COURSE: PRACTICAL MICROBIOLOGY V (SCT 320) TOPIC: HAEMATOLOGY

MARCH 13, 2012

DEPARTMENT: SCIENCE LABORATORY TECHNOLOGY (ACCRA POLYTECHNIC)

LECTURER: HENRY HACKMAN

SICKLE CELL SLIDE TEST

VALUE OF TEST It determines HbS in RBC. It does not differentiate between sickle cell disease and sickle cell triat.

SICKLE CELL SLIDE TEST

PRINCIPLE: Blood is mixed on a slide with a reducing agent such as sodium metabisulphite (di-sodium disulphite), covered with a cover slip and incubated at room temperature for up to 30 minutes or more. The reducing agent deoxygenates the haemoglobin in the red cells making the RBCs containing HbS to sickle.

SICKLE CELL SLIDE TEST

MATERIALS Sodium metabisulphite Whole blood sample Slide Cover slip Petri dish Birght-field microscope

SICKLE CELL SLIDE TEST

METHOD One drop of capillary blood is put on a slide. An equal volume of sodium metabisulphite is added, mixed and covered with a cover slip. Airs bubbles must be excluded. The slide is placed in a closed container (Petri dish) with a damp piece of blotting paper at the bottom to prevent drying of the preparation. It is incubated at room temperature for 10 20 minutes and examined for sickling using 40X objective. A negative and/or positive control should be prepared.

SICKLE CELL SLIDE TEST

A positive slide appear crescent shaped with pointed ends.

ERYTHROCYTE SEDIMENTATION RATE (ESR)

Value of test It is not a specific test. It is raised in infectious, inflammatory, degenerative and malignant conditions associated with increases in fibrinogen, immunoglobulins and C-reactive proteins. Test results must be interpreted in conjunction with clinical diagnosis and other laboratory tests.

ERYTHROCYTE SEDIMENTATION RATE (ESR)

Principle of Test When citrated blood in a vertically positioned Westergren pipette (capillary with ESR basket) is left undisturbed, red cells aggregate, stack together to form rouleaux and sediment through the plasma. The ESR is the rate at which the sedimentation occurs in 1 hour as indicated by the length of the column of clear plasma above the red cells, measured in mm/hr.

ERYTHROCYTE SEDIMENTATION RATE (ESR)

Materials Westergren ESR pipette (capillary tube) Westergren ESR stand ESR bucket with the capillary tube (pipette) ESR bucket containing tri-sodium citrate Venous blood sample Stop watch, gloves, tourniquet, syringe, cotton wool

ERYTHROCYTE SEDIMENTATION RATE (ESR)

Method 1.6ml of venous blood is dispensed into the ESR bucket containing the 0.4ml tri-sodium citrate. Insert the capillary tube (pipette) vertically into the ESR bucket and mix the blood and tri-sodium citrate by inverting the tube. Press the capillary tube into the ESR basket until the blood raises through the capillary tube to the graduated mark. Position the capillary tube with the ESR basket vertically for one hour. After I hour read the level at which the plasma meets the red cells in mm. Dispose of the blood safely and decontaminate the equipment.

ERYTHROCYTE SEDIMENTATION RATE (ESR)

Reference Ranges for tropical countries Children up to 10mm/hr Male Adults up to 15mm/hr Female Adults up to 20mm/hr

BLEEDING TIME TEST

Value of Test This is a sensitive test of endothelial function, platelet function and platelet numbers. Abnormal bleeding may be caused by damage to vascular endothelium (infections such as viral haemorrhagic fevers, septicaemia), reduction in platelets number (thrombocytopenia), defeective platelet function (VHF, liver cirrhosis, alcoholism, leukaemia, drugs (aspirin,NSAID) and disorders of blood coagulation (Haemophilia A & B, Vit K deficiency, severe liver disease, obstetric complications

BLEEDING TIME TEST

Principle of Test The finger is pricked for blood to drop until the blood stops. The time taken for the blood to stop is the bleeding time.

BLEEDING TIME TEST

Materials Syringe, alcohol, cotton wool, stop watch, filter paper

BLEEDING TIME TEST

Method Aseptically puncture the thump. Wipe the first blood and with less pressure press the thump on a filter paper at 30 seconds interval at different spots until the blood stop. Calculate the bleeding time by multiplying the number of blood spots with 30seconds. Convert the time to minutes.

ABO AND RHESUS BLOOD GROUPING

Value of Test To determine the blood group of an individual for purposes of blood transfusion and compatibility analysis

ABO AND RHESUS BLOOD GROUPING

Principle of Test RBC with antigen A will agglutinate (form clots) with Antisera-A . Antigen B RBC will agglutinate with Antisera- B. Rhesus antigen will agglutinate with Antisera-D. The vice versa is true (No agglutination if the antigen is absent in the RBC)

ABO AND RHESUS BLOOD GROUPING

Materials Capillary blood sample Lancet Anti-sera A Anti-sera B Anti-sera D

ABO AND RHESUS BLOOD GROUPING

Method

ABO AND RHESUS BLOOD GROUPING

Intepretation

METHAEMOGLOBIN REDUCING TEST (SCREENING FOR G6PD DEFICIENCY)

Value of test Reduced G6PD activity in red cells can cause acute intravascular haemolysis following exposure to oxidant agents, neonatal jaundice and less commonly, chronic haemolytic anaemia.

METHAEMOGLOBIN REDUCING TEST (SCREENING FOR G6PD DEFICIENCY)

Principle of Test Haemoglobin is oxidized to methaemoglobin (Hi) by sodium nitrite. The redox dye, methylene blue activates the pentose phosphate pathway, resulting in the enzymatic conversion of Hi back to haemoglobin in those red cells with normal G6PD activity. In G6PD deficien cells there is no enzymatic reconversion to haemoglobin.

METHAEMOGLOBIN REDUCING TEST (SCREENING FOR G6PD DEFICIENCY)

Materials Test tubes Methylene blue reagent Sodium nitrite-glucose reagent 6ml of EDTA anti-coagulated venous blood sample

METHAEMOGLOBIN REDUCING TEST (SCREENING FOR G6PD DEFICIENCY)

METHOD 3 test tubes are labelled Test (T), Normal (N), Deficient(D). 0.1ml of sodium nitrite-glucose reagent is pipetted into the test and deficient control tubes. 0.1ml of methylene blue reagent is pipetted into the test tube. 2ml of blood is pipetted into the test, normal and deficient test tubes. The tubes are mixed well and incubated at 35-37C for 90 minutes. Pipette 0.1ml of each mix tube into 10ml if distilled water in 3 separate tubes and mix the contents. Examine the colour of the solution

METHAEMOGLOBIN REDUCING TEST (SCREENING FOR G6PD DEFICIENCY)

Interpretation When colour of test solution is similar to the red colour of the N tube then it is a normal G6PD When the colour of the test solution is similar to the brown colour of the deficient tube then it is reduced G6PD activity or G6PD deficient.