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Reference ranges and normal values
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2 Reference ranges and normal values S. Mitchell Lewis Reference ranges Statistical procedures Confidence limits Normal reference values Physiological variations in the blood count Red cell components
number of factors affect haematological values in apparently healthy individuals. As described in Chapter 1, these include the technique and timing of blood collection, transport and storage of specimens, differences in the subject’s posture when the sample is taken, prior physical activity, or whether the subject is confined to bed. Variation in the analytic methods used may also affect the measurements. These can all be standardized. More problematic are the inherent variables as a result of sex, age, occupation, body build, genetic background, and adaptation to diet and to environment (especially altitude). These factors must be recognized when establishing physiologically normal values. Furthermore, it is difficult to be certain in any survey of a population for the purposes of obtaining data from which normal ranges may be constructed that the “normal” subjects are completely healthy and do not have nutritional deficiencies (especially iron deficiency, which is prevalent in many countries), mild chronic infections, parasitic infestations, or the effects of smoking. Haematological values for the normal and abnormal will overlap, and a value within the recognized normal range may be definitely pathological in a particular subject. For these reasons the concept of “normal values” and “normal ranges” has been
A
11 12 13 13 17 17
Transient changes Leucocyte count Platelet count Other blood constituents
19 20 20 21
replaced by reference values and the reference range, which is defined by reference limits and obtained from measurements on the reference population for a particular test. The reference range is also termed the reference interval.1,2 Ideally, each laboratory should establish a databank of reference values that take account of the variables mentioned earlier, so that an individual’s result can be expressed and interpreted relative to a comparable apparently normal population, insofar as normal can be defined.
REFERENCE RANGES A reference range for a specified population can be established from measurements on a relatively small number of subjects (discussed later) if they are assumed to be representative of the population as a whole.2 The conditions for obtaining samples from the individuals and the analytic procedures must be standardized, whereas data should be analyzed separately for different variables such as individuals who are in bed or ambulant, smokers or nonsmokers, and so on. The samples should be collected at about the same time of day, preferably in the morning before breakfast; the last meal should have been eaten not later than 9 p.m. on the previous
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evening and at that time alcohol should have been restricted to one bottle of beer or an equivalent amount of other alcoholic drink.3
STATISTICAL PROCEDURES In biological measurements it is usually assumed that the data will fit a specified type of pattern, either symmetric (Gaussian) or asymmetric with a skewed distribution (non-Gaussian). With a Gaussian –) can be obtained distribution, the arithmetic mean (χ by dividing the sum of all measurements by the number of observations. The mode is the value that occurs most frequently, and the median (m) is the point at which there are an equal number of observations above and below it. In a true Gaussian distribution they should all be the same. The standard deviation (SD) can be calculated as described on p. 698. If the data fit a Gaussian distribution, when plotted as a frequency histogram the pattern shown in Figure 2.1 is obtained. Taking the mode and the calculated SD as reference points, a Gaussian curve is superimposed on the histogram. From this curve, practical reference limits can be determined even if the original histogram included outlying results from some subjects not belonging to the normal
population. Limits representing the 95% reference range are calculated from arithmetic mean ±2SD (or more accurately ± 1.96SD). When there is a log normal (skew) distribution of measurements, the range to –2SD may even extend to zero (Fig. 2.2A). To avoid this anomaly, the data should be plotted on semilogarithmic graph paper to obtain a normal distribution histogram (Fig. 2.2B). To calculate the mean and SD the data should be converted to their logarithms by means of a calculator with the appropriate facility. The logmean value is obtained by adding the logs of all the measurements and dividing by the number of observations. The log SD is calculated by the formula on p. 698, and the results are then converted to their antilogs to express the data in the arithmetic scale. This process can also be carried out on a computer with an appropriate statistical program. When it is not possible to make an assumption about the type of distribution, a nonparametric procedure may be used instead to obtain the median and SD. For this, the data are sorted out and ranked according to increasing quantitative values, and the median is calculated as illustrated in Table 2.1. To obtain an approximation of the SD, the range that comprises the middle 50% spread (i.e., between 25% and 75% of results) is read and divided by 1.35. This represents 1SD.4
24
20
16
12
8
4
0 100
120 –3
–2
140 –1
0
160 +1
+2
Figure 2.1 Example of establishing a reference range. Histogram of data of haemoglobin measurements in a population, with Gaussian curve superimposed. The ordinate shows the 180 Hb g/l number that occurred at each reference point. The mean was 140 g/l; the reference +3 SD ranges at 1SD, 2SD, and 3SD are indicated.
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Range: mean 2 SD
15
15
10
10
Frequency
Frequency
Range: mean 2 SD
5
0 0
200 400 600 Serum B12 (pg/ml)
800
5
0 100
Linear scale
Table 2.1
Figure 2.2 Example of conversion to a log normal distribution. Data of vitamin B12 measurements in a population. A: Arithmetic scale: Mean 340; 2SD range 200 400 600 900 calculated as 10–665 pg/ml. B: Serum B12 (pg/ml) Geometric scale: Mean 308; 2SD range Logarithmic scale calculated as 120–780 pg/ml.
Illustration of calculation of median*
Rank no.
1
2
3
4
5
6
7
8
9
10
11
Haemoglobin g/l
110
112
113
115
115
118
120
122
124
126
127
(n+1) ; (i.e., position 6 = 118 g/l). 2
If the total (n) is an odd number, the rank position for the median is calculated from Rank no.
1
2
3
4
5
6
7
8
9
10
Haemoglobin g/l
110
112
113
115
115
118
120
122
124
126
If the total (n) is an even number, the rank position for the median lies between rank positions 5 and 6 =
n (n+2) and ; (i.e., between 2 2
(115+118) = 116.5 g/l). 2
*In practice a larger number would be required for meaningful statistics (see text).
Confidence Limits In any of the methods of analysis, a reasonably reliable estimate can be obtained with 40 values, although a larger number (120 or more) is preferable (Fig. 2.3).5 When a large set of reference values is unattainable and precise estimation is impossible, a smaller number of values may still serve as a useful clinical guide. Confidence limits define the reliability (e.g., 95% or 99%) of the established reference values when assessing the significance of a test result, especially when it is on the borderline between normal and abnormal. Calculation of confidence limits is described on p. 698. Another
important measurement is the coefficient of variance (CV) of the test because a wide CV is likely to influence its clinical utility (see p. 629).
NORMAL REFERENCE VALUES The data given in Tables 2.2, 2.3, and 2.4 provide general guidance to normal reference values that are applicable to most healthy adults and children, respectively, in industrialized countries. The data have been derived from personal observations as well as various published reports.6–11 However, slightly different ranges may be found in individual laboratories where different analyzers and methods
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60
70
Figure 2.3 Effect of sample size on reference values. A smoothed distribution graph was obtained for haemoglobin measurement from a group of normal women; the ordinate 0.95 shows the frequency distribution. The 95% reference interval is defined by the lower and higher reference limits, 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 which are 115 and 165 g/l, respectively. The confidence levels for g/l n = 165 these values are shown for three n = 40 sample sizes of 20, 40, and 165 values, respectively. n = 20
Table 2.2
Haematological values for normal adults expressed as a mean ±2SD (95% range)
Red blood cell count Men Women
5.0 ± 0.5 × 1012/l 4.3 ± 0.5 × 1012/l
Haemoglobin Men Women
150 ± 20 g/l 135 ± 15 g/l
Packed cell volume (PCV) or Haematocrit (Hct) Men 0.45 ± 0.05 (l/l) Women 0.41 ± 0.05 (l/l) Mean cell volume (MCV) Men and women
92 ± 9 fl
Differential white cell count Neutrophils 2.0–7.0 × 109/l (40–80%) Lymphocytes 1.0–3.0 × 109/l (20–40%) Monocytes 0.2–1.0 × 109/l (2–10%) Eosinophils 0.02–0.5 × 109/l (1–6%) Basophils 0.02–0.1 × 109/l (<1–2%) Lymphocyte subsets (approximations from ranges in published data) CD3 0.6–2.5 × 109/l (60–85%) CD4 0.4–1.5 × 109/l (30–50%) CD8 0.2–1.1 × 109/l (10–35%) CD4/CD8 ratio 0.7–3.5 Platelet count
280 ± 130 × 109/l
Mean cell haemoglobin concentration (MCHC) Men and women 330 ± 15 g/l
Bleeding time Ivy’s method Template method
2–7 min 2.5–9.5 min
Red cell distribution width (RDW) As coefficient of variation (CV) 12.8 ± 1.2% As standard deviation (SD) 42.5 ± 3.5 fl
Prothrombin time Recombinant thromboplastin
11–16 s 10–12 s
Red cell diameter (mean values) Dry films 6.7–7.7 mm
Activated partial thombo- 30–40 s plastin time (APTT)
Mean cell haemoglobin (MCH) Men and women 29.5 ± 2.5 pg
Red cell density
1092–1100 g/l
Thrombin time
15–19 s
Reticulocyte count
50–100 × 10 /l (0.5–2.5%)
White blood cell count
4.0–10.0 × 10 /l
Plasma fibrinogen Clauss Dry clot
2.0–4.0 g/l 1.5–4.0 g/l
9
9
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Table 2.2
Haematological values for normal adults expressed as a mean ±2SD (95% range)—cont’d
Fibrinogen titre
≥128
Transferrin saturation
Plasminogen
0.75–1.60 u/ml
Euglobulin lysis time
90–240 min
Ferritin Men
Antithrombin
0.75–1.25 u/ml
b-Thromboglobulin
<50 ng/ml
Platelet factor 4
<10 ng/ml
Protein C Function Antigen Protein S Total Free Activity Men Women Heparin cofactor II
0.70–1.40 u/ml 0.61–1.32 u/ml 0.78–1.37 u/ml 0.68–1.52 u/ml 0.60–1.35 u/ml 0.55–1.35 u/ml 0.55–1.45 u/ml
Median red cell fragility (MCF) (g/l NaCl) Fresh blood 4.0–4.45 g/l NaCl 24h at 37°C 4.65–5.9 g/l NaCl Cold agglutinin titre (4°C)<64 Blood volume (normalized Red cell volume Men Women Plasma volume Total blood volume
to “ideal weight”) 30 ± 5 ml/kg 25 ± 5 ml/kg 45 ± 5 ml/kg 70 ± 10 ml/kg
Red cell lifespan
120 ± 30 days
Serum iron Men and Women Total iron-binding capacity
10–30 μmol/(c 0.6–1.7 mg/l) 47–70 μmol/l (c 2.5–4.0 mg/l)
are used. The reference interval, which comprises a range of ±2SD from the mean, indicates the limits that should cover 95% of normal subjects; 99% of normal subjects will be included in a range of ±3SD. Age and sex differences have been taken into account for some values. Even so, the wide ranges that are shown for some tests reflect the influence
Women
16–50% 15–300 μgl/l (median 100 μg/l) 15–200 μg/l (median 40 μgl/l)
Serum vitamin B12
180–640 ng/l
Serum folate
3–20 μg/l (6.8–45 nmol/l)
Red Cell folate
160–640 μg/l (0.36–1.45 μmol/l)
Plasma haemoglobin
10–40 mg/l
Serum haptoglobin Radial immunodiffusion Haemoglobin binding capacity
0.8–2.7 g/l 0.3–2.0 g/l
Hb A2
2.2–3.5%
Hb F
<1.0%
Methaemoglobin
<2.0%
Sedimentation rate (mm in 1 hour at 20 ± 3°C) Men 17–50 yr 10 or < 51–60 yr 12 or < 61–70 yr 14 or < >70 yr 30 or < Women 17–50 yr 51–60 yr 61–70 yr >70 yr
12 or < 19 or < 20 or < 35 or <
Plasma viscosity 25°C 37°C
1.50–1.72 mPa/s 1.16–1.33 mPa/s
of various factors, as described below. Narrower ranges would be expected under standardized conditions. Because modern analyzers provide a high level of technical precision, even small differences in successive measurements may be significant. It is thus important to establish and understand the limits of physiological variation for various tests. The
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Table 2.3 Haematological values for normal infants (amalgamation of data derived from various sources; expressed as mean ±2SD or 95% Range)* Birth
Day 3
Day 7
Day 14
1 Month
2 Months
3–6 Months
Red blood cell count (RBC) × 1012/l
6.0 ± 1.0
5.3 ± 1.3
5.1±1.2
4.9±1.3
4.2 ± 1.2
3.7 ± 0.6
4.7 ± 0.6
Haemoglobin g/l
180 ± 40
80 ± 30
175±4
165±4
140 ± 25
112 ± 18
126 ± 15
Packed cell volume (PCV) l/l
0.60 ± 0.15 0.56 ± 0.11 0.54 ± 0.12 0.51 ± 0.2
0.43 ± 0.10 0.35 ± 0.07 0.35 ± 0.05
Mean cell volume (MCV) fl
110 ± 10
105 ± 13
107 ± 19
105 ± 19
104 ± 12
95 ± 8
76 ± 8
Mean cell Hb (MCH) pg
34 ± 3
34 ± 3
34 ± 3
34 ± 3
33 ± 3
30 ± 3
27 ± 3
Mean cell Hb conc (MCHC) g/l
330 ± 30
330 ± 40
330 ± 50
330 ± 50
330 ± 40
320 ± 35
330 ± 30
Reticulocytes × 109/l
120–400
50–350
50–100
50–100
20–60
30–50
40–100
White blood cell count (WBC) × 109/l
18 ± 8
15 ± 8
14 ± 8
14 ± 8
12 ± 7
10 ± 5
12 ± 6
Neutrophils × 109/l
4–14
3–5
3–6
3–7
3–9
1–5
1–6
Lymphocytes × 10 /l
3–8
2–8
3–9
3–9
3–16
4–10
4–12
Monocytes × 10 /l
0.5–2.0
0.5–1.0
0.1–1.7
0.1–1.7
0.3–1.0
0.4–1.2
0.2–1.2
Eosinophils × 10 /l
0.1–1.0
0.1–2.0
0.1–0.8
0.1–0.9
0.2–1.0
0.1–1.0
0.1–1.0
9
9
9
9
Lymphocyte subsets (× 10 /l)** CD3
3.1–5.6
2.4–6.5
2.0–5.3
CD4
2.2–4.3
1.4–5.6
1.5–3.2
CD8
0.9–1.8
0.7–2.5
0.5–1.6
1.1–4.5
1.1–4.4
1.1–4.2
210–650
200–550
CD4/CD8 ratio Platelets × 10 /l 9
100–450
210–500
160–500
170–500
200–500
*There have been some reports of WBC and platelet counts being lower in venous blood than in capillary blood samples, although still within these reference ranges. In one study venous blood from a newborn gave lower values for haemoglobin, RBC, and WBC than capillary blood but gave higher values for platelets and lymphocytes.60 **Approximations because wide variations have been reported in different studies.
blood count data and other test results can then provide sensitive indications of minor abnormalities that may be important in clinical interpretation and health screening. It should be noted that in Table 2.2 the differential white cell count is shown as percentages and in absolute numbers. Automated analysers
provide absolute counts for each type of leucocyte, and because proportional (percentage) counting is less likely to interpret correctly their absolute increase or decrease, the International Council for Standardization in Haematology has recommended that the differential leucocyte count should always be given as the absolute number of each cell type
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Table 2.4 Haematological values for normal children (amalgamation of data derived from various sources; expressed as mean ±2SD or 95% Range) 1 Year
2–6 Years
6–12 Years
Red cell count × 10 /l
4.5 ± 0.6
4.6 ± 0.6
4.6 ± 0.6
Haemoglobin g/l
126 ± 15
125 ± 15
135 ± 20
Packed cell volume (PCV) l/l
0.34 ± 0.04
0.37 ± 0.03
0.40 ± 0.05
Mean cell volume (MCV) fl
78 ± 6
81 ± 6
86 ± 9
Mean cell Hb (MCH) pg
27 ± 2
27 ± 3
29 ± 4
Mean cell Hb conc (MCHC) g/l
340 ± 20
340 ± 30
340 ± 30
Reticulocytes × 10 /l
30–100
30–100
30–100
White cell count × 109/l
11 ± 5
10 ± 5
9±4
Neutrophils × 109/l
1–7
1.5–8
2–8
Lymphocytes × 10 /l
3.5–11
6–9
1–5
Monocytes × 10 /l
0.2–1.0
0.2–1.0
0.2–1.0
0.1–1.0
0.1–1.0
0.1–1.0
CD3
1.5–5.4
1.6–4.2
0.9–2.5
CD4
1.0–3.6
0.9–2.9
0.5–1.5
CD8
0.6–2.2
0.6–2.0
0.4–1.2
CD4/CD8 ratio
1.0–3.0
0.9–2.7
1.0–3.0
Platelets × 10 /l
200–550
200–490
170–450
12
9
9
9
Eosinophils × 10 /l 9
9
Lymphocyte subsets (¥10 /l)*
9
*Approximations because wide variations have been reported in different studies.
per unit volume of blood.12 The neutrophil:lymphocyte ratio obtained from a differential leucocyte count should be regarded only as an approximation.
PHYSIOLOGICAL VARIATIONS IN THE BLOOD COUNT
Red Cell Components There is considerable variation in the red blood cell count (RBC) and haemoglobin concentration (Hb) at different periods of life. At birth the haemoglobin is higher than at any period subsequently (Table 2.3). The RBC is high immediately after birth,6,13 and values for haemoglobin greater than 200 g/l, RBC
higher than 6.0 × 1012/l, and a packed cell volume (PCV) of 0.65 are encountered frequently when the cord is tied late after delivery. Probably it is the cessation of pulsation of the umbilical artery in the cord as well as the uterine contractions that result in much of the blood contained in the placenta reentering the infant’s circulation. After the immediate postnatal period, the Hb falls fairly steeply to a minimum by about the second month (Fig. 2.4). The RBC and PCV also fall, although less steeply, and the cells may become microcytic with the development of iron deficiency. The changes in the mean cell haemoglobin (MCH), mean cell haemoglobin concentration, and mean cell volume (MCV) from the neonate through infancy to early childhood are shown in Tables 2.3 and 2.4.
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Hb g/l
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210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 1
2
3 4 6 Months after birth
The Hb and RBC normally increase gradually to almost adult levels by puberty.7 However, in a health survey of apparently normal men and women in Britain, mean haemoglobin values of 145 g/l for men and 128 g/l for women have been reported9; the lower normal limits for haemoglobin (i.e., 2SD below the mean) are usually taken as 130 and 120 g/l, respectively, but in some apparently normal men and women lower limits of 120 and 110 g/l, respectively, were noted. Statistically, at least 1% of a normal population have levels at 3SD below the mean, but in some studies there have been considerably larger numbers.9 It is possible that some have nutritional deficiencies, especially iron deficiency, without clinical effects. The levels in women tend to be significantly lower than those of men.9,14 Apart from a hormonal influence on haemopoiesis, iron deficiency is likely to be a factor influencing the difference; the extent to which menstrual blood loss is a significant factor is not clear because a loss of up to 100 ml of blood with each period may lead to iron depletion although without anaemia.15,16 Moreover, arrest of menstruation by oral contraceptives causes an increase in serum iron without affecting the haemoglobin level.17 There may be ethnic differences. A major national health survey in the United States over a 6-year period has shown that in socially comparable populations the haemoglobin in Black Americans is 5–10 g/l lower than their White counterparts at all ages and as much as 20 g/l lower in the first 2 years of life.18
12
Figure 2.4 Changes in haemoglobin values in the first 2 years after birth. The perpendicular lines show means and 2SD ranges.
24
Table 2.5
Haemoglobin values in pregnancy
1st trimester 2nd trimester 3rd trimester
124–135 g/l 110–117 g/l 106–109 g/l*
Mean values postpartum Day 2 Week 1 Week 3 Month 2
104 g/l 107 g/l 116 g/l 119 g/l
*Normal values (120 g/l or higher) may be found when supplementary iron is being given.
Pregnancy In normal pregnancy, there is an increase in erythropoietic activity.19 However, at the same time, an increase in plasma volume occurs,20,21 and this results in a progressive decrease in haemoglobin, PCV, and RBC (Table 2.5). The level returns to normal about a week after delivery. There is a slight increase in MCV during the second trimester.22 Serum ferritin decreases in early pregnancy and usually remains low throughout pregnancy, even when supplementary iron is given,23 although the decrease in haemoglobin is less marked.
Old Age In healthy men and women, haemoglobin, RBC, PCV and related parameters remain remarkably
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constant until the sixth decade. Aging is, however, a gradual process, the start of which is arbitrary. In many studies it is assumed to be 65 years, but anaemia becomes more common in those older than 70–75 years.24–26 This is less marked in women than in men, so that a difference of 20 g/l in younger age groups is reduced to 10 g/l or less in old age. There is a concomitant increase in serum iron, although serum ferritin levels remain higher in men than in women. The factors to be considered for the lower haemoglobin in the elderly include diminished erythropoietic reserves with decrease in erythroid progenitors in the bone marrow, cobalamin deficiency, and chronic inflammatory disease or chronic blood loss (which is often overlooked).24,25 Moderate or severe anaemia should never be attributed to aging per se until underlying disease has been excluded; however, a significant number of elderly subjects with anaemia have no identifiable clinical or nutritional causes.
Transient Changes In addition to the permanent effects of age and sex, there seem to be transient fluctuations, the significance of which is often difficult to assess.
Exercise It is not clear whether light exercise increases the RBC or haemoglobin significantly above the baseline observed with the subject at rest; the effects may be small enough to be submerged in the technical errors of estimation. More significant changes occur in endurance athletes—for example, long-distance runners who may develop so-called “sports anaemia” with a slightly lower haemoglobin and RBC, thought to be the result of increased plasma volume).27,28 Conversely, in sprinters who require a short burst of very strenuous muscular activity, the RBC increases by 0.5 × 1012/l and haemoglobin by 15 g/l, largely because of reduction in plasma volume and to a lesser extent to the re-entry into the circulation of cells previously sequestered in the spleen.29 This is a transient event that occurs in athletes immediately after a race, whereas at all other times there are no significant differences in haemoglobin and PCV between these athletes and nonathletic controls—a point to be aware of when checking athletes for “dope”-related effects.30,31
Decreased levels of serum iron and ferritin occur during endurance training, possibly associated with loss of iron in sweat.32 These effects of exercise must be distinguished from a form of haemolysis known as “runner’s anaemia” or “march haemoglobinuria,” which occurs as a result of pounding of the feet on the ground.33
Posture There is a small but significant alteration in the plasma volume with an increase in haemoglobin and PCV as the posture changes from lying to sitting, especially in women34; conversely, change from walking about to lying down results in a 5–10% decrease in the Hb and PCV. Thus, subjects should rest for 5–10 min before their blood is collected. The difference in position of the arm during venous sampling, whether dependent or held at atrial level, can also affect the PCV. These aspects highlight the importance of using a standardized method for blood collection. This is discussed in Chapter 1 and differences between venous and capillary blood are described on p. 5.
Diurnal and Seasonal Variation Changes in Hb and RBC during the course of the day are usually slight, about 3%, with negligible changes in the MCV and MCH. However, variation of 20% occurs with reticulocytes.8 Serum erythropoietin has a marked diurnal variation, being lowest at 8 a.m., with increases by 40% at 4 p.m. and 60% at 8 p.m.35 Pronounced, but variable, diurnal variations are also seen in serum iron and ferritin.36,37 Because fluctuations will also occur in patients taking ironcontaining supplements, the timing of specimen collection must also take this into account.37 It has been suggested that minor seasonal variations also occur, but the evidence for this is conflicting.38–40
Altitude The effect of altitude is to increase the Hb and PCV and increase the number of circulating red cells with a lower MCV. The magnitude of the polycythaemia depends on the degree of hypoxaemia.41 At an altitude of 2000 metres (c 6500 ft), haemoglobin is c 8–10 g/l and PCV is 0.025 higher than at sea level; at 3000 metres (c 10,000 ft), haemoglobin is c 20 g/l and PCV is 0.060 higher, and at 4000 metres (c 13,000 feet) haemoglobin is
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35 g/l and PCV is 0.110 higher. Corresponding increases occur at intermediate and at higher altitudes.42 These increases appear to be the result of both increased erythropoiesis as a result of the hypoxic stimulus and the decrease in plasma volume that occurs at high altitudes.
Smoking Cigarette smoking affects Hb, RBC, PCV, and MCV (see p. 21).
Leucocyte Count The effect of age is indicated in Tables 2.2, 2.3, and 2.4; at birth, the total leucocyte count is high; neutrophils predominate, reaching a peak of c 13.0 × 109/l at 12 hours, then falling to c 4.0 × 109/l over the next few weeks, and then to a level at which the count remains steady. The lymphocytes decrease during the first 3 days of life often to a low level of c 2.0–2.5 × 109/l and then rise up to the 10th day; after this time, they are the predominant cell (up to about 60%) until the 5th to 7th year when they give way to the neutrophils. From that age onward, the levels are the same as for adults.7 There are also slight sex differences; the total leucocyte count and the neutrophil count may be slightly higher in girls than in boys,7 and in women than in men.43 After menopause, the counts fall in women so that they tend to become lower than in men of similar age.15,43 People differ considerably in their leucocyte counts. Some tend to maintain a relatively constant level over long periods; others have counts that may vary by as much as 100% at different times. In some subjects, there appears to be a rhythm, occurring in cycles of 14 to 23 days, and in women this may be related to some extent to the menstrual cycle. Some forms of oral contraception have been reported to raise the leucocyte count.43 There is no clearcut diurnal variation, but minimum counts are found in the morning with the subject at rest, and during the course of a day there may be differences of 14% for the total leucocyte count, 10% for neutrophils, 14% for lymphocytes, and 20% for eosinophils8; in some cases this may result in a reversed neutrophil:lymphocyte ratio. Random activity may raise the count slightly; strenuous exercise causes increases of up to 30 × 109/l, chiefly because of
decreased splenic blood flow resulting in reduced pooling of neutrophils in the spleen and to some extent because of liberation into the bloodstream of neutrophils formerly sequestered in shutdown capillaries and in the spleen.44 Large numbers of lymphocytes and monocytes also enter the bloodstream during strenuous exercise. However, there have also been reports of neutropenia and lymphopenia in athletes undergoing daily training sessions.45,46 Adrenaline (epinephrine) injection causes an increase in the leucocyte count; here, too, increases in the numbers of all major types of leucocytes (and platelets) occur, possibly reflecting the extent of the reservoir of mature blood cells present not only in the bone marrow and spleen but also in other tissues and organs of the body. Emotion may possibly cause an increase in the leucocyte count in a similar way. A transient lymphocytosis with a reversed neutrophil:lymphocyte ratio occurs in adults with physical stress or trauma.47 This may also occur in patients when visiting their doctors. The effect of ingestion of food is uncertain. Cigarette smoking has an effect on the leucocyte count (see p. 21). A moderate leucocytosis of up to 15 × 109/l is common during pregnancy, owing to a neutrophilia, with the peak in the second trimester.22 The count returns to normal levels a week or so after delivery.48 The environment may influence the leucocyte count. Thus, in tropical Africa, there is a tendency for a reversal of the neutrophil:lymphocyte ratio in individuals with a low total leucocyte count.49 This may partly result from endemic parasitic and protozoal disease; however, genetics are also likely to play a part because significantly lower leucocyte counts, especially neutrophil counts, have also been observed in Africans living in Britain.50 In some tropical areas, reactive eosinophilia or monocytosis is sufficiently common to be regarded as a (normal?) reference value for that population. Elderly people receiving influenza vaccination show a lower total leucocyte count owing to a decrease in lymphocytes.51
Platelet Count There is a slight diurnal variation of about 5%8; this occurs during the course of a day as well as from
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day to day. Within the wide normal reference range, there are some ethnic differences, and in healthy West Indians and Africans platelet counts may on average be 10–20% lower than those in Europeans living in the same environment.52 There may be a sex difference; thus, in women, the platelet count has been reported to be about 20% higher than in men.53 A decrease in the platelet count may occur in women at about the time of menstruation. There are no obvious age differences; however, in the first year after birth the platelet count tends to be at the higher level of the adult normal reference range. Strenuous exercise causes a 30–40% increase in platelet count44; the mechanism is similar to that which occurs with leucocytes.
Other Blood Constituents As with the blood count, variations from normal reference values occur in respect of sex, age, stress, diurnal fluctuation, and so on. These are described in the relevant chapters.
Effects of Smoking Cigarette smoking has a significant effect on many haematological normal reference values.54 Some effects may be transient, and their severity varies between individuals as well as by the number of cigarettes smoked. Smoking 10 or more cigarettes a day results in slightly higher Hb, PCV, and MCV.40,55 This is probably a consequence of the accumulation of carboxyhaemoglobin in the blood together with a decrease in plasma volume. After a single cigarette, the carboxyhaemoglobin level increases by about 1%,56 and in heavy smokers the carboxyhaemoglobin may constitute c 4–5% of the total Hb. There may be polycythaemia.57 The leucocyte count increases, largely as a result of an increase in the neutrophils, and neutrophil function may be affected.14,54,58 Lymphocytes are also affected with an increase in CD4-positive and total lymphocyte count.54,58,59 Smokers tend to have higher platelet counts than nonsmokers, but the counts decrease rapidly on cessation of smoking.54 Studies of platelet aggregation and adhesiveness have given equivocal results, but there appears to be a consistent increase in platelet turnover with decreased platelet survival and increased plasma β-thromboglobulin. Elevated
fibrinogen concentration (with increased plasma viscosity) and reduced protein S have been reported, but smoking does not seem to have any consistent effects on the fibrinolytic system.54 The influence of smoking on the blood is summarized in Table 2.6.
Table 2.6
Effects of cigarette smoking*54–59
Increased
Decreased
Haemoglobin (Hb) Plasma volume Red cell count (RBC) Protein S Packed cell volume (PCV) Mean cell volume (MCV) Mean cell haemoglobin (MCH) White cell count (WBC) Neutrophil count Lymphocyte count T cells (CD4-positive) Monocyte count Carboxyhaemoglobin (>2 %) Platelet count (transient) Mean platelet volume Fibrinogen b-thromboglobulin von Willebrand factor Red cell mass Haptoglobins Plasma viscosity Whole blood viscosity Erythrocyte sedimentation rate (ESR) *Extent of change from normal reference values varies with individuals and amount smoked; some effects may occur only during and immediately after smoking. Some effects may be transient, and their severity varies between individuals as well as by the number of cigarettes smoked.
REFERENCES 1. International Committee for Standardization in Haematology 1981 The theory of reference values. Clinical and Laboratory Haematology 3:369–373. 2. International Federation of Clinical Chemistry and International Council for Standardization in Haematology 1987 The theory of reference values Part 6: Presentation of observed values related to reference values. Journal of Clinical Biochemistry 25:657–662.
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3. International Committee for Standardization in Haematology 1982 Standardization of blood specimen collection procedures for reference values. Clinical and Laboratory Haematology 4:83–86. 4. Tukey JW 1977 Exploratory data analysis. Addison-Wesley, Reading MA. 5. International Federation of Clinical Chemistry and International Council for Standardization in Haematology 1987 The theory of reference values Part 5: Statistical treatment of collected reference values. Determination of reference limits. Journal of Clinical Chemistry and Clinical Biochemistry 25:645–656. 6. Lilleyman JS, Hann IM, Blanchette VS 1999 Pediatric hematology, 2nd ed., Churchill Livingstone, London. 7. Taylor MRH, Holland CV, Spencer R, et al 1997 Haematological reference ranges for school children. Clinical and Laboratory Haematology 19:1–15. 8. Richardson Jones A, Twedt D, Swaim W, et al 1996 Diurnal change of blood count analytes in normal subjects. American Journal of Clinical Pathology 106:723–727. 9. White A, Nicolaas G, Foster K, et al 1991 Health Survey for England: Office of population census and surveys—Social Survey Division. HMSO, London. 10. Bellamy GJ, Hinchliffe RF, Crawshaw KC 2000 Total and differential leucocyte counts in infants at 2, 5 and 13 months of age. Clinical and Laboratory Haematology 22:81–87. 11. Handin RI, Lux SE, Stossel TP 2003 Blood— Principles and Practice of Hematology, 2nd ed., p. 2219. Lippincott Williams and Wilkins, Philadelphia. 12. International Council for Standardization in Haematology: Expert Panel on Cytometry 1995 Recommendation of the International Council for Standardization in Haematology on reporting differential leucocyte counts. Clinical and Laboratory Haematology 17:113. 13. Özbek N, Gürakan B, Kayiran SM 2000 Complete blood cell counts in capillary and venous blood of healthy term newborns. Acta Haematologica 103:226–228. 14. Helman N, Rubenstein LS 1975 The effects of age, sex and smoking on erythrocytes and leukocytes. American Journal of Clinical Pathology 63:35–44. 15. Cruickshank JM, Alexander MK 1970 The effect of age, parity, haemoglobin level and oral contraceptive preparations on the normal leucocyte count. British Journal of Haematology 18:541.
16. Hallberg L, Hogdahl AM, Nilsson L, et al 1966 Menstrual blood loss and iron deficiency. Acta Medica Scandinavica 180:639–650. 17. Burton JL 1967 Effect of oral contraceptives on haemoglobin, packed cell volume, serum-iron and total iron-binding capacity in healthy women. Lancet 1:978–980. 18. Houwen B, van Assendelft OW 2002 Haemoglobinometry: screening and routine practice. In: Rowan RM, van Assendelft OW, Preston FE (eds) Advanced Laboratory Methods in Haematology, pp 182–190, Arnold, London. 19. McMullin MF, White R, LappinT, et al 2003 Haemoglobin during pregnancy: relationship to erythropoietin and haematinic status. European Journal of Haematology 71:44–50. 20. Chesley LC 1972 Plasma and red cell volumes during pregnancy. American Journal of Obstetrics and Gynecology 112:440–450. 21. Large RD, Dynesius R 1973 Blood volume changes during normal pregnancy. Clinics in Haematology 2:433–451. 22. Balloch AJ, Cauchi MN 1993 Reference ranges for haematology parameters in pregnancy derived from patient populations. Clinical and Laboratory Haematology 15:7–14. 23. Howells MR, Jones SE, Napier JAF, et al 1986 Erythropoiesis in pregnancy. British Journal of Haematology 64:595–599. 24. Carmel R 2001 Anemia and aging: an overview of clinical, diagnostic and biological issues. Blood Reviews 15:9–18. 25. Nilsson-Ehle H, Jagenburg R, Landahl S, et al 2000 Blood haemoglobin declines in the elderly: implications for reference intervals from age 70 to 85. European Journal of Haematology 65:297–305. 26. Mattila KS, Kuusela V, Pelliniemi TT, et al 1986 Haematological laboratory findings in the elderly; influence of age and sex. Scandinavian Journal of Clinical and Laboratory Investigation 46:411–415. 27. Smith JA 1995 Exercise, training and red blood cell turnover. Sports Medicine 19:9–31. 28. Green HJ, Sutton JR, Coates G, et al 1991 Response of red cell and plasma volume to prolonged training in humans. Journal of Applied Physiology 70:1810–1815. 29. Allsop P, Peters AM, Arnot RN, et al 1992 Intrasplenic blood cell kinetics in man before and after brief maximal exercise. Clinical Science 83:47–54. 30. Marx JJM, Vergouwen PCJ 1998 Packed-cell volume in elite athletes. Lancet 352:451–452.
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31. Lippi G, Franchini M, Guidi G 2002 Haematocrit measurement and antidoping policies. Clinical and Laboratory Haematology 24:65–66. 32. Cook JD 1994 The effect of endurance training on iron metabolism. Seminars in Hematology 31:146–154. 33. Davidson RJL 1964 March or exertional haemoglobinuria. Seminars in Haematology 6:150–161. 34. Felding P, Tryding N, Hyltoft Petersen P, et al 1980 Effects of posture on concentration of blood constituents in healthy adults: practical application of blood specimen collection procedures recommended by the Scandinavian Committee on Reference Values. Scandinavian Journal of Clinical and Laboratory Investigation 40:615–621. 35. Wide L, Bengtsson C, Birgegård G 1989 Circadian rhythm of erythropoietin in human serum. British Journal of Haematology 72:85–90. 36. Romslo I, Talstad I 1988 Day-to-day variations in serum iron, serum iron binding capacity, serum ferritin and erythrocyte protoporphyrin concentration in anaemic subjects. European Journal of Haematology 40:79. 37. Dale JC, Burritt MF, Zinsmeister AR 2002 Diurnal variation of serum iron, iron-binding capacity, transferrin saturation and ferritin levels. American Journal of Clinical Pathology 117:802–808. 38. Costongs GMPJ, Janson PCW, Bas BM, et al 1985 Short-term and long-term intra-individual variations and critical differences of haematological laboratory parameters. Journal of Clinical Chemistry and Biochemistry 23:69–76. 39. Ross DW, Ayscue LH, Watson J, et al 1988 Stability of hematologic parameters in healthy subjects: intraindividual versus interindividual variation. American Journal of Clinical Pathology 90:262–267. 40. Kristal-Boneh E, Froom P, Harari G, et al 1997 Seasonal differences in blood cell parameters and the association with cigarette smoking. Clinical and Laboratory Haematology 19:177–181 41. Ruiz-Arguelles GJ, Sanchez-Medal L, Loria A, et al 1980 Red cell indices in normal adults residing at altitudes from sea level to 2670 meters. American Journal of Hematology 8:265–271. 42. Myhre LD, Dill DB, Hall FG, et al 1970 Blood volume changes during three week residence at high altitude. Clinical Chemistry 16:7–14. 43. England JM, Bain BJ 1976 Total and differential leucocyte count. British Journal of Haematology 33:1–7. 44. Allsop P, Arnot R, Gwilliam M et al 1988 Does splenic autotransfusion occur during high intensity
45.
46.
47.
48.
49. 50.
51.
52.
53.
54.
55.
56.
57.
cycle exercise in man. Journal of Physiology (London) 407:24P Watson HG, Meiklejohn DJ 2001 Leucopenia in professional football players. British Journal of Haematology 112:824–827. Bain BJ, Philllips D, Thomson K, et al 2000 Investigation of the effect of marathon running on leucocyte counts of subjects of different ethnic origins: relevance to the aetiology of ethnic neutropenia. British Journal of Haematology 108:483–487. Karandikar NJ, Hotchkiss EC, McKenna RW 2002 Transient stress lymphocytosis: an immunophenotypic characterization of the most common cause of newly identified adult lymphocytosis in a tertiary hospital. American Journal of Clinical Pathology 117:819–825. Cruickshank JM 1970 The effects of parity on the leucocyte count in pregnant and non-pregnant women. British Journal of Haematology 18:531–540. Woodliff HJ, Kataaha PK, Tibaleka AK, et al 1972 Total leucocyte count in Africans. Lancet 2:875. Bain BJ, Seed M, Godsland I 1984 Normal values for peripheral blood white cell counts in women of four different ethnic origins. Journal of Clinical Pathology 37:188–193. Cummins D, Wilson ME, Foulger KJ, et al 1998 Haematological changes associated with influenza vaccination in people aged over 65: case report and prospective study. Clinical and Laboratory Haematology 20:285–287. Bain BJ, Seed M 1986 Platelet count and platelet size in healthy Africans and West Indians. Clinical and Laboratory Haematology 8:43–48. Stevens RF, Alexander MK 1977 A sex difference in the platelet count. British Journal of Haematology 37:295–300. Bain BJ 1992 The haematological effects of smoking. Journal of Smoking-related Diseases 3:99–108. Whitehead TP, Robinson D, Allaway SL, et al 1995 The effects of cigarette smoking and alcohol consumption on blood haemoglobin, erythrocytes and leucocytes: a dose related study on male subjects. Clinical and Laboratory Haematology 17:131–138. Russell MA, Wilson C, Cole PV, et al 1973 Comparison of increases in carboxyhaemoglobin after smoking “extramild” and “non mild” cigarettes. Lancet 2:687–690. Smith JR, Landaw SA 1978 Smoker’s polycythemia. New England Journal of Medicine 298:6–10.
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58. Parry H, Cohen S, Schlarb J 1997 Smoking, alcohol consumption and leukocyte counts. American Journal of Clinical Pathology 107:64–67. 59. Bain BJ, Rothwell M, Feher MD et al 1992 Acute changes in haematological parameters on cessation of smoking. Journal of the Royal Society of Medicine 85:80–82.
60. Kayiran SM, Özbek N, Turan M, et al 2003 Significant differences between capillary and venous complete blood counts in the neonatal period. Clinical and Laboratory Haematology 25:9–16.
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2 Reference ranges and normal values S. Mitchell Lewis Reference ranges Statistical procedures Confidence limits Normal reference values Physiological variations in the blood count Red cell components
number of factors affect haematological values in apparently healthy individuals. As described in Chapter 1, these include the technique and timing of blood collection, transport and storage of specimens, differences in the subject’s posture when the sample is taken, prior physical activity, or whether the subject is confined to bed. Variation in the analytic methods used may also affect the measurements. These can all be standardized. More problematic are the inherent variables as a result of sex, age, occupation, body build, genetic background, and adaptation to diet and to environment (especially altitude). These factors must be recognized when establishing physiologically normal values. Furthermore, it is difficult to be certain in any survey of a population for the purposes of obtaining data from which normal ranges may be constructed that the “normal” subjects are completely healthy and do not have nutritional deficiencies (especially iron deficiency, which is prevalent in many countries), mild chronic infections, parasitic infestations, or the effects of smoking. Haematological values for the normal and abnormal will overlap, and a value within the recognized normal range may be definitely pathological in a particular subject. For these reasons the concept of “normal values” and “normal ranges” has been
A
11 12 13 13 17 17
Transient changes Leucocyte count Platelet count Other blood constituents
19 20 20 21
replaced by reference values and the reference range, which is defined by reference limits and obtained from measurements on the reference population for a particular test. The reference range is also termed the reference interval.1,2 Ideally, each laboratory should establish a databank of reference values that take account of the variables mentioned earlier, so that an individual’s result can be expressed and interpreted relative to a comparable apparently normal population, insofar as normal can be defined.
REFERENCE RANGES A reference range for a specified population can be established from measurements on a relatively small number of subjects (discussed later) if they are assumed to be representative of the population as a whole.2 The conditions for obtaining samples from the individuals and the analytic procedures must be standardized, whereas data should be analyzed separately for different variables such as individuals who are in bed or ambulant, smokers or nonsmokers, and so on. The samples should be collected at about the same time of day, preferably in the morning before breakfast; the last meal should have been eaten not later than 9 p.m. on the previous
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evening and at that time alcohol should have been restricted to one bottle of beer or an equivalent amount of other alcoholic drink.3
STATISTICAL PROCEDURES In biological measurements it is usually assumed that the data will fit a specified type of pattern, either symmetric (Gaussian) or asymmetric with a skewed distribution (non-Gaussian). With a Gaussian –) can be obtained distribution, the arithmetic mean (χ by dividing the sum of all measurements by the number of observations. The mode is the value that occurs most frequently, and the median (m) is the point at which there are an equal number of observations above and below it. In a true Gaussian distribution they should all be the same. The standard deviation (SD) can be calculated as described on p. 698. If the data fit a Gaussian distribution, when plotted as a frequency histogram the pattern shown in Figure 2.1 is obtained. Taking the mode and the calculated SD as reference points, a Gaussian curve is superimposed on the histogram. From this curve, practical reference limits can be determined even if the original histogram included outlying results from some subjects not belonging to the normal
population. Limits representing the 95% reference range are calculated from arithmetic mean ±2SD (or more accurately ± 1.96SD). When there is a log normal (skew) distribution of measurements, the range to –2SD may even extend to zero (Fig. 2.2A). To avoid this anomaly, the data should be plotted on semilogarithmic graph paper to obtain a normal distribution histogram (Fig. 2.2B). To calculate the mean and SD the data should be converted to their logarithms by means of a calculator with the appropriate facility. The logmean value is obtained by adding the logs of all the measurements and dividing by the number of observations. The log SD is calculated by the formula on p. 698, and the results are then converted to their antilogs to express the data in the arithmetic scale. This process can also be carried out on a computer with an appropriate statistical program. When it is not possible to make an assumption about the type of distribution, a nonparametric procedure may be used instead to obtain the median and SD. For this, the data are sorted out and ranked according to increasing quantitative values, and the median is calculated as illustrated in Table 2.1. To obtain an approximation of the SD, the range that comprises the middle 50% spread (i.e., between 25% and 75% of results) is read and divided by 1.35. This represents 1SD.4
24
20
16
12
8
4
0 100
120 –3
–2
140 –1
0
160 +1
+2
Figure 2.1 Example of establishing a reference range. Histogram of data of haemoglobin measurements in a population, with Gaussian curve superimposed. The ordinate shows the 180 Hb g/l number that occurred at each reference point. The mean was 140 g/l; the reference +3 SD ranges at 1SD, 2SD, and 3SD are indicated.
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Range: mean 2 SD
15
15
10
10
Frequency
Frequency
Range: mean 2 SD
5
0 0
200 400 600 Serum B12 (pg/ml)
800
5
0 100
Linear scale
Table 2.1
Figure 2.2 Example of conversion to a log normal distribution. Data of vitamin B12 measurements in a population. A: Arithmetic scale: Mean 340; 2SD range 200 400 600 900 calculated as 10–665 pg/ml. B: Serum B12 (pg/ml) Geometric scale: Mean 308; 2SD range Logarithmic scale calculated as 120–780 pg/ml.
Illustration of calculation of median*
Rank no.
1
2
3
4
5
6
7
8
9
10
11
Haemoglobin g/l
110
112
113
115
115
118
120
122
124
126
127
(n+1) ; (i.e., position 6 = 118 g/l). 2
If the total (n) is an odd number, the rank position for the median is calculated from Rank no.
1
2
3
4
5
6
7
8
9
10
Haemoglobin g/l
110
112
113
115
115
118
120
122
124
126
If the total (n) is an even number, the rank position for the median lies between rank positions 5 and 6 =
n (n+2) and ; (i.e., between 2 2
(115+118) = 116.5 g/l). 2
*In practice a larger number would be required for meaningful statistics (see text).
Confidence Limits In any of the methods of analysis, a reasonably reliable estimate can be obtained with 40 values, although a larger number (120 or more) is preferable (Fig. 2.3).5 When a large set of reference values is unattainable and precise estimation is impossible, a smaller number of values may still serve as a useful clinical guide. Confidence limits define the reliability (e.g., 95% or 99%) of the established reference values when assessing the significance of a test result, especially when it is on the borderline between normal and abnormal. Calculation of confidence limits is described on p. 698. Another
important measurement is the coefficient of variance (CV) of the test because a wide CV is likely to influence its clinical utility (see p. 629).
NORMAL REFERENCE VALUES The data given in Tables 2.2, 2.3, and 2.4 provide general guidance to normal reference values that are applicable to most healthy adults and children, respectively, in industrialized countries. The data have been derived from personal observations as well as various published reports.6–11 However, slightly different ranges may be found in individual laboratories where different analyzers and methods
13
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60
70
Figure 2.3 Effect of sample size on reference values. A smoothed distribution graph was obtained for haemoglobin measurement from a group of normal women; the ordinate 0.95 shows the frequency distribution. The 95% reference interval is defined by the lower and higher reference limits, 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 which are 115 and 165 g/l, respectively. The confidence levels for g/l n = 165 these values are shown for three n = 40 sample sizes of 20, 40, and 165 values, respectively. n = 20
Table 2.2
Haematological values for normal adults expressed as a mean ±2SD (95% range)
Red blood cell count Men Women
5.0 ± 0.5 × 1012/l 4.3 ± 0.5 × 1012/l
Haemoglobin Men Women
150 ± 20 g/l 135 ± 15 g/l
Packed cell volume (PCV) or Haematocrit (Hct) Men 0.45 ± 0.05 (l/l) Women 0.41 ± 0.05 (l/l) Mean cell volume (MCV) Men and women
92 ± 9 fl
Differential white cell count Neutrophils 2.0–7.0 × 109/l (40–80%) Lymphocytes 1.0–3.0 × 109/l (20–40%) Monocytes 0.2–1.0 × 109/l (2–10%) Eosinophils 0.02–0.5 × 109/l (1–6%) Basophils 0.02–0.1 × 109/l (<1–2%) Lymphocyte subsets (approximations from ranges in published data) CD3 0.6–2.5 × 109/l (60–85%) CD4 0.4–1.5 × 109/l (30–50%) CD8 0.2–1.1 × 109/l (10–35%) CD4/CD8 ratio 0.7–3.5 Platelet count
280 ± 130 × 109/l
Mean cell haemoglobin concentration (MCHC) Men and women 330 ± 15 g/l
Bleeding time Ivy’s method Template method
2–7 min 2.5–9.5 min
Red cell distribution width (RDW) As coefficient of variation (CV) 12.8 ± 1.2% As standard deviation (SD) 42.5 ± 3.5 fl
Prothrombin time Recombinant thromboplastin
11–16 s 10–12 s
Red cell diameter (mean values) Dry films 6.7–7.7 mm
Activated partial thombo- 30–40 s plastin time (APTT)
Mean cell haemoglobin (MCH) Men and women 29.5 ± 2.5 pg
Red cell density
1092–1100 g/l
Thrombin time
15–19 s
Reticulocyte count
50–100 × 10 /l (0.5–2.5%)
White blood cell count
4.0–10.0 × 10 /l
Plasma fibrinogen Clauss Dry clot
2.0–4.0 g/l 1.5–4.0 g/l
9
9
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Table 2.2
Haematological values for normal adults expressed as a mean ±2SD (95% range)—cont’d
Fibrinogen titre
≥128
Transferrin saturation
Plasminogen
0.75–1.60 u/ml
Euglobulin lysis time
90–240 min
Ferritin Men
Antithrombin
0.75–1.25 u/ml
b-Thromboglobulin
<50 ng/ml
Platelet factor 4
<10 ng/ml
Protein C Function Antigen Protein S Total Free Activity Men Women Heparin cofactor II
0.70–1.40 u/ml 0.61–1.32 u/ml 0.78–1.37 u/ml 0.68–1.52 u/ml 0.60–1.35 u/ml 0.55–1.35 u/ml 0.55–1.45 u/ml
Median red cell fragility (MCF) (g/l NaCl) Fresh blood 4.0–4.45 g/l NaCl 24h at 37°C 4.65–5.9 g/l NaCl Cold agglutinin titre (4°C)<64 Blood volume (normalized Red cell volume Men Women Plasma volume Total blood volume
to “ideal weight”) 30 ± 5 ml/kg 25 ± 5 ml/kg 45 ± 5 ml/kg 70 ± 10 ml/kg
Red cell lifespan
120 ± 30 days
Serum iron Men and Women Total iron-binding capacity
10–30 μmol/(c 0.6–1.7 mg/l) 47–70 μmol/l (c 2.5–4.0 mg/l)
are used. The reference interval, which comprises a range of ±2SD from the mean, indicates the limits that should cover 95% of normal subjects; 99% of normal subjects will be included in a range of ±3SD. Age and sex differences have been taken into account for some values. Even so, the wide ranges that are shown for some tests reflect the influence
Women
16–50% 15–300 μgl/l (median 100 μg/l) 15–200 μg/l (median 40 μgl/l)
Serum vitamin B12
180–640 ng/l
Serum folate
3–20 μg/l (6.8–45 nmol/l)
Red Cell folate
160–640 μg/l (0.36–1.45 μmol/l)
Plasma haemoglobin
10–40 mg/l
Serum haptoglobin Radial immunodiffusion Haemoglobin binding capacity
0.8–2.7 g/l 0.3–2.0 g/l
Hb A2
2.2–3.5%
Hb F
<1.0%
Methaemoglobin
<2.0%
Sedimentation rate (mm in 1 hour at 20 ± 3°C) Men 17–50 yr 10 or < 51–60 yr 12 or < 61–70 yr 14 or < >70 yr 30 or < Women 17–50 yr 51–60 yr 61–70 yr >70 yr
12 or < 19 or < 20 or < 35 or <
Plasma viscosity 25°C 37°C
1.50–1.72 mPa/s 1.16–1.33 mPa/s
of various factors, as described below. Narrower ranges would be expected under standardized conditions. Because modern analyzers provide a high level of technical precision, even small differences in successive measurements may be significant. It is thus important to establish and understand the limits of physiological variation for various tests. The
15
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Table 2.3 Haematological values for normal infants (amalgamation of data derived from various sources; expressed as mean ±2SD or 95% Range)* Birth
Day 3
Day 7
Day 14
1 Month
2 Months
3–6 Months
Red blood cell count (RBC) × 1012/l
6.0 ± 1.0
5.3 ± 1.3
5.1±1.2
4.9±1.3
4.2 ± 1.2
3.7 ± 0.6
4.7 ± 0.6
Haemoglobin g/l
180 ± 40
80 ± 30
175±4
165±4
140 ± 25
112 ± 18
126 ± 15
Packed cell volume (PCV) l/l
0.60 ± 0.15 0.56 ± 0.11 0.54 ± 0.12 0.51 ± 0.2
0.43 ± 0.10 0.35 ± 0.07 0.35 ± 0.05
Mean cell volume (MCV) fl
110 ± 10
105 ± 13
107 ± 19
105 ± 19
104 ± 12
95 ± 8
76 ± 8
Mean cell Hb (MCH) pg
34 ± 3
34 ± 3
34 ± 3
34 ± 3
33 ± 3
30 ± 3
27 ± 3
Mean cell Hb conc (MCHC) g/l
330 ± 30
330 ± 40
330 ± 50
330 ± 50
330 ± 40
320 ± 35
330 ± 30
Reticulocytes × 109/l
120–400
50–350
50–100
50–100
20–60
30–50
40–100
White blood cell count (WBC) × 109/l
18 ± 8
15 ± 8
14 ± 8
14 ± 8
12 ± 7
10 ± 5
12 ± 6
Neutrophils × 109/l
4–14
3–5
3–6
3–7
3–9
1–5
1–6
Lymphocytes × 10 /l
3–8
2–8
3–9
3–9
3–16
4–10
4–12
Monocytes × 10 /l
0.5–2.0
0.5–1.0
0.1–1.7
0.1–1.7
0.3–1.0
0.4–1.2
0.2–1.2
Eosinophils × 10 /l
0.1–1.0
0.1–2.0
0.1–0.8
0.1–0.9
0.2–1.0
0.1–1.0
0.1–1.0
9
9
9
9
Lymphocyte subsets (× 10 /l)** CD3
3.1–5.6
2.4–6.5
2.0–5.3
CD4
2.2–4.3
1.4–5.6
1.5–3.2
CD8
0.9–1.8
0.7–2.5
0.5–1.6
1.1–4.5
1.1–4.4
1.1–4.2
210–650
200–550
CD4/CD8 ratio Platelets × 10 /l 9
100–450
210–500
160–500
170–500
200–500
*There have been some reports of WBC and platelet counts being lower in venous blood than in capillary blood samples, although still within these reference ranges. In one study venous blood from a newborn gave lower values for haemoglobin, RBC, and WBC than capillary blood but gave higher values for platelets and lymphocytes.60 **Approximations because wide variations have been reported in different studies.
blood count data and other test results can then provide sensitive indications of minor abnormalities that may be important in clinical interpretation and health screening. It should be noted that in Table 2.2 the differential white cell count is shown as percentages and in absolute numbers. Automated analysers
provide absolute counts for each type of leucocyte, and because proportional (percentage) counting is less likely to interpret correctly their absolute increase or decrease, the International Council for Standardization in Haematology has recommended that the differential leucocyte count should always be given as the absolute number of each cell type
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Table 2.4 Haematological values for normal children (amalgamation of data derived from various sources; expressed as mean ±2SD or 95% Range) 1 Year
2–6 Years
6–12 Years
Red cell count × 10 /l
4.5 ± 0.6
4.6 ± 0.6
4.6 ± 0.6
Haemoglobin g/l
126 ± 15
125 ± 15
135 ± 20
Packed cell volume (PCV) l/l
0.34 ± 0.04
0.37 ± 0.03
0.40 ± 0.05
Mean cell volume (MCV) fl
78 ± 6
81 ± 6
86 ± 9
Mean cell Hb (MCH) pg
27 ± 2
27 ± 3
29 ± 4
Mean cell Hb conc (MCHC) g/l
340 ± 20
340 ± 30
340 ± 30
Reticulocytes × 10 /l
30–100
30–100
30–100
White cell count × 109/l
11 ± 5
10 ± 5
9±4
Neutrophils × 109/l
1–7
1.5–8
2–8
Lymphocytes × 10 /l
3.5–11
6–9
1–5
Monocytes × 10 /l
0.2–1.0
0.2–1.0
0.2–1.0
0.1–1.0
0.1–1.0
0.1–1.0
CD3
1.5–5.4
1.6–4.2
0.9–2.5
CD4
1.0–3.6
0.9–2.9
0.5–1.5
CD8
0.6–2.2
0.6–2.0
0.4–1.2
CD4/CD8 ratio
1.0–3.0
0.9–2.7
1.0–3.0
Platelets × 10 /l
200–550
200–490
170–450
12
9
9
9
Eosinophils × 10 /l 9
9
Lymphocyte subsets (¥10 /l)*
9
*Approximations because wide variations have been reported in different studies.
per unit volume of blood.12 The neutrophil:lymphocyte ratio obtained from a differential leucocyte count should be regarded only as an approximation.
PHYSIOLOGICAL VARIATIONS IN THE BLOOD COUNT
Red Cell Components There is considerable variation in the red blood cell count (RBC) and haemoglobin concentration (Hb) at different periods of life. At birth the haemoglobin is higher than at any period subsequently (Table 2.3). The RBC is high immediately after birth,6,13 and values for haemoglobin greater than 200 g/l, RBC
higher than 6.0 × 1012/l, and a packed cell volume (PCV) of 0.65 are encountered frequently when the cord is tied late after delivery. Probably it is the cessation of pulsation of the umbilical artery in the cord as well as the uterine contractions that result in much of the blood contained in the placenta reentering the infant’s circulation. After the immediate postnatal period, the Hb falls fairly steeply to a minimum by about the second month (Fig. 2.4). The RBC and PCV also fall, although less steeply, and the cells may become microcytic with the development of iron deficiency. The changes in the mean cell haemoglobin (MCH), mean cell haemoglobin concentration, and mean cell volume (MCV) from the neonate through infancy to early childhood are shown in Tables 2.3 and 2.4.
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Hb g/l
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210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 1
2
3 4 6 Months after birth
The Hb and RBC normally increase gradually to almost adult levels by puberty.7 However, in a health survey of apparently normal men and women in Britain, mean haemoglobin values of 145 g/l for men and 128 g/l for women have been reported9; the lower normal limits for haemoglobin (i.e., 2SD below the mean) are usually taken as 130 and 120 g/l, respectively, but in some apparently normal men and women lower limits of 120 and 110 g/l, respectively, were noted. Statistically, at least 1% of a normal population have levels at 3SD below the mean, but in some studies there have been considerably larger numbers.9 It is possible that some have nutritional deficiencies, especially iron deficiency, without clinical effects. The levels in women tend to be significantly lower than those of men.9,14 Apart from a hormonal influence on haemopoiesis, iron deficiency is likely to be a factor influencing the difference; the extent to which menstrual blood loss is a significant factor is not clear because a loss of up to 100 ml of blood with each period may lead to iron depletion although without anaemia.15,16 Moreover, arrest of menstruation by oral contraceptives causes an increase in serum iron without affecting the haemoglobin level.17 There may be ethnic differences. A major national health survey in the United States over a 6-year period has shown that in socially comparable populations the haemoglobin in Black Americans is 5–10 g/l lower than their White counterparts at all ages and as much as 20 g/l lower in the first 2 years of life.18
12
Figure 2.4 Changes in haemoglobin values in the first 2 years after birth. The perpendicular lines show means and 2SD ranges.
24
Table 2.5
Haemoglobin values in pregnancy
1st trimester 2nd trimester 3rd trimester
124–135 g/l 110–117 g/l 106–109 g/l*
Mean values postpartum Day 2 Week 1 Week 3 Month 2
104 g/l 107 g/l 116 g/l 119 g/l
*Normal values (120 g/l or higher) may be found when supplementary iron is being given.
Pregnancy In normal pregnancy, there is an increase in erythropoietic activity.19 However, at the same time, an increase in plasma volume occurs,20,21 and this results in a progressive decrease in haemoglobin, PCV, and RBC (Table 2.5). The level returns to normal about a week after delivery. There is a slight increase in MCV during the second trimester.22 Serum ferritin decreases in early pregnancy and usually remains low throughout pregnancy, even when supplementary iron is given,23 although the decrease in haemoglobin is less marked.
Old Age In healthy men and women, haemoglobin, RBC, PCV and related parameters remain remarkably
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constant until the sixth decade. Aging is, however, a gradual process, the start of which is arbitrary. In many studies it is assumed to be 65 years, but anaemia becomes more common in those older than 70–75 years.24–26 This is less marked in women than in men, so that a difference of 20 g/l in younger age groups is reduced to 10 g/l or less in old age. There is a concomitant increase in serum iron, although serum ferritin levels remain higher in men than in women. The factors to be considered for the lower haemoglobin in the elderly include diminished erythropoietic reserves with decrease in erythroid progenitors in the bone marrow, cobalamin deficiency, and chronic inflammatory disease or chronic blood loss (which is often overlooked).24,25 Moderate or severe anaemia should never be attributed to aging per se until underlying disease has been excluded; however, a significant number of elderly subjects with anaemia have no identifiable clinical or nutritional causes.
Transient Changes In addition to the permanent effects of age and sex, there seem to be transient fluctuations, the significance of which is often difficult to assess.
Exercise It is not clear whether light exercise increases the RBC or haemoglobin significantly above the baseline observed with the subject at rest; the effects may be small enough to be submerged in the technical errors of estimation. More significant changes occur in endurance athletes—for example, long-distance runners who may develop so-called “sports anaemia” with a slightly lower haemoglobin and RBC, thought to be the result of increased plasma volume).27,28 Conversely, in sprinters who require a short burst of very strenuous muscular activity, the RBC increases by 0.5 × 1012/l and haemoglobin by 15 g/l, largely because of reduction in plasma volume and to a lesser extent to the re-entry into the circulation of cells previously sequestered in the spleen.29 This is a transient event that occurs in athletes immediately after a race, whereas at all other times there are no significant differences in haemoglobin and PCV between these athletes and nonathletic controls—a point to be aware of when checking athletes for “dope”-related effects.30,31
Decreased levels of serum iron and ferritin occur during endurance training, possibly associated with loss of iron in sweat.32 These effects of exercise must be distinguished from a form of haemolysis known as “runner’s anaemia” or “march haemoglobinuria,” which occurs as a result of pounding of the feet on the ground.33
Posture There is a small but significant alteration in the plasma volume with an increase in haemoglobin and PCV as the posture changes from lying to sitting, especially in women34; conversely, change from walking about to lying down results in a 5–10% decrease in the Hb and PCV. Thus, subjects should rest for 5–10 min before their blood is collected. The difference in position of the arm during venous sampling, whether dependent or held at atrial level, can also affect the PCV. These aspects highlight the importance of using a standardized method for blood collection. This is discussed in Chapter 1 and differences between venous and capillary blood are described on p. 5.
Diurnal and Seasonal Variation Changes in Hb and RBC during the course of the day are usually slight, about 3%, with negligible changes in the MCV and MCH. However, variation of 20% occurs with reticulocytes.8 Serum erythropoietin has a marked diurnal variation, being lowest at 8 a.m., with increases by 40% at 4 p.m. and 60% at 8 p.m.35 Pronounced, but variable, diurnal variations are also seen in serum iron and ferritin.36,37 Because fluctuations will also occur in patients taking ironcontaining supplements, the timing of specimen collection must also take this into account.37 It has been suggested that minor seasonal variations also occur, but the evidence for this is conflicting.38–40
Altitude The effect of altitude is to increase the Hb and PCV and increase the number of circulating red cells with a lower MCV. The magnitude of the polycythaemia depends on the degree of hypoxaemia.41 At an altitude of 2000 metres (c 6500 ft), haemoglobin is c 8–10 g/l and PCV is 0.025 higher than at sea level; at 3000 metres (c 10,000 ft), haemoglobin is c 20 g/l and PCV is 0.060 higher, and at 4000 metres (c 13,000 feet) haemoglobin is
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35 g/l and PCV is 0.110 higher. Corresponding increases occur at intermediate and at higher altitudes.42 These increases appear to be the result of both increased erythropoiesis as a result of the hypoxic stimulus and the decrease in plasma volume that occurs at high altitudes.
Smoking Cigarette smoking affects Hb, RBC, PCV, and MCV (see p. 21).
Leucocyte Count The effect of age is indicated in Tables 2.2, 2.3, and 2.4; at birth, the total leucocyte count is high; neutrophils predominate, reaching a peak of c 13.0 × 109/l at 12 hours, then falling to c 4.0 × 109/l over the next few weeks, and then to a level at which the count remains steady. The lymphocytes decrease during the first 3 days of life often to a low level of c 2.0–2.5 × 109/l and then rise up to the 10th day; after this time, they are the predominant cell (up to about 60%) until the 5th to 7th year when they give way to the neutrophils. From that age onward, the levels are the same as for adults.7 There are also slight sex differences; the total leucocyte count and the neutrophil count may be slightly higher in girls than in boys,7 and in women than in men.43 After menopause, the counts fall in women so that they tend to become lower than in men of similar age.15,43 People differ considerably in their leucocyte counts. Some tend to maintain a relatively constant level over long periods; others have counts that may vary by as much as 100% at different times. In some subjects, there appears to be a rhythm, occurring in cycles of 14 to 23 days, and in women this may be related to some extent to the menstrual cycle. Some forms of oral contraception have been reported to raise the leucocyte count.43 There is no clearcut diurnal variation, but minimum counts are found in the morning with the subject at rest, and during the course of a day there may be differences of 14% for the total leucocyte count, 10% for neutrophils, 14% for lymphocytes, and 20% for eosinophils8; in some cases this may result in a reversed neutrophil:lymphocyte ratio. Random activity may raise the count slightly; strenuous exercise causes increases of up to 30 × 109/l, chiefly because of
decreased splenic blood flow resulting in reduced pooling of neutrophils in the spleen and to some extent because of liberation into the bloodstream of neutrophils formerly sequestered in shutdown capillaries and in the spleen.44 Large numbers of lymphocytes and monocytes also enter the bloodstream during strenuous exercise. However, there have also been reports of neutropenia and lymphopenia in athletes undergoing daily training sessions.45,46 Adrenaline (epinephrine) injection causes an increase in the leucocyte count; here, too, increases in the numbers of all major types of leucocytes (and platelets) occur, possibly reflecting the extent of the reservoir of mature blood cells present not only in the bone marrow and spleen but also in other tissues and organs of the body. Emotion may possibly cause an increase in the leucocyte count in a similar way. A transient lymphocytosis with a reversed neutrophil:lymphocyte ratio occurs in adults with physical stress or trauma.47 This may also occur in patients when visiting their doctors. The effect of ingestion of food is uncertain. Cigarette smoking has an effect on the leucocyte count (see p. 21). A moderate leucocytosis of up to 15 × 109/l is common during pregnancy, owing to a neutrophilia, with the peak in the second trimester.22 The count returns to normal levels a week or so after delivery.48 The environment may influence the leucocyte count. Thus, in tropical Africa, there is a tendency for a reversal of the neutrophil:lymphocyte ratio in individuals with a low total leucocyte count.49 This may partly result from endemic parasitic and protozoal disease; however, genetics are also likely to play a part because significantly lower leucocyte counts, especially neutrophil counts, have also been observed in Africans living in Britain.50 In some tropical areas, reactive eosinophilia or monocytosis is sufficiently common to be regarded as a (normal?) reference value for that population. Elderly people receiving influenza vaccination show a lower total leucocyte count owing to a decrease in lymphocytes.51
Platelet Count There is a slight diurnal variation of about 5%8; this occurs during the course of a day as well as from
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day to day. Within the wide normal reference range, there are some ethnic differences, and in healthy West Indians and Africans platelet counts may on average be 10–20% lower than those in Europeans living in the same environment.52 There may be a sex difference; thus, in women, the platelet count has been reported to be about 20% higher than in men.53 A decrease in the platelet count may occur in women at about the time of menstruation. There are no obvious age differences; however, in the first year after birth the platelet count tends to be at the higher level of the adult normal reference range. Strenuous exercise causes a 30–40% increase in platelet count44; the mechanism is similar to that which occurs with leucocytes.
Other Blood Constituents As with the blood count, variations from normal reference values occur in respect of sex, age, stress, diurnal fluctuation, and so on. These are described in the relevant chapters.
Effects of Smoking Cigarette smoking has a significant effect on many haematological normal reference values.54 Some effects may be transient, and their severity varies between individuals as well as by the number of cigarettes smoked. Smoking 10 or more cigarettes a day results in slightly higher Hb, PCV, and MCV.40,55 This is probably a consequence of the accumulation of carboxyhaemoglobin in the blood together with a decrease in plasma volume. After a single cigarette, the carboxyhaemoglobin level increases by about 1%,56 and in heavy smokers the carboxyhaemoglobin may constitute c 4–5% of the total Hb. There may be polycythaemia.57 The leucocyte count increases, largely as a result of an increase in the neutrophils, and neutrophil function may be affected.14,54,58 Lymphocytes are also affected with an increase in CD4-positive and total lymphocyte count.54,58,59 Smokers tend to have higher platelet counts than nonsmokers, but the counts decrease rapidly on cessation of smoking.54 Studies of platelet aggregation and adhesiveness have given equivocal results, but there appears to be a consistent increase in platelet turnover with decreased platelet survival and increased plasma β-thromboglobulin. Elevated
fibrinogen concentration (with increased plasma viscosity) and reduced protein S have been reported, but smoking does not seem to have any consistent effects on the fibrinolytic system.54 The influence of smoking on the blood is summarized in Table 2.6.
Table 2.6
Effects of cigarette smoking*54–59
Increased
Decreased
Haemoglobin (Hb) Plasma volume Red cell count (RBC) Protein S Packed cell volume (PCV) Mean cell volume (MCV) Mean cell haemoglobin (MCH) White cell count (WBC) Neutrophil count Lymphocyte count T cells (CD4-positive) Monocyte count Carboxyhaemoglobin (>2 %) Platelet count (transient) Mean platelet volume Fibrinogen b-thromboglobulin von Willebrand factor Red cell mass Haptoglobins Plasma viscosity Whole blood viscosity Erythrocyte sedimentation rate (ESR) *Extent of change from normal reference values varies with individuals and amount smoked; some effects may occur only during and immediately after smoking. Some effects may be transient, and their severity varies between individuals as well as by the number of cigarettes smoked.
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