Procedures, Assays, and Normal Values	9

Procedures, Assays, and Normal Values

Taking Blood Samples

Since cell counts are affected by the state of the blood circulation, the conditions under which samples are taken should be the same so far as possible if comparable values are desired.

This means that blood should always be drawn at about the same time of day and after at least eight hours of fasting, since both circadian rhythm and nutritional status can affect the findings. If strictly comparable values are required, there should also be half an hour of bed rest before the sample is drawn, but this is only practicable in a hospital setting. In other settings (i.e., outpatient clinics), bringing portable instruments to the relaxed, seated patient works well.

A sample of capillary blood may be taken when there are no further tests that would require venous access for a larger sample volume. A wellperfused fingertip or an earlobe is ideal; in newborns or young infants, the heel is also a good site. If the circulation is poor, the blood flow can be increased by warming the extremity by immersing it in warm water. Without pressure, the puncture area is swabbed several times with 70% alcohol, and the skin is then punctured firmly but gently with a sterile disposable lancet. The first droplet of blood is discarded because it may be contaminated, and the ensuing blood is drawn into the pipette (see below). Care should be taken not to exert pressure on the tissue from which the blood is being drawn, because this too can change the cell composition of the sample.

Obviously, if a venous blood sample is to be taken for the purposes of other tests, or if an intravenous injection is going to be performed, the blood sample for hematological analysis can be taken from the same site. To do this, the blood is allowed to flow via an intravenous needle into a specially prepared (commercially available) EDTA-treated tube. The tube is filled to the 1-ml mark and then carefully shaken several times. The very small amount of EDTA in the tube prevents the blood from clotting, but can itself be safely ignored in the quantitative analysis.

10 Physiology and Pathophysiology of Blood Cells

Erythrocyte Count

Up to 20 years ago, blood cells were counted “by hand” in an optical counting chamber. This method has now been almost completely abandoned in favor of automated counters that determine the number of erythrocytes by measuring the impedance or light dispersion of EDTA blood (1 ml), or heparinized capillary blood. Due to differences in the hematocrit, the value from a sample taken after (at least 15 minutes’) standing or physical activity will be 5–10% higher than the value from a sample taken after 15 minutes’ bed rest.

Hemoglobin and Hematocrit Assay

Hemoglobin is oxidized to cyanmethemoglobin by the addition of cyanide, and the cyanmethemoglobin is then determined spectrophotometrically by the automated counter. The hematocrit describes the ratio of the volume of erythrocytes to the total blood volume (the SI unit is without dimension, e.g., 0.4).

The EDTA blood is centrifuged in a disposable capillary tube for 10 minutes using a high-speed microhematocrit centrifuge (reference method). The automated hematology counter determines the mean corpuscular or cell volume (MCV, measured in femtoliters, fl) and the number of erythrocytes. It calculates the hematocrit (HCT) using the following formula:

HCT = MCV (fl)!number of erythrocytes (106/µl).

Calculation of Erythrocyte Parameters

The quality of erythrocytes is characterized by their MCV, their mean cell hemoglobin content (MCH), and the mean cellular hemoglobin concentration (MCHC).

MCV is measured directly using an automated hemoglobin analyzer, or is calculated as follows:

Hematocrit (l/l)

MCV " Number of erythrocytes (106/µl)

MCH (in picograms per erythrocyte) is calculated using the following formula:

Hemoglobin (g/l)

MCH (pg) " Number of erythrocytes (106/µl)

Procedures, Assays, and Normal Values	11

MCHC is determined using this formula:

MCHC (g/dl) " Hemoglobin concentration (g/dl) Hematocrit (l/l)

Red Cell Distribution Width (RDW)

Modern analyzers also record the red cell distribution width (cell volume distribution). In normal erythrocyte morphology, this correlates with the Price-Jones curve for the cell diameter distribution. Discrepancies are used diagnostically and indicate the presence of microspherocytes (smaller cells with lighter central pallor).

Reticulocyte Count

Reticulocytes can be counted using flow cytometry and is based on the light absorbed by stained aggregates of reticulocyte organelles. The data are recorded as the number of reticulocytes per mill (‰) of the total number of erythrocytes. Reticulocytes can, of course, be counted in a counting chamber using a microscope. While this method is not particularly laborious, it is mostly employed in laboratories that often deal with or have a special interest in anemia. Reticulocytes are young erythrocytes immediately after they have extruded their nuclei: they contain, as a remainder of aggregated cell organelles, a net-like structure (hence the name “reticulocyte”) that is not discernible after the usual staining procedures for leukocytes, but can be observed after vital staining of cells with brilliant cresyl blue or new methylene blue. The staining solution is mixed in an Eppendorf tube with an equal volume of EDTA blood and incubated for 30 minutes. After repeated mixing, a blood smear is prepared and allowed to dry. The sample is viewed using a microscope equipped with an oil immersion lens. The ratio of reticulocytes to erythrocytes is determined and plotted as reticulocytes per 1000 erythrocytes (per mill).

Normal values are listed in Table 2, p. 12.

Table 2 Normal ranges and mean values for blood cell components*

		Adults	Newborns	Toddlers	Children
		# 18 years old	1 months	2 years old	10 years old
Leukocytes/µl	MV	7000	11000	10000	8000
or 106/l**	NR	4300–10000
Band granulocytes %	MV	2	5	3	3
	NR	0–5
Segmented neutrophilic	MV	60	30	30	30
granulocytes	NR	35–85
absolute ct./µl	MV	3650	3800	3500	4400
or 106/l**	NR	1850–7250
Lymphocytes %	MV	30	55	60	40
absolute ct./µl	NR	20–50
	MV	2500	6000	6300	3100
or 106/l**	NR	1500–3500
Monocytes %	MV	4	6	5	4
absolute ct./µl	NR	2–6
or 106/l**	MV	450
	NR	70–840
Eosinophilic granulocytes (%)	MV	2	3	2	2
absolute ct./µl	NR	0–4
	MV	165
or 106/l**	NR	0–400

Cells Blood of Pathophysiology and Physiology 12

Basophilic granulocytes (%)	MW	0.5		0.5	0.5	0.5
	NR	0–1
		Male	Female
Erythrocytes 106/µl	MV	5.4	4.8	4.7	4.7	4.8
or 1012/l**	NR	4.6–5.9	4.2–5.4	3.9–5.9	3.8–5.4	3.8–5.4
Hb g/dl	MV	15	13	17	12	14
or 10 g/l**	NR	14–18	12–16	15–18	11–13	12–15
HKT	MV	0.45	0.42	44	37	39
	NR	0.42–0.48	0.38–0.43
MCH " HbE (pg)	MV		29	33	27	25
	NR		26–32
MCV/µm3	MV		87	91	78	80
or fl**	NR		77–99
MCHC g/dl	MV		33	35	33	34
or 10 g/l	NR		33–36
Erythrocyte, diameter (µm)	MV		7.5	8.1	7.3	7.4
Reticulocytes (%)	MV	16	24	7.9	7.1	7.6
	NR	8–25	8–40
Thrombocytes 103/µl	MV		180	155–566	286–509	247–436
	NR		140–440

MV " mean value, NR " normal range (range for 95% of the population, reference range), ct. count, ** SI units give the measurements per liter.

* For technical reasons, data may vary considerably between laboratories. It is therefore important also to consult the reference ranges of the chosen laboratory.

13 Values Normal and Assays, Procedures,