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1

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

2

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.

3

What does heparin do

Heparin anticoagulant
• cause clumping of cells
• gives blue background staining

4

What does citrate do

anticoagulant
• Dilutes blood due to volume of liquid
• Exception-can be used for platelet count only

5

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

6

Effects of old samples:

• Platelets clump
• Nucleus of WBCs deteriorate
• Cytoplasm of WBC has vacuoles and dark granules
• RBCs swell or lyse

7

Three important steps Blood Film Preparation

1. Making
2. Fixing
3. Staining

8

Three methods of Blood Film Preparation

1. Wedge-manual
2. Spun-automated slide spinner
3. Automated-Hematology analyzer

9

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

10

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

11

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)

12

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

13

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

14

Romanowsky Staining

• Wrights
• Giemsa or Wright/Giemsa mixture

15

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

16

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

17

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

18

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

19

Corrective actions: if slides are too blue

• decrease stain time
• increase wash time
• decrease buffer pH

20

Corrective actions: if slides are too pink

• increase stain time
• decrease wash time
• increase buffer pH

21

2. COAGULATION TESTS
Type of specimen

• Plasma is required
• Citrate is the anticoagulant for PT and PTT
• Sodium citrate binds calcium, preventing coagulation
• Tubes must be filled to it’s draw capacity
• Record if patient is on anticoagulant therapy or aspirin

22

Handling and storage coagulation samples

• Centrifuge sample immediately and separate plasma
• Some clotting factors are highly labile and deteriorate significantly at room temperature
• Freeze plasma if not tested within 4 hrs

23

Haematolgy QC

Activities that ensure that specific steps in the procedure meet acceptable standards or parameters

24

Controls and Calibrators

• Commercial control ( Low, High or Normal ) -Used to check precision for verifying known manufacturer’s values. Must fall within 2SD
• Analyze controls daily or in batch. Plot Levey- Jennings Chart
• Commercial controls or calibrators-Have a short shelf life and must be refrigerated between uses.
• Calibrators measure accuracy, verifying reference centre values.
• If 2 results are out of 2SD, run samples again, then get new controls to see if it’s the samples or the controls. IF neither works then you know its not the control, sample. Then change the reagent, do it again . If not then it’s the instrument .

25

For accuracy

controls and calibrators of known value are used

26

For precision:

An in lab prepared control with known value is used

27

For linearity:

A Calibrator is used to ensure that the instrument is sensitive to very low or high values. Patient results are never reported below or above the linearity values.

28

Cell Counters Manual

The manual procedure, using a diluting chamber/ hemocytometer, is no longer used except for white cell counts in body fluids with extremely few cells, e.g. cerebrospinal fluid

29

Disadvantage of Manual Counts

• Time consuming
• Cell identification error

30

Advantage of Automated Counts

• More efficient and cost effective