Chapter 18: Haematology tests

Objectives
On completion of this chapter you should be able to: explain the principle of the cyanmethaemoglobin method for haemoglobin estimation understand how to perform a manual haematocrit describe the procedure for manual white cell and platelet counts calculate red cell indices to evaluate red cell size understand the usefulness of the erythrocyte sedimentation rate describe the preparation of a blood film and the performance of a white cell differential count define the significance of the reticulocyte count and the method employed to calculate absolute numbers understand the principle of the latex test used to detect infectious mononucleosis.

Introduction
In the haematology laboratory there are various techniques, both automated and manual, to determine the components of the Full Blood Count (FBC), otherwise known as the Complete Blood Count (CBC). Automated methods are used in large laboratories where rapid analysis of large numbers of tests are required. The instrumentation is expensive, but requires only a small number of trained staff to operate. Once calibrated, the instrument gives accurate, precise, reproducible results. The majority of laboratories use automated analysers exclusively. The mechanics of these instruments will be discussed in a later chapter of this Study Guide. The need for manual techniques, as part of the FBC, is still evident in todays modern haematology laboratory. Manual techniques are invaluable in the following instances: Instrument breakdownSpecialised instrumentation requires trained engineers (based in capital cities) to perform major repairs. Urgent specimens already in the laboratory need to be processed without delay, manually, if no back up analyser is available. Leukaemic patients with grossly elevated white blood cell counts require manual haemoglobins to be performed. Excessive white blood cells cause turbidity, falsely elevating haemoglobin levels. Testing of body fluids (such as Cerebrospinal fluid) require manual cell counts. Low numbers of white blood cells will be below the instrument counting threshold. Paediatric specimens have insufficient volume for some automated tests, requiring manual procedures, such as Erythrocyte Sedimentation Rate, to be performed.

Haemoglobin
The haemoglobin concentration (Hb) of a sample may be estimated in the laboratory colourimetrically, by the Cyanmethaemoglobin method. This method is recommended by the World Health Organisation, due to the accuracy of the procedure and stability of the reagent. The sample of choice for the cyanmethaemoglobin method is whole blood with EDTA anticoagulant. The method uses a reagent called Drabkins Reagent. Drabkins reagent contains potassium cyanide and potassium ferricyanide. Haemoglobin is converted to methaemoglobin by the potassium ferricyanide. The methaemoglobin is then converted to cyanmethaemoglobin by the potassium cyanide (Scheme 181).
Textbook Biomedical Science Rodak 2007 Chs 14 & 15

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The cyanmethaemoglobin is measured using a spectrophotometer. The concentration of haemoglobin is directly proportional to the absorbance of the cyanmethaemoglobin at 540 nm. Scheme 181: Principle of Drabkins method for haemoglobin estimation Haemoglobin (Fe 2 +) + K 3 Fe(CN)6 Methaemoglobin (Fe3 +)

Methaemoglobin + KCN Cyanmethaemoglobin A standard control sample, of known concentration, is tested at the same time as the unknown sample. Both samples are diluted 1 in 201 with the Drabkins reagent. The tubes are mixed and allowed to stand for 10 minutes at room temperature. Using the Drabkins reagent as a blank, the absorbance of the tubes are read at 540 nm on the spectrophotometer. The concentration of the unknown sample is calculated using the absorbance and concentration of the standard sample as shown in the equation below.
Hb (g/dL ) = Abs of test sample Concentration of standard Abs of standard

Haematocrit
The Haematocrit (Hct) is also known as the Packed Cell Volume (PCV). This test measures the red blood cell concentration in whole blood as a percentage. It is performed on whole blood. The test is performed by filling a glass capillary tube with whole blood. One end of the tube is plugged with plasticine-type putty. The tube is then centrifuged in a Microhaematocrit centrifuge at 10,000 rpm for 5 minutes. The blood separates into three formed elements layers (Figure 181): Red blood cells, white blood cells/platelets (buffy coat layer) and plasma.

Figure 181

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The haematocrit value is determined simply by using a Microhaematocrit Reader (Figure 182) to calculate the percentage. Alternately, if a reader is unavailable, the total blood volume and red blood cell volumes may be measured with a ruler and the percentage calculated.

Figure 182: Microhaematocrit reader

(Source: Clements Medical Equipment)

Red blood cell count

The red blood cell count (RCC) is a determination of the number of red blood cells in each microlitre of blood. Manual red blood cell counts are regarded as unnecessary and often inaccurate, and so, are not usually performed. An estimation of Haemoglobin and Packed cell volume will give the clinician an accurate picture of the patients red blood cell population.

White blood cell count

The white blood cell count (WCC) is a determination of the number of white blood cells in each litre of blood. In the haematology laboratory, manual counts are performed on EDTA anticoagulated whole blood, using a haemocytometer. The Improved Neubauer Haemocytometer is a calibrated counting chamber with visible rulings (Figure 183). For white blood cell countscount the four large corner squares of the figure (16 squares in each). For platelet cell countscount the 25 (darkly ruled) central small squares.

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Figure 183: Improver neubauer haemocytometer counting areas Whole blood is diluted 1 in 20 with 2% Acetic acid (coloured a pale mauve colour by gentian violet). After placing a coverslip on top of the haemocytometer chamber, the diluted blood is introduced into the counting chamber. The haemocytometer is placed in a moist chamber and the cells are allowed to settle for at least 2 minutes. The haemocytometer is mounted on a microscope and the cells are counted using the 40X objective (Figure 18 3). The cell count per litre is calculated based on the size of the area counted (mm3) as shown in the equation below.
number cells counted dilution factor 10 Area (mm 2 ) This amount is divided by 1000 to report the number of cells x 109/L. Total cell count =

Platelet cell count

The platelet cell count (PCC) is the determination of the number of platelets in each litre of blood. Before commencing, the sample must be examined to ensure there are no clots. If the specimen is clotted it must be rejected and recollected, otherwise the platelet count will be inaccurate. Anticoagulated whole blood is diluted in 1 in 100 with 1% aqueous ammonium oxalate, which lyses the red blood cells. The haemocytometer is filled and placed in a moist chamber for 15 minutes to allow the cells time to settle. The haemocytometer is then placed on the microscope and the cells counted using the 40X objective. (Figure 35) As platelets are highly refractile, examination of the platelets is easier if the microscope condenser is lowered. The platelet cell count per litre is calculated on the size of the area counted (mm3) (Figure 183).

Red cell indices

Red cell indices are part of the full blood count that are calculated from the values for the red cell count, haematocrit and haemoglobin. They are MCV, MCH and MCHC. These indices indicate the size and haemoglobin content of the red blood cells.

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1. Mean Corpuscular Volume (MCV) This test describes the average volume in a red blood cell. Since red blood cells are very small, the unit used is femtolitre (fL) or 10-15 L. Hct(%) 10 RCC MCV is used to determine the size of the red blood cells. MCV = 2. Mean Corpuscular Haemoglobin (MCH) This test measures the average weight of haemoglobin in a red cell. Since the red blood cells are very small, the unit used is picogram (pg) or 10-12 g. Hb RCC MCH is also used as an indicator of red blood cell size. MCH = 3. Mean Corpuscular Haemoglobin Concentration (MCHC) This test measures the concentration of haemoglobin in a red blood cell. The unit of measure is grams/ decilitre or g/100mL. Hb 10 Hct The smaller the red blood cell, the higher the concentration of haemoglobin, that is the higher the MCHC. MCHC =

Erythrocyte sedimentation rate

In assessing patients, it is helpful to measure the activation of acute phase response. The acute phase response suggests a physical cause for symptoms. Causes may include infections, trauma, myocardial infarction, degenerative disorders or autoimmune diseases. Serial measurements are used to monitor both the disease and the effects of the treatment prescribed. The Erythrocyte Sedimentation Rate (ESR) is one of the most widely used measurements of acute phase response. The rate of the fall of the red blood cells depends on the specific gravity between the red blood cells and the plasma. The proteins produced during an acute phase response buffer the electrostatic repellent forces on the membranes of the red cells, causing them to aggregate together and have a quicker fall rate through the plasma. Hence, patients in an acute phase response will have a high ESR. This test is only accurate if the blood is less than 24 hours old. Whole blood is diluted 4 parts with 1 part of sodium citrate anticoagulant. Once mixed thoroughly, the blood is drawn up into a calibrated tube (Figure 184) and allowed to stand for 1 hour at room temperature. After 1 hour, the distance the red blood cells have fallen is measured to the nearest 1 mm. The result is expressed as mm/hr.

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Figure 184: Erythrocyte sedimentation rate

(Source: Sediplast, LP Italiana SPA)

White blood cell differential

The white blood cell differential is performed on a freshly prepared blood film. The blood film is stained with Wrights stain enabling distinction between the cells. The stain enhances the properties of the nucleus, cytoplasm and cytoplasmic granules as well as other cellular inclusions. A blood film is prepared from fresh blood with either EDTA or no anticoagulant. EDTA blood has a time advantage, allowing films to be made satisfactorily within the first 4 hours of collection. With uncoagulated blood, the film must be made immediately before the clotting process commences. To make a blood film (Figure 185), a small drop of blood is placed at one end of the centre of a clean slide. A blood spreader is then used to make contact with the blood and spread the blood in an even motion towards the other end of the slide. The spreader, which is held at a 45 angle, must not be lifted off the slide until the last trace of blood has been spread out. The thickness of the blood film is regulated by pressure, speed and the angle of the spreader.

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Step 1

Step 2

Step 3
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Step 4 Figure 185: Making a blood smear

(Source: Radil Research Animal Diagnostic Laboratory)

Once dried, the blood film is stained and then, allowed to dry at room temperature. The blood film is first examined macroscopically to assess the quality of the smear. The smear should be finger shaped, with a rounded end and no irregularities. The rounded end of the smear should have a rainbow appearance when held up to the light. The slide is placed on the microscope and using the 10X objective, the smear is examined for quality and distribution of cells. An area of even cell distribution, with cells in a single plane, is chosen. Increasing to the 40X objective, an area is selected where the majority of red blood cells separate from each other, and do not overlap. Using a back and forth, zigzag motion (Figure 186), a total of 100 white blood cells are counted and recorded. In health, the five categories of white blood cells found are neutrophils, lymphocytes, monocytes, eosinophils and basophils (Figure 33). In cases of disease, immature cells are released in to peripheral blood. These cells are identified and included in the white cell differential. The 100X oil immersion objective is used to examine red blood cell inclusions. Red cell morphology and platelet morphology are evaluated when performing the white blood cell differential. The red cell changes and platelet descriptions are recorded to complete the peripheral blood picture. A haematological diagnosis is made by the analysis of the full blood count parameters and microscopic examination of the blood film.

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Figure 186: Blood film area counted for a white blood cell differential

Reticulocyte count
Reticulocytes are juvenile red cells, containing remnants of ribosomal RNA. The ribosome material reacts with new methylene blue to form blue granules (Figure 187).

Figure 187: Reticulocytes stained in new methylene blue (as indicated by arrows) The number of reticulocytes in the peripheral blood is an accurate reflection of bone marrow production. The reticulocyte spends 1 day in the peripheral blood before developing into a mature red blood cell. An increase in the number of reticulocytes in peripheral blood indicates increased bone marrow stimulus, leading to their premature release.
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Equal volumes of 1% New Methylene Blue and EDTA anticoagulated whole blood are mixed together in a small glass test tube. Mixing gently every 5 minutes, incubate at room temperature for 20 minutes. Prepare a blood film of the preparation and allow to air dry. Once dry, the reticulocytes are counted as a percentage of the red blood cells. The absolute number of reticulocytes in then calculated as shown below. % reticulocytes red blood cell count 100 Reticulocytes are released excessively by the bone marrow to compensate for anaemia. Abs reticulocytes count =

Latex tests for infectious mononucleosis

Infectious mononucleosis is caused by the Epstein-Barr virus (EBV). Every infected cell contains viral antigens. These viral antigens give rise to IgM antibodies specific to the Epstein-Barr virus. Commercially available screening kits, to detect EBV IgM, are used to confirm the diagnosis of Infectious Mononucleosis after indication by blood film examination. The kits are based on antigen-coated latex particles to which the IgM antibody binds, causing agglutination. A positive and negative control must be run, at the same time as the patient sample, to assure the validity of the test. These slide kits are both sensitive and specific, with an overall accuracy or a 9095%. Antibodies to EBV are often present from the 56th day of infection and are always found by the 21st day. They generally disappear within 45 months.

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Review questions
Review question 181 Give examples of where manual tests can be invaluable. Review question 182 Describe the principle of Drabkins method for haemoglobin estimation. Review question 183 What does the haematocrit measure and how is it performed? Review question 184 Name two types of blood cells that can be counted manually using a haemocytometer. Review question 185 MCV, MCH and MCHC are referred to as red cell indices. Describe what the abbreviations stand for and what they describe. Review question 186 What does ESR stand for and what does it measure? Review question 187 What is a white blood cell differential? Review question 188 What is the best type of blood to use when making a blood smear? Review question 189 What are reticulocytes and why are they released prematurely from the bone marrow?

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