Doctors Revision

Doctors Revision

RED BLOOD CELL COUNT & WBC COUNT

DETERMINATION OF RED BLOOD CELL COUNT

Principle

The methods generally used are based on the estimation of the number of cells in a small volume of diluted blood. The counting is carried out in a glass counting chamber. The volume of the fluid over each square is calculated from the area of a square and the depth of the fluid layer over it.

Core Concept: The average number of cells lying on one square is found from the counts of a series of squares. The product of this average number by the dilution gives the average number of cells in the undiluted blood.

Aim: To enumerate the number of RBCs per cubic millimeter of blood.

Student Objectives

After completion of this experiment, you should be able to:

  • Describe the relevance determining the red cell count.
  • Identify the different equipment and reagents used in this experiment.
  • List the normal red cell count in different age groups.
  • Outline the common physiological and pathological conditions that cause an increase or decrease in the red cell count.

Materials and Apparatus

1. RBC Diluting Pipette

Specifications:
  • Bulb type.
  • Graduated to give a dilution of 1 in 100 or 1 in 200.
  • Stem Markings: 0.5 and 1.0.
  • Upper Line: 101 (immediately above the bulb).

Red Bead: Located in the bulb to facilitate mixing of the blood and diluting fluid.

2. Hayem’s Fluid (Diluting Fluid)

Properties:

Must be isotonic; causes neither hemolysis nor crenation. Contains a fixative to preserve shape and prevent autolysis. Prevents agglutination/rouleaux.

Composition (per 200ml)
  • NaCl (3.8%): 1.0 g
  • Sodium Sulfate (Na₂SO₄): 5.0 g
  • Mercuric Chloride (HgCl₂): 0.5 g
  • Distilled Water: 200 ml
Function of Ingredients
  • NaCl & Na₂SO₄: Provide isotonicity (prevents shape change) and anticoagulant properties (prevents rouleaux).
  • Mercuric Chloride: Fixative, antifungal, and antimicrobial agent.

3. Neubauer Haemocytometer

Consists of a thick glass slide with a central platform 0.1mm lower than the side platforms (Depth of chamber = 0.1mm).

The Ruled Area (Center):
  • Divided into 16 medium sized squares.
  • Each medium square is subdivided into 16 small squares.
  • Area of smallest square = 1/400 sq. mm.
  • Counting Area: The red cells lying in 5 of the medium squares (E1, E2, E3, E4, and E5) are counted.

NB: For use, the haemocytometer as well as the diluting pipette must be clean, dry and absolutely grease free.

Procedure

  1. Sampling: Fill the pipette up to mark 0.5 on the scale with blood from the finger tip.
  2. Diluting: Wipe the outside of the pipette. Draw Hayem's fluid up to mark 101. Close the tip, detach sucker, and mix well (shaking 3-4 mins).
  3. Chamber Prep: Place coverslip on counting chamber. Apply gentle pressure until Newton rings (rainbow colors) appear.
  4. Discarding: Discard the first few drops (they contain no cells).
  5. Charging: Fill the chamber by holding the pipette tip against the edge of the coverslip. Do not overfill into troughs.
  6. Settling: Allow several minutes for cells to settle.
  7. Counting: Count red cells in 80 small squares (5 groups of 16 squares: E1-E5).
    Rule: Include cells touching top and right border lines only.

CALCULATION

Dimensions

Area of 1 small square = 1/400 sq mm

Depth of chamber = 1/10 mm

Volume of 1 square = 1/4000 cu mm

Variables

N = Total cells counted in 80 squares

Dilution Factor = 200

Squares Counted = 80

Final Formula (Cells per cu mm):

Total = N × 10,000

Derivation: (N × 4000 × 200) / 80

QUESTIONS

  1. When blood is taken to the mark 0.5 and diluent to mark 101, why is the dilution 1 in 200 and not 1 in 202?
  2. Why is blood diluted 200 times for red cell count?
  3. What is the function of the bead in the bulb?
  4. If Hayem’s solution is not available, can you use any other?
  5. How will you differentiate red cells from dust particles?
  6. What is the fate of leukocytes in this experiment?
  7. Since the mature red cells do not contain ‘nuclei’, are they dead cells? Explain your answer.
  8. Explain the possible errors that could arise in obtaining and diluting blood, due to uneven distribution of cells in the counting chamber, due to mechanical causes and from other sources.

DETERMINATION OF THE DIFFERENTIAL WHITE BLOOD CELL COUNT

Student Objectives

At the end of this experiment, you should be able to:

  • Identify all equipment and reagents used in the determination of the differential WBC count.
  • Describe the relevance and importance of preparing and staining a blood smear and doing a differential leukocyte count.
  • Prepare satisfactory blood films, fix and stain them and describe the features of a well stained film.
  • Identify different blood cells in a film and indicate the identifying features of each type of leukocyte.
  • Differentiate between neutrophils, eosinophils, basophils, monocytes, and lymphocytes.
  • Describe the functions of each type of the different leukocytes.
  • Outline the conditions in which the leukocyte numbers increase or decrease.

Relevance & Principle

Relevance:

Many hematological and other disorders can be diagnosed by a careful examination of a stained blood film. A physician may order a differential leukocyte count (always along with the total leukocyte count) to differentiate between the different causes of infection (e.g. bacterial vs. viral causes) depending on which sub-category of leukocyte is greatly affected. The differential leukocyte count is also done to monitor blood diseases like leukemia, or to detect allergic or parasitic infection.

Principle:

A blood film is stained with Leishman’s stain and scanned under oil immersion, from one end to the other. As each WBC is encountered, it is identified until 100 leukocytes have been examined. The percentage distribution of each type of WBC is then calculated.

Procedure

  1. Wipe the punctured finger with a piece of cotton wool soaked in alcohol, and allow a fresh drop of blood to accumulate.
  2. Hold a clean, dry microscope slide between the thumb and forefinger of the left hand. The slide is held by the corners of its right hand end so that its length extends at an approximate angle of 45 degrees above the left thumb and forefinger.
  3. Rotate the left hand inward, and touch the former upper surface of the slide to the drop of blood on the subject’s finger. A small drop of blood should be deposited onto the center of the slide about 1/3 of the length from the end held by the fingers of the left hand.
  4. Rotate the left hand outward until the surface of the slide with the deposited blood is uppermost and horizontal.
  5. A second clean, dry slide is held near its right hand end by the thumb and forefinger of the right hand. The free end should extend downward and to the left (away from the thumb and forefinger of the right hand). The edge of the lower end of this slide is brought onto contact with the slide held by the left hand at an angle of 45 degrees. The site of contact should be just ahead of the blood drop.
  6. The right hand slide (the spreader) is pulled back so that the edge on the inner side of the angle formed between the two slides just touches the blood drop. Capillarity at the inner apex of this 45 degrees angle distributes blood evenly across the width of the slides.
  7. A smooth, fairly fast sliding motion of the spreader (maintaining the 45 degrees angle of contact) along the length of the horizontal slide, deposits a thin, uniform film of blood. Several trials should produce an acceptable blood smear for staining.
  8. The slides which are to be stained are then laid smear side up on a staining and allowed to air dry.
  9. When the thin film of blood has air dried, Wright’s or Leishman’s stain is dripped from a dropping bottle onto the slide. The entire surface is covered until the stain is standing up from the edges of the glass but not running off the sides.
  10. The stain is allowed to stand on the slide from 1 to 3 minutes. The actual period of time depends upon the properties of each different batch of stain. Next, an equal volume of buffer solution should be added to the dye on the slide. If the buffer is dripped onto the dye, the entire fluid volume stands up from the edge of the slide without spilling.
  11. The buffer and stain are mixed by blowing lightly on the slide. A glossy sheen soon appears on the surface of the mixed liquid, which is allowed to remain on the slide for 4 to 5 minutes.
  12. Then the slide is flushed by flooding with distilled water or by holding one end of the slide horizontally under a slow stream of tap water. After the slide is well washed, place it in a slightly inclined position to drain and air dry.
  13. When the slide is dry, examine it first under the 4mm objective of the microscope to note the distribution of leukocytes. Since the distribution is often quite uneven and large leukocytes are carried to the edges of the smear, the differential count should sample the entire smear.
  14. The oil immersion objective of the microscope is required to identify the white cell types. Each white cell, as it is identified, is entered by a tally mark in the appropriate space on the data sheet.
  15. Proceed till 100 cells are counted, no cell will be seen twice in this way.
  16. Record the percent contributed to the total by each of the white cell types.
  17. After completion of the white count, observe the red cells on the slide. Record their shapes, sizes and color.

Focusing under Oil-Immersion Lens

  1. Examine the appearance of the slide for the general quality of staining. A good smear is roughly rectangular with a rather dense and straight ‘head end’ and a thinner and convex ‘tail end’. It is light purplish in color and translucent.
  2. Focus under the lowest power in the microscope and inspect the slide quickly for the distribution and appearance of the cells.
  3. Focus under the high power (40) and inspect the different areas of the smear. First distinguish between the numerous pink-colored red blood cells and the fewer large blue stained white blood cells.
  4. Then observe the distribution and appearance of the cells in different parts of the slide. At the head end the red cells are crowded and the white cells are poorly stained. At the extreme tail the cells are wide apart and white cells are distorted. The cells are stained well and seen clearly in the body of the smear near the tail end. Identify the best area (the body of the smear) for further study.
  5. The detail structure of the individual cells can only be seen through the oil immersion objective (magnification 100). Utmost care is needed when focusing under this objective as the focal distance is less than 2mm. Lower the stage of the microscope further down and switch on (turn) the oil immersion objective to position while watching the stage and the slide to avoid any damage. If the objective lens is likely to touch the slide, lower the stage further down.
  6. Place a drop of immersion oil on the blood smear and move the slide so that the oil (immersion oil) on the blood smear is directly under the objective. While watching the slide and the objective from the side and NOT through the eye-piece of the microscope, raise the stage until the oil touches the objective.
  7. Now look through the eye-piece and adjust the illumination (bright light is needed for clear vision). Looking through the eye-piece, raise the stage slowly until suddenly the cells come under focus. If clear image has not appeared within two or three turns of the knob, lower the stage and start focusing once again after ensuring that the illumination is adequate and that the slide contains cells (sometimes if the fixation was not properly done or if the slide was washed vigorously, the cells may be washed away. The slide may also be upside down). The oil between the objective and the slide serves as a concave lens to increase magnification and reduces aberration of light and facilitates the entry of all light into the microscope.
  8. Keep the cells under focus (by constant adjustment of the knob because the slightest alteration in the depth can affect the image) and move the slide about and study the structure of various types of cells and their size in relation to red cells.
  9. The red cells can be easily identified because they are pink non-nucleated discs found all over the field.
  10. You have to search for the white cells which will be seen as distinct cells with nucleus stained purple with clear or granulated cytoplasm. Remember that the cells are spheres and at any time the microscope will be focused only in one plane of the cell. Therefore, it will be necessary to adjust the focus up and down to see the cell in full.

Identification of Leucocytes

Note the following points with regard to any leucocyte:

  • The size and shape of the nucleus.
  • Presence or absence of cytoplasmic granules.
  • When present- the size, number and staining reaction of the granules.

a) If the nucleus occupies only a small portion of the cell and it is lobulated, the cell is a polymorpho-nuclear leucocyte.

b) If there are three more clear lobes then the cell may be Neutrophil; if the lobes are clearly defined and arranged like spectacles then it is probably an eosinophil; but if the two lobes lie on top of each other because of the position of the cell, only one small lobe can be seen. The nucleus of the basophil is elongated and poorly divided into three lobes.

c) If the nucleus is not lobulated but spherical and fills almost all the cell then the cell is a lymphocyte.

d) If the cell has a large kidney shaped nucleus, it is a monocyte; the nucleus of the monocyte can appear circular or even oval shaped depending on the orientation of the cell on the slide.

e) If cytoplasm is clear and light purplish in color, the cell is an agranulocyte.

f) If there is only scanty cytoplasm then the cell is a lymphocyte. Lymphocytes can be found is sizes equal to red cells (small lymphocytes) or much larger than the red cells (large lymphocyte).

Table 1: Appearance of White Blood Corpuscles in a Stained Blood Film

Cell type Diameter (μm) Nucleus Cytoplasm Cytoplasmic granules
Granulocytes
Neutrophils
(40-70%)		 10-14
(1.5-2X a RBC)		 Blue-violet
2-5 lobes, connected by chromatin threads
Seen clearly through cytoplasm		 Slate-blue in color Fine, closely-packed violet pink
Not seen separately
Give ground-glass appearance
Do not cover nucleus
Eosinophils
(1-6%)		 10-15 Blue-violet
2-3 lobes, often bi-lobed, lobes connected by thick or thin chromatin band
Seen clearly through cytoplasm		 Eosinophilic
Light pink-red		 Granular
Large, coarse
Uniform-sized
Brick-red to orange
Seen separately
Do not cover nucleus
Basophils
(0-1%)		 10-15 Blue-violet
Irregular shape, may be S-shaped, rarely bilobed
Not clearly seen, because overlaid with granules		 Basophilic
Bluish		 Granular
Large, very coarse
Variable-sized
Deep purple
Seen separately
Completely fill the cell, and cover the nucleus
Agranulocytes
Monocytes
(5-10%)		 12-20
(1.5-3 X a RBC)		 Pale blue-violet
Large single
May be indented horse-shoe, or kidney shaped (can appear oval or round, if seen from the side)		 Abundant
‘Frosty’
Slate-blue		 Amount may be larger than that of nucleus
No visible granules
Small Lymphocytes
(20-40%)		 7-9 Deep blue-violet
Single, large, round, almost fills cell.
Condensed, lumpy chromatin, gives ‘ink-spot’ appearance		 Hardly visible
Thin crescent of clear, light blue cytoplasm		 No visible granules
Large Lymphocytes
(5-10%)		 10-15 Deep blue-violet
Single, large, round or oval, almost fills cell
May be central or eccentric		 Large, crescent of clear, light blue cytoplasm
Amount larger than in small lymphocyte		 No visible granules

Exercise

Draw each type of the white blood cell as you see in the microscope and label them.

Neutrophil Drawing Area
Eosinophil Drawing Area
Basophil Drawing Area
Monocyte Drawing Area
Lymphocyte Drawing Area

RED BLOOD CELL MORPHOLOGY

Student Objectives

  • Identify various cell morphologies in relation to size, shape, and colour.
  • Identify normal RBCs and indicate their identifying features.
  • Identify abnormal RBCs and indicate the identifying features of each.
  • Discuss the conditions involved in each of RBC abnormalities.

Introduction

Usually, only normal, mature or nearly mature cells are released into the bloodstream, but certain circumstances can induce the bone marrow to release immature and/or abnormal cells into the circulation. When a significant number or type of abnormal cells are present, it can suggest a disease or condition and prompt a health practitioner to do further testing.

Characteristics of Normal RBCs (Normocytes):
  • Size: Uniform, 7 - 8 μm in diameter.
  • Nucleus: Absent (anucleated).
  • Shape: Round, biconcave discs (flattened like a donut with a depression in the middle).
  • Color: Pink to red with a pale center (central pallor).
  • Terminology: Often reported as normochromic and normocytic.

Aim: To study the colour and different morphologies of red blood cells in a stained film.

Procedure

Use a stained film (from the previous procedure) and study:

  • Shape and Size: Note the moderate variation in size around the diameter of about 7.5 μm.
  • Staining: Note the size of the central pallor (it normally occupies the central third) and compare the depth of colour in different cells. Look out for any granules in some cells.

Abnormal Red Blood Cells

1. Characteristics Related to Size

Term Morphology Description
Anisocytosis An increase in the variability of red cell size.
Microcytosis Decrease in the red cell size. Smaller than ± 7 μm.
Comparison: The nucleus of a small lymphocyte (± 8 μm) is a useful guide.
Macrocytosis Increase in the size of a red cell. Larger than 9 μm. May be round or oval.

2. Characteristics Related to Color

Term Morphology Description
Hypochromia Increase in the central pallor, occupying more than the normal third of the red cell diameter.
Hyperchromia Decrease in the central pallor and more dense staining.
Polychromasia Red cells stain shades of blue-gray. Due to uptake of both eosin (Hb) and basic dyes (residual ribosomal RNA). Often slightly larger (round macrocytosis).

3. Characteristics Related to Shape

Term Morphology Description
Poikilocytosis General term referring to an increase in abnormal red blood cells of any shape.
Acanthocytes Spherical cells with 2 - 20 spicules of unequal length, distributed unevenly over the surface.
Spherocytosis Red cells are more spherical. Lack the central area of pallor on a stained blood film.
Schistocytosis Fragmentation of the red cells.
Sickle Cells Sickle shaped (crescent) red cells.
Elliptocytosis Red cells are oval or elliptical. Long axis is twice the short axis.

EXERCISE

Draw the type of red blood cell as you see in the microscope and label them here.

DISCUSSION

1. Differential Count Analysis
  • Describe the possible errors in the determination of the differential count.
  • Describe the importance of total white cell count in interpreting the differential count.
  • Describe the importance of the Differential White cell count in clinical practice.
2. RBC Abnormalities

Discuss the different conditions related to the abnormalities of size, shape, and colour of red blood cells.

Physiology Steeplechase: Blood Cell Count

Blood Cell Steeplechase

Hemocytometry & WBC Differential

What to master:

  • Pipettes: RBC (Red bead) vs WBC (White bead).
  • The Grid: Where do you count RBCs vs WBCs?
  • WBC ID: Distinguish Eosinophils (Red granules) from Lymphocytes (Round nucleus).
  • Morphology: Sickle cells and Anisocytosis.
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