Chart 8.5 Preliminary laboratory findings of common anaemias in tropical countries
8.6 Counting white cells and platelets
WHITE CELL COUNT
Value of test: A white blood cell (WBC) count is used to investigate HIV/AIDS, infections and unex- plained fever, and to monitor treatments which can cause leukopenia. In most situations when a total WBC count is requested it is usual to perform also a differential WBC count as described in subunit 8.7.
Principle of test
Whole blood is diluted 1 in 20 in an acid reagent which haemolyzes the red cells (not the nucleus of nucleated red cells), leaving the white cells to be counted. White cells are Summary of red cell indices in common anaemias Reference
range:
Anaemias Normocytic
normochromic N N N
Microcytic
hypochromic ↓ ↓ ↓
e.g. Iron deficiency Macrocytic
normochromic N ↑ ↑
e.g. Folate deficiency
N Normal, ↓Reduced value,
↑Increased value
MCH 27–32 pg MCV
80–98 fl MCHC
315–360 g/l
required to measure blood samples. A safe pipette/capillary filler should be used to aspirate and dispense the blood. This can be a simple bulb filler or device as shown on pp. 134–135 in Part 1 of the book.
Bulb pipettes: These pipettes are still supplied with haemocytometers by some manufacturers. They should notbe used for counting WBCs because it is not possible to obtain reliable mixing of the blood and diluting fluid inside the bulb of the pipette. Bulb pipettes are also diffi- cult to clean and expensive to replace.
Note: Safe systems for measuring and dispens- ing blood samples and diluting fluid are described in subunit 4.6 in Part 1 of the book.
● Hand counter
To count white cells accurately, a simple inex- pensive mechanical hand tally counter as shown in Plate 8.3 is required. Turning the knob on the side returns the counter to zero after each count.
Availability of supplies for WBC counting:All of the above mentioned items for counting white cells are available at low cost from Developing Health Technology (see Appendix 11).
Reagent
WBC diluting fluid* Reagent No. 88
*This is a weak acid solution to which gentian violet is added which stains the nucleus of white cells.
Test method
1 Measure 0.38 ml of diluting fluid and dispense it into a small container or tube.
2 Add 20 l (0.02 ml, 20 cmm) of well-mixed EDTA anticoagulated venous blood or free- flowing capillary blood and mix.
Important: The volume of blood used in the test must be correct. This can be achieved by using the technique illustrated on page 302.
3 Assemble the counting chamber:
– Make sure the central grid areas of the chamber and the special haemocytometer cover glass are completely clean and dry.
– Slide the cover glass into position over the grid areas and press down on each side until rainbow colours (Newton’s rings) are seen.
Prior moistening of the chamber surface on each side of the grid areas will help the cover glass to adhere to the chamber.
counted microscopically using an Improved Neubauer ruled counting chamber (haemocytometer) and the number of WBCs per litre of blood calculated.
Note: When after examining a stained blood film, many nucle- ated red cells are present (more than 10%), the WBC count should be corrected (see end of Test Method).
Electronic counting of WBCs: This is described briefly at the end of this subunit.
Blood sample:EDTA anticoagulated blood or cap- illary blood can be used for counting white cells. Heparin or sodium citrate anticoagulated blood must not be used. The count should be performed within 6 hours (blood should not be refrigerated).
Note: The correct and safe collection of capillary and venous blood is described in subunit 8.3.
Equipment
● Counting chamber (haemocytometer)
The counting chamber recommended for cell counts is a metallized surface (‘Bright-line’) double cell Improved Neubauer ruled chamber.
Non-metallized haemocytometers: While these are less expensive, they are not recommended. It is more difficult to count WBCs reliably using this type of chamber because the background rulings and cells are not as easily seen. Non-metallized chambers are also more difficult to fill.
The rulings of an Improved Neubauer ruled chamber are shown in Fig. 8.6. The chamber grid has an area of 9 mm2and the depth of the chamber (space between cover glass and grid) is 0.1 mm.
Bürker ruled counting chamber
This chamber is occasionally found in laboratories and may be the only one available for counting cells. To count white cells using a Bürker Chamber, the four large corner squares are used (4 mm2) and the same calculation as described for the Improved Neubauer ruled chamber is used.
● Counting chamber cover glasses
Special optically plane cover glasses of defined thickness (designed for use with haemocytome- ters) are required. Other cover glasses must not be used. Manufacturers of counting chambers provide two cover glasses with each chamber.
The laboratory should always keep in stock spare cover glasses.
● Pipettes/calibrated capillaries and safe filling device
A 20 l (0.02 ml, 20 cmm) micropipette e.g.
white shellback type, or calibrated capillary is
4 Re-mix the diluted blood sample. Using a capil- lary, Pasteur pipette, or plastic bulb pastette held at an angle of about 45, fill one of the grids of the chamber with the sample, taking care not to overfill the area.
Important: The chamber must be refilled if the sample overfills into the channel beyond the grid or an air bubble forms in the grid area.
5 Leave the chamber undisturbed for 2 minutes to allow time for the white cells to settle.
Note: To prevent drying of the fluid, place the chamber in a petri dish or plastic container on dampened tissue or blotting paper and cover with a lid as shown in Fig. 8.6(c) on p. 317.
6 Dry the underside of the chamber and place it on the microscope stage.
Using the 10 objective with the condenser iris closed sufficientlyto give good contrast, focus the rulings of the chamber and white cells. Focus the cells until they appear as small black dots.
7 Count the cells in the four large corner squares of the chamber marked W1, W2, W3, W4 in Fig.
8.6 (total area of 4 mm2). Include in the count the cells lying on the lines of two sides of each large square as shown in Fig. 8.6(b).
8.6
8 Report the number of white cells per litre of blood using the following simple calculation:
– Divide the total number of cells counted by 2.
– Divide the number obtained by 10.
The number obtained109is the white cell count.
Example
Cells counted in 4 squares 84
84 2 42
42 10 4.2
WBC count 4.2 cells 109/l
WBC calculation details WBC count (per litre)
where* 1 in 20 dilution of blood, ‡ 4 mm2 area counted, ả0.1 mm depth of chamber.
9 After performing the count, before the sample dries, dismantle the chamber, wash and dry it.
Store it with the cover glass in a safe place.
Cells counted 20* 106 4‡ 0.1ả
Fig 8.6a Improved Neubauer ruled counting chamber. The four large squares marked W1, W2, W3, W4 are used for counting WBCs and the five small squares marked Pare used for counting platelets.
W
1
W
2
W
3 W
4
P P
P
P P
Fig 8.6b Cells lying on two sides of the square are included in the count.
Plate 8.3 Hand tally counter used to count blood cells.
Counts higher than 50.0 109/l
When a count is higher than 50.0/109/l, repeat the count using 0.76 ml of diluting fluid and 20 l of blood. Multiply the result by 2. Very high WBC counts are found in some forms of leukaemia.
Always examine a stained thin blood film.
Counts lower than 2.0 109/l
When a count is lower than 2.0 109/l, repeat the count using 0.38 ml of diluting fluid and 40 l of blood. Divide the result by 2.
Correcting a WBC count when there are many nucleated RBCs
When more than 10 nucleated red blood cells (RBCs) per 100 WBC are present in the blood film, correct the WBC count as follows:
Corrected WBC count
*Number of nucleated RBCs per 100 WBC as seen in stained blood film.
Quality control of WBC counts
Whenever possible perform WBC counts in duplicate. The difference between the two counts (as a percentage of the mean) should not be more than 20%.
How to calculate the % difference between two counts 1 Record the number of cells counted in Count 1 and
Count 2.
2 Calculate:
– the difference in the number of cells counted between the two counts.
– the mean of the two counts
3 Calculate the difference of the two counts as a per- centage of the mean.
Example
Cells counted in Count 1 88, Count 2 76 – Difference in numbers of cells between the two
counts: 88–76 12
– Mean of Count 1 and Count 2: 82 – Difference of the two counts as a % of the mean:
14.6%
Note: When the difference between the two counts is more than 20%, repeat the counts.
Check that the diluting fluid is free from particles which could be mistaken for WBCs. To do this, fill a counting chamber with a sample of the diluting fluid and examine the grid areas micro- scopically using the 10 objective with greatly reduced condenser iris. If the fluid contains par-
12 100 82
88 76 2
Uncorrected WBC count 100
Nucleated RBCs* 100
ticles resembling WBCs, filter it and recheck or discard the fluid and prepare fresh diluting fluid.
When examining the blood film, check that there is no major discrepancy between the total white cell count and white cells seen in the blood film.
External quality assessment
Whenever possible the Regional or Central Haematology Laboratory should send control blood samples to district laboratories for analysis.
Sources of error in manual WBC counts
● Incorrect measurement of blood due to poor technique (see Text Method) or using a wet or chipped pipette.
● When using anticoagulated blood, not mixing the blood sufficiently or not checking the sample for clots.
● Inadequate mixing of blood with diluting fluid.
● Not checking whether the chamber and cover glass are completely clean.
● Not using a haemocytometer cover glass.
● Over-filling a counting chamber or counting cells when the sample contains air-bubbles.
● Not allowing sufficient time (2 minutes) for the cells to settle in the chamber.
● Using too intense a light source or not reducing the iris diaphragm sufficiently to give good contrast (poor focusing and difficulty in seeing clearly the cells and rulings are common when using non-metallized haemocytometers, see previous text).
● Not completing counting of the cells before the sample begins to dry in the chamber.
● Counting too few cells (see previous text).
Precision increases with the number of cells counted.
● Not correcting a count when the sample contains many nucleated RBCs (see previous text).
Interpretation of WBC counts
Reference ranges for white cell counts vary with age with higher counts being found in children. There are also gender differences with higher total WBC and neutrophil counts being found in women of child-bearing age and during pregnancy. Counts also vary in different populations with lower total WBC and neutrophil counts being found in Africans and people of African descent.
WBC reference range*
*These are guideline figures which should be checked locally.
Children at 1 y . . . 6.0–18.0 109/l Children 4–7 y . . . 5.0–15.0 109/l Adults . . . 4.0–10.0 109/l Adults of African origin . . . 2.6–8.3 109/l Pregnant women . . . Up to 15 109/l Leukocytosis
The main causes of a raised WBC count are:
● Acute infections
e.g. pneumonia, meningitis, abscess, whooping cough, tonsillitis, acute rheumatic fever, septicaemia, gonor- rhoea, cholera, septic abortion.
Note: Acute infections in children can cause a sharp rise in WBC count.
● Inflammation and tissue necrosis
e.g. burns, gangrene, fractures and trauma, arthritis, tumours, acute myocardial infarction.
● Metabolic disorders
e.g. eclampsia, uraemia, diabetic coma and acidosis.
● Poisoning
e.g. chemicals, drugs, snake venoms.
● Acute haemorrhage
● Leukaemias and myeloproliferative disorders
● Stress, menstruation, strenuous exercise.
Leukopenia
The main causes of a reduced WBC count are:
● Viral, bacterial, parasitic infections
e.g. HIV/AIDS, viral hepatitis, measles, rubella, influenza, rickettsial infections, overwhelming bacterial infections such as miliary tuberculosis, relapsing fever, typhoid, paratyphoid, bucellosis, parasitic infections including leishmaniasis and malaria.
● Drugs (e.g. cytotoxic) and reactions to chemicals
● Hypersplenism
● Aplastic anaemia
● Folate and vitamin B12 deficiencies (megaloblas- tic anaemia)
● Bone marrow infiltration (e.g. lymphomas, myelo- fibrosis, myelomatosis)
● Anaphylactic shock
● Ionizing radiation
PLATELET COUNT
Value of test:A platelet count may be requested to investigate abnormal skin and mucosal bleeding which can occur when the platelet count is very low (usually below 20109/l). Platelet counts are also performed when patients are being treated with cytotoxic drugs or other drugs which may cause thrombocytopenia.
8.6
Fig 8.6c Counting chamber in petri dish to prevent drying of the preparation.
Principle of test
Blood is diluted 1 in 20 in a filtered solution of ammonium oxalate reagent which lyzes the red cells. Platelets are counted microscopically using an Improved Neubauer ruled counting chamber and the number of platelets per litre of blood calcu- lated.
Blood sample: Use EDTA anticoagulated venous blood. The collection of venous blood is described in subunit 8.3. Capillary blood should not be used because platelets clump as the blood is being collected.
Equipment
An Improved Neubauer ruled Bright-line counting chamber and other equipment as described pre- viously for WBC counting are required for counting platelets.
Platelet haemocytometers:Thin glass chambers for counting platelets by phase contrast microscopy are available. Such chambers are expensive and break easily. They are not essen- tial for counting platelets.
Reagent
Ammonium oxalate 10 g/l Reagent No. 13 (1% w/v) diluting fluid.
Important: Always filter the fluid before use.
Test method
Perform a platelet count within 2 hours of collecting the blood.
1 Measure 0.38 ml of filtered ammonium oxalate diluting fluid and dispense it into a small con- tainer or tube.
2 Add 20 l (0.02 ml, 20 cmm) of well-mixedanti- coagulated venous blood and mix.
3 Assemble the counting chamber and fill it with well-mixed sample as described previously in steps 3 and 4 of the method for counting white cells.
4 Leave the chamber undisturbed for 20 minutes.
To prevent drying of the fluid, place the chamber in a petri dish or plastic container on dampened tissue or blotting paper and cover with a lid as shown in Fig. 8.6c.
5 Dry the underside of the chamber and place it on the microscope stage. Using the 10 objec- tive, focus the rulings of the grid and bring the central square of the chamber into view. Change to the 40 objective and focus the small platelets. They will be seen as small bright frag- ments (refractile).
Note: If available, use phase contrast microscopy.
6 Count the platelets in the small squares marked P as shown in Fig. 8.6 on p. 315.
7 Report the number of platelets in 1 litre of blood.
This is the actual number of platelets counted 109.
Example
If 150 platelets are counted, the platelet count is 150 109/l.
Platelet calculation details Platelet count (per litre)
where * 1 in 20 dilution of blood, †0.2 mm2area counted ả0.1 mm depth of chamber
Quality control
In district laboratories the most feasible quality control of platelet counts is to follow the test pro- cedure exactly, perform duplicate counts, and examine the platelet fluid microscopically (at the time of performing the count) to ensure it does not contain refractile particles resembling platelets. The mean of the two counts should be calculated as described previously for WBC counts.
Sources of error in counting platelets
Sources of error when counting platelets are similar to those mentioned previously for WBC counts.
Special care must be taken when counting platelets:
Cells counted 20* 106 0.2† 0.1ả
● To check there are no clots in the blood sample.
● To ensure the blood is well mixed with the diluting fluid.
● Not to mistake debris from haemolyzed red cells or particles in the diluting fluid for platelets.
● To ensure the platelets are evenly distributed and not in small clumps (if clumps are present, obtain a new blood sample).
● Not to use too intense an illumination.
Note: In some disorders, large platelets may be present.
Interpretation of platelets counts
In health there are about 150–400 109 platelets/litre of blood. Platelet counts are lower in Africans.
Thrombocytopenia
The main causes for a reduction in platelet numbers are:
REDUCED PRODUCTION OF PLATELETS
● Infections, e.g. typhoid, brucellosis
● Deficiency of folate or vitamin B12
● Aplastic anaemia
● Drugs (e.g. cytotoxic, quinine, aspirin), chemicals (e.g. benzene), some herbal remedies, alco- holism
● Leukaemias, lymphoma, myeloma, myelofibrosis, carcinoma
● Hereditary thrombocytopenia (rare condition).
INCREASED DESTRUCTION OR CONSUMPTION OF PLATELETS
● Infections, e.g. acute falciparum malaria, dengue, trypanosomiasis, visceral leishmaniasis
● Disseminated intravascular coagulation (DIC)
● Hypersplenism
● Immune destruction of platelets, e.g. idiopathic thrombocytopenic purpura (ITP), onyalai*, systemic lupus erythematosus (SLE), other con- nective tissue disorders, chronic lymphatic leukaemia, lymphomas and HIV/AIDS. Also, exposure to drugs, e.g. quinine, mefloquine, penicillin, and some herbal remedies.
*Onyalai: This is an acquired immune thrombocytopenia mainly found in young people in parts of Southern Africa (e.g.
Southern Angola, Northern Namibia). The condition is char- acterized by haemorrhagic bullae in the buccal mucosa, bleeding from other sites and very low platelet count (20 109/l or below). Bleeding may last for several days or longer.
Mortality is about 10% with death resulting from haemor-
8.6–8.7
rhagic shock (can be reduced with supportive management, including blood transfusion). Most patients recover with a slow return to normal platelet numbers but relapse is common and the condition may become chronic. The cause of onyalai is not known (mycotoxins from fungal contamination of grain are suspected).
Thrombocytosis
Causes of an increase in platelet numbers include:
● Chronic myeloproliferative diseases, e.g. essential thrombocythaemia, polycythaemia vera, chronic myeloid leukaemia, myelofibrosis.
● Carcinoma (disseminated)
● Chronic inflammatory disease, e.g. tuberculosis
● Haemorrhage
● Sickle cell disease associated with a non- functioning spleen or after splenectomy.
● Iron deficiency anaemia, associated with active bleeding.
Automated blood cell counting using an electronic blood cell analyzer
Most electronic blood cell analyzers including those manufactured by Beckman Coulter and Sysmex (Toa Medical), count blood cells by impedance.
Principle of impedance analyzers
Blood cells are diluted in a buffered electrolyte solution. A measured volume of the sample passes through an aperture tube (e.g. 100 m in diameter) between two electrodes.
Interruption of the current by the non-conducting blood cells alters the electrical charge and a pulse is produced. The ampli- tude of each pulse is proportioned to the volume of the cell which caused it. A threshold circuit ensures only those pulses that exceed the pre-set threshold level are counted. The cell count is determined from the total number of pulses obtained from a measured volume of blood.
Note: Analysis of the pulse heights enables mean cell volume (MCV) to be measured and the haematocrit to be calculated from the MCV value and red cell count. In Sysmex impedance analyzers, the haematocrit is determined from voltage pulse data and the MCV calculated from the haematocrit value. The haemoglobin concentration is used with the red cell count, MCV, and haematocrit, to calculate the MCH and MCHC.
Electronic blood cell analyzers are available as semi- automated instruments which require blood samples to be externally diluted (with separate dilu- tions for counting RBCs and WBCs) and fully automated instruments with internal dilution and simultaneous counting of white cells, red cells, and platelets. The more advanced analyzers in addition to determining haemoglobin, WBC, RBC, platelets, haematocrit, MCV, MCHC, and MCH, also provide red cell distribution width (RDW), platelet distri- bution width (PDW), and a white cell differential.
Use of an electronic blood cell analyzer
An electronic blood cell analyzer is appropriate to use when:
● The work load is sufficiently high.
● The capital cost and running costs of the analyzer are affordable.
● Local engineers (trained by the manufacturer of the analyzer) are available to assist with instal- lation of the analyzer, its calibration, regular servicing (about twice a year), and on-site- training and repairs.
● Technologists are trained in the correct use, quality control and maintenance of the analyzer and are able to interpret the data and error codes.
● Essential reagents, calibrants, and control materials are locally and reliably available and also replacement parts can be easily accessed.
● Electricity supplies are sufficiently stable and the analyzer can be protected from excessive dust and extremes of temperature and humidity.
Note: Further information on electronic blood cell analyzers, their use, and sources of error can be found in practical haematology textbooks and from analyzer manufacturers and their agents.