Lecture 1 Flashcards
What is EDTA
(ethylene –diamine tetra acetic acid) is the anticoagulant of choice
• It maintains the cell’s shape and size.
• Prevents blood from clotting by binding with calcium
What is CBC
evaluation of the formed elements of blood: red cells, white cells and platelets. A CBC includes counting these elements and then inspecting them on a blood film microscopically.
What does heparin do
Heparin anticoagulant
• cause clumping of cells
• gives blue background staining
What does citrate do
anticoagulant
• Dilutes blood due to volume of liquid
• Exception-can be used for platelet count only
Handling and storage of CBC samples
- Refrigerate at 4ºC until testing
- Referrals –ice pack
- Mix Samples well before use x 6
- Test Samples the same day
- Blood Film/ Smears must be made the same day
- Samples >24 hrs old: No good for testing
Effects of old samples:
- Platelets clump
- Nucleus of WBCs deteriorate
- Cytoplasm of WBC has vacuoles and dark granules
- RBCs swell or lyse
Three important steps Blood Film Preparation
- Making
- Fixing
- Staining
Three methods of Blood Film Preparation
- Wedge-manual
- Spun-automated slide spinner
- Automated-Hematology analyzer
Thickness of the film
- If the hemoglobin/hematocrit is increased the angle of the spreader slide should be decreased to 30 degrees
- If the hemoglobin/hematocrit is decreased the angle of the spreader slide should be increased to 40 degrees
Area used for microscopic evaluation
- Cell morphology and differential are done in the mono layer area
- Cells overlap at the start (thick area)
- Cells are distorted at the end of the blood film
Features of a well-made peripheral blood film
- Correct size of drop
- The film is 2/3 to 3/4 the length of the slide
- The film is finger shaped with slightly rounded or feathered edge
- The film is smooth without irregularities, holes or streaks
- The whole drop of blood is picked up and spread
- The blood film should not touch the ends of the slide
- The angle of the spreader should be about 35º ( the greater the angle the thicker and shorter the blood film. The lower the angle the longer and thinner the blood film)
Common causes of unacceptable blood film
- Chipped or rough end spreader slide
- Drop of blood too big or too small
- Dirt or grease on the slide
- Holes in the film – Lipemic
- Too long or too short
- Too thick or too thin
- Dried up drop of blood
- Ripples
- Jazzed edge
Staining and fixing
- Allow blood film to dry completely before staining. Do not blow to dry as moisture from breath will cause RBC artifact
- Methanol is the fixative of choice
- Stain has methanol
- Timing and pH are critical
Romanowsky Staining
- Wrights
* Giemsa or Wright/Giemsa mixture
Stain composition
• A Cationic or basic dye (methylene blue), stains the cell nucleus and granules of basophils blue –grey.
• An anionic or acidic dye (eosin), stains haemoglobin and eosinophil granules red –orange
Azure B
Effects of poorly stained blood films - RBC’s too pink or red
- Staining time /buffering time too short
- Over –rinsing
- Stain or buffer pH too acidic
Effects of poorly stained blood films - RBC’s grey-blue, WBC’s too dark or Eosinophil granules are grey
- Stain or buffer pH too alkaline
- Inadequate rinsing
- Prolonged staining
- Heparinized blood
Effects of poorly stained blood films - Stain Deposit
- Dirty slide
- Stain solution not filtered
- Inadequate rinsing
- Stain dried up on slide ( excess timing)
- Staining rack not levelled