KOHLER ILLUMINATION

Published in Microbiology
Tuesday, 25 July 2017 13:43
What is Kohler illumination?

Kohler illumination is a method of adjusting a microscope in order to provide optimal illumination by focusing the light on the specimen. When a microscope is in Kohler, specimens will appear clearer, and in more detail.

Process of setting Kohler
 
Materials required
 
  • Specimen slide (will need tofocus under 10× power)
  • Compound microscope.
 
Kohler illumination
 
  1. Mount the specimen slide onthe stage and focus under 10×.
  2. Close the iris diaphragm completely.
  3. If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
  4. Using the condenser adjustment knobs, raise or lower the condenser until the edges of the field becomes sharp (see Figure 797.1 and Figure 797.2).
  5. Open the iris diaphragm until the entire field is illuminated.
 
Note the blurry edges of the unfocused light
Figure 797.1 Note the blurry edges of the unfocused light
 
Adjusting the condenser height sharpens the edges of the ball of light
Figure 797.2 Adjusting the condenser height sharpens the edges of the “ball of light.”
 
When should you set/check Kohler?
 
  • During regular microscope maintenance
  • After the microscope is moved/transported
  • Whenever you suspect objects do not appear as sharp as they could be.
 
Further Reading:
 

PROCEDURES FOR THE COLLECTION OF BLOOD FOR HEMOTOLOGICAL INVESTIGATIONS

Published in Hemotology
Monday, 24 July 2017 10:21
COLLECTION OF BLOOD
 
It is necessary to follow a standard procedure for specimen collection to get the most accurate and trustworthy results of the laboratory test. The blood sample can be collected from the venipuncture or skin puncture for the hematological investigations.
 
SKIN PUNCTURE

This method is most common and mostly used in infants and small children and if the small amount of blood is required. This method is suitable for the estimation of hemoglobin, cell counts, determination of hematocrit (HCT) or packed cell volume (PCV) by micro method and preparation of blood films. Blood obtained by this method is also called as capillary blood. However, it is the mixture of blood from arterioles, venules, and capillaries. It also contains small amount of tissue fluid. In infants, blood is collected from the heel (the medial or lateral aspect of plantar surface or great toe). In adults, it is collected from the side of a middle or ring finger (distal digit) or from the earlobe. (see Figure 796.1).
 
A. Blood lancet and sites of B. finger puncture cross and C. heel puncture shaded areas
Figure 796.1 (A) Blood lancet and sites of (B) finger puncture (cross) and (C) heel puncture (shaded areas)

The puncture site is cleansed with the 70% ethanol or another suitable disinfectant. After drying, a puncture is made with a sterile, dry, disposable lancet, in deep to allow free flow of blood. The first drop of blood is wiped away with the dry and sterile cotton as it contains tissue fluid. After wiping the first drop of blood, next few drops of blood are collected. Excessive pressing should be avoided, as it may dilute the blood with the tissue fluid. After collection of blood, a piece of dry and sterile cotton is pressed over the puncture site till the bleeding ends. Hemoglobin, red cell count and hematocrit (HCT) or packed cell volume (PCV) are moderately higher in the blood collected from skin puncture, as compared to the venous blood. The reason behind this scenario is that platelets adhere to the puncture site and cause the lower count of platelet, and due to small sample size, instant repeat testing is not possible if the result is abnormal or unusual. Avoid collecting blood from cold, cyanosed skin since the false elevation of values of red blood cells, white blood cells and hemoglobin will be obtained.

VENOUS BLOOD COLLECTION

Venous blood is obtained when the larger quantity of blood is needed to perform multiple tests. Different test tubes are filled with blood as per requirement of anticoagulant and blood ratio for the test. Anticoagulant is not required for the test performed by the serum.

Method
 
  1. Common sites of venepuncture in antecubital fossaThe best site for obtaining blood is the veins of antecubital fossa. A rubber tourniquet is applied to the upper arm (see Figure; Common sites of venepuncture in antecubital fossa (red circles)). It should not be too much tight and should not remain in a place for more than 120 seconds. To get veins more palpable and prominent, the patient is asked to make a fist.
  2. The puncture site is cleansed with the 70% ethanol or other suitable disinfectant and allowed to dry.
  3. The preferred vein is anchored by squeezing and pulling the soft tissues below the prick site with the left hand.
  4. Sterile, dry, disposable needles and syringes should be used for the collection of blood. Needle size should be 23-gauge in children and 19- to 21-gauge in adults. Venepuncture is made along with the direction of the vein and with the bevel of the needle up. Blood is withdrawn slowly. Pulling the piston quickly can cause hemolysis and collapse the vein. The tourniquet should be released as soon as the blood begins to flow into the syringe.
  5. When the required blood is collected, the patient is asked to open his/her fist. The needle is removed from the vein. A sterile alcohol swab is pressed over the puncture site. The patient is asked to press the alcohol swab over the site till the bleeding ends.
  6. The needle is removed from the syringe and the required amount of blood is carefully transferred into the test tube containing anticoagulant as per requirement of the laboratory test. If the blood is forced through the syringe without removing the needle, hemolysis can occur. Containers may be glass bottles or disposable plastic tubes with corks and flat bottom.
  7. Blood is mixed with the anticoagulant in the container thoroughly by gently inverting the container several times. The container should not be shaken strenuously as it can cause hemolysis and fizzing.
  8. Check whether the patient is dizzy and bleeding has stopped. Cover the site of puncture with a sticky bandage strip. Recapping the needle by hand can cause needle-prick injury. After the usage of disposable syringe, needles are crashed by the syringe needle destroyer and the syringe is disposed into the biohazard box. The blood container is labeled properly with the patient’s name, age, gender and the time of collection. The sample should be sent without delay to the laboratory with accompanying properly filled laboratory requisition form.
 
Precautions
 
  1. The tourniquet should not be too tight and should not be applied for more than 120 seconds as it will cause hemoconcentration and variation of test results.
  2. The tourniquet should be released before removing the needle from the vein to prevent the formation of a hematoma.
  3. Blood is never collected from the arm being used for the intravenous line since it will dilute the blood sample.
  4. Blood is never collected from an area with hematoma and from a sclerosed vein.
  5. A small bore needle should not be used, blood is withdrawn gradually and the needle is removed from the syringe before transferring blood into the container to avoid hemolysis.
  6. Proper precautions should be noticed while collecting blood either from a skin or a vein puncture since all blood samples are considered as infectious.
  7. The anticoagulated blood sample should be tested within 1-2 hours of collection. If this is not possible, the sample can be stored for 24 hours in a refrigerator at 4-6° C. After the sample is taken out of the refrigerator, it should be allowed to return to room temperature, mixed properly, and then laboratory test is performed.
 
Complications
 
  1. Failure to obtain blood: This is very common and usually painful for the patient. This happens if the vein is missed, or excessive pull is applied to the piston causing collapse of the vein.
  2. Formation of hematoma, abscess, thrombosis, thrombophlebitis, or bleeding.
  3. Transmission of infection like human immunodeficiency virus (HIV) or hepatitis B virus (HBV) if reusable syringes and needles, which are not properly sterilized, are used.
 
Further Reading:
 

SEQUENCE OF FILLING OF TUBES FOR HEMOTOLOGICAL INVESTIGATIONS

Published in Hemotology
Saturday, 22 July 2017 12:14
SEQUENCE OF FILLING OF TUBES
 
Following order of filling of tubes should be followed after withdrawal of blood from the patient if multiple investigations are ordered:
 
  1. First tube: Blood culture.
  2. Second tube: Plain tube (serum).
  3. Third tube: Tube containing anticoagulant (EDTA, citrate, or heparin).
  4. Fourth tube: Tube containing additional stabilizing agent like fluoride.
 
Further Reading:
 

USE OF PLASMA VS. SERUM [DIFFERENCE BETWEEN PLASMA AND SERUM]

Published in Hemotology
Saturday, 22 July 2017 11:59
Plasma is the supernatant liquid obtained after centrifugation of anticoagulated whole blood.
 
Serum is the liquid obtained after clotting of whole blood sample collected in a plain tube.
 
Some of the differences between the two are as follows:
 
  1. Plasma contains fibrinogen as well as all the other proteins, while serum does not contain fibrinogen.
  2. Plasma can be obtained immediately after sample collection by centrifugation, while minimum of 30 minutes are required for separation of serum from the clotted blood.
  3. Amount of sample is greater with plasma than with serum for a given amount of blood.
  4. Use of anticoagulant may alter the concentration of some constituents if they are to be measured like sodium, potassium, lithium, etc.

PLAIN TUBES (Without any anticoagulant) AND FLUORIDE TUBES FOR COLLECTION OF BLOOD

Published in Hemotology
Saturday, 22 July 2017 11:43
Plain tubes (i.e. without any anticoagulant) are used for chemistry studies after separation of serum: liver function tests (total proteins, albumin, aspartate aminotransferase, alanine aminotransferase, bilirubin), renal function tests (blood urea nitrogen, creatinine), calcium, lipid profile, electrolytes, hormones, and serum osmolality. Fluoride bulb is used for collection of whole blood for estimation of blood glucose. Addition of sodium fluoride (2.5 mg/ml of blood) maintains stable glucose level by inhibiting glycolysis. Sodium fluoride is commonly used along with an anticoagulant such as potassium oxalate or EDTA.

International Council for Standardization in Haematology (ICSH)

Published in Hemotology
Saturday, 22 July 2017 11:23
The International Council for Standardization in Haematology (ICSH) was initiated as a standardization committee by the European Society of Haematology (ESH) in 1963 and officially constituted by the International Society of Hematology (ISH) and the ESH in Stockholm in 1964. The ICSH is recognised as a Non-Governmental Organisation with official relations to the World Health Organisation (WHO).
 
The ICSH is a not-for-profit organisation that aims to achieve reliable and reproducible results in laboratory analysis in the field of diagnostic haematology.
 
The ICSH coordinates Working Groups of experts to examine laboratory methods and instruments for haematological analyses, to deliberate on issues of standardization and to stimulate and coordinate scientific work as necessary towards the development of international standardization materials and guidelines.

USES OF ANTICOAGULANTS FOR HEMOTOLOGICAL INVESTIGATIONS

Published in Hemotology
Saturday, 22 July 2017 10:04
Anticoagulants used for hematological investigations are ethylene diamine tetra-acetic acid (EDTA), heparin, double oxalate, and trisodium citrate (Table 791.1).
 
Table 791.1 Salient features of three main anticoagulants used in the hematology laboratory
Salient features of three main anticoagulants used in the hematology laboratory
 
Ethylene Diamine Tetra-acetic Acid (EDTA)
 
Changes occurring due to prolonged storage of blood in EDTAThis is also called as Sequestrene or Versene. This is the recommended anticoagulant for routine hematological investigations. However, it cannot be used for coagulation studies. Disodium and dipotassium salts of EDTA are in common use. International Committee for Standardization in Hematology recommends dipotassium EDTA since it is more soluble. It is used in a concentration of 1.5 mg/ml of blood. Dried form of anticoagulant is used as it avoids dilution of sample. Its mechanism of action is chelation of calcium. Proportion of anticoagulant to blood should be maintained. EDTA in excess of 2mg/ml causes shrinkage of and degenerative changes in red and white blood cells, decrease in hematocrit, and increase in mean corpuscular hemoglobin concentration. Excess EDTA also causess welling and fragmentation of platelets, which leads to erroneously high platelet counts. Prolonged storage of blood in EDTA anticoagulant leads to alterations as shown in Figure 791.1 and Box 791.1. EDTA is used for estimation of hemoglobin, hematocrit, cell counts, making blood films, sickling test, reticulocyte count, and hemoglobin electrophoresis.
 
Preparation
 
Dipotassium EDTA 20 gm
Distilled water 200 ml
 
Mix to dissolve. Place 0.04 ml of this solution in a bottle for 2.5 ml of blood. Anticoagulant should be dried on a warm bench or in an incubator at 37°C before use. For routine hematological investigations, 2-3 ml of EDTA blood is required.
 
Changes in blood cell morphology crenation of red cells separation of nuclear lobes of neutrophil vacuoles in cytoplasm and irregular lobulation of monocyte and lymphocyte nuclei due to storage of blood in EDTA anti
Figure 791.1 Changes in blood cell morphology (crenation of red cells, separation of nuclear lobes of neutrophil, vacuoles in cytoplasm, and irregular lobulation of monocyte and lymphocyte nuclei) due to storage of blood in EDTA anticoagulant for prolonged time
 
Heparin
 
Heparin prevents coagulation by enhancing the activity of anti-thrombin III (AT III). AT III inhibits thrombin and some other coagulation factors. It is used in the proportion of 15-20 IU/ ml of blood. Sodium, lithium, or ammonium salt of heparin is used. Heparin should not be used for total leukocyte count (since it causes leukocyte clumping) and for making of blood films (since it imparts a blue background). It is used for osmotic fragility test (since it does not alter the size of cells) and for immunophenotyping.
 
Double Oxalate (Wintrobe Mixture)
 
This consists of ammonium oxalate and potassium oxalate in 3:2 proportion. This combination is used to balance the swelling of red cells caused by ammonium oxalate and shrinkage caused by potassium oxalate. Mechanism of anticoagulant action is removal of calcium. It is used for routine hematological tests and for estimation of erythrocyte sedimentation rate by Wintrobe method. As it causes crenation of red cells and morphologic alteration in white blood cells, it cannot be used for making of blood films.
 
Preparation
 
Ammonium oxalate 1.2 gm
Potassium oxalate 0.8 gm
Distilled water upto 100 ml
 
Place 0.5 ml of this solution in a bottle for 5 ml of blood. Anticoagulant should be dried in an incubator at 37°C or on a warm bench before use.
 
Trisodium Citrate (3.2%)
 
This is the anticoagulant of choice for coagulation studies and for estimation of erythrocyte sedimentation rate by Westergren method.
 
Preparation
 
Trisodium citrate 3.2 gm
Distilled water upto 100 ml
 
Mix well to dissolve. Store in a refrigerator at 2-8°C.
 
Use 1:9 (anticoagulant: blood) proportion for coagulation studies; for ESR, 1:4 proportion is recommended.
 
ESR should be measured within 4 hours of collection of blood, while coagulation studies should be performed within 2 hours.
 
Further Reading:
 

ABO GROUPING AND Rh D GROUPING

Published in Hemotology
Friday, 21 July 2017 11:48
ABO Grouping

There are two methods for ABO grouping:
 
  • Cell grouping (forward grouping): Red cells are tested for the presence of A and B antigens employing known specific anti-A and anti-B (and sometimes anti-A, B) sera.
  • Serum grouping (reverse grouping): Serum is tested for the presence of anti-A and anti-B antibodies by employing known group A and group B reagent red cells.

Both cell and serum grouping should be done since each test acts as a check on the other.
 
There are three methods for blood grouping: slide, tube and microplate. Tube and microplate methods are better and are employed in blood banks.
 
Further Reading:
 

FALSE REACTION IN ABO GROUPING

Published in Hemotology
Friday, 21 July 2017 11:19
  1. Autoagglutination: Presence of IgM autoantibodies reactive at room temperature in patient’s serum can lead to autoagglutination. If autocontrol is not used, blood group in such a case will be wrongly typed as AB. Therefore, for correct result, if autocontrol is also showing agglutination, cell grouping should be repeated after washing red cells with warm saline, and serum grouping should be repeated at 37°C.
  2. Rouleaux formation: Rouleux formation refers to red cells adhering to each other like a stack of coins and can be mistaken for agglutination. Rouleaux formation is caused by high levels of fibrinogen, immunoglobulins, or intravenous administration of a plasma expander such as dextran. Rouleaux formation (but not agglutination) can be dispersed by addition of normal saline during serum grouping.
  3. False-negative result due to inactivated antisera: For preservation of potency of antisera, they should be kept stored at 4°-6°C. If kept at room temperature for long, antisera are inactivated and will give false-negative result.
  4. Age: Infants start producing ABO antibodies by 3-6 months of age and serum grouping done before this age will yield false-negative result. Elderly individuals also have low antibody levels.

Rh D GROUPING METHOD

Published in Hemotology
Friday, 21 July 2017 10:47
D antigen is the most immunogenic after ABO antigens and therefore red cells are routinely tested for D. Individuals are called as Rh-positive or Rh-negative depending on presence or absence of D antigen on their red cells. Following transfusion of Rhpositive blood to Rh-negative persons, 70% of them will develop anti Rh-D antibodies. This is of particular importance in women of childbearing age as anti-D antibodies can crosss the placenta during pregnancy and destroy Dpositive fetal red cells and cause hemolytic disease of newborn. In other sensitized individuals, reexposure to D antigen can cause hemolytic transfusion reaction.
 
In Rh D grouping, patient’s red cells are mixed with anti-D reagent. Serum or reverse grouping is not carried out because most Rhnegative persons do not have anti-D antibodies; anti-D develops in Rh-negative individuals only following exposure to Rh-positive red cells.
 
Rh typing is done at the same time as ABO grouping. Method of Rh D grouping is similar in principle to ABO grouping. Since serum or reverse grouping is not possible, each sample is tested in duplicate. Dosage effect (stronger antigenantibody reaction in homozygous cells i.e. stronger reaction with DD) is observed with antigens of the Rh system. Autocontrol (patient’s red cell + patient’s serum) and positive and negative controls are included in every test run. Monoclonal IgM anti-D antiserum should be used for cell grouping, which allows Rh grouping to be caried out at the same time as ABO grouping at room temperature. With monoclonal antisera, most weak and variant forms of D antigen are detected and further testing for weak forms of D antigen (Du) is not required. Differences between ABO and Rh grouping are shown in Table 788.1.
 
Table 788.1 Comparison of ABO grouping and Rh typing
Comparison of ABO grouping and Rh typing
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