All the tests discussed in this chapter can be performed on venous or free-flowing capillary blood that has been anticoagulated with ethylenediaminetetra-acetic acid (EDTA) (see p. 6). Thorough mixing of the blood specimen before sampling is essential for accurate test results. Ideally, tests should be performed within 6 h of obtaining the blood specimen because some test results are altered by longer periods of storage. However, results that are sufficiently reliable for clinical purposes can usually be obtained on blood stored for up to 24 h at 4°C (see p. 7).

Haemoglobinometry

The haemoglobin concentration (Hb) of a solution may be estimated by measurement of its colour, by its power of combining with oxygen or carbon monoxide or by its iron content. The methods to be described are all colour or light-intensity matching techniques, which also measure, to a varying extent, any methaemoglobin (Hi) or sulphaemoglobin (SHb) that may be present. The oxygen-combining capacity of blood is 1.34 ml O₂ per g haemoglobin. Ideally, for assessing clinical anaemia, a functional estimation of Hb should be carried out by measurement of oxygen capacity, but this is hardly practical in the routine haematology laboratory. It gives results that are at least 2% lower than those given by the other methods, probably because a small proportion of inert pigment is always present. The iron content of haemoglobin can be estimated accurately,¹ but again the method is impractical for routine purposes. Estimations based on iron content are generally taken as authentic, but iron bound to inactive pigment is included. Iron content is converted into haemoglobin by assuming the following relationship: 0.347 g iron = 100 g haemoglobin.²

Measurement of haemoglobin concentration using a spectrometer (spectrophotometer) or photoelectric colorimeter

Two methods are in common use: (1) haemiglobincyanide (HiCN; cyanmethaemoglobin) method and (2) oxyhaemoglobin (HbO₂) method. There is little to choose in accuracy between these methods, although a major advantage of the HiCN method is the availability of a stable and reliable reference preparation.

Although the HiCN reagent contains cyanide, there is only 50 mg of potassium cyanide per litre and 600–1000 ml would have to be swallowed to produce serious effects. However, the use of potassium cyanide has been viewed as a potential hazard; alternative nonhazardous reagents that have been proposed are sodium azide ³ and sodium lauryl sulphate,^4,⁵ which convert haemoglobin to haemiglobinazide and haemiglobinsulphate, respectively. They are used in some automated systems, but no stable standards are available and they, too, are toxic substances that must be handled with care.

Other methods that have been used include Sahli’s acid-haematin method, which is less accurate because the colour develops slowly, is unstable and begins to fade almost immediately after it reaches its peak. The alkaline-haematin method gives a true estimate of total Hb even if carboxyhaemoglobin (HbCO), Hi or SHb is present; plasma proteins and lipids have little effect on the development of colour, although they cause turbidity. The original method was more cumbersome and less accurate than the HiCN or HbO₂ methods, but a modified method has been developed in which blood is diluted in an alkaline solution with non-ionic detergent and read in a spectrometer at an absorbance of 575 nm against a standard solution of chlorohaemin.^6,⁷ One evaluation has given encouraging results,⁸ although another study has shown a bias of 2.6% when compared with the reference method, with non-linearity in the relationship between haemoglobin concentration and absorbance at high and low haemoglobins.⁹

Haemiglobincyanide (cyanmethaemoglobin) method

The haemiglobincyanide (cyanmethaemoglobin) method is the internationally recommended method ² for determining the haemoglobin concentration of blood. In some countries cyanide reagents are no longer available. The basis of the method is dilution of blood in a solution containing potassium cyanide and potassium ferricyanide. Haemoglobin, Hi and HbCO, but not SHb, are converted to HiCN. The absorbance of the solution is then measured in a spectrometer at a wavelength of 540 nm or a photoelectric colorimeter with a yellow-green filter (e.g. Ilford 625, Wratten 74, Chance 0 Gr1).

Diluent

The original (Drabkin’s) reagent had a pH of 8.6. The following modified solution listed in Table 3.1, Drabkin-type reagent, as recommended by the International Committee for Standardization in Haematology (ICSH),² has a pH of 7.0–7.4. It is less likely to cause turbidity from precipitation of plasma proteins and requires a shorter conversion time (3–5 min) than the original Drabkin’s solution, but it has the disadvantage that the detergent causes some frothing.

Table 3.1 Drabkin-type reagent

Potassium ferricyanide (0.607 mmol/l)	200 mg
Potassium cyanide (0.768 mmol/l)	50 mg
Potassium dihydrogen phosphate (1.029 mmol/l)	140 mg
Non-ionic detergent^a	1 ml
Distilled or deionized water	To 1 litre

a Suitable non-ionic detergents include Nonidet P40 (VWR International, Merck, Eurolab) and Triton X-100 (Aldrich).

The pH should be 7.0–7.4 and must be checked with a pH meter at least once a month. The diluent should be clear and pale yellow in colour. When measured against water as a blank in a spectrometer at a wavelength of 540 nm, absorbance must be zero. If stored at room temperature in a brown borosilicate glass bottle, the solution keeps for several months. If the ambient temperature is higher than 30°C, the solution should be stored in the refrigerator but brought to room temperature before use. It must not be allowed to freeze. The reagent must be discarded if it becomes turbid, if the pH is found to be outside the 7.0–7.4 range or if it has an absorbance other than zero at 540 nm against a water blank.

Haemiglobincyanide Reference Standard

With the advent of HiCN solution, which is stable for many years, other standards have become outmoded.¹⁰ The International Committee for Standardization in Haematology² has defined specifications on the basis of a relative molecular mass (molecular weight) of human haemoglobin of 64 458 (i.e. 16 114 as the monomer) and a millimolar area absorbance (coefficient extinction) of 11.0 (that is, the absorbance at 540 nm of a solution containing 55.8 mg of haemoglobin iron per litre).

Some standards are prepared from ox blood, which has the same coefficient extinction but a molecular weight of 64 532 (16 133 as the monomer). These specifications have been widely adopted; a World Health Organization (WHO) International Standard has been established and a comparable reference material is available from the ICSH. A new lot of the haemoglobincyanide or haemoglobin standard was released in 2008.¹¹ This newly released standard replaces the previous lot and was produced using the same methodology previously specified by ICSH.² The current standard has an assigned concentration value of 574.2 (± 5.1) mg/l or 35.63 (± 0.32) μmol/l; the exact concentration is indicated on the label. The stability expectation of this standard is 15 years¹¹ but it will continue to be monitored on a twice-yearly basis over the lifetime of this lot of reference material. The haemoglobin standard provides a reference material from which both laboratory-based cell counters and point-of-care instruments calibrate their haemoglobin methods.²

The HiCN solution is dispensed in 10 ml sealed ampoules and is regarded as a dilution of whole blood. The original Hb that it represents is obtained by multiplying the figure stated on the label by the dilution to be applied to the blood sample. Thus, if the standard solution contains 800 mg (0.8 g) of haemoglobin per litre, it will have the same optical density as a blood sample containing 160 g/l of haemoglobin if diluted 1 to 200 or as one containing 200 g/l of haemoglobin if diluted 1 to 250. Within the SI system, haemoglobin may be expressed in terms of substance concentration as μmol/l or in mass concentration as g/l (or g/dl) or μmol/l = g/l × 0.062. For clinical purposes, there are practical advantages in expressing haemoglobin in mass concentration per litre or per decilitre (dl).

The HiCN reference preparation is intended primarily for direct comparison with blood that is converted to HiCN. It can also be used for the standardization of a whole-blood standard in the HbO₂ method (discussed later).

Method

Make a 1 in 201 dilution of blood by adding 20 μl of blood to 4 ml of diluent. Stopper the tube containing the solution and invert it several times. Let the test sample stand at room temperature for at least 5 min (to ensure the complete conversion of haemoglobin to haemiglobincyanide) and then pour it into a cuvette and read the absorbance in a spectrometer at 540 nm or in a photoelectric colorimeter with a suitable filter (e.g. Ilford 625, Wratten 74, Chance 0 Gr1) against a reagent blank. The absorbance of the test sample must be measured within 6 h of its initial dilution. The absorbance of a commercially available HiCN standard (brought to room temperature if previously stored in a refrigerator) should also be compared to a reagent blank in the same spectrometer or photoelectric colorimeter as the patient sample. The standard should be kept in the dark and, to ensure that contamination is avoided, any unused solution should be discarded at the end of the day on which the ampoule is opened.

Calculation of Haemoglobin Concentration

*†

Preparation of Standard Graph and Standard Table

When many blood samples are to be tested, it is convenient to read the results from a standard graph or table relating absorbance readings to haemoglobin in g/l for the individual instrument. This graph should be prepared each time a new photometer is put into use or when a bulb or other component is replaced. It can be prepared as follows.

Prepare five dilutions of the HiCN reference standard (or equivalent preparation) (brought to room temperature) with the cyanide-ferricyanide reagent according to Table 3.2. Because the graph will be used to determine the haemoglobin measurements, it is essential that the dilutions are performed accurately.

Table 3.2 Dilutions of haemiglobincyanide (HiCN) reference solution for preparation of standard graph

The haemoglobin concentration of the reference preparation in each tube should be plotted against the absorbance measurement. For example, if the label on the reference preparation states that it contains 800 mg/l (i.e. 0.8 g/l) and the method for haemoglobin measurement uses a dilution of 1:201, the respective haemoglobin concentrations of tubes 1–5 would be 160 g/l, 120 g/l, 80 g/l, 40 g/l and zero.

Using linear graph paper, plot the absorbance values on the vertical axis and the haemoglobin values (absorbance; formerly called optical density). In some instruments, measurements are read as percentage transmittance) on the horizontal axis. (If the readings are in percentage transmittance, use semilogarithmic paper with the transmittance recorded on the vertical or log scale.) The points should fit a straight line that passes through the origin. Providing that the standard has been correctly diluted, this provides a check that the calibration of the photometer is linear. From the graph, it is possible to construct a table of readings and corresponding haemoglobin values. This is more convenient than reading values from a graph when large numbers of measurements are made. It is important that the performance of the instrument does not vary and that its calibration remains constant in relation to haemoglobin measurements. To ensure this, the reference preparation should be measured at frequent intervals, preferably with each batch of blood samples.

The main advantages of the HiCN method for haemoglobin determination are that it allows direct comparison with the reference standard and that the readings need not be made immediately after dilution so batching of samples is possible. It also has the advantage that all forms of haemoglobin, except SHb, are readily converted to HiCN.

The rate of conversion of blood containing HbCO is markedly slow. This difficulty can be overcome by prolonging the reaction time to 30 min before reading.¹² The difference between the 5 and 30 min readings can be used as a semiquantitative method for estimating the percentage of HbCO in the blood.

As referred to earlier, lauryl sulphate ⁵ or sodium azide³ can be used as non-hazardous substitutes for potassium cyanide. However, no stable standards are available for these methods so a sample of blood that has first had a haemoglobin value assigned by the HiCN method needs to be used as a secondary standard.

Abnormal plasma proteins or a high leucocyte count may result in turbidity when the blood is diluted in the Drabkin-type reagent. The turbidity can be avoided by centrifuging the diluted sample or by increasing the concentration of potassium dihydrogen phosphate to 33 mmol/l (4.0 g/l).¹³

Oxyhaemoglobin method

The HbO₂ method is the simplest and quickest method for general use with a photometer. Its disadvantage is that it is not possible to prepare a stable HbO₂ standard, so the calibration of these instruments should be checked regularly using HiCN reference solutions or a secondary standard of preserved blood or lysate (see p. 25). The reliability of the method is not affected by a moderate increase in plasma bilirubin, but it is not satisfactory in the presence of HbCO, Hi or SHb.

Method

Wash 20 ml of blood into a tube containing 4 ml of 0.4 ml/l ammonia (specific gravity 0.88) to give a ×201 dilution. Use a tightly fitting stopper and mix by inverting the tube several times. The solution of HbO₂ is then ready for matching against a standard in a spectrometer at 540 nm or a photometer with a yellow-green filter (e.g. Ilford 625) against a water blank. If the absorbance of the haemoglobin solution exceeds 0.7, dilute the blood further with an equal volume of water and read again. Fresh ammonia solution must be made up each week. Once diluted, the blood sample is stable at 20°C for about 2 days.

Standard

A standard should be prepared from a specimen of normal anticoagulated whole blood. Its haemoglobin concentration is first determined by the HiCN method (see p. 25). The blood is then diluted 1:201 by pipetting 20 ml of the well-mixed blood into 4 ml of ammonia; sequential dilutions are made in ammonia and absorbance is read in a spectrometer at 540 nm or photometer using a yellow-green filter (Ilford 625, Wratten 74 or Chance 0 Gr 1). The readings are plotted on arithmetic graph paper. Linearity of response is checked and absorbance is related to haemoglobin from the measurement obtained in the original sample by the HiCN method.

Colorimeters and light filters unfortunately, differ sufficiently to make it essential to check the chosen standard at frequent intervals against a HiCN reference preparation in the photometer in which it is going to be used. It is probably preferable to use a new fresh whole-blood sample each day as a secondary standard after measuring its haemoglobin concentration by the HiCN method. Preserved blood (see p. 599) or lysate (see p. 599) can be used instead.

Direct calculation from a standard:

If the HiCN method is not available, a neutral grey filter of 0.475 density (Ilford or Chance) can be used as a calibration standard. This corresponds to a 1:201 dilution of blood with 146 g/l haemoglobin concentration in a 1 cm cuvette at a wavelength of 540 nm.

Direct spectrometry

The haemoglobin concentration of a diluted blood sample can be determined by spectrometry without the need for a standard, provided that the spectrometer has been correctly calibrated. The blood is diluted 1:201 (or 1:251) with cyanide-ferricyanide reagent (see p. 25) and the absorbance is measured at 540 nm. Haemoglobin concentration is calculated as follows:

where A⁵⁴⁰ = absorbance of solution at 540 nm; 16 114 = monomeric molecular weight of haemoglobin; dilution = 201 when 20 ml of blood are diluted in 4 ml of reagent; 11.0 = millimolar coefficient extinction; d = layer thickness in cm; and 1000 = conversion of mg to g.

When assigning a value to a haemoglobin solution that may be used as a reference preparation, it is necessary first to calibrate the spectrometer. This requires checking wavelength with a holmium oxide filter, absorbance with a set of calibrated neutral density filters and stray light with a neutral density filter at 220 nm (National Physical Laboratory, Teddington, UK). Matched optical or quartz glass cuvettes with a transmission difference of <1% at 200 nm should be used. Subsequently, the calibration of the spectrophotometer can be checked by verifying that it gives an accurate reading of the HiCN standard. Slight deviations from the expected A⁵⁴⁰ HiCN value for the standard may be used to correct the results of test samples for a bias in measurement.²

Direct reading portable haemoglobinometers

Colour Comparators

These are simple clinical devices that compare the colour of blood against a range of colours representing haemoglobin concentrations. They are intended for anaemia screening in the absence of laboratory facilities and are described in Chapter 26.

Portable Haemoglobinometers

Portable haemoglobinometers have a built-in filter and a scale calibrated for direct reading of haemoglobin concentration in g/dl or g/l. They are generally based on the HbO₂ method. A number of instruments are now available that use a light-emitting diode of appropriate wavelength and are standardized to give the same results as with the HiCN method.

The HemoCue system (HemoCue AB, Ängelsholm, Sweden) is a well-established method for haemoglobinometry. It consists of a precalibrated, portable, battery-operated spectrometer; no dilution is necessary because blood is run by capillary action directly into a cuvette containing sodium nitrite and sodium azide, which convert the haemoglobin to azidemethaemoglobin. The absorbance is measured at wavelengths of 565 and 880 nm. Measurements are not affected by high levels of bilirubin, lipids or white cells and it is sufficiently reliable for use as a laboratory instrument; it is easy for non-technical personnel to operate and is thus also suitable for use at point-of-care. The cuvettes must be stored in a container with a drying agent and kept within the temperature range of 15–30°C. Some devices are now available that use reagent-free cuvettes that will not deteriorate in adverse climatic conditions.¹⁴ HemoCue have recently released a portable system that measures both haemoglobin and the white blood cell count (WBC), the HemoCue WBC.¹⁵

Chempaq (Chempaq A/S, Hirsemarken 1B, Farum, Denmark) produce two different portable multiplatform haematology analysers that use impedance cell counting and measurement of haemoglobin by a spectrophotometric method on 20 μl of blood. The Chempaq XBC uses a disposable cartridge to measure three different test profiles, Hb alone or WBC, with three-part differential, plus Hb or Hb with red blood cell count (RBC), haematocrit (Hct), mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC). The Chempaq XDM701 uses the same principles but also reports a platelet count.

The DiaSpect Haemoglobinometry system is a newly developed technology for measuring haemoglobin concentration in unaltered whole blood in a special plastic cuvette that also serves as the sampling device.¹⁶ The instrument is a portable spectrophotometer powered by 3.6 V integrated lithium-ion rechargeable batteries or 100–240 V adaptor. As the cuvettes do not contain any reagents, they are not affected by temperature or humidity and no special storage conditions are required. They have a shelf life of at least 2 years. Haemoglobin fractions are measured from absorbance wavelengths between 400 and 800 nm. A patented method eliminates the impact of scattering from the blood cells while possible background turbidity from interfering substances is measured and compensated for at high wavelength. The results are displayed in <5 seconds. Preliminary studies have shown an accuracy within ± 3 g/l for measurements between 10 and 200 g/l.

Non-invasive Screening Tests

Methods are being developed for using near infrared spectroscopy at body sites, mainly a finger, to identify the spectral pattern of haemoglobin in an underlying blood vessel and derive a measurement of haemoglobin concentration. Early studies have shown an approximate correlation with blood haemoglobinometry.^17,¹⁸

Range of Haemoglobin Concentration in Health

See Chapter 2, Tables 2.1, 2.2 and 2.3. It should be noted that there are sex differences, diurnal variations and environmental and physiological factors that must also be taken into account.

Packed cell volume or haematocrit

The packed cell volume (PCV) can be used as a simple screening test for anaemia, as a reference method for calibrating automated blood count systems and as a rough guide to the accuracy of haemoglobin measurements. The PCV × 1000 is about three times the Hb expressed in g/l. In conjunction with estimations of Hb and RBC, it can be used in the calculation of red cell indices. However, its use in under-resourced laboratories may be limited by the need for a specialized centrifuge and a reliable supply of capillary tubes.

Microhaematocrit Method

The microhaematocrit method ¹⁹ is carried out on blood contained in capillary tubes 75 mm in length and having an internal diameter of about 1 mm. The tubes may be plain for use with anticoagulated blood samples or coated inside with 1 iu of heparin for the direct collection of capillary blood. The centrifuge used for the capillary tubes provides a centrifugal force of c12 000 g and 5 min centrifugation results in a constant PCV. When the PCV is >0.5, it may be necessary to centrifuge for a further 5 min.

Allow blood from a well-mixed specimen, or from a free flow of blood by skin puncture, to enter the tube by capillarity, leaving at least 15 mm unfilled. Then seal the tube by a plastic seal (e.g. Cristaseal, Hawksley, Lancing, Sussex). Sealing the tube by heating is not recommended because the seals tend to be tapered and there is the likelihood of lysis. After centrifugation for 5 min, measure the proportion of cells to the whole column (i.e. the PCV) using a reading device.

International Council for Standardization in Haematology Reference Method

Haemoglobin concentration is measured by the routine method on blood specimens with a range of haemoglobin concentrations. Samples of the same specimens are then taken into special borosilicate glass capillary tubes, which are centrifuged for 5 min or longer to achieve full red cell packing. The tubes are then broken at the midpoint of the packed red cells, blood is extracted with a micropipette and its haemoglobin concentration is measured. PCV is calculated as the ratio of the Hb of whole blood to that of the packed cells. This method ²⁶ is appropriate for instrument and reagent manufacturers, but it is time-consuming and requires significant expertise, which makes it impractical for occasional use in routine laboratories. Accordingly, the International Council for Standardization in Haematology (ICSH) has developed a ‘surrogate reference method’.²³

Surrogate Reference Method

Method

1. Take up duplicate samples of well-mixed blood into the specified capillary tubes and centrifuge as described on p. 29.

2. Promptly remove the tubes from the centrifuge, position each in turn against the edge of the 25 mm slide and place this on the stage of the microscope.

3. Ensure that the capillary tube is aligned in a true horizontal position relative to the field of view and, using low power, note on the vernier scale the lengths of the tube at the interfaces of (a) red cells and seal, (b) red cells and leucocytes and (c) plasma and air.

4. Calculate the spun PCV = (B–A)/(C–A). Determine the acceptability of paired measurements – duplicates must agree within 0.007 units; if they do not, the paired tests must be repeated.

5. Calculate the surrogate reference PCV from the formula:

This formula applies only to the specified capillary tubes; other tubes require specific validation by the ICSH reference method ²² so that an appropriate formula can be derived. If the surrogate reference measurements are to be used to validate equipment or methods, a minimum of six different blood samples are required, at least two in each of the ranges of PCV 0.20–0.25, 0.40–0.45 and 0.60–0.65. If necessary, the PCV of normal samples may be adjusted by the appropriate addition or removal of autologous plasma.

Range of Packed Cell Volume in Health

See Chapter 2, Tables 2.1, 2.2 and 2.3.

Manual cell counts and red cell indices

The principles of manual cell counts, the use of the haemocytometer counting chamber for manually counting white cells and platelets and the limitations of these measurements are described in Chapter 26.

An accurate RBC enables the MCV and MCH to be calculated. In well-equipped laboratories, where these indices are provided by an automated system (see p. 41), they are of considerable clinical importance and are widely used in the classification of anaemia. Where automated analysers are not used, manual RBCs (and consequently, calculations of these red cell indices) are so inaccurate and time-consuming that they have become obsolete.

The only measurement that can be obtained with reasonable accuracy by manual methods is MCHC because this is derived from Hb and PCV from the following formula:

Range of MCHC in Health

See Chapter 2, Tables 2.1, 2.2 and 2.3.

Manual differential leucocyte count

Differential leucocyte counts are usually performed by visual examination of blood films that are prepared on slides by the spread or ‘wedge’ technique. Unfortunately, even in well-spread films, the distribution of the various cell types is not totally random (see below).

For a reliable differential count on films spread on slides, the film must not be too thin and the tail of the film should be smooth. To achieve this, the film should be made with a rapid movement using a smooth glass spreader. This should result in a film in which there is some overlap of the red cells, diminishing to separation near the tail, and in which the white cells in the body of the film are not too badly shrunken. If the film is too thin or if a rough-edged spreader is used, many of the white cells, perhaps even 50% of them, accumulate at the edges and in the tail (Fig. 3.1). Moreover, a gross qualitative irregularity in distribution is the rule: polymorphonuclear neutrophils and monocytes predominate at the margins and the tail; lymphocytes predominate in the middle of the film (Fig. 3.2). This separation probably depends on differences in stickiness, size and specific gravity of the different types of cells.

Figure 3.1 Badly spread film. Two areas of a badly spread film from a patient with a white blood cell count of 20 × 10⁹/l showing (A) many leucocytes in the tail and (B) very few leucocytes in body of film.

Figure 3.2 Schematic drawing of a blood film made on a slide. The film has been spread from left to right. An indication is given of the way the white blood cells are distributed.

Differences in distribution of the various types of cells are probably always present to a small extent even in well-made films. Various systems for performing the differential count have been advocated, but none can compensate for the gross irregularities in distribution in a badly made film. On well-made films, the following technique of counting is recommended.

Method

Count the cells using a ×40 objective in a strip running the whole length of the film. Avoid the lateral edges of the film. Inspect the film from the head to the tail and if fewer than 100 cells are encountered in a single narrow strip, examine one or more additional strips until at least 100 cells have been counted. Each longitudinal strip represents the blood drawn out from a small part of the original drop of blood when it has spread out between the slide and spreader (Fig. 3.3). If all the cells are counted in such a strip, the differential totals will closely approximate the true differential count. This technique is liable to error if cells in the thick part of the film cannot be identified; also, it does not allow for any excess of neutrophils and monocytes at the edges of the film, but this preponderance is slight in a well-made film and in practice makes little difference to the result.

Figure 3.3 Schematic drawing illustrating the longitudinal method of performing differential leucocyte counts. The original drop of blood spreads out between spreader and slide (D–D₁). The film is made in such a way that representative strips of films, such as A–A₁ and B–B₁, are formed from blood originally at A and B, respectively. To perform an accurate differential count, all the leucocytes in one or more strips, such as A–A₁ and B–B₁, should be inspected and classified.

This technique is easy to carry out; with high counts (10–30 × 10⁹/l) a short, 2–3 cm, film is desirable. In patients with very high counts (as in leukaemia), the method has to be abandoned and the cells should be counted in any well-spread area where the cell types are easy to identify. Other systems of counting, such as the ‘battlement’ count, are more elaborate but may minimize error owing to variation of distribution of cells between the centre and the edge of the film. The results of the differential count can be recorded using a multiple manual register or they can be directly entered onto a computer.

The variance of the differential count depends not only on artefactual differences in distribution owing to the process of spreading but also on ‘random’ distribution; together they are by far the most important cause of unreliable differential counts. The random distribution means that, if a total of 100 cells are counted, with a true neutrophil proportion of 50%, the range (± 2SD) within which 95% of the counts will fall is of the order of ± 14% (i.e. 36–64%) neutrophils. A 200-cell count can provide a more accurate estimate; in the previous example, the ± 2SD range will be about 40–60%. In a 500-cell count, the range would be reduced to 44–56% neutrophils. In practice, a 100- or 200-cell count is recommended as a routine procedure. However, if abnormal cells are present in small numbers, they are more likely to be detected when 200–500-cell counts are performed than with a 100-cell count.

Basophil and eosinophil counts

A manual basophil or eosinophil count may be necessary to validate an automated count or when abnormal characteristics of the cells render an automated count unreliable, e.g. with degranulated eosinophils. Count the percentage of eosinophils or basophils in a differential count of all the leucocytes on a stained blood film. If the cells of interest are infrequent, a 500-cell differential count should be performed. If fewer than 500 cells are seen in the film, continue the count on a second film. However, if the eosinophil count is markedly elevated a conventional 100-cell count will suffice for most purposes. Calculate the eosinophil or basophil count per litre from the total leucocyte count. It is essential to have thin, preferably short, films with the leucocytes evenly distributed throughout the film and readily identified (see p. 31).

Range of Eosinophil Count in Health

See Chapter 2, Tables 2.1, 2.2 and 2.3.

There is normally considerable diurnal variation in the eosinophil count and differences amounting to as much as 100% have been recorded. The lowest counts are found in the morning (10 a.m. to noon) and the highest at night (midnight to 4 a.m.).^27,²⁸ For a review of the causes of eosinophilia, see Brito-Babapulle.²⁹

Range of Basophil Count in Health

See Chapter 2, Table 2.1.

Gilbert and Ornstein ³⁰ reported a 95% distribution in normal subjects of 0.01–0.08 × 10⁹/l. There are no age or sex differences, although serial counts have shown lower levels during ovulation.³¹

Reporting the Differential Leucocyte Count

The differential count, expressed as the percentage of each type of cell, should be related to the total leucocyte count and the results should be reported in absolute numbers (× 10⁹/l). Myelocytes and metamyelocytes, if present, are recorded separately from neutrophils. Band (stab) cells are generally counted as neutrophils, but it may be useful to record them separately. They normally constitute <6% of the neutrophils; an increase may point to an inflammatory process even in the absence of an absolute leucocytosis.³² However, the band cell count is imprecise and, although it is sometimes recommended in infants, it has been found to be unhelpful in predicting occult bacteraemia in this group.³³

Correcting the Count for Nucleated Red Blood Cells

When nucleated red blood cells (NRBCs) are present, they will be included in the total WBC, which is really a ‘total nucleated cell count’ (TNCC). They should also be included in the differential count, as a percentage of the TNCC and reported in absolute numbers (× 10⁹/l) in the same way as the different types of leucocytes. If they are present in significant numbers, the TNCC should be corrected to obtain the true total WBC. Thus, for example, if total WBC is 8.0 × 10⁹/l and the percentage of NRBCs on the differential count is 25%, then

Care should be taken to differentiate small lymphocytes from nucleated red blood cells (e.g. Chapter 5, Fig. 5.64).

Reference Differential White Cell Count

A reference method is required to validate the accuracy of automated systems ³⁴ (described later). The method that has been used widely for this purpose is essentially similar to the routine manual procedure on stained blood films, but to ensure adequate precision a 200-cell count is carried out by two independent observers, each on two films prepared from the same sample. However, this is still too imprecise for cells with a low frequency; attempts have been made to establish a reference method using flow cytometry with specific monoclonal-antibody labelling of the specific cell types including immature leucocytes.^35,³⁶ More recent flow cytometric protocols also include blast cells, reactive lymphocytes, differentiation between B and T lymphocytes and nucleated red cells.^37,³⁸

Range of Differential White Cells in Health

See Chapter 2, Tables 2.1, 2.2 and 2.3.

Platelet count

The method for manual counting of platelets using a counting chamber is described on p. 610. If an RBC by a semiautomated counter is available, it is possible to obtain an approximation of the platelet count by counting the proportion of platelets to red cells in a thin part of a film made from an EDTA-anticoagulated blood sample, using the ×100 oil-immersion objective and, if possible, eyepieces provided with an adjustable diaphragm, as for a reticulocyte count.