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1

What are manual WBC counts used for?

Mainly used in today’s modern lab for CSF , sperm count, peri –cardial, synovial and pleural count.

2

Describe WBC

-approx 10-20 µm diameter (2-3 x size of RBC)
-can count under 400 x magnification (high dry)
-can see detail under 1000 x magnification (oil immersion)
- cells are nucleated,
- flexible membrane with variable shape, resists lysis

3

Lymphocytes

Bcells - antibody production
T cells - T helper and T cytotoxic

25-33% of wbcs
only a little larger than wbcs. the nucleus almost fills the whole cell leaves a little sliver of cytoplasm
provides immunity

4

Neutrophils

Phagocytic against bacteria coated with antibody
Receptors specific for bacteria
Kill with Lysosomes
they are 60% of WBC
Nucleus with three to five lobes and have phagocytic qualities
Destruction of bacteria and their numbers increase in the early stage of acute inflammation.
stains light purple in acid and base stains

5

Eosinophils

Attracted to the site of allergy
IgE receptors
Granules with allergic response (not histamiine)

(3%) of WBCs
Fight parasitic infections and are also increased in number in people with acute or active allergies
nucleus is bilobed- stain red in acid stain

6

Basophils

Contain vacoactive amines, histamine and serotonin
<1% of WBCs
Release substances such as histamine, which promotes inflammation
Release heparin, which is an anticoagulant and histamine
nucleus is lobed takes up the whole cell, stains blue in basic stain

7

Monocytes

phagocytic and present bacterial antigen to T helper cells
8% of wbcs - very big
Agranulocytes - very fine granules
Active and quite large (macrophages) when they leave the blood vessel and enter tissues
2-3 times larger than RBC. Nucleus can be spherical or lobed.
phagocytizes large particles and old RBC
bean shaped

8

What happens at the beginning of a bacterial infection or acute inflammation?

Number of neutrophils increases

9

What happens 2 weeks after a bacterial infection?

Monocytes increase

10

What happens during a parasitic infection

Eosinophil numbers increase

11

What happens in viral infections and chronic illness?

Lymphocytes increase

12

What happens in a person with AIDS

T Lymphocytes decrease

13

What happens during allergic reactions?

Basophils increase

14

Principle of manual count

- dilute EDTA blood with fluid that lyses RBCs and stains WBCs
- count the WBCs in a known volume of blood
- calculate cells/L

15

Dilution factor

• Dilute blood 1/100 (1.98ml diluent +in 0.02ml blood) – count all 9 squares
• 1/20 (180 ul diluents + 20 ul blood) – count 4 large squares
• Diluent -2% - 3% acetic acid (CH3COOH) with dye
count with neubauer hemocytometer
= 20

16

Neubauer Chamber / Haemocytometer

• The surface of the chamber contains two square ruled areas separated by an H-shaped moat.
• The distance between the bottom of the coverslip and the surface of the counting area of the chamber is 0.1 mm.
• Each side has a total area of 9 mm2 (1 mm on each side)
• There are nine large squares with an area of 1mm2 each
• The four corner squares are used when counting leukocytes.
• If the WBC is very low, you would count all 9 squares.
• These 4 large corner squares contain 16 smaller secondary squares, each with an area of 0.25 mm2.

17

Calculation of WBC

Total Area = 3x3= 9mm2
Volume of one large square=1 mm2
Depth=0.1mm
T Volume =W xH x depth
(1x1x0.1)= 0.1 mm3
Count both sides and average the count

18

How are manual counts done

• Cells which lie on LEFT and TOP boundary lines of the squares are counted.
• Cells which lie on the RIGHT and BOTTOM boundary lines of the squares are NOT counted. (This is to make cell counts statistically accurate.)

19

Normal Values:

adult 4.5 – 11.0 x109 /L (same for men and women)
- infant 9.0 - 30.0 x109/L (higher than adult)

20

Critical Values:

< 1 x 109 /L and >50 x 109 /L

21

Leukocytosis

(high WBC)
• caused by bacterial infection, leukemia, polycythemia, exercise, pregnancy

22

Leukopenia (

(low WBC)
• caused by viral infection, chemotherapy, some drugs, overwhelming bacterial infection

23

QC for WBC counts

need even distribution of cells in chamber,
- chamber should not be scratched
- chamber should be lint free
- counts from both chambers should be close (~10%)
- use commercial controls
- blood must be properly mixed

About 20% difference to automated count

24

Safety

standard precautions: gloves, goggles and coat
- disinfect (soak for 10mins) haemocytometer in 10% Javex or 70% alcohol
- use disposable chamber

25

RBC Count why important

The red blood cell count is an important test because the number of red blood cells (RBCs) can affect how much oxygen the tissues receive

26

Principle of RBC count

Dilute well mixed EDTA blood with isotonic fluid
>Count RBCs
>Calculate cells x10^12/L

27

Dilution factor for RBC counts

(1:200)
=200

The large square in the middle of the hemacytometer grid has been subdivided into twenty five (25) smaller squares. Each square is further divided into 16 smaller tertiary squares
The four corner and centre squares are used to count RBC’s
The volume of a small square
= 0.004 mm3. (.04 mm2)

calculation is # of cell x Dilution factor / 9mm (if 9 squares or 4 for WBC) or (0.2 for RBC) x 0.1

28

Normal RBC Values

male 4.5 – 6.0 x1012/L
female 4.0 – 5.5 x1012/L

29

What happens when nucleated RBC are found

Nucleated RBCs if present are counted along with WBCs resulting in falsely high WBC.
If nRBCs are found during differential, correction is performed

30

SYMPTOMS OF LOW RBC COUNT

hematocrit or hemoglobin will be low
• fatigue
• shortness of breath
• feeling dizzy, weak, or lightheaded, particularly when you change positions quickly
• increased heart rate
• headaches
• pale skin