Total Leukocyte Count

Specimens

• Double oxalated or EDTA blood
• Capillary blood (specimen need not be a fasting sample).

Requirements

Microscope

Improved Neubauer Chamber

WBC pipette

WBC diluting fluid: It is prepared as follows:

• Glacial acetic acid: 2.0 ml
• 1 % (w/v) gentian violet: 1.0 ml
• Distilled water: 97 ml

This solution is stable at room temperature (25°C ± 5°C). A pinch of thymol maybe added as preservative.

Principle

• The glacial acetic acid lyses the red cells while the gentian violet slightly stains the nuclei of the leukocytes. 
• The blood specimen is diluted 1:20 in a WBC pipette with the diluting fluid and the cells are counted under low power of the micro scope by using a counting chamber.
• The numbers of cells in undiluted blood are reported per cu mm (μl) of whole blood.

 

Procedure

• Draw blood up to 0.5 mark of a WBC pipette.
• Carefully, wipe excess blood outside the pipette by using cotton. Draw diluting fluid up to 11 marks.
• Mix the contents in the pipette and after five minutes by discarding few drops, fill the counting chamber and allow the cells to settle for two to three minutes.
• Since Bulb pipettes are not recommended following procedure is performed
• Make a 1:20 dilution of blood by adding 20 μl of blood to (1 38 ml of diluting fluid in a glass tube (10 * 75 mm) Cork the tube tightly and mix the suspension by rotating in a cell-suspension mixer for at least 1 minute. Fill the Neubauer counting chamber by means of a Pasteur pipette or glass capillary.
• Focus on one of the ‘W’ marked areas (each having 16 small squares) by turning objective to low power. (10 X).
• Count cells in all four W marked corners.

Calculations

Number of white cells/cu mm (μl) of whole blood = (number of white cells counted * dilution) / (area counted * depth of fluid)

Where: 

• Dilution = 20
• Area counted 4 * 1 sq.mm = 4 sq.mm
• Depth of fluid = 0.1 mm (constant)

Hence number of white cells per cu mm (μl) of whole blood = (No. of cells counted * 20) / (4 * 0.1) = No of cells counted * 50

No. of leukocyte per liter of blood = No. of cells per cu mm (ml) * 106 or

Use the following formula

WBC count / liter = [No. of cells counted (per 1mm2 area) / volume counted (μl)] * dilution * 106

 

Precautions taken are exactly the same as for REC counting technique.

• The sources of error are also same as for RBC counting technique. However; in the case of WBC counting extra care is taken during the preparation and storage of WBC diluting fluid. 
• It should be perfectly free from dust particles and yeast cells, otherwise falsely high counts are obtained due to the presence of yeast cells and dust particles.

Normal values

• Adults: 4,000-10,000/cu mm (μl)
• At birth: 10,000-25,000/cu mm (μl)
• 1 to 3 years: 6,000-18,000/cu mm (μl)
• 4 to 7 years: 6,000-15,000/cu mm (μl)
• 8 to 12 years: 4,500-13,500/cu mm (μl)

Error of the total white cell count

• The inherent distribution error = λ1/2, here λ = total number of cells in each area.
• The error as high as 20% may make difference between 5.0 and 6.0 x 109 cells per liter, which is of little practical significance.
• The error can be reduced by counting more cells. If 400 cells are counted the error is reduced to 5%.
• Error may also be caused due to dirt clumped RBC debris or due to clumping of leukocytes.

Interpretation of Results:

Clinical Significance

Increase in total leukocyte count of more than 10,000/cu mm (μl) is known as

leukocytosis and decrease of less than 4 000 cu mm (μl) as leukopenia.

Causes of leukocytosis

Pathological

• It is common for a transient period in infections. The degree of rise in leukocytes depends on the type and severity of the infection and the response of the body.
• The infection may be 1) bacterial 2) viral 3) protozoal (malaria) or 4) parasitic (filaria, hookworm infection).
• Leukocytosis is also observed in severe hemorrhage and in Leukemia.

Physiological

• Age: At birth the total leukocyte count is about 18,000/cu mm (μl). It drops gradually to adult level.
• Pregnancy: At ‘full term’ the total count tends to be about 12,000 to 15,000/cu mm (μl). It rises soon after delivery and then gradually returns to normal.
• High temperature
• Severe pain
• Muscular exercise

Causes of Leukopenia

Certain viral and bacterial infections (typhoid) lead to leukopenia rather than leukocytosis.

Infections

• Bacterial (typhoid. paratyphoid, tuberculosis, etc)
• Viral (hepatitis, influenza, measles, etc)
• Protozoal (malaria)

Functions of WBCs:

Neutrophil

• Neutrophils defend against bacterial or fungal infection and other very small inflammatory processes that are usually first responders to microbial infection; their activity and death in large numbers forms pus.
• Neutrophils are very active in phagocytosing bacteria and are present in large amount in the pus of wounds.
• These cells are not able to renew their lysosomes used in digesting microbes and die after having phagocytosed a few pathogens. Most common cell seen in acute inflammation comes in and kills foreign substance.
• They stick to walls of the blood vessels of the infected or injured area and prevent the entry of any foreign particles into the blood stream. They can be found in the pus of wounds.

Eosinophil

• Eosinophils primarily deal with parasitic infections and an increase in them may indicate such.
• Eosinophils are also the predominant inflammatory cells in allergic reactions.
• The most important causes of eosinophilia include allergies such as asthma, hay fever, and hives; and also parasitic infections.
• Disorder in the eosinophils may lead to over active response to a disease in the Immune system in an attempt to destroy foreign organisms

Basophil

• Basophils are chiefly responsible for allergic and antigen response by releasing the chemical histamine causing inflammation.
• They secrete anti coagulant and anti bodies which mediate hypersensitivity reaction within the blood. They are the reason for immediate immune reaction against external germs and diseases.

Lymphocyte

They produce antibodies against toxins secreted by bacteria and infecting germs. These antibodies will be excreted into the plasma to kill bacteria in the blood and also act as anti toxins.

The blood has three types of lymphocytes:

B cells:

B cells make antibodies that bind to pathogens to enable their destruction. (B cells not only make antibodies that bind to pathogens, but after an attack, some B cells will retain the ability to produce an antibody to serve as a 'memory' system.)

T cells:

CD4+ (helper) T cells co-ordinate the immune response and are important in the defense against intracellular bacteria. In acute HIV infection, these T cells are the main index to identify the individual's immune system activity.

CD8+ cytotoxic T cells are able to kill virus-infected and tumor cells.

Natural killer cells: Natural killer cells are able to kill cells of the body which are displaying a signal to kill them, as they have been infected by a virus or have become cancerous.

Monocyte

• Monocytes share the "vacuum cleaner" (phagocytosis) function of neutrophils, but are much longer lived as they have an additional role: they present pieces of pathogens to T cells so that the pathogens may be recognized again and killed, or so that an antibody response may be mounted.
• Monocytes eventually leave the bloodstream to become tissue macrophages which remove dead cell debris as well as attacking microorganisms.
• Neither of these can be dealt with effectively by the neutrophils. Unlike neutrophils, monocytes are able to replace their lysosomal contents and are thought to have a much longer active life. 

Leukogram

A tabulation of the leukocytes present in a blood sample.

Leukergy

The clumping of white blood cells that accompanies some inflammations and infections

Lupus Erythematosus

• Lupus erythematosus is a category for a collection of diseases with similar underlying problems with immunity. This is an autoimmune disease in which any part of the body may be attacked by the immune system resulting in inflammations and tissue damage.
• Symptoms of these diseases can affect many different body systems, including joints, skin, kidneys, blood cells, heart, and lungs.
• Four main types of lupus exist — systemic lupus erythematosus, discoid lupus erythematosus, drug-induced lupus erythematosus and neonatal lupus erythematosus. Of these, systemic lupus erythematosus is the most common and serious form of lupus.

Total Erythrocyte Count..Clinical Pathology

An RBC count is a blood test that tells how many red blood cells (RBCs) you have.

Why the Test is Performed

• The RBC count is almost always part of the CBCCBC (complete blood count) test.
• The test can help diagnose anemia and other conditions affecting red blood cells.
• Additional conditions under which an RBC count may be performed:
• Alport syndromeAlport syndrome
• Drug-induced immune hemolytic anemia
• Hemolytic anemia due to G6PD deficiencyHemolytic anemia due to G6PD deficiency
• Hereditary anemias, such as thalassemia
• Idiopathic autoimmune hemolytic anemiaIdiopathic autoimmune hemolytic anemia
• Immune hemolytic anemiaImmune hemolytic anemia
• Macroglobulinemia of WaldenstromMacroglobulinemia of Waldenstrom
• Paroxysmal nocturnal hemoglobinuria (PNH)Paroxysmal nocturnal hemoglobinuria (PNH)
• Primary myelofibrosisPrimary myelofibrosis 

Normal red blood cells values at various ages are:

• Newborns: 4.8 - 7.2 million
• Adults: (males): 4.6 - 6.0 million
• (Females): 4.2-5.0 million
• Pregnancy: slightly lower than normal adult values
• Children: 3.8 – 5.5 million

Procedure:

• A blood collection and diluting system is first assembled.
• To gain access to diluting solution, the reservoir seal needs is punctured.
• A finger is lanced and a small drop of blood is allowed to accumulate.
• Blood is drawn into the pipette.
• Blood in the pipette is transferred to diluting reservoir, which contains a solution (0.9% saline) which is isotonic for blood cells and prevents them from rupturing. Usually, there is enough fluid in the reservoir to dilute the blood by a factor of 200-400 times and the dilution factor is provided by the manufacturer.
• The reservoir and pipette are converted into a dropper assembly by removing the pipette from the reservoir and reseating it in the reverse position.
• A cover slip is placed on a hemacytometer and the dropper assembly is carefull positioned next to the coverslip. A few drops of the solution are squeezed from the reservoir.
• The hemacytometer draws a set amount of solution under the coverslip. Usually this is limited to a depth of 0.1mm.
• Etched in the hemacytometer is a counting grid which appears under magnification.
• Five grid squares are used to count RBCs;
• All RBCs in each grid square are counted.
• RBCs count = RBCs counted in 5 squares*volume correction factor*dilution factor
• Correction factor = desired volume/ calculated volume

What Abnormal Results Mean

Decreased RBC

• Trauma
• Burns
• Pregnancy
• Hemolytic anemia
• Hemorrhagic infections
• Gastrointestinal (GI) or other vascular bleed
• Iron deficiency anemia
• Vitamin B12 or folate deficiency
• bone marrow damage
• Metabolic disorders
• Chronic inflammation

Increased RBC

• Dehydration
• Pulmonary disease
• Congenital heart disease
• Polycythemia vera
• Renal problems
• Over-transfusion of whole blood
• Tissue hypoxia

Polycythemia vera

• Polycythemia vera is a disease of unknown origin that results in an abnormal increase in red blood cells. Polycythemia vera is referred to as a "primary polycythemia" because the overproduction of red blood cells does not result from hypoxia.
• The term "vera" means true; thus polycythemia vera refers specifically to overproduction of red blood cells in the bone marrow not caused by a physiologic need.
• Polycythemia vera is treated by radioactive phosphorus to slow down bone marrow overproduction of red blood cells.
• Hydration is an important consideration when caring for patients with abnormally high red blood cell counts.
• Very high RBC mass will slow blood velocity and increase the risk of intravascular clotting.
• Examples of "secondary polycythemias", that occur in response to hypoxia, are chronic lung disease in adults and children with congenital heart defects characterized by cyanosis.

Anemia:

• Anemia describes the condition in which the number of red blood cells in the blood is low. Anemia is of three types depending upon the size of the red blood cells. Size is reflected in mean corpuscular volume (MCV).
• The mean corpuscular volume, or "mean cell volume" (MCV), is a measure of the average red blood cell volume that is reported as part of a standard complete blood count.
• In patients with anemia, it is the MCV measurement that allows classification as either a microcytic anemia (MCV below normal range), normocytic anemia (MCV within normal range) or macrocytic anemia (MCV above normal range).

Calculation

• It can be calculated (in litres) by dividing the hematocrit by the red blood cell count (number of red blood cells per litre). The result is typically reported in femtolitres.
• If the MCV was determined by automated equipment, the result can be compared to RBC morphology on a peripheral blood smear. Any deviation would be indicative of either faulty equipment or technician error.
• For further specification, it can be used to calculate red blood cell distribution width (RDW).

Interpretation

The normal reference range is typically 80-100 fL[1].

High

In presence of hemolytic anaemia, presence of reticulocytes can increase MCV. In pernicious anemia (macrocytic), MCV can range up to 150 femtolitres. An elevated MCV is also associated with alcoholism[2] (as are an elevated GGT and a ratio of AST:ALT of 2:1). Vitamin B12 and/or Folic Acid deficiency has also been associated with macrocytic anemia (high MCV numbers).

Low

• The most common causes of microcytic anemia are iron deficiency (due to inadequate dietary intake, gastrointestinal blood loss, or menstrual blood loss), thalassemia, or chronic disease.
•  
• A low MCV number in a patient with a positive stool guaiac test (bloody stool) is highly suggestive of GI cancer.
• In iron deficiency anemia (microcytic anemia), it can be as low as 60 to 70 femtolitres. In cases of thalassemia, the MCV may be low even though the patient is not iron deficient.

Worked example

• If the red blood cells are smaller than normal, this is called microcytic anemia. The major causes of this type are iron deficiency (low level iron) anemia and thalassemia (inherited disorders of hemoglobin).
• If the red blood cells size are normal in size (but low in number), this is called normocytic anemia, such as anemia that accompanies chronic disease or anemia related to kidney disease.
• If red blood cells are larger than normal, then it is called macrocytic anemia. Major causes of this type are pernicious anemia and anemia related to alcoholism.

PCV(Hematocrit Method)..Path-504

It is proportion of blood cells occupied by the RBCs. It is normally 48% for men and 38% for women. It is considered integral part of a person’s complete blood count (CBC).

Hematocrit stands for hema=”blood” and crit=”judgment”. There are two methods for performing hematocrit method. Indicators of anemia include Hb estimation, RBC count and PCV. Microhematocrit and Macrohematocrit Methods:

Interpretation of Results:

Polycythemia and Doping:

Polycythemia and doping can both be detected by PCV at regular intervals. 

Acute and Chronic Blood Loss:

Hemoglobin Estimation..clinical pathology

Hemoglobin is a protein present in RBCs for exchange of gases with tissues.  Blood is a specialized type of connective tissue in the form of fluid. It has two components called as plasma and cells.

• Plasma contains 12 coagulation factors. Serum is formed by the removal of these coagulation factors. Serum is collected in red color vacuotainer. It does not let the blood clot.
• The ratio of cells in the blood is RBCs, WBCs and Platelets as 500:1:30 respectively.

There are four methods for the determination of hemoglobin in the blood;

1. Sahli’s method..Outdated
2. Cyanmet Hb method..Updated
3. Spectrophotometer
4. Haden Haussen method

Sahli’s Method of Hemoglobin Determination:

Apparatus:

• Sahli’s tube which is having red and yellow scales on two sides. Red scale is percentage scale and yellow scale is gram percentage or g/100ml scale.
• Heamometer which is having two standards.
• Sahli’s pipette.
• Error percentage is 3%.

Procedure:

• In sahli’s tube, take N/10 HCL(1/10^th of the original HCL) up to 10^th level of scale.
• In sahli’s pipette, take 0.02ml(20microleter) blood.
• Add blood from pipette into tube.
• HCL will cause the lysis of the blood cells and hemoglobin is released.
• Hb after combining with HCL, forms acid hematin which is of tan color.
• Put tube in the hemometer and continuously add drops of distilled water and shake with the stirrer until color matches. Then, take the reading.

Interpretation of the results:

Heam portion of hemoglobin is a porphyrin type pigment and contains iron.

Types of Hemoglobin:

Heam Cycle: