained in such a way to allow the various blood cells to be examined microscopically. Blood films are usually examined to investigate hematological problems (disorders of the blood) and, occasionally, to look for parasites within the blood such as malaria and filaria.
Blood films are made by placing a drop of blood on one end of a slide, and using a spreader slide to disperse the blood over the slide's length. The aim is to get a region, called a monolayer, where the cells are spaced far enough apart to be counted and differentiated. The monolayer is found in the "feathered edge" created by the spreader slide as it draws the blood forward.
The slide is left to air dry, after which the blood is fixed to the slide by immersing it briefly in methanol. The fixative is essential for good staining and presentation of cellular detail. After fixation, the slide is stained to distinguish the cells from each other.
Routine analysis of blood in medical laboratories is usually performed on blood films stained with Romanowsky, Wright's, or Giemsa stain. Wright-Giemsa combination stain is also a popular choice. These stains allow for the detection of white blood cell, red blood cell, and platelet abnormalities. Hematopathologists often use other specialized stains to aid in the differential diagnosis of blood disorders.
After staining, the monolayer is viewed under a microscope using magnification up to 1000x. Individual cells are examined and their morphology is characterized and recorded.
 
USES
(1) Blood smear is helpful in suggesting the cause of anemia or thrombocytopenia, identifying and typing of leukemia, and in diagnosing hemoparasitic infections (malaria, filaria, and trypanosomiasis). It is also helpful in the management of these conditions.
(2) To monitor the effect of chemotherapy and radiotherapy on bone marrow.
(3) To provide direction for further investigations that will help in arriving at the correct diagnosis (e.g. in infections, drug toxicity, etc.). Blood smear examination is therefore indicated in clinically suspected cases of anemia, thrombocytopenia, hematological malignancies (leukemia, lymphoma, multiple myeloma), disseminated intravascular coagulation, parasitic infections (like malaria or filaria), infectious mononucleosis, and various inflammatory, or malignant diseases.
 
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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.

STAGES OF 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)

COMPONENTS INFLUENCING ON ERYTHROCYTE SEDIMENTATION RATE

  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, a1-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.

IMPORTANCE OF 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.

METHODS FOR ESTIMATION OF ERYTHROCYTE SEDIMENTATION RATE

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 METHOD

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.

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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 METHOD

Equipment

Westergren ESR tube and Westergren stand.

Reagent

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.

Specimen

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.

Procedure

  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.

Precautions

  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.

MICRO-ESR

Capillary blood is uses for the estimation of Micro-ESR. This method is recommended for the estimation of Erythrocyte Sedimentation Rate in small children.

ZETA SEDIMENTATION RATIO

In this method a special device is uses named zetafuge. Zeta Sedimentation Ratio is not pretended due to anemia, unlike Westergren method.

REFERENCE RANGES

Erythrocyte Sedimentation Rate by Wintrobe Method

  • Male 0-9 mm in an hour
  • Female 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 0-33 mm in an hour
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). wikipedia.org

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.

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.

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

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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.

Leukocytosis

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.

Leukopenia

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)

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.
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.

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.

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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 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

  • 29 Apr 2016
Platelets

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
 
MANUAL METHOD
 
Principle
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.)
 
Equipment
Hemocytometer with cover glass, compound microscope. Unopette capillary pipette, lint-free wipe, alcohol pads,  hand counter, petri dish with moist filter paper.
 
Reagent
Ammonium oxalate: 11.45 gm
Sorensen’s phosphate buffer: 1.0 gm
Thimerosal: 0.1 gm
Distilled water: 1000 ml
 
Specimen
EDTA-anticoagulated blood or capillary blood is preferred.
 
Method
(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.
 
Calculation
 
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.
 
Example:
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
 
REFERENCE RANGES
• 150,000 - 450,000/µL
• 150 - 450 x 109/L
 
REFERENCES
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.
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.
About one thousand species of fishes are found in marine and fresh water in Pakistan. Majority of these are edible. And very few are examined for their nematode parasites.
 
Most of marine fishes are included among the group of edible fishes. Some of these including, Scomberomorus guttatus, Pomadasys olivaceum, Pomadasys maculatum, Pomadasys stridens, Otolithus ruber, Sphyraena forsteri, Sphyraena jello, Lates calcarifer and Sillago sihama are popular edible fishes in Pakistan, due to their delicious taste and are full of nourishment such as proteins and vitamins particularly vitamin E and vitamin D.

Total leukocyte count (TLC) refers to the number of white blood cells in 1 μl of blood (or in 1 liter of blood if the result is expressed in SI units). There are two methods for estimation of TLC:

  • Manual or microscopic method
  • Automated method

A differential leukocyte count should always be performed along with TLC to obtain the absolute cell counts.

The purpose of carrying out TLC is to detect increase or decrease in the total number of white cells in blood, i.e. leukocytosis or leukopenia respectively. TLC is carried out in the investigation of infections, any fever, hematologic disorders, malignancy, and for follow-up of chemotherapy or radiotherapy.

MANUAL METHOD

Principle

A sample of whole blood is mixed with a diluent, which lyses red cells and stains nuclei of white blood cells. White blood cells are counted in a hemocytometer counting chamber under the microscope and the result is expressed as total number of leukocytes per μl of blood or per liter of blood.

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Equipment

(1) Hemocytometer or counting chamber with coverglass: The recommended hemocytometer is one with improved Neubauer rulings and metalized surface. There are two ruled areas on the surface of the chamber. Each ruled area is 3 mm × 3 mm in size and consists of 9 large squares with each large square measuring 1 mm × 1 mm. When the special thick coverglass is placed over the ruled area, the volume occupied by the diluted blood in each large square is 0.1 ml. In the improved Neubauer chamber, the central large square is divided into 25 squares, each of which is further subdivided into 16 small squares. A group of 16 small squares is separated by closely ruled triple lines. Metalized surface makes background rulings and cells easily visible. The 4 large corner squares are used for counting leukocytes, while the central large square is used for counting platelets and red blood cells. Only special coverglass, which is intended for use with hemocytometer, should be used. It should be thick and optically flat. When the special coverglass is placed on the surface of the chamber, a volumetric chamber with constant depth and volume throughout its entire area is formed. Ordinary coverslips should never be employed since they do not provide constant depth to the underlying chamber due to bowing.

When the special cover glass is placed over the ruled area of the chamber and pressed, Newton’s rings (colored refraction or rainbow colored rings) appear between the two glass surfaces; their formation indicates the correct placement of the cover glass.

(2) Pipette calibrated to deliver 20 μl (0.02 ml, 20 cmm): WBC bulb pipettes, which have a bulb for dilution and mixing (Thoma pipettes) are no longer recommended. This is because blood and diluting fluid cannot be mixed adequately inside the bulb of the pipette. Bulb pipettes are also difficult to calibrate, costly, and charging of counting chamber is difficult. Tips of pipettes often chip easily and unnecessarily small volume of blood needs to be used.

  1. Graduated pipette, 1 ml.
  2. Pasteur pipett
  3. Test tube (75 × 12 mm).

Reagent

WBC diluting fluid (Turk’s fluid) consists of a weak acid solution (which hemolyzes red cells) and gentian violet (which stains leucocyte nuclei deep violet). Diluting fluid also suspends and disperses the cells and facilitates counting. Its composition is as follows:

  • Acetic acid, glacial 2 ml
  • Gentian violet, 1% aqueous 1 ml
  • Distilled water to make 100 ml

Specimen

EDTA anticoagulated venous blood or blood obtained by skin puncture is used. (Heparin should not be used since it causes leukocyte clumping). While collecting capillary blood from the finger, excess squeezing should be avoided so as not to dilute blood with tissue fluid.

Method

(1) Dilution of blood: Take 0.38 ml of diluting fluid in a test tube. To this, add exactly 20 μl of blood and mix. This produces 1:20 dilution. Alternatively, 0.1 ml of blood can be added to 1.9 ml of diluting fluid to get the same dilution.

(2) Charging the counting chamber: Place a coverglass over the hemocytometer. Draw some of the diluted blood in a Pasteur pipette. Holding the Pasteur pipette at an angle of 45° and placing its tip between the coverglass and the chamber, fill one of the ruled areas of the hemocytometer with the sample. The sample should cover the entire ruled area, should not contain air bubbles, and should not flow into the side channels. Allow 2 minutes for settling of cells.

(3) Counting the cells: Place the charged hemocytometer on the microscope stage. With the illumination reduced to give sufficient contrast, bring the rulings and the white cells under the focus of the low power objective (× 10). White cells appear as small black dots. Count the number of white cells in four large corner squares. (To reduce the error of distribution, counting of cells in all the nine squares is preferable). To correct for the random distribution of cells lying on the margins of the square, cells which are touching the left-hand lines or upper lines of the square are included in the count, while cells touching the lower and right margins are excluded.

(a) Calculation of TLC:

TLC/μl = Nw x Cd x Cv
                    NLS
          = Nw x 20 x  10
                      4
          = Nw x 50
                    

Where Nw is the number of WBCs counted, Cd is the correction of dilution, Cv is the correction of volume and NLS is the number of large squares counted.

(b) TLC/L = Number of WBCs counted × 50 × 106 (106 is the correction factor to convert count in 1 μl to count in 1 liter). Example: If 200 WBCs are counted in 4 large squares, TLC/μl will be 10,000/μl and TLC/liter will be 10.0 × 109/liter.

If TLC is more than 50,000/ml, then dilution of blood should be increased to 1:40 to increase the accuracy of the result.

If TLC is less than 2,000/ml then lesser dilution should be used.


Expression of TLC: Conventionally, TLC is expressed as cells/μl or cells/cmm or cells/mm3. In SI units, TLC is expressed as cells × 109/liter. Conversion factors for conventional to SI units is 0.001 and SI to conventional units is 1000.

Correction of TLC for nucleated red cells: The diluting fluid does not lyse nucleated red cells or erythroblasts. Therefore, they are counted as leukocytes in hemocytometer. If erythroblasts are markedly increased in the blood sample, overestimation of TLC can occur. To avoid this if erythroblasts are greater than 10 per 100 leukocytes as seen on blood film, TLC should be corrected for nucleated red cells by the following formula:

CTLC =    TLC x 100 
             NRBC + 100

Where CTLC is the Corrected TLC/μl, TLC is the Total Leukocyte Count and NRBC is the Nucleated RBCs per 100 WBCs.

REFERENCE RANGES

  • Adults 4000-11,000/μl
  • At birth 10,000-26000/μl
  • 1 year 6,000-16,000/μl
  • 6-12 year 5,000-13,000/μl
  • Pregnancy up to 15,000/μl

CRITICAL VALUES

  • TLC < 2000/μl or > 50000/μl
An acetylcholine receptor (AChR) antibody test is used to help diagnose myasthenia gravis (MG) and to distinguish it from other conditions that may cause similar symptoms, such as chronic muscle fatigue and weakness.
 
AchR antibodies hinder the action of acetylcholine, a chemical (neurotransmitter) that transmits messages between nerve cells. The antibodies do this in three major ways:
  • "Binding" antibodies attach to the acetylcholine receptors on nerve cells and may initiate an inflammatory reaction that destroys them.
  • "Blocking" antibodies may sit on the receptors, preventing acetylcholine from binding.
  • "Modulating" antibodies may cross-link the receptors, causing them to be taken up into the muscle cell and removed from the neuromuscular junction.
Three different types of tests are available to determine which of these may be the problem in a particular individual. However, the test that measures "binding" antibodies is most commonly used because it is generally rare for the other two tests to be positive without the "binding" test being positive as well. These other tests may be used when a doctor strongly suspects myasthenia gravis and the "binding" test is negative.
 
One or more of the AChR antibody tests may be ordered as part of a panel of tests that may also include a striated muscle antibody test to help establish a diagnosis. Depending upon results, an anti-MuSK (muscle-specific kinase) antibody test may also be ordered. The AChR antibody test may be ordered initially as a baseline test and then as indicated to evaluate MG disease activity and/or response to therapy.
 
People with MG often have an enlarged thymus gland and may have thymomas (typically benign tumors of the thymus). Located under the breastbone, the thymus is an active part of the immune system during childhood but normally becomes less active after puberty. If a thymoma is detected, such as during a chest computed tomography (CT) scan done for a different reason, then an AChR antibody test may sometimes be used to determine whether the person has developed these antibodies.
 
When is it ordered?
The AChR antibody test may be ordered when a person has symptoms that suggest MG, such as:
  • Drooping eyelid
  • Double vision
  • Decreased eye movement control
  • Difficulty swallowing, chewing, with choking, drooling and gagging
  • Slurred speech
  • Weak neck muscles
  • Trouble holding up head
  • Difficulty breathing
  • Difficulty walking and an altered gait
  • Specific muscle weakness but normal feelings/sensations
  • Muscle weakness that worsens with sustained effort and improves with rest
When a person has been diagnosed with MG, an AChR antibody test may be ordered occasionally to evaluate MG disease activity and/or response to therapy.
 
An AChR antibody test may sometimes be ordered when a thymoma is detected.
 
What does the test result mean?
AChR antibodies are not normally present in the blood. They are autoantibodies and their presence indicates an autoimmune response.
 
If a person has AChR antibodies and symptoms of MG, then it is likely that the person has this condition.
 
AChR antibodies may be seen with some thymomas, in people who are being treated with drugs such as penicillamine, with some small cell lung cancers, with autoimmune liver disease, and with Lambert-Eaton myasthenic syndrome (a condition associated with interference with the release of acetylcholine from the nerve ending).
 
A negative test result does not rule out MG. Up to 50% of those with ocular MG (affecting only eye-related muscles) and about 10-15% of those with generalized MG will be negative for AChR antibodies.
 
In general, the greater the quantity of AChR antibody, the more likely a person is to have significant symptoms, but the test results cannot be used to evaluate the severity of symptoms in a specific person.
Bacterial genetics is the subfield of genetics devoted to the study of bacteria. Bacterial genetics are subtly different from eukaryotic genetics, however bacteria still serve as a good model for animal genetic studies. One of the major distinctions between bacterial and eukaryotic genetics stems from the bacteria's lack of membrane-bound organelles (this is true of all prokaryotes. While it is a fact that there are prokaryotic organelles, they are never bound by a lipid membrane, but by a shell of proteins), necessitating protein synthesis occur in the cytoplasm.
 
Like other organisms, bacteria also breed true and maintain their characteristics from generation to generation, yet at same time, exhibit variations in particular properties in a small proportion of their progeny. Though heritability and variations in bacteria had been noticed from the early days of bacteriology, it was not realised then that bacteria too obey the laws of genetics. Even the existence of a bacterial nucleus was a subject of controversy. The differences in morphology and other properties were attributed by Nageli in 1877, to bacterial pleomorphism, which postulated the existence of a single, a few species of bacteria, which possessed a protein capacity for a variation. With the development and application of precise methods of pure culture, it became apparent that different types of bacteria retained constant form and function through successive generations. This led to the concept of monomorphism.
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