PHYSIOLOGY LAB REVIEWER

RBC Counting

Erythrocytes – most numerous of blood cells

- Responsible for providing oxygen to tissues and partly for recovering CO2 produced as

waste

- 4-6 millions/cubic mm

- Devoid of nucleus (man, mammals) w/ a shape of a biconcave lens

- Nucleated (fishes, birds, reptilians and birds)

- Shapes: Normal (discocyte), Berry (crenated), burr (echinocyte), target (codocyte), oat,

sickled, helmet, pinched, pointed, indented, poikilocyte, etc.

Hemoglobin – protein able to bind in a reversible manner to oxygen

Materials:

1. Thoma pipette with red bead (0.5 and 101 mark)

2. Hayem’s solution

3. Hemocytometer

Procedure:

a. Filling the pipette

1. Get fresh, unclotted blood from the animal subject

2. Using the thoma pipette with attached rubber tubing apply gentle suction on the

mouthpiece to draw blood exactly to the 0.5 mark of the pipette

3. Wipe the tip of the pipette then thoroughly steady suction draw the diluting fluid up to

101 mark

4. Bring the pipette in a horizontal position and place the finger at the tip then remove the

rubber tubing

5. Keep the pipette in horizontal position by holding it between the thumb and middle

finger then carefully shake it using a figure of 8 for atleast 2-3 mins

b. Counting of RBC

1. Clean the hemocytometer and the coverglass make sure these are free from grease

before using

2. Place the coverglass laterally to and on the supporting tibs of the counting chamber

3. Discard at least one third of the contents of the pipette in a clean tissue paper and wipe

off adhered excess fluid

4. Carefully touch tip of the pipette to the space between the chamber and the coverglass.

Fill the space with the mixture up to three fourths of the chamber withdraw the pipette

and let the fluid flow to completely fill the entire area

5. Stand the mixture for about 3 mins to allow the cells to settle care should be taken to

avoid evaporation

6. Focus under LPO locate the central square of the nine primary large squares of the

counting chamber

7. Shift to HPO observe all RBC in the area within the 5 squares of the 25 small squares in

the center of the 9 primary squares

8. Note for even distribution of cells in the specified areas (4 corners and the center of the

25 secondary squares)

9. Each of the five small squares to be used for counting is lined by double or triple lines

and further subdivided into 16 smaller squares (5X16=80 small squares)

10. To avoid any duplication in counting just count the cells following the sequence from

the upper left corner to right then next row of 4 squares front right to left and so on

Triple ruling count cells touching top and left center lines while cells touching bottom

and right center lines are not

Calculations: Sum of number of cells in 5 small squares X 10,000 = total RBC/ml of blood

Questions

1. Define:

a. Erythropoiesis - the production of red blood cells.

- *hypoxia is the best stimulus for erythropoiesis

b. Polycythemia - : An abnormally increased concentration of hemoglobin in the blood.

c. Polycythemia vera - is a blood disorder in which the bone marrow makes too many

red blood cells. Polycythemia vera may also result in the overproduction of white

blood cells and platelets.

d. Anemia - A condition marked by a deficiency of red blood cells or of hemoglobin in

the blood, resulting in pallor and weariness

Pernicious anemia – deficiency of VitB12

Megaloblastic anemia – deficiency of Folic Acid

2. Diagram of erythropoiesis

Bone Marrow metarubricyte (nucleated) ; mammalian erythrocyte (non-nucleated)

nucleus of metarubricyte is lost by extrusion or by absorption before it enters

bloodstream with supravital stain the RNA is precipitated, giving a network of blue fibers

known as reticulocyte during hemorrhage, parasitism, most animals respond w/ a

release of young nucleated RBC and reticulocyte from bone marrow in circulating blood

3. What are the important factors needed for maturation of RBC

Vit B12 & Folic Acid (DNA synthesis)

4. Give factors that may cause on invasion erythrocyte count

a. Sex – female have less RBC; they undergo estrus

- Male have more RBC count; they are more active compared to

females

b. Age – immature has increase RBC count

c. Physical activities

d. Altitude – lower oxygen in air which causes erythropoiesis to occur

e. Diet/nutrition – obesity may have higher count of RBC because of increase degree of

abdominal pressure

f. Species – poultry have higher level of RBC because of egg formation

5. Importance of erythropoietin

Erythropoietin stimulates red bone marrow to produce more RBC. Thus, when O2 levels in

the blood decrease the production of erythropoietin increases red blood cells production

the increased number of RBC increases the ability of the blood transport oxygen. This

mechanism returns blood O2 levels to normal and maintains homeostasis by increasing the

delivery of tissues.

“Platelet Counting”

Platelets (thrombocytes) – not true cells

- Geminate from big leukocytes called megakaryocytes

- Appear purple color and are more intense than red cells

Materials

1. Hemocytometer

2. Thoma pipette w/ red bead (0.5 101 mark)

3. Rees & Ecker solution

Procedure:

1. Make a venipuncture and draw blood into an RBC diluting pipette to the 0.5 mark and

then draw Rees and Ecker solution up to the 101 mark

2. Mix vigorously using the figure 8 direction for 5 minutes

3. Discard the 4 first drops and then fill both chambers of the hemocytometer similara to

erythrocyte count

4. Let it stand for 15 minutes to allow cells to settle

5. Focus the 9 primary squares under LPO then locate the center square (w/ 25 squares)

6. Count the thrombocytes in the entire central rules area on both chambers

Results: total number of platelets X 10,000 = thrombocytes/microliter of blood

Questions

1. Differentiate thrombocytopenia and thrombocytosis

Thrombocytopenia is any disorder in which there is an abnormally low amount of

platelets. Platelets are parts of the blood that help blood to clot

Thrombocytosis increase in the number of platelets in the blood which tends to cause clots

to form; associated with many neoplasms and chronic infections and other diseases.

2. Discuss importance of platelets

Platelets play a great role in the prevention of blood loss. This prevention is accomplished in

2 ways:

a. Formation of platelet plus

b. Formation of clots

3. What will happen to the individual w. thrombocytopenia

An individual w/ thrombocytopenia will undergo excessive bleeding time because of the

loss of platelets

Leukocyte Count

Leukocyte – density 5000-7000/cu. mm

- Two categories: granulocytes, agranulocytes

- Responsible for the defense of the organism B lymphocyte is the originator of 6 types of wbc (plasma cells, monocyte, lymphocyte, basophil, neutrophil, eosinophil) Materials:

1. Thoma pipette w/ white bead (0.5-11 mark) 2. WBC diluting fluid 3. hemocytometer

Procedure:

a. filling the technique 1. follow the technique described under RBC except for the diluting pipette that has 11

mark above the bulb. 2. Draw blood exactly to the 0.5 mark and wipe the tip of the tube with a clean tissue

paper 3. With the attached rubber tubing suck the wbc diluting fluid up to the 11 mark

above the bulb 4. Shake well for 3 minutes

b. Counting of leukocytes 1. Discard 2-3 drops from the pipette before filling the hemocytometer 2. Stand for about a minute to allow lysis of erythrocyte and for the wbc to settle 3. Under LPO count the number of cells in each of the 4 large corners of the 9 primary

squares c. Calculation

Cells counted in 4 large squares multiply by 10 then X 20 then divided by 4 which equals to leukocyte per microliter

Questions 1.

Morphology Function Distribution Neutrophil Nucleus w/ 2-4 lobes

connected by thin filaments; cytoplasmic granules stain purple, a light pink or reddish purple;

Phagocytizes microorganisms and other substances;

54-62%

Monocyte largest leukocyte; nucleus is round, kidney or horse shoe shaped

diapedesis; become the macrophage and is the weakest WBC

2-10%

Eosinophil “telephone” shaped nucleus; often bilobed

Releases chemicals that reduce inflammation; attacks certain worm or parasites

1-6%

Lymphocyte Immune response Produces antibodies and other chemicals responsive for destroying

28-33%

microorganisms;contributes to allergic reactions, graft rejection, tumor control and regulation of immune system

Basophil Nucleus w/ 2 lobes cytoplasmic stain blue-purple

Vasodilators. Release heparin, histamine and bronchodilators; promotes inflammation

Less than 1%

2. Leukemia - A malignant progressive disease in which the bone marrow and other blood-

forming organs produce increased numbers of immature or abnormal leukocytes, suppressing the production of normal blood cells.

3. Leukocytosis vs. Leukemia Leukocytosis - An increase in the number of white cells in the blood, esp. during an infection Leukemia - A malignant progressive disease in which the bone marrow and other blood-forming organs produce increased numbers of immature or abnormal leukocytes, suppressing the production of normal blood cells.

4. Leukocytosis vs. leucopenia Leukopenia - A reduction in the number of white cells in the blood, typical of various diseases. Leukocytosis - - An increase in the number of white cells in the blood, esp. during an infection

Erythrocytes Leukocytes Non-mobile Mobile Presence of hemoglobin Absent Smaller than wbc Larger than rbc Non-nucleated (mammals); nucleated (birds) Polymorphonucleated Red in color Color depends on the staining granules Segmented vs. Non-segmented neutrophil Nucleus of each mature cell is generally divided into lobes or segmented connected on filaments. The cells that are immature appear as curved or called bands, reduce or even deeply indented but w/ segmentation are known as band cells