1. Practical Hematology Manual

2. Whole blood 8% of body weight
Plasma
55%
Cells
45%
Water
91.5 %
Plasma protein 7%
Other solutes 1.5%
Albumin 54%
Globulin 38%
Fibrinogen 7%
Other 1%
Enzymes & Hormones
Nutrient
Electrolytes &Gases
Wastes
RBCs
4.5-6 million/mm3
Platelets
150, ,000/mm3
WBCs
/mm3
Neutrophils %
Lymphocytes 20-25%
Monocytes 4-8%
Eosinophils 2-4 %
Basophils 0-1%

3. Erythrocyte Count Normal ranges: Male 5 - 6 million/mm3 or μl
Female million/mm3 or μl
New born million/mm3 or μl
Erythrocyte count increased in case of polycythemia and decreased in anemia 

4. How to collect blood sample to do Complete blood count (CBC)
When blood come in contact with wettable surface e.g. glass (tube), it will normally clot (by intrinsic pathway) as follow
We need to prevent this blood clotting to be able to proceed through the intended CBC,
To do this we have to add anti-coagulants.

6. Thus, it is an instrument used to count the blood cells.
Hemocytometer
Hemo / cyto / Meter
Blood / cell / counter
Thus, it is an instrument used to count the blood cells.
The hemocytometer counting chamber is used for cell counting.
It is constructed so that the distance between the bottom of the coverslip and the surface of the counting area of the chamber is 0.1 mm.
The surface of the chamber contains two square ruled areas separated by an H-shaped moat.

7. It includes: - Neubauer’s slide - Cover slip - RBC pipette - WBC pipette

8. Whole blood using EDTA or heparin as anticoagulant. Equipments:
Erythrocyte Count
Principle:
In order to facilitate RBCs count a specified volume of blood is diluted with a specified volume of isotonic fluid
Diluting fluid:
One of the following solutions may be used:
Isotonic saline: 0.9 % NaCl in distilled water.
Hayam’s solution
Sample:
Whole blood using EDTA or heparin as anticoagulant.
Equipments:
RBCs pipettes.
Neubauer's chamber.
Cover slips.
Conventional light microscope.
Clean gauze.

9. haemocytometer chamber
Pipette
Rubber sucking tube

10. Each scale is 3mm wide and 3mm long = 3×3.
The whole scale is divided into 9 big squares (9 primary squares).
Each primary square is 1mm long and 1mm width = 1×1
Each primary square is further divided into 25 secondary square
Each secondary square is further subdivided into 16 tertiary square
So the primary square is divided into 400 tertiary square

11. Primary, secondary and tertiary square

12. DILUTION FACTORS For RBC counting Blood is filled till mark 0
DILUTION FACTORS For RBC counting Blood is filled till mark 0.5 and Hayam's fluid is then filled till mark 101. Both are thoroughly mixed and then few drops are discarded which contain just the diluting fluid in the stem. Thus, 1 portion out of 101 is discarded. So, 0.5 part of blood is in 100 parts of fluid or, 1 part of blood is mixed in 200 parts of fluid Thus, dilution factor for RBC counting is 200.

14. Procedure
Carefully charge hemocytometer with diluted blood by gently squeezing sides of reservoir to expel contents until chamber is properly filled.

15. FOCUSING
4X to see the general formation of slide. 10X for WBC counting 40X for RBC counting

16. Adjust the microscopic objective lens 40X for RBC counting

17. X = number of RBCs in 5 secondary squares
RBC COUNTING
Total no. of RBCs in 5 secondary squares = X
No. of RBCs in 1 secondary squares = X/ 5
X/ 5 × 25 = No. of RBCs in the primary sq (1mm2) of diluted blood
X/ 5 × 25 × 200 = No. of RBCs in the primary sq (1mm2) of pateint blood
Because the depth between the cover and slide was only mm
X/ 5 × 25 × 200 × 10 = No. of RBCs in the primary sq (1mm3) (= 1 μl of blood)
X = number of RBCs in 5 secondary squares
RBC COUNT = X × 10,000/mm³ or µL

18. Principle of WBCs count
The diluting fluid lyses the RBCs but preserves leukocytes and platelets. The diluted blood is added to the hemocytometer chamber.
Sample:
EDTA- anticoagulated blood or capillary blood is preferred.
WBCs count diluting fluid :
Acetic acid 1% (v/v) in distilled water.
HCl 1% (v/v) in distilled water.
Turk's solution.

19. Equipment White blood cells count diluting fluid WBCs pipette
Hemocytometer and coverslip
Microscope
Lint-free wipe
Alcohol pads
For WBC counting
0.5 part of blood is mixed in 10 parts of fluid
So, 1 part of blood is in 20 parts of fluid
Thus, dilution factor for WBC counting is 20.

21. Normal Value: 5000 - 11000/mm3 or µL
WBC COUNTING Total no. of WBCs in 4 Primary squares = X No. of WBCs in one Primary square (1 mm2) = X/4 (diluted) X/4 × 20 = No. of WBCs in 1 mm2 of undiluted blood X/4 × 20 x 10= No. of WBCs in 1 mm3 of undiluted blood. While X is the No of WBCs counted in the 4 primary square
No. of WBCs = X × 50/mm³ or µl
Normal Value: /mm3 or µL

22. This test assess the factors involved in intrinsic pathway
Coagulation time
Definition :-
It is the time needed
for formation of a hard clot.
Material:-
Clean dry glass slide, stop watch , water bath 37ºC, syringe, alcohol , cotton .
Procedure:-
Put a drop of the blood on the slide.
The slide is placed on a warm plate at 37ºC.
Every 30 seconds put a needle in the middle of the blood drop and try to elevate it
blood coagulation start when fibrin threads appear attached to the needles.
Normal : 5-10 minutes.
This test assess the factors involved in intrinsic pathway

23. To assess the platelet functions.
Bleeding time
The time needed for bleeding to stop.
Materials:
Filter paper, sterile lancets, stop watch, sphygmomanometer , ethyl alcohol.
Method:
A sphygmomanometer cuff is placed on the upper arm and inflated to 40mm Hg .
A sterilized area of forearm without visible superficial veins is selected and is pierced by sterilized lancet in 3 points .
Wipe the blood every 30sec from the 3 points alternatively until no blood comes to filter paper .
Normal : 3-5 min.
To assess the platelet functions.

24. Hematocrite value (H.V.) packed cell volume (PCV)
Definition:-
H.V. is the volume of RBCs in 100 ml blood or it is the percentage of the volume of RBCs to the volume of whole blood.
Volume of RBCs
H.V. = × 100
Volume of blood
Materials:-
Microhematocrite tube (75 mm long, 1 mm pore, heparinized )
Microhematocrite centrifuge .
Microhematocrite tube reader.
Sterile lancet.
70 % ethyl alcohol.

25. Procedure:- Normal values:- Obtain blood drop by pricking the thumb.
Micro hematocrite tube is filled up to it's 2/3 with blood sample by touch the drop of blood by one end of the tube.
Close the empty end of the tube by plasticine .
Centrifuge the tube at per minute for 5 minutes .
Remove the tube and read H.V. by putting the tube on special micro hematocrite scale.
Normal values:-
Adult male % - 47 %
Adult female % - 44%
Higher in new born 45 % - 49%

26. Hematocrite (PCV) determination
Capillary methods

28. Female:
36% - 48%
Male:
45% - 47%

29. Hematocrite (PCV) determination
Wintrobe method

32. Significance of the haematocrite
The Buffy coat normally 1%
Increased in leukocytosis and leukemia
Plasma layer
Colour : Normally faint yellow
Deep yellow color indicate Jaundice
Red color indicate Hemolysis
The PCV layer normally 45% - 47%
Increased in Polycythemia and dehydration
Decreased in anemia and overhydration

33. Determination of Hb values
Haemoglobin can be measured by manual or automatic.
Two method are common use:
Oxyhaemoglobin (HbO2method).
Haemiglobincyanid. (HicN;Cyanmethaemoglobin).
A major a advantage of the HicN method is availability of stable and reliable reference preparation.
Other methods: Sahli’s acid haematin and alkalin-haematin method.

34. Cyanmethaemoglobin method
Use Drabkins solution which contain potassium cyanide and potassium ferricynide at pH
Hb oxidation to methaemoglobin by potassium ferricynide then combine to potassium cyanide to form cyanmethaemoglobin which measure at 540 nm by spectrophotometer.
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35. Normal range of Hb in g/dl
Hb g/dl=
Men 15 ± 2 g/dl
Women 13.5 ± 1.5 g/dl
At birth 18 ± 4 g/dl
Principle of Hb cyanide method
Blood is diluted in a solution:
Contains potassium cyanide + potassium ferricyanide (Drabkin solution)
As a result this reaction will occur:
Hgb, HbCO  methemoglobin HiCN (cyanmethemoglobin)
HbS will not be converted using this method

36. Methods
Set spectrophotometer to 0.0 reading at 540 wave length, using diluted drubkin’s solution as blank
Test the reading of the cyanohemoglobin standard solution
Dilute blood sample in the working solution as 1:250 dilution (blood: diluents)
10µL of blood in 2.5 mL of working solution in the cuvet, Cover and invert the tube several times
Let it stand at R.T for 5-10 minutes to insure complete conversion
Put the cuvet in the hemoglobinometer
Continue reading patient samples and determin Hb value of sample

37. RBCs indices Red Cell Indices includes Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Hemoglobin Concentration (MCHC)
These calculation requires the presence of
RBCs count
Hb value
PCV

38. Mean Corpuscular Volume (MCV)
The MCV indicates the average volume of the red blood cells.
MCV =
= (fl)
Normal value for the MCV : 86±8 fl
If the MCV is less than 78 fl, the RBCs are Microcytic.
If the MCV is greater than 94 fl, the RBCs are Macrocytic.
If the MCV is within the normal range, the RBCs are Normocytic.
Volume of RBC in femtoliters (fl) / μl of blood
1 μl = 109 fl
RBC / μl of blood
Hematocrit x 10
RBC count in millions

39. Mean Corpuscular Hemoglobin (MCH)
The MCH indicates the average weight of hemoglobin in the red blood cells.
MCH =
Normal value for the MCH : 27~31 pg
If MCH is lower than 27 pg the condition is called Hypochromic
If MCH is higher than 31 pg the condition is called Hyperchromic
If MCH is within the rang of 27~31 pg condition is called Normochromic
Weight of hemoglobin in 1 μl of blood
Number of red blood cells in 1 μl of blood
Hemoglobin * 10
(pg)
Red blood cell count in millions
1 g = 1012 pg
1 ml = 103 μl

40. Mean Corpuscular Hemoglobin Concentration (MCHC)
The MCHC is an expression of the average concentration of hemoglobin in the red blood cells. It gives the ratio of the weight of hemoglobin to the volume of the red blood cells.
MCHC =
Hemoglobin in g/dl
* (to convert to %)
Hematocrit /dl
Hb (g/dl)
Hct (%)
MCHC (g/dl) =
150 45 = 15 45 = X 10
* 100
Normal value for the MCHC : 32~36 %
An MCHC below 32% indicates hypochromia, an MCHC above 36% indicates hyperchromia, and red blood cells with a normal MCHC are termed normochromic.

41. AA or AO = Type A BB or BO = Type B OO = Type O AB = Type AB
What are blood types?
Blood Types
AA or AO = Type A BB or BO = Type B OO = Type O AB = Type AB
There are 3 alleles or genes for blood type: A, B, & O. Since we have 2 genes, there are 6 possible combinations.

42. Average Percents…
Type O—46%
Type A—40%
Type B—10%
Type AB—4%

43. Rh Factors A+ A- B+ B- AB+ AB- O+ O-
Scientists sometimes study Rhesus monkeys to learn more about the human anatomy because there are certain similarities between the two species. While studying Rhesus monkeys, a certain blood protein was discovered. This protein is also present in the blood of some people. Other people, however, do not have the protein.
The presence of the protein, or lack of it, is referred to as the Rh (for Rhesus) factor.
If your blood does contain the protein, your blood is said to be Rh positive (Rh+). If your blood does not contain the protein, your blood is said to be Rh negative (Rh-).
A+ A- B+ B- AB+ AB- O+ O-

44. How common is your blood type?
46.1%
38.8%
11.1%
3.9%

45. Blood Typing Test
Determines blood type and compatibility
Figure 19–7

46. Red blood cell compatibility table  
AB+
AB- B+ B- A+ A- O+ O-
Donor
Recipient

47. Differential leukocytic Count blood smear examination
Specimen : EDTA blood within 2 to 3 hours & collected to the mark on tube.
Procedure
placing a drop of blood from mixed sample on a clean glass slide.
Spreader slide using another clean glass slide at degree angle.
Control thickness of the smear by changing the angle of spreader slide
Allow the blood film to air-dry completely before staining. (Do not blow to dry. The moisture from your breath will cause RBC artifact.)

48. STEPS FOR BLOOD FILM

50. Characteristics of a Good Smear
Thick at one end, thinning out to a smooth rounded feather edge.
Should occupy 2/3 of the total slide area.
Should not touch any edge of the slide.
Should be margin free, except for point of application.
Normal peripheral blood smear