Clinical Laboratory Haematology - June 1988 - England - The Assignment of Values To Fresh Blood Used For Calibrating

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Clin. lab. Haemat. 1988, 10,203-212
The assignment of values to fresh blood

used for calibrating automated blood
cell counters
INTERNATIONAL COMMITTEE FOR

STANDARDIZATION IN HAEM A TOLOGY;
EXPERT PANEL ON CYTOMETRY
Members: J.M. England (chairman), R.M. Rowan (secretary),
M. Bins, B.S. Bull, W.H. Coulter, W. Groner, A.R. Jones, J.A.
Koepke, S.M. Lewis, N.K. Shinton, R. Thorn, O. W. van
Assendelft, R.L. Verwilghen
Accepted for publication 2 December 1987
Introduction
CALIBRA TION AND CONTROL OF AUTOMATED BLOOD CELL COUNTERS
Most modern automated instruments used to measure the various components of

the routine blood count are not direct measurement devices but comparators
which require careful calibration and internal quality control. To obtain
measurements which are comparable with those obtained by other systems and
methods, it is necessary to adjust the instrument by means of calibrators with
assigned values of defined accuracy. Controls, to which values mayor may not
have previously been assigned, are then used to ensure continued satisfactory
performance of the instrument after it has been calibrated.
Patients' mean indices can also be used to supplement a classical scheme of
calibration and internal quality control (Bull et al. 1974; Korpman & Bull 1976;
Bull & Korpman 1982). However, before this technique could be instituted, it
would be necessary to obtain a good estimate of population mean and SD values
by testing 500 blood specimens with the automated counter very carefully
calibrated and controlled.
Thus for all types of counters and for various operating principles it is
essential to have calibrators and controls.
USE OF FRESH BLOOD, PRESER VED BLOOD AND AR TIFICIAL MATERIALS AS

CALIBRA TORS AND CONTROLS
Calibrators can be produced from fresh human and animal blood, preserved
blood, artificial materials (e.g. latex particles) or mixtures of some or all of these.
Correspondence: Dr R.M. Rowan, University Department of Haematology, Western Infirmary,
Dumbarton Road, Glasgow Gil 6NT.
203
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204 ICSH Expert Panel on Cytometry
The method for assigning values to the calibrator will, in general, depend upon
the nature of the calibrator and the system which is to be calibrated.
Fresh blood as a calibrator

Standardized haematological techniques are available for assigning values directly
to fresh blood calibrators. The instability of fresh blood restricts its use to short-
term procedures over a few hours.
Preserved blood and artificial materials as calibrators and controls

Haematological techniques cannot necessarily be directly applied to preserved
blood or artificial materials since fresh blood may behave differently from the
calibrator on an automated instrument. Under such circumstances values have to
be assigned indirectly to preserved blood or artificial materials which are to be
used as calibrators. The principles and procedure have been described previously
(Crosland-Taylor et al. 1979; England et al. 1982, 1983) and involve two steps:
(i) Direct determination of Hb, PCV, RBC, WBC and platelet count (PLT)
on fresh blood, using carefully defined methods.
(ii) Use of an automated blood counter to compare the Hb, PCV, RBC,
WBC, PLT and red cell indices of the preserved blood or artificial material
with those of the fresh bloods.
The Panel is currently preparing ICSH recommendations for the assignment
of values to preserved blood and artificial materials used as calibrators and
controls. This document deals only with the assignment of values to fresh blood
calibrators.
Assignment of values to fresh blood calibrators
Fresh blood calibrators are useful for those diagnostic laboratories that wish to
calibrate their automated counters themselves without obtaining preserved blood
calibrators from other sources.
BLOOD SAMPLES
Sample collection
Fresh blood samples should be obtained from healthy subjects known to have a
mean cell volume (MCV) of 86-96 fl when calculated from the PCV and RBC
values obtained by the method described in the sections headed PACKED CELL
VOLUME MEASUREMENT and RED CELL COUNT respectively. Their mean cell
haemoglobin concentration (MCHC) should be 33.0-34.5 g/dl when calculated
from the Hb and the PCV values obtained by the methods described under
HAEMOGLOBIN MEASUREMENT and PACKED CELL VOLUME MEASUREMENT respectively.
Assignment of values to fresh blood calibrators 205
The samples should be anticoagulated with K1EDTA or K 3EDTA in a final

concentration of 1.4-1.6 mg/ml and placed in containers which allow an equal
volume of air remaining to enable proper mixing and oxygenation of the blood.
If K 3EDTA is used there is a shrinkage of cells resulting in an approximate
2% decrease of PCV value. Fresh blood samples should, therefore, be obtained
from healthy subjects with MCV 84-94 fl and MCHC 33.7-35.2 g/dl.
Sampling may be undertaken by syringe or by evacuated container. Samples
should be rejected if there is visible haemolysis or if microdots are present.
Sample handling prior to testing

The samples must be stored at room temperature (circa 20°C) and tested within
4 h. Vortex mixers must not be used. Before any test is performed the samples
must be well mixed by gently inverting the container 20 times.
GLASSWARE
Pipettes
Pipettes used should be officially tested Class A glassware, clean and free from
chipping. The inaccuracy of pipetting should be less than 0.5%. To achieve this
level of accuracy the following specifications are mandatory:
The bore must be sufficiently narrow so that a I mm difference in height of
the fluid contained in the pipette in the region of the calibration mark will result
in a measurement difference of at least I %. At its tip the pipette must be tapered
to about half the diameter of the bore at the level of the calibration mark. Before
being put into service the calibration of the pipette must be checked by a
gravimetric method using certified weights. The pipette must then never be heated
above 50°C. The following pipettes are required:
Maclean (white shell-back) blood pipettes (ml) 0.05 ± 0.0005
0.2 ±0.002
0.5 ±0.005
Volumetric pipettes with controlled draining
time and prescribed waiting time (ml) 20±0.03
25±0.03
Disposable glass pipettes may be used instead provided that they are individually
calibrated.
Capillary tubes for haematocrit determination

Use should be made of disposable capillary tubes satisfying the specifications of a
national standards body (BSI 1968; American Society for Testing and Materials
1980). The capillary tubes should be straight and any taper of the capillary bore,
measured over the full length of the tube, should not exceed 2% of the internal
diameter. A procedure for verification of absence of appreciable taper on

representative tubes from each batch is described by Crosland-Taylor (1982).
Microhaematocrit capillary tubes should be sealed with a clay-like sealing
compound which should be dispensed 5-7 mm deep in small trays.
Volumetric flasks
Grade A volumetric flasks, made of borosilicate glass, should be used, each with
a stated volume which has been tested by an appropriate national standards
body, or which has been checked by the individual user by means of a
gravimetric method using certified weights and corrections for buoyancy and
temperature.
Volumetric flask (ml) 100 ± 0.1.
Counting vials
The counting vial (plastic or glass) should have a minimum volume of 10 ml. It
should be of sufficient height so that the input aperture is at approximately half
the depth of the fluid before counting commences and there should be more than
I em of fluid above the aperture after the completion of counting. Before use, the
vial should be cleaned free of chemical contaminants and adventitious particles.
It is necessary to confirm that cells do not adhere to the counting vial. This is
ascertained by testing a number of vials from each batch. Red and white cell
counts are made at measured intervals after the dilutions have been placed in the
counting vials (see sections headed RED CELL COUNT and WHITE CELL COUNT). The
dilution should be mixed in the vial before counting. Adherence is demonstrated
by a decline in the count.
EXPERIMENTAL DESIGN
Previous work suggests that considerable experience is needed to eliminate inter-

technologist biases in the direct measurements.
The methods used to make direct measurements on each fresh blood are:
(i) Haemoglobin by cyanmethaemoglobin method.
(ii) Packed cell volume by micro PCV method.
(iii) Red cell counts using a single channel semi-automated electronic
counter.
(iv) Red cell indices (MCV, MCH and MCHC) by calculation.
(v) White cell counts using a single channel semi-automated electronic
counter.
(vi) Platelet count derived from the results on an instrument capable of
analysing whole blood.
Table I lists the number of tests per specimen and the maximum permissible
bias.
Table 1. Direct measurements in the fresh blood
Maximum permissible
bias (%)
No. of tests (±2 CV)
Haemoglobin Two dilutions 1.0

Packed cell volume Four tubes 1.0
Red cell count Two dilutions each counted twice on 3.5
each polarity", i.e. four counts per
dilution
White cell count As for red cell count 4.0
Red cell/platelet count ratio Four replicates 15.0
• If counter changes polarity.
HAEMOGLOBIN MEASUREMENT
In accordance with the ICSH Reference Method (lCSH 1987) 0.5 ml of blood is
diluted, using reagent, to 100 ml in a volumetric flask and the haemoglobin
concentration estimated spectrophotometrically. Occasionally specimens may be
turbid (A 750 > 0.004) and filtration is necessary through a low protein binding
membrane filter, mean pore diameter 0.20-0.25 j.lm. The first 0.5 ml of the filtrate
should be discarded.
PACKED CELL VOLUME MEASUREMENT
The ICSH (1988) selected method for microhaematocrit determination, without

correction for trapped plasma, is used.
RED CELL COUNT
Introduction
Red cell counts should be performed using a semi-automated single channel
counter of design specifications which permit counting, by electronic means, of all
the cells in a known displaced volume of the diluted blood sample. Whilst light-
scatter counters or aperture-impedance counters could be used for this purpose,
the only commonly available counters, working on a known displaced volume
basis, are aperture-impedance counters without sheath flow. The instrument
specification which follows would have to be modified appropriately for any
other type of counter.
Instrument specification
The design should be such that the probability of recirculation of cells inside the
orifice tube is low. Cavitation (trapped air bubbles) and turbulence must be
avoided. The sample flow lines must not change volume in response to changes in
fluid pressure nor should their materials contribute any measurable cell loss due
to adherence.
The displaced volume must be known within an accuracy of 1% traceable to a
national or international metrological standard. This displacement can be
achieved by the motion of a mercury column or other suitable means. The
differential displacement due to thermal effects shall not be greater than 0.1% per
0c.
The detection electronics should have a signal to noise ratio of greater than
100 : 1 for red cells of 90 fl and a signal amplitude roughly proportional to cell
volume. The lower signal threshold should be adjusted to an incremental
accuracy equivalent to ± 5 fl cell volume or less over the range 0-100 fl. The total
electronic dead time for a signal pulse exceeding the lower threshold and not
exceeding the signal level associated with a red cell volume of 140 fl must be
< 50 J1S.
Coincidence correction may be provided automatically or by means of a
coincidence correction chart. The total difference between raw count and
coincidence corrected count must not exceed 15% at approximately 1 x 108 red
cells/I in the diluted sample and the counting error after coincidence must not
exceed 2%.
Method
Diluent specification. A sterile, non-toxic, buffered salt solution should be used
which contains < 5 x 104 particles/l in the size range 20-120 fl. It should neither
crenate nor lyse red blood cells nor alter MCV by > 2 fl over a 30-min period.
Dilution. Make a primary dilution (0.05 ml blood plus 25 ml diluent) and from
this make a secondary solution (0.2 ml primary dilution plus 20 ml diluent)
giving an overall 50 601-fold dilution.
Transfer to counting vial. The diluted sample must be transferred to the counting
vial and the counts performed within 5 min from completing dilution.
Counting procedure. The counting procedure must be performed on an instrument
which is set up to appropriately discriminate red blood cells. The orifice tube
must be filled with cell-free diluent. It is expected that thresholding will be
verified on a sample-by-sample basis but that the coincidence correction will only
require to be verified on installation of the counter or following aperture or major
component change.
Validating thresholds. Pulse height analysis should be used to establish that
particles in the range 20-35 fl (volumes in fl are for red cell not latex calibration
of the pulse height analyser) are < 0.5% of those of volume > 35 fl. If this is so
then the lower threshold is set at 35 fl and the upper at iX.
Verifying coincidence correction. To test coincidence correction effectiveness,
concentrate a normal blood specimen by centrifuging at about 1000 gn for 5 min.
Remove half the supernatant plasma and thoroughly mix the remaining plasma
and cells. Using plasma from the specimen, prepare doubling dilutions of the
concentrated specimen over the range 1 : 1-1 : 16. Doubling dilution is to be
preferred to decimal increments since it can be done with pipettes calibrated to a
single fiduciary mark, independently of absolute volumes. The concentrated
specimen and each of the dilutions prepared from it are diluted as described
above and counted as described below. The red cell count at each dilution (when
corrected for coincidence and dilution) should not have ± 2 CV > 3.5%. If the
manufacturer's coincidence correction is found to be inadequate, then further
advice should be sought.
Counting. Counting must be accomplished within 5 min of the last dilution step.
The counting vial should be placed on the sample platform and the sampling
aperture immersed. Ideally 0.5 ml should be drawn by displacement through the
sampling aperture within 11-13 s. The counting aperture shall be cylindrical
having a nominal diameter of 80-100 pm and a length of 70-100% of diameter.
The result is then corrected for coincidence and dilution.
Errors
The potential sources of error in counting procedures are listed below:
Sampling error. This error arises if the measured sample is not sufficient nor
representative of the original whole blood specimen. Factors to be considered
include poor mixing, haemorrheological effects and the inaccuracy and
imprecision of dilution.
Transport error. Errors due to transport occur if the cells counted do not indicate
each and every cell in a known volume of diluted blood sample. Such errors are
caused by sedimentation, cell loss and/or recirculation, inaccuracy and
imprecision of displaced volume.
Counting error. Errors can arise if the final count is distorted due to improper
discrimination, spurious counts or inaccurate correction for coincidence loss.
Using the specifications described, the maximum permissible bias is 3.5%
(Table 1).
RED CELL INDICES
These are calculated from the measurements already described, i.e.

MCV = PCV/RBC; MCH = Hb/RBC; MCHC = Hb/PCV.
WHITE CELL COUNT
The instrument principle and specifications are as for red cell counting.
Method
Add the recommended volume of lytic agent to a measured volume of the red cell
primary dilution.
Lytic agent specification. The lytic agent must be capable of completely lysing red
cells, leaving no residual material capable of contributing to the count. The signal
from the leucocytes should fall into the size range equivalent to 45-450 fl
(volumes for red cell not latex calibration) and the count should be stable for 15
min after preparation.
Counting procedure. It is necessary to establish by means of pulse height analysis
that particles in the size range 35-45 fl are < 0.5% of those of volume> 45 fl. If
this is so then the lower threshold is set at 45 fl and the upper at a. Verification of
coincidence correction is as for red cell counting.
The count result is corrected for coincidence. The dilution correction must
allow for that in the primary dilution and for the dilution by the lytic agent which
is added.
Analysis of error
Sources of error are similar to those inherent in red cell counting. The maximum
permissible bias is 4% (Table 1).
PLA TELET COUNTING
Due to biases which occur when preparing platelet-rich plasma it is unacceptable

to use other than a whole-blood method. However, there does not appear to be
any blood counter available which aspirates a known volume of diluted blood
through the sensing zone and directly counts platelets in the presence of red cells.
For this reason it is only possible to use an instrument for determining the ratio
of red cell to platelet count. The platelet count can then be calculated since RBC
is known already (see RED CELL COUNT).
Determination of the ratio of the red cell to the platelet count

Using a sheathed flow instrument (validated as explained in Appendix) which
electronically discriminates platelets from other blood cells or debris, the signals
from both red cells and platelets are processed, sorted and totalled in separate
registers. The red cell register should contain approximately 50000 counts and
the platelet register approximately 3000 counts. Since the registers contain the
counts obtained from the same, but unknown, volume of blood the ratio of the
red cell count to the platelet count is known. This manoeuvre eliminates errors
caused by variations in sample flow rate and by dilution errors.
Coincidence effects should also be eliminated with adequate circuitry in the
counter. A complex coincidence correction is required when more than one cell
type is being enumerated by the same detector. This is because when cells of
different types are in coincidence, one type is usually favoured by the
classification process and hence the coincidence correction must be different for
each. The proper corrections are determined by calculating the probability of
detection for each cell type specifically. This involves calculating the conditional
probabilities of coincidence (e.g. RBC with RBC, RBC with PLT, PLT with PLT
etc.).
Method
Each blood is tested in quadruplicate on the counter and the mean red cell to
platelet ratio calculated. Given that the RBC is known, the platelet count can be
calculated.
References
AMERICAN SOCIETY FOR TESTING AND MATERIALS (1980) Standard specification for disposable glass
blood sample capillary tube (microhaematocrit) designation E734-80
BRITISH STANDARDS INSTITUTE BS4316 (1968) British standard for apparatus for measurement of
packed red cell volume
BULL B.S., ELASHOFF R.M., HEILBRON D.G. & COUPERUS J. (1974) A study of various estimators
for the derivation of quality control procedures from patient erythrocyte indices. Am. J. din.
Path. 61,473-481
BULL B.S. & KORPMAN R.A. (1982) Intralaboratory quality control using patient's data. In:
Methods in Hematology, Vol. 4, pp 121-150. Churchill Livingstone, London
CROSLAND-TAYLOR PJ. (1982) The Micro-PVe. In: Advances in Hematological Methods: The Blood
Count (eds O.W. van Assendelft & J.M. England) pp 85-92. CRC Press, Boca Raton
CROSLAND-TAYLOR P.J., ALLEN R.W.B., ENGLAND J.M., FIELDING J.F., LEWIS S.M., SHINTON N.K.
& WHITE J.M. (1979) Draft protocol for testing calibration and quality control material used
with automatic blood counting apparatus. Clin. lab. Haemat. 1,61-64
DACIE J.V. & LEWIS S.M. (1984) Practical Haematology, 6th edn, pp 43-44, Churchill Livingstone,
Edinburgh
ENGLAND J.M., CHETTY M.e., GARVEY B., CROSLAND-TAYLOR P., BARNARD D., LEWIS S.M. &
WARDLE J. (1982) Value assignment to quality control material using the draft protocol
published by the British Committee for Standardisation in Haematology. In: Advances in
Haematology Methods: The Blood Count (eds O.W. van Assendelft & J.M. England) pp
239-247. CRC Press, Boca Raton
ENGLAND J.M., CHETTY M.e., GARVEY B., LEWIS S.M., WARDLE J., COUSINS S., CROSLAND-TAYLOR
P.J. & SYNDERCOMBE-COURT D. (1983) Testing of calibration and quality control materials
used with automatic blood counting apparatus: application of the Protocol devised by the
British Committee for Standardisation in Haematology. Clin. lab. Haemat. 5, 83-92
INTERNATIONAL COMMITTEE FOR STANDARDIZATION IN HAEMATOLOGY (1987) Recommendations for
reference method for haemoglobinometry in human blood and specification for international
haemiglobincyanide reference preparation (3rd edition). Clin lab. Haemat. 9, 73-79
INTERNATIONAL COMMITTEE FOR STANDARDIZATION IN HAEMATOLOGY (1988) Recommended
methods for the determination of packed cell volume: prepared on behalf of WHO by ICSH
Expert Panel in Cytometry (in press)
KORPMAN R.A. & BULL B.S. (1976) The implementation of a robust estimator of the mean for
quality control on a programmable calculator or laboratory computer. Am. J. din. Path. 65,
252-253
Appendix
METHOD FOR VALIDATING THAT A SHEATHED FLOW COUNTER CAN ACCURATELY

MEASURE THE RATIO OF THE RED CELL TO THE PLATELET COUNT
This procedure should be undertaken as a formal check following purchase of an instrument or

change in aperture or in any other major component. The following measurements are made on
20-30 specimens of fresh blood:
(I) RBC as in section headed RED CELL COUNT.
(2) Platelet counts (PLT) performed by visual haemocytometry using the ammonium oxalate
method and phase control microscopy (Dacie & Lewis 1984).
(3) The red cell/platelet count ratio on sheathed flow instrument to be tested.
To validate the instrument the following calculation is performed:
[Ratio - (RBC/PLT)] x 100%
(RBC/PLT)
For> 95% of the specimens the value calculated must be within ± 10%.

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13652257, 1988, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2257.1988.tb01172.x, Wiley Online Library on [22/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.

The assignment of values to fresh blood

INTERNATIONAL COMMITTEE FOR

Accepted for publication 2 December 1987

CALIBRA TION AND CONTROL OF AUTOMATED BLOOD CELL COUNTERS

Most modern automated instruments used to measure the various components of

USE OF FRESH BLOOD, PRESER VED BLOOD AND AR TIFICIAL MATERIALS AS

Fresh blood as a calibrator

Preserved blood and artificial materials as calibrators and controls

Assignment of values to fresh blood calibrators

The samples should be anticoagulated with K1EDTA or K 3EDTA in a final

Sample handling prior to testing

Capillary tubes for haematocrit determination

diameter. A procedure for verification of absence of appreciable taper on

Previous work suggests that considerable experience is needed to eliminate inter-

Table 1. Direct measurements in the fresh blood

Haemoglobin Two dilutions 1.0

• If counter changes polarity.

PACKED CELL VOLUME MEASUREMENT

The ICSH (1988) selected method for microhaematocrit determination, without

RED CELL COUNT

RED CELL INDICES

These are calculated from the measurements already described, i.e.

WHITE CELL COUNT

PLA TELET COUNTING

Due to biases which occur when preparing platelet-rich plasma it is unacceptable

Determination of the ratio of the red cell to the platelet count

METHOD FOR VALIDATING THAT A SHEATHED FLOW COUNTER CAN ACCURATELY

This procedure should be undertaken as a formal check following purchase of an instrument or

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