Chart 8.5 Preliminary laboratory findings of common anaemias in tropical countries
8.5 PCV and red cell indices
Value of test: The packed cell volume (PCV), also referred to as haematocrit* is used to screen for anaemia when it is not possible to
measure haemoglobin accurately and mains elec- tricity is available to operate a microhaematocrit centrifuge.
*Terminology:The International Council for Standardization in Haematology (ICSH) recommends that PCV be used when blood is centrifuged in a capillary tube, and the word haematocrit be used when an autoanalyzer is used to compute the value.
The PCV is also used in the investigation of dehydration, burns, dengue haemorrhagic fever (see subunit 7.18.53) and polycythaemia. In the investigation of anaemia, the PCV with haemoglobin value is used to calculate the mean cell haemoglobin concentration (MCHC), described later in this subunit. In African trypanosomiasis endemic areas, examination of the plasma above the buffy coat layer following centrifugation can help to detect trypanosomes as described in subunit 5.8 in Part 1
Value of examining plasma following centrifugation Plasma from normal blood appears straw-coloured.
In severe iron deficiency the plasma appears colour- less. When the blood contains an increased amount of bilirubin, as in haemolytic anaemia, the plasma will appear abnormally yellow. When the plasma is pink-red this indicates a haemolyzed sample (less commonly, haemoglobinaemia). A further blood sample should be tested.
When white cell numbers are significantly increased, this will be shown by an increase in the volume of buffy coat layer. When this is seen, perform a total white cell count and differential count. When lipids are raised, the plasma appears white and cloudy.
Principle of test
The packed cell volume is that proportion of whole blood occupied by red cells, expressed as a ratio (litre/litre).
Anticoagulated blood in a glass capillary of specified length, bore size, and wall-thickness is centrifuged in a microhaema- tocrit centrifuge at RCF 12 000–15 000 xg for 3–5 minutes to obtain constant packing of the red cells. A small amount of plasma remains trapped between the packed red cells. The PCV value is read from the scale of a microhaematocrit reader or calculated by dividing the height of the red cell column by the height of the total column of blood.
Note: Due to trapped plasma, PCV values using a centrifuga- tion technique are 1–3% higher than those obtained when using an electronic cell analyzer which computes the value from the MCV and red cell count.
Specimen:To measure the PCV, either well mixed well oxygenated EDTA anticoagulated blood can be used or capillary blood collected into a heparinized capillary. The correct and safe collection of capillary and venous blood are described in subunit 8.3.
Equipment
● Microhaematocrit centrifuge
An example of a microhaematocrit centrifuge is the Hawksley Haematospin 1300 model shown in Plate 8.2 with essential accessories. It is a robust, stable, fixed speed (11 800 rpm, 13 000 xg RCF) microhaematocrit centrifuge with essential safety features which include a lid interlock, metallic casing and internal rotor lid and imbalance detector. It is fitted with a digital timer, has short acceleration and braking rates, and low noise level when operating. In the event of mains electricity failure, the lid locking mechanism can be easily over ridden using the release key provided.
The Hawksley Haematospin 1300 measures 235 mm deep 262 mm wide 272 mm high, weighs 8 Kg (less rotor), and has a power consumption of 380 watts at 200/240 V, 50/60 Hz AC (brushed drive motor). The available rotors include a 24 place haematocrit rotor, (see Plate 8.2a and a useful multi-combination rotor which holds 8 haematocrits and 16 microtubes, 1.5–4 ml volumes, (see Plate 8.2b).
Availability: The Hawksley Haematospin 1300 micro- haematocrit centrifuge 220/240 V model, code 01300-00 or 110 V model, code 01301-00 is manufactured by and avail- able from Hawksley Company (see Appendix 11). The rotor is supplied separately. For the 24 place haematocrit rotor with lid, order code 01971-00, and for the multi-combination rotor with lid, order 01985-00. A micro- haematocrit reader is also needed (see following text).
Important: Also order extra rim gaskets (code 01504-00, 20 gaskets for haematocrit rotor, code 01989-00, 10 gaskets for combination rotor), and spare pair of motor brushes, code 01978-00. Capillaries and sealant are also required (see following text).
Centurion microhaematocrit centrifuge (model CEN. E20B0): This low cost fixed speed (12 000 xg RCF) microhaematocrit centrifuge is fitted with a brushless drive motor and essential safety features. The haematocrit rotor takes 24 capillaries. It is available from Developing Health Technology (see Appendix 11).
● Capillary tubes for measuring PCV
These need to be plain or heparinized capillaries, measuring 75 mm in length with an internal diameter of 1 mm and wall thickness of 0.2–0.25 mm. Plain capillaries are often blue- tipped and heparinized capillaries, red-tipped.
Availability: Heparinized capillaries (box 1000), code 01605-00 and plain capillaries (box 1000) code 01604-00 are available from Hawksley (see Appendix 11).
● Sealant
Capillaries are best sealed using a plastic sealant e.g. Cristased, Miniseal, or Seal-Ease.
Availability: Cristaseal sealant (box 10 plates), code 01503-00 is available from Hawksley (see Appendix 11).
When not in use, the sealant should be kept in a plastic bag to prevent it from drying out.
● Microhaematocrit reader
There are two types of microhaematocrit PCV reader, i.e. a spiral reader with magnifier which fits inside the centrifuge allowing PCV measure- ments to be made after centrifuging with the capillaries in place in the rotor, and a hand-held scale or graph (see Plate 8.2c). A hand-held PCV scale reader can be used to read samples cen- trifuged in any microhaematocrit centrifuge, whereas a spiral PCV reader can usually be used only with the centrifuge for which it has been designed. The spiral reader cannot be left in place during centrifugation.
Test method
Whenever possible perform the test in duplicate.
1 About three quarters fill* either:
– a plain capillary with well mixedEDTA antico- agulated blood (tested within 6 hours of collection), or
– a heparinized capillary with capillary blood.
*Leave 10–15 mm of the capillary unfilled.
2 Seal the unfilled end of the capillary using a sealant material (see previous text).
Heat sealing capillaries: Avoid using the flame from a spirit lamp or pilot flame from a Bunsen burner to seal a capillary because this can distort the glass, causing breakage when the internal lid is screwed down on the rotor. Red cells may also be lyzed by the heated glass. Use of an open flame is also a fire hazard.
3 Carefully locate the filled capillary in one of the numbered slots of the microhaematocrit rotor with the sealed end against the rim gasket (to prevent breakage). Write the number of the slot on the patient’s form.
Position the inner lid carefully to avoid dislodging the tubes.
4 Centrifuge for 5 minutes (RCF 12 000–
15 000 xg).
Note: If the PCV is more than 0.50, centrifuge for a further 3 minutes to ensure complete packing of the red cells.
5 Immediately after centrifuging, read the PCV. First check that there has been no leakage of blood from the capillary or breakage.*
*If this has occurred, wearing protective gloves, clean the area using a rag soaked in ethanol 70% v/v. Make sure all the glass fragments are removed from the slot and rim.
Plasma
Buffy coat (Platelets and WBC)
Sealant PCV{
To read the PCV in a hand-held microhaemato- crit reader, align the base of the red cell column (above the sealant) on the 0 line and the top of the plasma column on the 100 line. Read off the PCV from scale. The reading point is the top of the red cell column, just below the buffy coat layer (consisting of WBCs and platelets).
When no reader is available: Use a ruler to measure the length of the total column of blood (top of plasma to bottom of red cell column) in mm and the length of the red cell column (base to below buffy coat layer). Calculate the PCV as follows:
PCV Length of red cell column (mm)
Length of total column (mm)
8.5
8.2(a)
8.2(b)
8.2(c)
8.2(d)
Plate 8.2(a) Hawksley Haematospin 1300 haematocrit centrifugewith24placehaematocritrotor.(b)Combinationrotor to take 8 haematocrits and 16 microtubes (1.2–4.0 ml). (c) PCV graph header and spiral reader with magnifier. (d) Sealant used to seal capillaries in numbered tray.Courtesy Hawksley Ltd
Caution: Immediately after reading a PCV, discard the capillary into a puncture resistant container for incineration or burial in a deep covered pit. NEVER leave used capillary tubes on the bench from where they can easily roll to the floor, causing injury from broken glass and a serious biohazard risk.
6 Report any abnormal appearance of the plasma (see previous text).
Quality control of PCV
Tests should be performed in duplicate to check for imprecision (duplicate tests should not differ by more than 5%).
Sources of error in measuring PCV
● Centrifuging at too low an RCF or for an insuffi- cient length of time resulting in a PCV value being higher than it should be.
● Delay in reading the PCV after centrifugation, allowing plasma to evaporate.
● Using an anticoagulated blood sample contain- ing excess EDTA (e.g. too little blood added to anticoagulant). This will cause the red cells to shrink, resulting in a PCV value lower than it should be. The opposite occurs if anticoagulated blood is left for more than 6 hours before being tested (the red cells swell, causing a falsely raised value).
● Clots in an anticoagulated blood sample can result in a falsely low PCV value. False values will also be obtained when venous blood samples are not mixed adequately.
● Rises in PCV (up to 6% error) can occur when there is an increase in trapped plasma due to changes in red cell size or shape, e.g. in sphero- cytosis, microcytosis and macrocytosis. Increases up to 20% of the PCV value can occur in sickle cell disease due mainly to the abnormal shape and rigidity of sickle cells.
● Using capillary tubes that are not designed for measuring PCV.
● Not cleaning and maintaining the microhaema- tocrit centrifuge as recommended by the manufacturer.
Interpretation of PCV
In a similar way to haemoglobin levels, PCV values vary according to age, gender, and altitude.
PCV reference range (guideline figures), l/l*
Children at birth . . . 0.44–0.54 Children 2–5 y . . . 0.34–0.40
Children 6–12 y . . . 0.35–0.45 Adult men . . . 0.40–0.54 Adult women . . . 0.36–0.46
*Reference ranges vary in different populations and in dif- ferent laboratories. District laboratories should check the above figures with their nearest Haematology Reference Laboratory.
PCV values are reduced in anaemia. Increased values are found when there is loss of plasma as in severe burns, dehydration and in dengue haemorrhagic fever (see subunit 7.18.53). Also in all forms of polycythaemia.
RED CELL INDICES
Red cell indices most frequently used in the investi- gation of anaemia are:
Mean cell haemoglobin concentration (MCHC) Mean cell volume (MCV)
Mean cell haemoglobin (MCH)
Measuring red cell indices in district laboratories
MCHC: Providing a laboratory is able to measure a PCV as previously described and perform an accurate haemoglobin test, an MCHC can be calculated (see following text).
MCV and MCH: To calculate these indices, an accurate red blood cell (RBC) count is required. To perform an accurate RBC count, an electronic cell analyzer is needed. Most district laboratories will not therefore be able to calculate these indices, however, examining a well-stained blood film can help to detect macrocytosis or microcytosis.
MCHC
The MCHC gives the concentration of haemoglobin in g/l in 1 litre of packed red cells. It is calculated from the haemoglobin (Hb) and PCV as follows:
MCHC g/l Example
If the Hb of an anaemic patient is 81 g/l and PCV is 0.34, the MCHC is:
238 g/l*
*If using g/dl divide the g/l figure by 10.
Interpretation of MCHC values
A guideline reference range for MCHC in health is 315–360 g/l (31.5–36.0 g/dl). These figures should be checked locally.
● Low MCHC values are found in iron deficiency anaemia and other conditions in which the red 81
0.34 Hbg/l PCV (l/l)
cells are microcytic and hypochromic (MCHC may be normal in thalassaemia trait).
Electronically derived MCHCs are not as sensi- tive to early iron deficiency.
● An increased MCHC can occur in marked spherocytosis but this is a rare condition. A raised MCHC is more often due to a calculation error or an incorrect haemoglobin or PCV.
MCV
The mean red cell volume (MCV) provides infor- mation on red cell size. It is measured in femtolitres (fl) and is determined from the PCV and electroni- cally obtained RBC count. It can be calculated as follows:
MCV fl*
*A femtolitre (fl) is 10–15of a litre.
Note: Most electronic blood cell analyzers measure or calculate MCV as one of their parameters, pro- viding a print out of the value in femtolitres.
Interpretation of MCV values
There is some variation in reference ranges for MCV depending on the method used by manufacturers of blood cell analyzers to obtain the MCV value and how an instrument has been calibrated. A guideline reference range is 80–98 fl.
● Low MCV values: are found in microcytic anaemias particularly iron deficiency, anaemia of chronic disease and thalassaemia. The MCV is low in infancy (about 70 fl at 1 year of age).
● Raised MCV values: are found in macrocytic anaemias, marked reticulocytosis, and chronic alcoholism. The MCV is raised in newborn infants.
MCH
The MCH gives the amount of haemoglobin in picograms (pg) in an average red cell. It is calculated from the haemoglobin and electronically obtained RBC count:
MCH pg*
*A picogram (pg) is 10–12of a gram.
Note: Most electronic blood cell analyzers calculate the MCH as one of their parameters, providing a print-out of the value in pg.
Hb in g/l RBC 1012/l
PCV (l/l) RBC 1012/l
8.5–8.6
Interpretation of MCH values
A guideline reference range for MCH in health is 27–32 pg.
● Low MCH values: are found in microcytic hypochromic anaemias and also when red cells are microcytic and normochromic. In thalas- saemia minor the MCH is low even when anaemia is mild (MCHC is often normal).
● Raised MCH values: are found in macrocytic nor- mochromic anaemias. MCH is also raised in newborns.