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.
Thursday, 09 June 2016 20:14

Prothrombin Time (PT)

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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.
Tuesday, 07 June 2016 20:33

Bleeding Time (BT) and Clotting Time (CT)

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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.

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.

Monday, 06 June 2016 13:58

Thrombin Time (TT)

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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).
Monday, 06 June 2016 10:16

Platelet Function Analyzer (PFA-100)

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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.

Monday, 06 June 2016 09:27

Laboratory Tests in Bleeding Disorders Featured

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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.
Tuesday, 24 May 2016 19:54

Life Cycle of Malaria Parasite Featured

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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.

(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.

Monday, 23 May 2016 19:40

How to Count Reticulocytes (Manual Method) Featured

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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.

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
Monday, 23 May 2016 19:16

Reticulocyte

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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
 
Useful Links:
Description: The book Radioisotopes-Applications in Bio-Medical Science contains two sections: Radioisotopes and Radiations in Bioscience and Radioisotopes and Radiology in Medical Science. Section I includes chapters on radioisotope production, radio-labeled nano-particles, radioisotopes and nano-medicine, use of radiations in insects, drug research, medical radioisotopes and use of radioisotopes in interdisciplinary fields etc.

In Section II, chapters related to production of metal PET (positron emission tomography) radioisotopes, 3-dimensional and CT (computed tomography) scan, SS nuclear medicine in imaging, cancer diagnose and treatments have been included. The subject matter will by highly useful to the medical and paramedical staff in hospitals, as well as researchers and scholars in the field of nuclear medicine medical physics and nuclear bio-chemistry etc.

Additional Info

  • File Name Radioisotopes – Applications in Bio-Medical Science
  • Edition 1st
  • Year 2011
  • Editor(s) Nirmal Singh
  • ISBN 978-953-307-748-2
  • Publisher InTech
  • Size 19.0 MB
  • File Format .pdf
  • Password bioscience.pk
Tuesday, 17 May 2016 11:40

FORMATION OF CORALS AND TYPES

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INTRODUCTION
 
  • Corals are developed by the coelenterate organisms.
  • Corals are a deposit of calcium car­bonate.
  • The coelenterate animal produces calcareous skeleton.
  • But most coral pro­ducing polyps belong to the class Anthozoa.
  • The true stony corals belong to the order Madreporaria of class Anthozoa. Many Solitary and colonial anthozoans produce corals which are sometimes brilliantly colored.
  • The corals develop into coral rocks and coral islands.
 
CORAL PRODUCING POLYP STRUCTURE:
 
  • A coral-polyp resembles sea-anemone in its shape.
  • The coral is its external shell.
  • It is a product of the ectodermal cells, called Calicoblasts.
  • The formation of the coral is not clearly followed, so far Calcareous crystals may be precipitated in the matrix and secreted outside the epidermis.
  • Thus outer protective shell may be formed.
  • A coral polyp will not show a pedal disc as its basal part is surrounded by the calcareous skeleton.
  • The oral disc bears tentacles in cycles of 6.
  • The circular mouth leads into a short stomodeaum which will not show siphonoglyphs.
  • The mesenteries are restricted to the upper part of the polyp.
  • The muscles are poorly developed.
 
FORMATION OF CORALS
 
  • The basal region of the coral polyp is fixed in a cup-shaped calcareous exoskeleton secreted by the epidermis of the base.
  • On this basal plate a large number of radially arranged vertical septa are formed.
  • They grow in height and they push the ectoderm up.
  • An external wall is formed because of the circular up growth of the plate.
  • The single cup like exoskeleton formed by an individual polyp is called a coralitte.
  • It has the shape of the polyp.
  • The majority of the corals are colonial, and the skeleton of an entire colony is termed coralium it may contain thousands of corallites.
 
Example for Coral formation
 
  1. In "Flabellum" the coral formation is based on these lines.
  2. The corallite is disc like. It is 5 mm to 25 cm. in length.
  3. The outer wall of the cup is made by stony calcium carbonate. It is called theca.
  4. The flattened bottom of the cup beneath the polyp is called the basal plate. The cavity of the cup develops a number of vertical septa or sclero-septa, proceeding form theca towards the center of the cup.
  5. Like mesenteries, they are typically arranged in cycles of six (6 primaries extending towards the center, 6 secondary’s, 12 tertiaries, 24 quartemaries etc.)
  6. The sclerosepta alternates with mesenteries Corallite lies entirely outside the polyp body.
  7. The polyp body is pushed up into ridges over the sclerosepta.
  8. Each ridge is covered by an internal portion of the body-wall Thus, the sclerosepta are external and lie outside the enteric cavity.
  9. Between the sclerosepta and the mesenteries, the polyp base is depressed into pockets called loculi.
  10. The inner ends of the primary sclerosepta are fused to form central column called columella.
  11. In many corals, the theca is covered by a second calcareous wall called epitheca. The space separating the epitheca and theca, will show projections called costae.
 
Types of Corals and Examples: The true corals belong to Anthozoa but in class hydrozoa also some corals are seen.

I. Hydrozoan Corals:
 
1. Millepora:
 
  • It is a massive, calcareous skeleton with two types opening a) Large gastropores, b) Smaller dactylopores.
  • Millipora is called stinging coral because it is the only coral with nematocysts, which causes pain to man.
 
2. Stylasterina:
 
  • It is found in warm tropical water.
  • The colony is tree like. It is also similar to millepora but has cup like gastropores. There is a pointed style in the centre of each cup.
 
II. Authozoan Corals:
 
1. Alcyonium (dead man's fingers):
 
  • It is a marine colonial form living attached to stones.
  • It is tree like. It grows to 4 to 10 cm height.
  • The simplest form of skeleton is seen in this animal, which contains minute calcareous spicules.
 
2. Tubipora (organ-pipe coral):
 
  • It is a marine colony distributed in warm waters The spicules are fused to form a tube around the polyp.
  • Many such tubes are united by horizontal platforms Again new polyps are formed They develop vertical tubes.
  • This process is repeated. A big coral is formed.
 
3. Corallium (red coral):
 
  • It is a marine sedentary form.
  • It grows to 30 cm. It is highly branched.
  • The polyps are white in colour. Autozooids'and siphonozooids are present.
  • Herd and branched skeletal axis is formed by the union of many calcareous spicules in a matrix.
  • Thus a coral is developed which is red in colour.
 
4. Gorgonia (Sea fan):
 
  • It is also called sea-whip.
  • It is seen in tropical waters.
  • It is tree like colony, it grows 80 cm. height.
  • A central, rod like branched skeleton is formed by the ectoderm.
  • Mesogloea also contains many calcareous spicules.
 
III. The corals belong to Madreporaria are true corals.
 
The skeleton is entirely calcareous and is secreted by the ectoderm. The following are good examples.
 
1. Fungia (Mushroom coral):
 
  • It is marine form. The coral is discoid.
  • The septa are numerous and are connected by small calcareous rods called synapticula.
  • This coral is true coral.
 
2. Meandrina (Or Brain coral):
 
  • It is very big and grows up to 8 feet in diameter.
  • It weighs in tons.
  • The surface of the colony is marked by long curved grooves; hence it looks like human brain.
  • In a living brain coral the polyps do not occupy separate cups.
  • The mouths of these compound polyps will be separate.
Description: With cancer-related deaths projected to rise to 10.3 million people by 2020, the need to prevent, diagnose, and cure cancer is greater than ever. This book presents readers with the most up-to-date imaging instrumentation, general and diagnostic applications for various cancers, with an emphasis on lung and breast carcinomas--the two major worldwide malignancy types. This book discusses the various imaging techniques used to locate and diagnose tumors, including ultrasound, X-ray, color Doppler sonography, PET, CT, PET/CT, MRI, SPECT, diffusion tensor imaging, dynamic infrared imaging, and magnetic resonance spectroscopy. It also details strategies for imaging cancer, emphasizing the importance of the use of this technology for clinical diagnosis. Imaging techniques that predict the malignant potential of cancers, response to chemotherapy and other treatments, recurrence, and prognosis are also detailed.
  • Concentrates on the application of imaging technology to the diagnosis and prognosis of lung and breast carcinomas, the two major worldwide malignancies 
  • Addresses the relationship between radiation dose and image quality
  • Discusses the role of molecular imaging in identifying changes for the emergence and progression of cancer at the cellular and/or molecular levels

Additional Info

  • File Name Cancer Imaging: Lung and Breast Carcinomas
  • Volume 1
  • Edition 1st
  • Year 2007
  • Author(s) M.A. Hayat
  • ISBN-10 0123704685
  • ISBN-13 978-0123704689
  • Size 21.1 MB
  • File Format .pdf
  • Password bioscience.pk
Friday, 13 May 2016 07:55

PREPARATION OF BLOOD SMEAR (WEDGE METHOD)

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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
Thursday, 12 May 2016 21:04

Blood Smear

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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.
 
Useful Links:
 
Use the Reference manual for in-depth information on the principles of flow cytometry, information about what your instrument does, the methods it uses, its specifications, and information on installation, safety, and system options.
Use the Getting Started manual to become familiar with the controls and indicators for your system and to learn about protocols, regions, panels, and the basic skills you need to operate the system. This manual also has an overview of the software.
Use the Operator’s Guide for the day-to-day running of your instrument. Go through the detailed step-by-step procedures of startup, quality control (QC), running samples, analyzing data, printing reports, reviewing QC data, and shutdown.
Use the Data Management manual for instructions on how to export, save, copy, move, archive, and delete files. It also has information about the types of files your system creates and uses, instructions for working with QC features, and instructions for setting up the report template that you need to create your patient reports.
Use the Special Procedures and Troubleshooting manual to clean, replace, or adjust a component of the instrument. The Troubleshooting tables and error messages appear at the back of the manual.
Use the Operating Summary as a quick reference for basic procedures.
Use the Master Index to easily locate a topic in any of your manuals.
Use the User's Comment Card in the Reference manual to give us your comments about the manual and ways to improve it.

The erythrocyte sedimentation rate (ESR) measures the rate of settling (sedimentation) of erythrocytes in anticoagulated whole blood. Anticoagulated blood is allowed to stand in a glass tube for 1 hour and the length of column of plasma above the red cells is measured in millimeters; this corresponds to ESR. There are four different methods for the estimation of ESR.

Description: This book Edited by Larry C. James, Wright State University, Dayton, OH, USA John Linton, West Virginia University School of Medicine, Charleston Environmental, genetic, psychological, and societal factors interact to produce obesity, a chronic condition of epidemic proportions. The Handbook of Obesity Intervention for the Lifespan guides professionals in meeting this complex challenge with a multidisciplinary palette of evidence-based interventions that can be tailored to men and women across the lifespan, regardless of background.
 
This unique reference combines salient research data and hands-on clinical applications for use with overweight patients, from the very young to the very old, and includes a “treatment resources” section with extra materials to bolster therapy-all geared toward respectful, encouraging treatment and lasting weight-loss results. Key features of the Handbook: Review of the obesity literature, both pediatric and adult. Exercise interventions for children and adults. Lifestyle interventions for adolescents, adults, and seniors. Weight-related cultural issues throughout the lifespan. Bariatric surgery: when is it indicated? How successful is it? The ten critical steps to healthy weight loss. Clinical resources, patient self-help materials, and useful websites. With excess weight contributing to some 300,000 deaths in the U.S. each year, the Handbook of Obesity Intervention for the Lifespan will be welcomed-and consulted frequently-by clinical and health psychologists, psychiatrists, primary care providers, and nutritionists.

Additional Info

  • File Name Handbook of Obesity Intervention for the Lifespan
  • Year 2008
  • Editor(s) Larry James, John C. Linton
  • DOI 10.1007/978-0-387-78305-5
  • ISBN-10 0387783040
  • ISBN-13 978-0387783048
  • Publisher Springer Science+Business Media, LLC 2009
  • Size 1.70 MB
  • File Format .pdf
  • Password bioscience.pk
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