Displaying items by tag: Hemotological Tests

This is a newly introduced screening test for platelet function that assesses both platelet adhesion and aggregation. This method uses an instrument called as PFA-100 in which anticoagulated whole blood is passed at a high shear rate through small membranes that have been coated with either collagen and epinephrine or collagen and ADP.

Life history of malaria parasite consists of two cycles of development: asexual cycle or schizogony that occurs in humans and sexual cycle or sporogony that occurs in mosquitoes.

Asexual cycle (human cycle, schizogony)

This occurs in the liver cells and red blood cells of infected humans, and therefore humans are the intermediate hosts of the malaria parasite (Schizogony refers to the process of reproduction in protozoa in which there is production of daughter cells by fission). The human cycle begins when infected female Anopheles mosquito bites a person and sporozoites are injected into the circulation. There are four stages of human cycle.

(a) Pre-erythrocytic schizogony (Hepatic schizogony):

Inoculated sporozoites rapidly leave the circulation to enter the liver cells where they develop into hepatic (pre-erythrocytic) schizonts (Schizonts are cells undergoing schizogony). One sporozoite produces one tissue form. Hepatic schizonts rupture to release numerous merozoites in circulation (Merozoites are daughter cells produced after schizogony). Up to 40,000 merozoites are produced in the hepatic schizont.

In P. falciparum infection, all of the hepatic schizonts mature and rupture simultaneously; dormant forms do not persist in hepatocytes. In contrast, some of the sporozoites of P. vivax and P. ovale remain dormant after entering liver cells and develop into schizonts after some delay. Such persistent forms are called as hypnozoites; they develop into schizonts at a later date and are a cause of relapse.

(b) Erythrocytic schizogony:

Merozoites released from rupture of hepatic schizonts enter the red blood cells via specific surface receptors. These merozoites become trophozoites that utilize red cell contents for their metabolism. A brown-black granular pigment (malaria pigment or hemozoin) is produced due to breakdown of hemoglobin by malaria parasites. The fully formed trophozoite develops into a schizont by multiple nuclear and cytoplasmic divisions. Mature schizonts rupture to release merozoites, red cell contents, malarial toxins, and malarial pigment. (This pigment is taken up by monocytes in peripheral blood and by macrophages of reticulo-endothelial system. In severe cases, organs which are rich in macrophages like spleen, liver, lymph nodes, and bone marrow become slate-gray or black in color due to hemozoin pigment). Rupture of red cell schizonts corresponds with clinical attack of malaria. Released merozoites infect new red cells and enter another erythrocytic schizogony cycle. This leads to rapid amplification of plasmodia in the red cells of the human host. In P. falciparum, P. vivax, and P. ovale infections, cycle of schizogony lasts for 48 hours, while in P. malarie infection it lasts for 72 hours. Merozoites of P. vivax and P. ovale preferentially invade young red cells or reticulocytes while those of P. falciparum infect red cells of all ages. Senescent red cells are preferred by P. malariae.

P. vivax, P. ovale, and P. malariae complete the erythrocyte schizogony in general circulation. Schizonts of P. falciparum induce membrane changes in red cells, which causes them to adhere to the capillary endothelial cells (cytoadherence). Therefore, in P. falciparum infection, erythrocyte schizogony is completed in capillaries of internal organs and usually only ring forms are seen in circulation.

(c) Gametogony:

After several cycles of erythrocytic schizogony, some merozoites, instead of developing into trophozoites and schizonts, transform into male and female gametocytes. These sexual forms are infective to mosquito and the person harboring them is called as a “carrier”. Gametocytes are not pathogenic for humans.

(d) Exoerythrocytic schizogony:

In P. vivax and P. ovale infections, some of the sporozoites in liver cells persist and remain dormant. These dormant forms in liver cells are called as hypnozoites. They become active and develop into schizonts a few days, months, or even years later. These schizonts rupture, release merozoites, and cause relapse. Exoerythrocytic schizogony is absent in P. falciparum infection and therefore relapse does not occur. Hence, P. vivax and P. ovale are called as relapsing plasmodia while P. falciparum and P. malariae are known as non-relapsing plasmodia.

Sexual cycle (mosquito cycle, sporogony)

The sexual cycle begins when a female Anopheles mosquito ingests mature male and female gametocytes during a blood meal. First, 4-8 microgametes are produced from one male gametocyte (microgametocyte) in the stomach of the mosquito; this is called as exflagellation. The female gametocyte (macrogametocyte) undergoes maturation to produce one macrogamete. By chemotaxis, microgametes are attracted toward the macrogamete; one of the microgametes fertilizes the macrogamete to produce a zygote. The zygote becomes motile and is called as ookinete. Ookinete penetrates the lining of the stomach and comes to lie on the outer surface of the stomach where it develops into an oocyst. On further growth and maturation, multiple sporozoites are formed within the oocyst. After complete maturation, oocyst ruptures to release sporozoites into the body cavity of the mosquito. Most of the sporozoites migrate to the salivary glands. Infection is transmitted to the humans by the bite of the mosquito through saliva when it takes a blood meal.

Reticulocytes

Reticulocytes are young or juvenile red cells released from the bone marrow into the bloodstream and that contain remnants of ribonucleic acid (RNA) and ribosomes but no nucleus. After staining with a supravital dye such as new methylene blue, RNA appears as blue precipitating granules or filaments within the red cells. Following supravital staining, any nonnucleated red cell containing 2 or more granules of bluestained material is considered as a reticulocyte (The College of American Pathology). Supravital staining refers to staining of cells in a living state before they are killed by fixation or drying or with passage of time. Reticulocyte count is performed by manual method.

PRINCIPLE

A few drops of blood (collected in EDTA) are incubated with new methylene blue solution which stains granules of RNA in red cells. A thin smear is prepared on a glass slide from the mixture and reticulocytes are counted under the microscope. Number of reticulocytes is expressed as a percentage of red cells.

REAGENT

New methylene blue solution is prepared as follows:

• New methylene blue: 1.0 gm
• Sodium citrate: 0.6 gm
• Sodium chloride: 0.7 gm
• Distilled water: 100 ml

Reagent should be kept stored in a refrigerator at 2-6°C and filtered before use.
Suitable alternatives to new methylene blue are brilliant cresyl blue and azure B.

SPECIMEN

Capillary blood or EDTA anticoagulated venous blood can be used.

METHOD

(1) Take 2-3 drops of filtered new methylene blue solution in a 12 × 75 mm test tube.

(2) Add equal amount of blood and mix well.

(3) Keep the mixture at room temperature or at 37°C for 15 minutes.

(4) After gentle mixing, place a small drop from the mixture on a glass slide, prepare a thin smear, and allow to dry in the air.

(5) Examine under the microscope using oil-immersion objective. Mature red cells stain pale green blue. Reticulocytes show deep blue precipitates of fine granules and filaments in the form of a network (reticulum). Most immature reticulocytes show a large amount of precipitated material in the form of a mass, while the most mature reticulocytes show only a few granules or strands. Any nonnucleated red cell is considered as a reticulocyte if it contains 2 or more blue-stained particles of ribosomal RNA.

(6) Count 1000 red cells and note the number of red cells that are reticulocytes. Counting error is minimized if size of the microscopic field is reduced. This is achieved by using a Miller ocular disk inserted in the eyepiece; it divides the field into two squares (one nine times larger in size than the other). Reticulocytes are counted in both the squares and the red cells are counted in the smaller square.

REPORTING THE RESULT

(1) Reticulocyte percentage: The most common method of reporting is reticulocyte percentage which is calculated from the following formula:

Where NR is the Number of reticulocyte counted and NRBC is number of red blood cell counted.

Reference range is 0.5%-2.5% in adults and children. Reticulocyte count is higher in newborns.

(2) Absolute reticulocyte count = Reticulocyte percentage × Red cell count
Normal: 50,000 to 85,000/cmm

(3) Corrected reticulocyte count (Reticulocyte index)

Corrected reticulocyte count > 2% indicates reticulocyte release appropriate for the degree of anemia. If < 2%, reticulocyte release is inappropriate.

(4) Reticulocyte maturation production index

REFERENCE RANGES

• Reticulocyte percentage: 0.5 2.5%
• Absolute reticulocyte count: 50,000-85,000/cmm

• As one of the baseline studies in anemia with no obvious cause
• To diagnose anemia due to ineffective erythropoiesis (premature destruction of red cell precursors in bone marrow seen in megaloblastic anemia and thalassemia) or due to decreased production of red cells: In hypoplastic anemia or in ineffective erythropoiesis, reticulocyte count is low as compared to the degree of anemia. Increased erythropoiesis (e.g. in hemolytic anemia, blood loss, or specific treatment of nutritional anemia) is associated with increased reticulocyte count. Thus reticulocyte count is used to differentiate hypoproliferative anemia from hyperproliferative anemia.
• To assess response to specific therapy in iron deficiency and megaloblastic anemias.
• To assess response to erythropoietin therapy in anemia of chronic renal failure.
• To follow the course of bone marrow transplantation for engraftment
• To assess recovery from myelosuppressive therapy
• To assess anemia in neonate

The erythrocyte sedimentation rate (ESR) measures the rate of settling (sedimentation) of erythrocytes in anticoagulated whole blood. Anticoagulated blood is allowed to stand in a glass tube for 1 hour and the length of column of plasma above the red cells is measured in millimeters; this corresponds to ESR. There are four different methods for the estimation of ESR.

This method conceived and formulated by Stott and Lewis in 1995. This method is much similar in principle to the now outdated Tallqvist method. Positive technical changes have been made to improve the validity, accuracy and reliability. This method is simple, swift, reliable and inexpensive. This method is reliable and trustworthy within 1 gram/dl for diagnosis of anemia. The World Health Organization (WHO) Hemoglobin Color Scale consists of a printed set of colors corresponding to the hemoglobin value from 4 to 14 grams/dl. On a strip of chromatography paper, a drop of blood is placed and then the developed color is matched visually against the printed color scale. Research has proven that performance is greater than 90% in detecting anemia and 86% in classifying the grade of anemia. The World Health Organization (WHO) has developed hemoglobin color scale after extensive and vast field trails. It is mainly planned for the detection, treatment and control of anemia in under-resourced countries. It is especially use for the screening of blood donors, for screening women and children in health scheme, examine iron therapy, selection-making concerning referral to a hospital, and as a point of care tool.

Variations of leukocyte count occur in many infectious, hematologic, inflammatory, and neoplastic diseases.

Therefore, laboratory evaluation of almost all patients begins with the examination of the patient’s blood for total leukocyte count and examination of the peripheral blood smear for the differential leukocyte count as well as blood cells picture. Usually, some clinical interpretations may be made from the total leukocyte count and differential leukocyte count.

From total leukocyte count and differential leukocyte count, the absolute count of each leukocyte type can be calculated. The absolute leukocyte count provides a more accurate picture than the differential leukocyte count. (For example, a chronic lymphocytic leukemia patient has a total leukocyte count of 100 x 10³ cells µI and a differential leukocyte count of 7 percent neutrophils and 90 percent lymphocytes. By looking at the differential leukocyte count of 7 percent neutrophils alone one get the impression of very low neutrophils in this patient But if the absolute count of neutrophils is calculated, one will be surprised to see a normal neutrophil number in this patient.

Absolute neutrophil count = Total leukocyte count x neutrophil percent
=100 x 10³ x 7/100 = 7 x 10³ = 7000 / µl

At times of acute bacterial infection enormous numbers of neutrophils are required. Therefore, large number of neutrophils is released from bone marrow to cope with this requirement. Consequently, the number as well as the percentage of neutrophils in the blood increases several fold. Hence, an increase in total leukocyte count with increase in percentage of neutrophil is taken as an important indication of acute bacterial infection.

Leukocytosis

The terms leukocytosis and leukopenia indicate increase or decrease in the total number of leukocytes, respectively. Increase in numbers of neutrophils, eosinophils, lymphocytes, and monocytes are known as neutrophilia, eosinophilia, lymphocytosis, and monocytosis respectively.

i. Neutrophilia is the increase in peripheral blood absolute neutrophil count, above the upper limit of normal of 7.5 x 10⁹/L (in adults). Bacterial infection is one of the common causes of neutrophilia. Neutrophilia is not a feature of viral infections (However, the development of neutrophila late in the course of a viral infection may indicate the emergence of secondary bacterial infection). There is a storage pool of mature neutrophils in the bone marrow (Such storage pool does not occur for other leukocytes).

In response to stress, such as infections the neutrophils from storage pool are released into circulation, resulting in a rise in total leukocyte count and neutrophil percentage. Moreover, there is increased neutrophil production in the bone marrow. Increased neutrophil production during bacterial infection is usually associated with the entry of less mature neutrophils from bone marrow into blood. This is indicated by the appearance of neutrophils with lesser number of nuclear segmentation in the peripheral blood picture. Band cells may also be seen in the peripheral blood smear.

This is referred to as a ‘shift to the left’. The leukemoid reaction is a reactive and excessive leukocytosis, wherein the peripheral blood smear shows the presence of immature cells (e.g. myeloblasts, promyelocytes, and myelocytes). Leukemoid picture occurs in severe or chronic infections and hemolysis. Another most common change that occurs in neutrophils during infection is the presence of toxic basophilic inclusions in the cytoplasm.

Eosinophilia is an increase in peripheral blood absolute eosinophil count beyond 0.4 x 10⁹/L (in adults). Eosinophilia is usually associated with allergic conditions such as asthma and hay fever and parasitic infections. Eosinophilia may also occur in reactions to drugs.

Leukopenia

Decrease in total leukocyte count is known as leukopenia. Reduction in the number of neutrophils is the most frequent cause of leukopenia.

i. Neutropenia is the decrease in peripheral blood absolute neutrophil count below the lower limit of normal of 2 x 10⁹/L (in adults). Neutropenic patients are more vulnerable to infection.

ii. Lymphopenia in adults is the decrease in peripheral blood absolute lymphocyte count below the lower limit of normal of 1.5 x 10⁹/L. Lymphopenia is common in the leukopenic prodromal phase of many viral infections. A selective depletion of helper T lymphocytes (CD4⁺) with or without absolute lymphopenia occurs in acquired immunodeficiency disease (AIDS).

REFERENCES
1. Brown, B.A., Haemotology, Principles and Procedures, Lea & Febiger, U.S.A., 1976.
2. Hoffbrand, A. V. and Pettit, 1. E., Essential Haemotology, Blackwell Scientific Publication, U.S.A., 1980.
3. Kassirsky, I. and Alexeev, G., Clinical Haemotology, Mir Publishers, U.S.S.R., 1972.
4. Widmann, F.K., Clinical interpretation of Laboratory tests, F.A. Davis Company, U.S.A., 1985.
5. Kirk, C.J.C. et al, Basic Medical Laboratory Technology, Pitman Book Ltd., U.K. 1982.
6. Green, J.H., An Introduction to human Physiology, Oxford University Press, U.K., 1980.

Total leukocyte count (TLC) refers to the number of white blood cells in 1 μl of blood (or in 1 liter of blood if the result is expressed in SI units). There are two methods for estimation of TLC:

• Manual or microscopic method
• Automated method

A differential leukocyte count should always be performed along with TLC to obtain the absolute cell counts.

The purpose of carrying out TLC is to detect increase or decrease in the total number of white cells in blood, i.e. leukocytosis or leukopenia respectively. TLC is carried out in the investigation of infections, any fever, hematologic disorders, malignancy, and for follow-up of chemotherapy or radiotherapy.

MANUAL METHOD

Principle

A sample of whole blood is mixed with a diluent, which lyses red cells and stains nuclei of white blood cells. White blood cells are counted in a hemocytometer counting chamber under the microscope and the result is expressed as total number of leukocytes per μl of blood or per liter of blood.

Equipment

(1) Hemocytometer or counting chamber with coverglass: The recommended hemocytometer is one with improved Neubauer rulings and metalized surface. There are two ruled areas on the surface of the chamber. Each ruled area is 3 mm × 3 mm in size and consists of 9 large squares with each large square measuring 1 mm × 1 mm. When the special thick coverglass is placed over the ruled area, the volume occupied by the diluted blood in each large square is 0.1 ml. In the improved Neubauer chamber, the central large square is divided into 25 squares, each of which is further subdivided into 16 small squares. A group of 16 small squares is separated by closely ruled triple lines. Metalized surface makes background rulings and cells easily visible. The 4 large corner squares are used for counting leukocytes, while the central large square is used for counting platelets and red blood cells. Only special coverglass, which is intended for use with hemocytometer, should be used. It should be thick and optically flat. When the special coverglass is placed on the surface of the chamber, a volumetric chamber with constant depth and volume throughout its entire area is formed. Ordinary coverslips should never be employed since they do not provide constant depth to the underlying chamber due to bowing.

When the special cover glass is placed over the ruled area of the chamber and pressed, Newton’s rings (colored refraction or rainbow colored rings) appear between the two glass surfaces; their formation indicates the correct placement of the cover glass.

(2) Pipette calibrated to deliver 20 μl (0.02 ml, 20 cmm): WBC bulb pipettes, which have a bulb for dilution and mixing (Thoma pipettes) are no longer recommended. This is because blood and diluting fluid cannot be mixed adequately inside the bulb of the pipette. Bulb pipettes are also difficult to calibrate, costly, and charging of counting chamber is difficult. Tips of pipettes often chip easily and unnecessarily small volume of blood needs to be used.

1. Graduated pipette, 1 ml.
2. Pasteur pipett
3. Test tube (75 × 12 mm).

Reagent

WBC diluting fluid (Turk’s fluid) consists of a weak acid solution (which hemolyzes red cells) and gentian violet (which stains leucocyte nuclei deep violet). Diluting fluid also suspends and disperses the cells and facilitates counting. Its composition is as follows:

• Acetic acid, glacial 2 ml
• Gentian violet, 1% aqueous 1 ml
• Distilled water to make 100 ml

Specimen

EDTA anticoagulated venous blood or blood obtained by skin puncture is used. (Heparin should not be used since it causes leukocyte clumping). While collecting capillary blood from the finger, excess squeezing should be avoided so as not to dilute blood with tissue fluid.

Method

(1) Dilution of blood: Take 0.38 ml of diluting fluid in a test tube. To this, add exactly 20 μl of blood and mix. This produces 1:20 dilution. Alternatively, 0.1 ml of blood can be added to 1.9 ml of diluting fluid to get the same dilution.

(2) Charging the counting chamber: Place a coverglass over the hemocytometer. Draw some of the diluted blood in a Pasteur pipette. Holding the Pasteur pipette at an angle of 45° and placing its tip between the coverglass and the chamber, fill one of the ruled areas of the hemocytometer with the sample. The sample should cover the entire ruled area, should not contain air bubbles, and should not flow into the side channels. Allow 2 minutes for settling of cells.

(3) Counting the cells: Place the charged hemocytometer on the microscope stage. With the illumination reduced to give sufficient contrast, bring the rulings and the white cells under the focus of the low power objective (× 10). White cells appear as small black dots. Count the number of white cells in four large corner squares. (To reduce the error of distribution, counting of cells in all the nine squares is preferable). To correct for the random distribution of cells lying on the margins of the square, cells which are touching the left-hand lines or upper lines of the square are included in the count, while cells touching the lower and right margins are excluded.

(a) Calculation of TLC:

Where Nw is the number of WBCs counted, Cd is the correction of dilution, Cv is the correction of volume and NLS is the number of large squares counted.

(b) TLC/L = Number of WBCs counted × 50 × 10₆ (10₆ is the correction factor to convert count in 1 μl to count in 1 liter). Example: If 200 WBCs are counted in 4 large squares, TLC/μl will be 10,000/μl and TLC/liter will be 10.0 × 10₉/liter.

If TLC is more than 50,000/ml, then dilution of blood should be increased to 1:40 to increase the accuracy of the result.

If TLC is less than 2,000/ml then lesser dilution should be used.

Expression of TLC: Conventionally, TLC is expressed as cells/μl or cells/cmm or cells/mm³. In SI units, TLC is expressed as cells × 10₉/liter. Conversion factors for conventional to SI units is 0.001 and SI to conventional units is 1000.

Correction of TLC for nucleated red cells: The diluting fluid does not lyse nucleated red cells or erythroblasts. Therefore, they are counted as leukocytes in hemocytometer. If erythroblasts are markedly increased in the blood sample, overestimation of TLC can occur. To avoid this if erythroblasts are greater than 10 per 100 leukocytes as seen on blood film, TLC should be corrected for nucleated red cells by the following formula:

Where C_TLC is the Corrected TLC/μl, TLC is the Total Leukocyte Count and N_RBC is the Nucleated RBCs per 100 WBCs.

REFERENCE RANGES

• Adults 4000-11,000/μl
• At birth 10,000-26000/μl
• 1 year 6,000-16,000/μl
• 6-12 year 5,000-13,000/μl
• Pregnancy up to 15,000/μl

CRITICAL VALUES

• TLC < 2000/μl or > 50000/μl