174014

APPLICATION MANUAL
!"#$%&"'(
Hematology Analyzer
1.0 Release
Cat.-No.: 17401/4
!
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1 INTRODUCTION 3
2 BLOOD 4
2.1 Function of Blood 4
2.2 Composition of Blood 4
2.3 Blood Cell Parameters 5
2.3.1 Red Blood Cells, Hemoglobin 5
2.3.2 White Blood Cells 6
2.3.3 Platelets 7
2.4 Normal Hematology Ranges of Human Blood 7
3 MEASUREMENT METHODS 8
3.1 Volumetric Impedance Method 8
3.1.1 Diluting whole blood 9
3.1.2 Hemolysing blood cells, 3-part WBC differential method 9
3.2 Hemoglobin Determination by Photometry 11
4 ROUTINE UTILIZATION 12
4.1 Sample Handling 12
4.1.1 Anti-coagulant 12
4.1.2 Taking blood 12
4.1.3 Storage of Samples 12
4.1.4 Handling Blood Controls and Calibrators 13
4.2 Reagents and Environmental Conditions 13
4.3 Aperture Blockage - Clogging 14
4.4 Calibration 15
4.5 Quality Control 15
4.6 Measuring Patient Blood 15
4.7 Using Pre-dilution Mode 16
4.7.1 Sample preparation for 1:10 Pre-dilution Mode 16
4.7.2 Sample preparation for 1:3 Pre-dilution Mode 16
4.7.3 Calibrating Pre-dilution Mode 16
5 MEASUREMENT RESULTS, HISTOGRAMS 17
5.1 Typical histogram 17
5.2 Warning Flags 18
6 HUMAN CASES 20
6.1 Neonate 20
6.2 Three year-old Child 21
6.3 WBC clogging 22
6.4 WBC histogram with Nucleated RBCs 23
6.5 Lymphocytosis: WBC out of linearity 24
6.6 Lymphocytosis: measured in 1:10 pre-diluted mode 25
6.7 High MID and MID% 26
6.8 Granulocytosis 27
6.9 Thrombocytopenia 28
6.10 Thrombocytosis, Anisocytosis 29
6.11 Microcytosis (Low MCV) 30
6.12 Macrocytosis (High MCV) 31
6.13 Under-lysed Sample 32
6.14 Over-lysed Sample 33
Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 1/49

7 VETERINARY CASES 34
7.1 Normal Hematology Ranges of Animals 34
7.2 Effect of Lyse setting to 3-part WBC differential 35
7.3 Dog 36
7.3.1 Dog: Normal Sample 36
7.3.2 Dog: High LYM%, low GRA% 37
7.3.3 Dog: Flag D, high PLT 38
7.3.4 Dog: Flag W 39
7.4 Cat 40
7.4.1 Cat: Normal Sample 40
7.4.2 Cat: High LYM%, low GRA%, high RBC and HGB 41
7.4.3 Cat: Flag M, low RBC, HGB and very low PLT 42
7.4.4 Cat: Flag R 43
7.5 Horse 44
7.5.1 Horse: Normal Sample 44
7.5.2 Horse: Low LYM%, high GRA and GRA% 45
7.5.3 Horse: Low PLT 46
7.5.4 Horse: Low RBC and HGB 47
7.6 Rabbit 48
7.7 Rat 49

+,-'(.&/"%(-&'
This !""#$%&'$()*+&),&# is a supplement to the "01230,#45647.
The, "01230, #45647 describes the features and the use of the HUMACOUNT on a functional basis. In this
!""#$%&'$()* +&),&# we try to give an overview to the HUMACOUNT users how to measure, understand,
evaluate and validate the results, how to cope with warning flags.
We really believe that 819912,65:120945:;5<,=>,9?1,=@1249;=5,A;77,210679,;5,?;<?12,B=5>;:15B1 in our analyzer.
We have to keep in mind that machines and instruments can help our work, but 9?1,6012,?40,9=,C4D1,9?1,:1B;E
0;=50, accept the results, and the overall responsibility is ours. If we know the ability of our analyzer, we will
make the necessary decisions easier, accept the good results even though there are warning flags or repeat
measurement if necessary.
We tried to avoid complicated descriptions and definitions of the hematology terms. You will find several simple
figures and drafts describing the basic functionality, because our scope is to make you understand the basics
easily, and by doing so, you will have better control over the analyzer.
In B?4@9120,F,9=,G all data are referenced to human samples and blood.
In B?4@912,F we discuss the function of blood, and describe the target of hematology cell counting (blood cells).
Then we define the hematology parameters from the characteristics of blood cells (with human samples).
In B?4@912,H we typify different methods of cell counting by giving an overview of todays technology.
%?4@912, I deals with the rules of routine utilisation of the analyzer from taking the blood to the results, and
measurement reports.
In B?4@912,J,a typical normal histogram and the overview of warning flags can be found.
%?4@912, G contains human samples and their evaluation with histograms and description of the results. Every
case is different, so we try to give an overview of the basic cases you can meet in your practice.
%?4@912, K was created to our Veterinary users. We go through the most commonly studied three animal spe-
cies: :=<L,B49,and,?=201, their characteristics, and then a few additional animals. Even though you are a veteri-
narian user, we suggest you to go through the human cases as well, since they contain a lot of other information
not detailed among the Veterinary cases.
The information in this !"#$"% is based on the knowledge of the "01230,#45647. So, please, keep it at hand if
you need it for reference.
&'()#$'-.&',.-)/(,.%-/)*+),"-"),./0%*/(,).#)-'./)!""#$%&'$()*+&),&#)"1(2
34) 0%1&#23/)4-'-.$)&.5)6-2&'(#(755)6-')(,.-.*#5)78)8(%,9"#5):;)<.#=%5)>?):".#)@A,.-*1/B5)C.DD.#0*--)E.%F
%."9/)G)E.%=.#/5)HIII
H4) 8#$)$%&#*#&9(.&'(.5*:$&7)(/'$%/5)3/-)(,.-.*#5*:$'J/K)L4)F)M$N)C45)OD1.#P(1)O0.(#0(5)HIII

F,MN&&/
FO+,P65B9;=5,=>,M7==:
Blood circulates in the body and acts as a transport medium to carry the optimum supply of essential materials to
and remove waste products from all the cells of the body. Neurones, muscle cells, connective tissue cells and
epithelial cells draw their nourishment from the interstitial space and respond to the glucose and oxygen content
of that environment. Fresh supplies of oxygen and glucose are obtained by exchange with the blood circulating
in the capillaries. Blood receives its oxygen from lungs and glucose from intestines and liver.
Normal function of the cells depends on the rapid removal of toxic metabolic product (CO2 and NH3) from the
interstitial fluid environment. These subtonics are taken up by the plasma and 21:,87==:,B1770 and eliminated as
the blood passes trough the kidneys and lungs. Blood also delivers hormones, lipids, amino acids, salts and
vitamins and removes urea and conjugated acids. Moreover, blood ensures the normal water content of intersti-
tial space.
The heat, generated by metabolising body cells, is distributed by the blood circulation system, so that body tem-
perature is maintained practically at a constant level. Blood @74917190 and plasma coagulation mechanisms pre-
vent blood loss in the event of vascular injury. They aggregate with other platelets to form large hemostatic
plugs.
Q?;91,87==:,B1770 protect us against infections by identifying and killing invasive bacteria.
(?1,56C812L,0;R1,45:,:;092;869;=5,=>,87==:,B1770,@2=S;:1,;C@=29459,;5>=2C49;=5,>=2,C1:;B47,:;4<5=0;0,45:
9?124@TO (?1,4;C,=>,?1C49=7=<T,;0,9=,B=771B9,9?;0,;5>=2C49;=5O
FOF,%=C@=0;9;=5,=>,M7==:
The three groups of particles in whole blood are:
1. .1:,M7==:,%1770 (erythrocytes, .M%)
2. Q?;91,M7==:,%1770 (leukocytes, QM%)
3. U74917190 (thrombocytesL,UN()
;-:*<#((:*8-##/ =1$'-*<#((:*8-##/ >#&'-#-'/

12 9 9
'=2C47,:150;9T 4.5 5.5 x10 cells/l 5 10 x10 cells/l 150 450 x10 cells/l
'6B71491:V No* Yes No
GRAnulocytes: 65
*68E@=@6749;=50 RBC: 99.9% NEUtrophil: 60%
45:,9?1;2,W *NRBC (Nucleated RBC) EOSinophil: 4%
0.1% BASophil: 1%
LYMphocytes: 30
MONOcytes:
Biconcave (donut) shape GRAnulocytes: 13-16 m Fragments with a
*?4@1L,0;R1 diameter: 7-8 m LYMphocytes: 8-15 m diameter of 2-4 m
thickness: 1.8-2 m
MONocytes: 15-25 m
X=76C1,=>,B177 80 100 fl 50 1500 fl (native) 5 15 fl
X=76C192;B,W,;5 45 % 0.1 % 0.3 %
A?=71,87==:
?&9#-*@A*8-##*%(2"(/$'$()*(B*C1(#-*1,2&)*9#((:**@O*$10(2)HB

FOH,M7==:,%177,U424C19120
FOHO+,.1:,M7==:,%1770L,!1C=<7=8;5
.1:,87==:,B1770 Y,.M% Y,are formed in the bone marrow. A full-grown human red cell is non-nucleated and its
C145, B=2@60B6742, S=76C1 Y,#%X, Y is approx. 90 fl. RBCs are the most numerous cells in blood. There are
+F,
approx. J, Z+[ B1770\7 in the blood of a healthy person. RBC and MCV are measured primary parameters, so
they have calibration factor: CALRBC and CALMCV.
!1C49=B2;9, Y, !%( Y,measurement gives the proportion of RBCs to plasma in peripheral blood. It is the most
accurate and simplest way to measure the degree of anemia. It is calculated from the RBC and MCV values.
(Some instruments measures HCT and calculates MCV.)
HCTpercent = RBC x MCV/10 %, HCTabsolute = HCTpercent / 10, typically HCT = 0,45 = 45%.
Histogram of RBCs,in a healthy sample show 5=2C47,(Gaussian),:;092;869;=5O,It can be characterised by stan-
dard deviation Y,./QE*/,Y and coefficient of variation Y ./QE%X,Y,=>,4@@2=ZO,+IW. They have common cali-
bration factor of CALRDW .
The distribution width of RBC population derived from the histogram at 20% of peak. Definition of RDW depends
on the manufacturer of analyzers. We use the formula below.
Graphical definition of RDW (RBC Distribution Width):
RDW-SD = CALRDW x (P2 - P1) in fl unit,
RDW-CV = CALRDW x 0.56 x (P2 - P1) / (P2 + P1) %
By the factor of 0.56 CV is corrected to the 60% cut.
(The small population on the left is PLT the large is RBC).
D$7,.-*@A*E-B$)$'$()*(B*;E=
!1C=<7=8;5,Y,!]M,Y,is the main component of RBCs. It is conjugated protein (with Fe). Its main function is to
transport oxygen from the lungs to tissues and to transport carbon dioxide from the tissues back to the lungs.
Normal HGB concentration of human samples is approximately 14 g/dl = 140 g/l = 87 mmol/l*. The unit is select-
able in the Settings menu.
#145,%=2@60B6742,!1C=<7=8;5,Y #%!, Y,is the average hemoglobin content of RBCs. Calculated from RBC
and HGB values.
MCH = HGB / RBC in pg or fmol unit*.
#145,%=2@60B6742,!1C=<7=8;5,%=5B159249;=5,Y #%!% is the concentration of HGB in average RBC, cal-
culated from the HGB and HCT values:
MCHC = HGB / HCTabsolute in g/dl, g/l or mmol/l*.
*The HGB unit specifies MCH and MCHC units as well.

FOHOF,Q?;91,M7==:,%1770
Q?;91,87==:,B1770,Y,QM%,Y,are formed in the bone marrow. During their maturation sequence they differentiate
from the same parent-cell to 5 sub-populations. WBCs are nucleated and comprise granulocytes, lymphocytes
and monocytes. WBCs are equipped with all cell organelles necessary for life to carry out vital protective func-
tions in the body. The 068E@=@6749;=50 of QM%0 in a blood sample >=77=A,5=2C47,:;092;869;=5.
^,
The normal QM% is near KZ+[ B1770\7O It is very small compared to RBCs. In pathological conditions, the WBC
count can increase dramatically, e.g. to 500,000/l in extreme leukemia. In this case, pre-dilution of the sample
is required (more about this later).
3-part differential histograms (volume distribution curves) of WBCs can be used as a 0B2115;5<, 9109, to deter-
mine the absolute number and relative percentage of 7TC@?=BT910,Y,N_#L,N_#W,Y,C=5=BT910 Y,#-/L,#-/W
Y and granulocytes Y,].$L,].$WO
The total WBC count has calibration factor:,CALWBC
The definition of the WBC-related parameters:
WBC = LYM+MID+GRA, and LYM% = LYM/WBC, MID% = MID/WBC, GRA% = GRA/WBC.
If the differential values are out of the normal ranges, a complete differential count under a microscope can be
necessary.
We have to know, that during WBC counting cells are treated so that they lose their native volume, and the de-
rived histogram will show much smaller cells. This histogram contains cells shrunk to their nucleus (after he-
molysis), but it depends on the used lyse and amount.
Percentages of WBC sub-populations are represented by their area under the WBC histogram. The operation of
3-part WBC differential is illustrated by the following figure.
To determine WBC sub-populations, first the software

puts :;0B2;C;549=2, +O to the border of hemolysed
RBCs + PLTs (on the left) and LYM population, then
fits normal distribution curves to the remaining WBC
histogram (they are shown in different hatching).
After identifying the MID (middle) population the soft-
ware will set :;0B2;C;549=2,FO,45:,HO to the CV point
(60%) of the normal distribution of the MID population
(shown in grey). In other words, the software shows
where it found the MID population.
D$7,.-*FA*E-B$)$'$()*(B*=<8*"(",#&'$()/
You should remember this way of operation during the verification/validation process, when you accept the WBC
and 3-part differential results. Do not forget, MID population contains monocytes and some part of the eosino-
phils, sometimes giant lymhocytes, but this is patient dependent.

FOHOH,U74917190
U74917190,Y,UN(,Y,are non-nucleated fragments of the megakaryocyte. Note, that platelets are formed by frag-
mentation and not by normal maturation sequence like RBCs and WBCs. This means that the @7491719, ?;09=E
<24C on the left side has a logarithmic shape and on the right side has normal shape `a7=<E5=2C47b
:;092;869;=5c.
^,
Normal PLT concentrations range from +J[EIJ[Z+[ B1770\7 depending on mean platelet volume (MPV). PLT can
vary from 0 to 1 million cells/l in different conditions.
PLTs are relatively small compared to RBCs. The mean platelet volume Y,#UX,Y,is approx. 12 fl, so that they
can be separated from RBCs by their size. PLT and MPV are measured primary parameters, so they have cali-
bration factor: CALPLT and CALMPV.
Like RBC, we calculate the volumetric ratio of PLTs in whole blood by:
U%( @12B159 = PLT x MPV/10 %, U%(480=7691 = PCTpercent / 10, typically PCT = 0,003 = 0,3%.
The definition of the U/QE%X and U/QE*/ are similar to the RBC definition (see above).
The display and print of these xDW-SD and xDW-CV parameters depend on the actual setting: either CV or SD
parameters can be selected at once (for RDW and PDW, too).
FOI,'=2C47,!1C49=7=<T,.45<10,=>,!6C45,M7==:
The following table shows a summary of normal ranges of blood cell parameters of healthy adult human patients.
Normal values vary from population to population and can be slightly different in your location.
>&.&2-'-. G)$' +&#- D-2&#-

9
QM% 10 cells/l 7.0 3.0 7.0 3.0
N_#W % 32.5 7.5 32.5 7.5
#-/W % 5.0 3.0 5.0 3.0
].$W % 67.5 7.5 67.5 7.5
12
.M% 10 cells/l 5.0 0.5 4.5 0.5
!%( % 45.0 5.0 41.0 5.0
#%X fl 90.0 5.0 90.0 5.0
./QE%X % 14.0 2.0 14.0 2.0
!]M g/dl 15.0 2.0 13.0 2.0
#%! pg 30.0 2.0 30.0 2.0
#%!% g/dl 33.5 2.0 33.5 2.0
9
UN( 10 cells/l 275 125 275 125
#UX fl 12.0 2.0 12.0 2.0
?&9#-*FA*H(.2&#*6,2&)*6-2&'(#(75*;&)7-/*@O*$10()H4B

H,#)$*".)#)'(,#)(!&/*
In this chapter we make an overview of hematology measurement methods. It is important to know how your
system works, because this way it is easier to handle if you get unexpected results during routine utilisation.
HO+,X=76C192;B,-C@1:45B1,#19?=:
At the beginning only manual counting of blood cells under microscope was available to determine the cell con-
centrations of a blood sample. Because of the inaccuracy and high time consumption of the manual counting of
blood cells, the claim arose to a more accurate and quicker method. The volumetric impedance method is an
automated electronic way of counting blood cells.
D$7,.-*IA*0%1-2&'$%*(B*4(#,2-'.$%*J2"-:&)%-*+-'1(:
The principle of this measurement is that the blood diluted with isotonic solution (diluent) can conduct current by
ionic conduction. A counting chamber made of insulating material (plastics) holds this dilution of the blood, while
there is a small circular hole (aperture) on this chamber, which makes flow of diluted blood possible.
If we place two electrodes on the two sides of this aperture, and we apply constant electric current, the isotonic
solution will begin to conduct electricity, and a voltage can be measured on the aperture.
Applying pressure to the dilution it begins to flow trough the aperture. Once a cell is passing the aperture, a small
change in electric impedance will occur, making the voltage a little bit higher, i.e. a small electric pulse will be
present. The amplitude of the pulse will be proportional to the ratio of the cell volume (size) and the aperture
volume, so the bigger the cell is, the higher pulse we get. The typical aperture size diameter and length is 80 m.
For proper counting differentiation of cells, we need high likelihood (more than 90%) of passing one cell the
aperture at once. Since cell concentration is high in whole blood, we have to :;7691,A?=71,87==: to reduce con-
centration.

Although we dilute blood, in extremely
high concentration cases (e.g. leuke-
mia) WBC density can be 100x higher
than in normal cases causing two or
more cells passing the aperture at the
same time, generating one pulse in-
stead of two (or more). It is called coin-
cidence, and it will result in non-linear
counting of cells. Flags C and # show
this case.
The WBC linearity range is:
KJZ+[^ cells/lL, +[[Z+[^ cells/l for the
HUMACOUNT.
In this case you should pre-dilute the
sample and repeat measurement.
D$7,.-*KA*LBB-%'*(B*8($)%$:-)%-*M*)()N#$)-&.$'5
The applicable pre-dilution ratio depends on the setting of your analyzer (1:3 or 1:10). See section 4.7 for details
on usage.
HO+O+,/;769;5<,A?=71,87==:
Automated analyzers make the following steps automatically, to reduce the cell concentration to the desired
level.
Typical sample preparation sequence:

1. The analyzer takes 25 l of whole blood treated
with EDTA. This will be diluted to MIX chamber with
4ml of diluent to form +d+G[,#-e, :;769;=5 of whole
blood.
2. Another 25 l of the MIX dilution is sampled, and
put into the RBC chamber with 5ml of diluent to
form an overall +dHF,[[[,.M%,:;769;=5. This is suit-
able to measure RBC/PLT.
D$7,.-*OAE$#,'$()*>.(%-//*B(.*<#((:*8-##*8(,)'$)7
3. Then 0.8ml of lyse is added to the MIX dilution (practically while it is moved to the WBC counting chamber
to avoid contamination of MIX chamber with lyse) to form an overall +dF[[,QM%\!]M,:;769;=5. This liquid is
suitable to measure WBC and HGB.
HO+OF,!1C=7T0;5<,87==:,B1770L,HE@429,QM%,:;>>12159;47,C19?=:
Since RBCs are typically 1000 times more concentrated in normal blood, they would interfere with WBC count-
ing, i.e. the high number of RBCs would prevent counting of WBCs, because the aperture containing RBCs
would be always busy.
On the other hand we have to measure HGB, which is inside the RBCs, so we have to call it out somehow from
the cells.
A ?1C=7T0;5<,214<159,`7T01c can be used to dissolve the cell membrane of blood cells, destroying the RBCs,
and creating a complex solution suitable for photometry of HGB.

D$7,.-*PA*81&)7-/*$)*9#((:*%-##*%1&.&%'-.$/'$%/*:,.$)7*1-2(#5/$/*QIN"&.'*:$BB-.-)'$&#R
The membrane of WBCs become selectively permeable so in the slightly hypertonic lyse solution they begin to
shrink to their nucleus. The properly hemolysed liquid contains WBC particles in the 30-400 fl region.
*D$7,.-*SA
?5"$%&#*IN"&.'*:$BB-.-)'$&#*=<8*1$/'(7.&2*(B*/-#-%'$T-*1-2(#5/$/
Selective (3-part differential) hemolysis of WBCs depends on

lyse reagent type, lysing time and the amount of lyse used.
If we add too much lyse or we wait too much, the result will not
be selective, all WBCs will be shrunk to their nucleus, i.e. we
get a histogram with all WBCs in one population in 30-120 fl
region (see the histogram on the left). We call this case 9=9477T
or =S12E7T01:,04C@71.
Using 3-part WBC differential lyse with and HUMACOUNT,
lyse amount is the only parameter which can be modified.
D$7,.-*UA*6$/'(7.&2*(B*'('&##5*#5/-:*=<8/
If the software gets over-lysed histogram, it will put flag ) No WBC 3-part differential or flag Q, Y, 3-part
WBC differential is not reliable Y to the results.

With the default lyse setting (0.7 0.9 ml) most of the measurements will give satisfactory results. In some cases
=S12E7T0;5<, =2, 65:12E7T0;5< of samples may occur, because of patient blood characteristics. Under-lysing
means that too much RBC remains intact, i.e. the left side of the histogram starts with a high peak near 20 fl.
and HUMACOUNT allows sample adaptive measurement by increasing or decreasing lyse reagent depending
on the results. If the histogram shows over-lysed result, you can repeat the measurement after decreasing the
lyse amount by pressing the down arrow ( !"# $%&'(&# )*+,# -.# %./*&01+'*/# $2'*3# 4&*''# %4# 2&&(5# 6 7"# 8(# 9.$&*2'*
lyse, and measure again.
You can adjust lyse quantity in Y[OFL,Y[O+,=2,f[O+L,f[OF,C7 steps (refer to Users Manual).
There is no way to measure all the 5 different WBC types with this method, using one dilution and one lyse only.
There are JE@429,QM%,:;>>12159;47,4547TR120 employing flow-cytometers, selective lyses or other techniques to
get information from internal cell structure of WBCs for better qualification.
We can sort the hematology analyzers according to their differentiating capabilities:
U424C19120 '=,QM%,:;>>O HE@429,QM%,:;>>O JE@429,QM%,:;>>O
QM% V-/ V-/ V-/

N_#L,#&'L,].$ >* V-/ V-/
N_#L,#&'L,')"L,)&*L,M$* >* >* V-/
?&9#-*IA*81&.&%'-.$/'$%/*(B*:$BB-.-)'*'5"-/*(B*1-2&'(#(75*&)&#5W-./
-9, ;0, ;C@=29459, 9=, D5=A that independently of the manufacturer of the analyzer volumetric impedance
method (HE@429,QM%,:;>>12159;47) can differentiate the type of cells only on the basis of their sizes, but not ac-
cording to their internal, structural differences. Consequently this method is only a 7;C;91:, :;>>12159;49;=5
method for making qualitative hematology report.
HOF,!1C=<7=8;5,/1912C;549;=5,8T,U?=9=C192T
HGB determination is one of the first methods developed and is one of the most important hematology parame-
ters as it relates to the oxygen carrying capacity of blood.
Several HGB determination methods are known, but the cyanmethemoglobin method is commonly used. It is
possible to get a two-component neutralising reagent for the waste of lyses containing cyanide. (There are cya-
nide-free lysing reagents on the market in order to avoid environmental pollution.)
The lyse reagent containing potassium ferrocyanide and potassium cyanide converts the hemoglobin to cy-
anmethemoglobin. This substance absorbs green light of 540 nm.
HGB measurement is made by passing light of 540 nm
wave-length through the WBC dilution, and measuring
the light transmitted with a photo detector.
The light intensity (I) of the sample liquid is logarithmi-
cally proportional to the concentration of HGB:
HGB :#1(;#6-reference / Isample)
D$7,.-*UA*6X<*+-&/,.-2-)'*+-'1(:
The analyzer takes reference HGB measurements on clean diluent in each cycle during the cleaning phase to
ensure stability over time and temperature.

I,.&"(-'),"(-N-g$(-&'
IO+,*4C@71,!45:7;5<
IO+O+,$59;EB=4<67459
Since some time will usually elapse between collection of samples and counting, it is necessary to preserve the
sample with an anti-coagulant to prevent large groups of cells forming into clots or lumps of cell matter that will
clog the cell counter. Choice of anti-coagulant is very important, as some anticoagulants will affect the shape
and size of blood cells. In general )/($, preferably sodium or potassium based, is the only anti-coagulant rec-
ommended for use with electronic blood counters.
Care must be taken when using home-made containers pre-dosed with EDTA. If the container is not filled com-
pletely with blood, the ratio of EDTA to blood may reach a level, which results in osmotic transfer from the RBCs,
shrinking them. (?1, 249;=, =>, )/($, 9=, 87==:, 0?=67:, 5=9, 1ZB11:, H, C<\C7. Generally, we recommend using
manufactured sample tubes containing the necessary amount of EDTA.
IO+OF,(4D;5<,87==:
After filling the sample tubes to the required level, do not forget to invert the sample tube 51S12,0?4D1 sev-
eral times to activate the anti-coagulant by correct mixing. It can be a real problem especially when taking a
small amount of blood from babies or small animals.
Try to keep the 3 mg/ml maximum EDTA concentration rule as well.
(See section 4.7 for details on pre-dilution mode.)
IO+OH,*9=24<1,=>,*4C@710
Anti-coagulant requires time to take its effect on the sample. On the other hand, it will destroy the cells by time.
The fresh samples require at least JE+[,C;56910 to stabilise after taking, and they should be measured A;9?;5,G
?=620 at standard room temperature (25C).
This rule basically applies for the 3-part WBC differential. The granulocytes are destroyed first, after 12-16 hours
you get a much different histogram with much higher MID, because ageing will result in histogram looking over-
lysed. The correct time of collapsing of white blood cells depends on the patient blood.
If you want to measure later, put the samples into refrigerator to extend their stability. Care must be taken in this
case, because the samples must warm up to room temperature before measuring them. Roll the sample tubes
between your palms to speed up warming. This will help mixing the sample, too. If the sample is not mixed cor-
rectly, PLTs and large WBCs can stuck together forming much bigger particles, which will result in distorted
(lower) cell counts and the appearance of non-existing populations (e.g. in high region of WBC histogram).

IO+OI,!45:7;5<,M7==:,%=592=70,45:,%47;8249=20
M7==:,B=592=70 must be handled correctly to ensure their reliability. During transport and handling they should
be maintained at a temperature of FEhi%. Protect from heat and freezing. Bring to the room temperature by roll-
ing the vial between the palms of the hands until the red cell sediment is completely suspended (approx. +EF
C;56910). The control should look red, not black. If it is black do not use.
Mix gently the sample by inverting the tube JE+[,9;C10. ')X).,0?4D1,04C@71,96810O
After sampling, wipe the vial rim cap with lint-free gauze or tissue. Replace the cap immediately. Replace vial to
refrigerator within H[, C;56910 of use. When opening a vial for the first time note the date on vial label as the
open vial stability is limited.
Stability of an open control is usually +I,:4T0 only (check the manual of the manufacturer).
IOF,.14<1590,45:,)5S;2=5C15947,%=5:;9;=50
Always 601, 9?1, 21B=CC15:1:, and best quality, 214<1590 possible. The use of poor quality reagent can de-
crease analyzer performance, especially for PLT by increasing blank value and 3-part WBC differential by
modifying hemolysing characteristics.
The analyzer will automatically 265 8745D,C140621C159 to ensure instrument and reagent cleanliness. If blank
values are high, repeat blank measurement to ensure whether it is a stable high background or just some tempo-
rary contamination.
Stable ?;<?,84BD<2=65: can be caused by several 2140=50d
1. %=594C;5491:,214<159 if the reagent caps are open on the tanks, and dust or bacteria could get into
the reagent. Replace the diluent, if high PLT blank does not disappear even after several cleaning cy-
cles. Replace lyse, if high WBC blank is present, but PLT blank is good.
2. U12;=:;B,5=;01, can appear on the WBC channel due to protein build-up on the WBC draining tube.
Ask for service to decrease automatic cleaning cycle counter.
3. )71B92;B47,5=;01 can interfere with measurement by causing false pulses. Do not forget, our aperture
generates very small electrical signals in the several hundred micro-Volts (100 V) region. If there is a
device generating high-energy signals near the analyzer, it can harm counting by generating electrical
noise.
The main protection against electromagnetic noise is <==:,<2=65:;5< and shielding. It is therefore very
important that the power supply has a solidly grounded earth lead. If this is not sufficient to overcome the
problem, separate ground wire connected to the rear grounding point (marked) may be needed.
If you face to a high background problem:
! Replace the reagents to check their condition.
! Try to find out what could change in the environment of the analyzer causing interference.
! Try to operate the instrument from another power outlet, or relocate it completely (into another
room, if possible), then try again.
! Ask for the help of qualified service personnel, if necessary.
You should run blank measurement if you replace either reagent during the daily work to ensure cleanliness.
You have to know, that the analyzer will store these values, and e.g. PLT result will be compensated by the
blank value: UN(210679,j,UN(C140621:,Y,UN(8745D.

IOH,$@129621,M7=BD4<1,E,%7=<<;5<
Some build-up of protein, etc. on the aperture, which is not removed may cause blockage. This reduces the
cross-section of the aperture.
Clogging can be detected by increased measuring time. The basic measuring time is 8 seconds for WBC and
RBC/PLT. If there is a higher counting time, the corresponding aperture must have been blocked, repeating of
measurement is recommended.
Your analyzer is equipped with a three-fold clogging prevention cleaning system:
1. High-pressure back-flush (against big clogging particles),
2. High-voltage burning (against protein build-up), and
3. Chemical cleaning of apertures (to maintain stability).
A clog can also be detected electronically by analysing the base impedance of the aperture. In case of any clog-
ging the base impedance of the aperture increases causing a higher value. This feature is used in the
HUMACOUNT analyzer. You can monitor the probe voltage range at RPrV and WPrV under the PLT histogram
on the screen.
If clogging is detected, B or % flag appears. You should repeat the measurement.
D$7,.-*YA*LBB-%'*(B*>&.'$&#*8#(77$)7*'(*+84
If the aperture is partially clogged, its volume (V) decreases.

Since the measuring aperture generates pulses proportional to the ratio of cell volume (x) to the aperture volume
(V), the resulting pulse is higher if the aperture is partially clogged. In this case the size-dependent parameters
(MCV, MPV) are higher, consequently HCT is higher, too. We have to remove clogging for proper results.
To avoid hard clogging, do not let the analyzer dry out (i.e. formation of salt and reagent residuals) in case of
longer shut-down times. Run Preparing for shipment procedure to remove reagents and drain the system.

IOI,%47;8249;=5
Calibration is used to set the absolute accuracy of the analyzer. For this sake, we measure control blood having
known results. If there are differences between the reference values and the measured values, we modify the
factors to get the most corresponding results.
The HUMACOUNT offers automatic calibration where the user must enter the reference (or target) values, and
then measure the control blood. After accepting the results, the new factors will be calculated automatically.
%47;8249;=5,C609,81,@12>=2C1:d
1. If the analyzer has been relocated (change in altitude, temperature, etc.).
2. After a big service repair (changing of main components, e.g. dilutors, needle, etc.).
3. If the Q.C. measurement results show sloping of some results (be careful: most of the time it is due to the
ageing of the control).
Be sure to run calibration only in perfect conditions, if there is no clogging, contamination, etc. otherwise you can
have much worse results. It is recommended to run a cleaning and a blank measurement before calibrating, to
ensure optimal conditions.
IOJ,k647;9T,%=592=7
Quality control (kO%O) is a feature to monitor the operation and stability of the instrument.
The HUMACOUNT offers 6 levels of separate Q.C. measuring capability, i.e. you can use 6 different blood con-
trols in the same time to check stability.
The manufacturers of blood control usually offer at least 3 different levels: +, 7=A abnormal, +,5=2C47 (suitable
for calibration as well), and +,?;<? abnormal blood controls.
If you program them to levels 1, 2 and 3, you can measure them every day before starting daily routine meas-
urements to ensure stability of the system. In some laboratories working under quality assurance system
there is a regulation to do so.
You can use the N1STEl155;5<0 chart to get visual information about the everyday results.
If 0=C1,=>,9?1,2106790,421,=69,=>,9?1,@12C;00;871,245<1, try another vial of blood control to see if the results
are outside too on this control. If the second control confirms that the results are outside the permissible range,
you can 21B47;82491,9?1,4547TR12.
Do not forget, the 0948;7;9T,=>,45,=@15,S;47,=>,87==:,B=592=7,depends on the manufacturer, but it is usually,+I
:4T0 only. The closed vials can be used longer (typically for 3 months).
IOG,#14062;5<,U49;159,M7==:
m11@,9?1 04C@7;5<,@210B2;@9;=50 of the Users Manual.
You can make big mistakes if the sample is not correctly homogenised (all counted parameters will be higher),
contains micro-bubbles (lower effective sample value, lower counted parameters), or cold (PLT count will be
bad). At least 30 minutes is required for a sample tube to reach the room temperature if it was in refrigerator
before.
It is recommended to use a 2=942T,C;Z12 to keep samples homogenous during the measuring session. If you do
not have sample mixer, ;5S129,9?1,96810,6@0;:1,:=A5,01S1247, 9;C10 before sampling. '1S12, 0?4D1 them to
avoid formation of micro-bubbles inside, because 0C477, 8688710, @21S159, B=221B9, 04C@7;5< (air is sampled
instead of blood).
After measurement, B?1BD,9?1,2106790,45:,?;09=<24C0 to identify any sort of disorder. .1@149, 9?1, C140621E
C159 in case of any doubt, you can adjust the lyse setting if necessary.
X47;:49;=5 (acceptance of the results) is a very important process. During validation the hematologist or clinician
can sort out those reports, which require confirmation by manual counting under microscope. Do not forget, use

the 3-part differential results to identify the abnormal cases, and make manual differential count on them if you
need better WBC qualification.
IOK,"0;5<,U21E:;769;=5,#=:1
Pre-dilution mode is used in two cases:
1. if B4@;7742T,9681 (e.g. 25 l of volume) is used for taking blood from the patient,
2. in case of 7;5142;9T,122=2 due to high cell concentration.
In both cases the sample must be prepared before measurement. Create the necessary dilution of whole blood
(or capillary blood) sample according to the set-up pre-dilution mode (you can check it by selecting pre-dilution
mode in Measurement menu, the ratio appears on the screen +dH or +d+[)
U21@421,49,71409,+[[,n7,=>,@21E:;7691:,04C@71, because in pre-dilution mode the analyzer samples double vol-
ume (approx. 60 l).
IOKO+,*4C@71,@21@4249;=5,>=2,+d+[,U21E:;769;=5,#=:1
1. Put,FJ[n7o of clean diluent or isotonic solution into a clean cup or test tube.
2. Add FJn7o,of whole or capillary blood and homogenise it by rotating the cup circularly
The above sample is suitable for measurement in +d+[,@21E:;769;=5,C=:1.
IOKOF,*4C@71,@21@4249;=5,>=2,+dH,U21E:;769;=5,#=:1
1. Put,+J[n7o of clean diluent or isotonic solution into a clean cup or test tube.
2. Add,J[n7o,of whole or capillary blood and homogenise it by rotating the cup circularly
The above sample is suitable for measurement in +dH,@21E:;769;=5,C=:1.
IOKOH,%47;8249;5<,U21E:;769;=5,#=:1
1. %47;82491 the analyzer in 5=2C47,C=:1 using blood control.
2. %21491 the desired @21E:;769;=5 of blood control (1:10 or 1:3) with the method you wish to use for patient
samples as well.
3. $B9;S491,@21E:;769;=5,C=:1 in Calibration menu.
4. U12>=2C,B47;8249;=5 with the prepared sample.
5. After accepting the pre-dilution factors :14B9;S491,@21E:;769;=5,C=:1 in Calibration menu to avoid mix-
up of calibration factors.
6. Make sure to 601,9?1,04C1,C19?=:,45:,@2=B1:621 (tools, settings of pipettes, dispenser volume, etc.)
9=,B2149;5<,04C@710,>=2,C140621C159, too. By doing so you will be able to compensate the inaccura-
cies of your dilution procedure.
oExact volumes may vary. Keep the ratio: e.g. +,65;9 of sample + H,65;90 of diluent (for 1:3 mode) constant.

J,#)$*".)#)'(,.)*"N(*L,!-*(&].$#*
JO+,(T@;B47,?;09=<24C
Evaluation:
QM% .M% UN(

All cells larger than the RBC/WBC All cells above the RBC/PLT PLT histogram is a magnified
discriminator (near 30 fl) are discriminator (near 30 fl) are portion of the beginning of the
counted as WBCs. counted as RBCs. RBC histogram.
WBC histogram has three The RBC histogram follows normal PLT value is near 250.
populations: distribution.
PLT cell population is approx.
1. lymphocytes The RDW value is within the normal between 2-30 fl
2. MID population range, so the width of the RBC
PLT histogram follows a log-
(monocytes and some histogram is normal
normal distribution, with a clear
eosinophils), and The whole histogram is normalised separation from RBCs.
3. granulocytes (100% fit) to the RBC peak.
Lymphocytes can be found approx.
between 30-90 fl.
The two other discriminators show
the place of MID population (90-
140 fl).
Granulocyte population is above
discriminator 3 (140 fl)

JOF,Q425;5<,P74<0
In this section we summarise the error flags and we give an explanation of their possible cause and a few hints
to overcome the problem.
"@@12B401,7199120,21>12,9=,QM%,=2,!]M,@2=871C0d
P74< #145;5< .1B=CC15:1:,6012,4B9;=5
WBC three part Possibly lyse problem. Try reducing the lyse amount and repeat the
warning or WBC three measurement. In extreme Lymphocytosis case you can get this flag.
Q part diff. unsuccessful Check the discriminators in the WBC histogram. If the discriminators are in the
0--*8&/-*OZ*@I*&): proper place (the populations can be separated even by eye) then the results
E(7*%&/-/ are correct and acceptable.
Possibly lyse problem, but in some pathological samples (too high
) No WBC three part
lymphocytes), it can happen.
Repeat the blank measurement. If HGB blank is not stable there are probably
HGB blank is high, or
! bubbles in the WBC chamber. Run a cleaning and try blank again. Close the
no HGB blank
side door if open during measurement.
WBC blank is high, or Repeat blank measurement, or run prime lyse and try blank again.
M
no WBC blank Possibly lyse contamination, or noise problem.
Check the 1. RBC-LYM discriminator. If it is in the minimum point (or close to
it), accept the results. Otherwise repeat the measurement.
WBC/RBC limit
N If the repeated measurement gives similar results and discriminator 1. is in
warning
wrong place then the MID and GRA results are OK, but the WBC and LYM
results can be higher because of the RBCs.
Too many RBC cut
Repeat the measurement with increased lyse amount.
from WBC
. If the WBC measuring time is too high (more than 8 sec. in ) it could be
0--*8&/-*@I*&):*8&'
aperture clogging. In this case perform cleaning and repeat the measurement.
%&/-/
WBC coincidence is
The results are out of the linearity range. Make a dilution with an external
too high. Linearity
dilutor with the set-up (1:10 or 1:3) dilution rate.
# error.
Do not forget to calibrate pre-dilution mode before using it for measuring
0--*8&/-*OZ*@I*&):
samples.
8&'*%&/-/
WBC data package

Perform cleaning and repeat the measurement (aperture clogging).
/ errors
If it is a general problem, please call your Service Personnel.
0--*E(7*%&/-/
* WBC time error Same action as in case of warning flag,/.
Aperture clogging. Same action as in case of warning flag,/.
WBC clogging
% Low temperature reagents can caused it as well (mainly diluent), in this case
0--*8&/-*I
you will have to wait until they reach room temperature.
Run cleaning and repeat the blank measurement.
PLT blank is high, or
U Diluent or system cleanliness problem. If it is stable high, replace the diluent
no PLT blank
by opening a new tank.
RBC blank is high, or
M Same action as in case of warning flag,U.
no RBC blank
?&9#-*KA*0,22&.5*(B*C&.)$)7*B#&7/*.-#&'-:*'(*=<8[6X<

Q425;5<,>74<0,;5,7=A12B401,21>12,9=,.M%,=2,UN(,@2=871C0d
P74< #145;5< .1B=CC15:1:,6012,4B9;=5
RBC/PLT valley is too high, or the PLT peak is not high enough.
RBC/PLT limit flag
7 If the discriminator is in a wrong place (in the PLT or RBC histogram) then
0--*8&/-*Y
repeat the measurement for a correct PLT result.
Perform cleaning and repeat the measurement (probably clogging).
D RBC peak warning
If it becomes a general problem, change the RBC aperture.
RBC/PLT coincidence
C is too high. Same action as in case of warning flag,#.
Linearity error.
RBC/PLT data
: Same action as in case of warning flag,/.
package errors
0 RBC/PLT time error Same action as in case of warning flag,/.
B RBC/PLT clogging Same action as in case of warning flag,%.
?&9#-*OA*0,22&.5*(B*C&.)$)7*B#&7/*.-#&'-:*'(*;<8[>\?
Warning flags can be grouped according to measurement conditions and according to the problems relating to
the blood sample.
Measurement conditions: when the flags are related to clogging (BL, %L, :L, /L, D), probably hemolysing problems
()L,8L,ML,@), time out (0L,*), and pressure problems (Fatal pressure error). In this case we suggest repeating the
measurement.
Problems related to blood sample: few or overlapped thrombocytes (7), possible presence of nucleated RBCs
(N), 3-part WBC differential problems (Q). In this case we suggest to change lyse setting and repeat measure-
ment (you can use the arrow keys before measuring to adjust the default lyse setting in 0.1 ml steps). If it gives
the same result, you can accept the results, keeping in mind that parameters marked by asterisk (o) are not
100% reliable.
For PLT (7), lyse setting is not applicable, so if the PLT/RBC discriminator is in correct place, accept PLT result.
The third case is when repeating is required in pre-dilution mode. There is linearity error cell concentration is
too high causing coincidence (CL,#L,.) (see section 3.1).
The asterisk flag (o) near a parameter shows some doubt suspected during the evaluation of that parameter. The
reasons can be: a high PLT blank (PLT value will be marked), a case of indefinite discriminator setting (default
location is used for some reasons, related parameters will be marked), etc.
Another flagging method is evaluation against the normal ranges. If some of the parameters is out of range it
gets a (E) flag if under the range, or gets (f) if over the range. You can customise ranges for all kind of patients
by setting the corresponding lower and upper ranges. If you set 0 for a range limit, it will be not verified (see
Users Manual: Limits).

G,!"#$',%$*)*
In the following section you will see several printed results with histograms, and a short explanation. Try to iden-
tify the different populations, their position and ratio for better understanding of the meaning of the different his-
tograms.
Do not forget, the first WBC discriminator separates PLTs and hemolysed RBCs (on the left) from WBCs. If you
see here high peaks it shows particles here:
! either nucleated RBCs,
! or under-lysed sample, (bigger pieces of hemolysed RBCs in WBC solution),
! or some sort of contaminated reagent, noise, etc.
The second and third WBC discriminator shows the place of the MID population.
The RBC/PLT discriminator shows the separation of the two populations. You can easily decide whether it is in
good position or not. Normally it should appear in the valley between the PLT and RBC populations.
GO+,'1=5491
This sample comes from a sick new-born baby.
$BB1@9,9?1,2106790,(there are no flags, and all discriminators are in correct position).
Evaluation:
QM% .M% UN(

high LYM, but this is normal for
low RBC, anemia normal
baby blood

GOF,(?211,T142E=7:,%?;7:
As children grow, the amount of LYM becomes lower (in normal case).
$BB1@9,9?1,2106790O
Evaluation:
QM% .M% UN(

! high LYM
! low GRA low HGB & MCH high PLT
! slightly high WBC

GOH,QM%,B7=<<;5<
In cases of aperture clogging we can see a histogram magnified to the right (the smaller aperture has higher
sensitivity, so we see the particles to be bigger). In case of RBC clogging MCV and consequently HCT will
be higher.
.1@149,C140621C159O
Evaluation:
QM% .M% UN(

! flag C shows WBC aperture clogging, the
sensitivity of the aperture increases, speed of
flow decreases
! we can see that WBC measuring time low RBC normal
(WBCt) is high, the instrument does not give
any WBC values and the histogram is magni-
fied to the right (all discriminators are put
higher)

GOI,QM%,?;09=<24C,A;9?,'6B71491:,.M%0
You can have some more information about the composition of the sample even though the analyzer cannot give
N-RBC result.
This case shows a possible result with Nucleated RBCs. This is just a suspect, since contaminated reagents,
noise, etc. and under-lysed sample can generate similar histograms.
$BB1@9,B=221B91:,2106790 (see evaluation below).
possibly N-RBCs
Evaluation:
QM% .M% UN(

There are probably N-RBCs in the LYM region,
so WBC, LYM and LYM% is higher.
Since this additional number is proportional to
the histogram area shown with the arrow, we
can be sure, this extra amount is approx. 0.5, normal thrombocytopenia: low PLT
so we can subtract this from WBC and LYM.
The estimated parameters: WBC = 6.5, LYM =
2.7.
This is the way you can correct the results.

GOJ,NTC@?=BT9=0;0d,QM%,=69,=>,7;5142;9T
Due to coincidence (see Section 3.1) WBC linearity is limited. This sample is a typical Lymphocytosis and it
demonstrates linearity error as well.
In order to obtain better WBC values C140621,9?1,04C1,87==:,;5,@21E:;7691: C=:1 +d+[ or +dH,depending on
which is set up in your system.
(See the next case as well.)
Evaluation:
QM% .M% UN(

! WBC is very high: leukocytosis
! #, flag means that WBC coincidence is too high, the re-
sults are out of the linearity range. The high amount of
coincidence pulses cause overlapping of populations, normal normal
therefore their clear identification is not possible (flag Q).
The absolute and percentage values are marked with as-
terisks (o), because the software is not sure that the dis-
criminators are put in the right places.

GOG,NTC@?=BT9=0;0d,C140621:,;5,+d+[,@21E:;7691:,C=:1
In order to increase the linearity of the instrument, you should pre-dilute the sample in a clean tube for repeated
measurement. (Refer to section 4.7 and the Users Manual for details, since operation can be slightly different
depending on software versions).
The main conception of pre-dilution: by reducing the concentration of the cells we can put measurement back in
the linear range.
We suggest the use of +dH,@21E:;769;=5,C=:1 because the cell concentration is better there than in +d+[ mode.
1:10 mode may be better for use with capillary blood.
(?1,210679,=>,9?1,@21S;=60,87==:,04C@71,C140621:,;5,+d+[,@21E:;7691:,C=:1O
You can see the higher WBC result. All asterisks disappeared from differential parameters.
Another effect comes from the accuracy of pre-dilution.
Since we did not calibrate pre-dilution mode before measurement of pre-diluted sample, there are significant
differences in RBC and PLT results. It is due to the non-calibrated pre-dilution mode. _=6,0?=67:,B47;82491,@21E
:;769;=5,C=:1,4>912,B47;8249;5<,5=2C47,C=:1 in order to compensate the differences between the ideal (cal-
culated) and real (created) dilution (see section 4.7.3).
In situations like this, you can accept WBC values from this measurement, while RBC and PLT values are used
from the previous measurement (case 5).

GOK,!;<?,#-/,45:,#-/W
In case of Monocytosis or Eosinophilia the MID population will have a significant peak in the WBC histogram
between 100 and 180 fl.
$BB1@9,9?1,2106790O
Evaluation:
QM% .M% UN(

! high MID and MID%
! MID and GRA populations are low RBC, HGB, HCT normal
overlapped

GOh,]24567=BT9=0;0
This case shows Granulocytosis. The peak of Granolucyte population is so high that the software is normalizing
the WBC histogram to it, so the LYM population looks much smaller.
$BB1@9,9?1,2106790O
Evaluation:
QM% .M% UN(

! Leukocytosis: high WBC
anemia: low RBC and HGB normal
! GRA is high, LYM is low

GO^,(?2=C8=BT9=@15;4
In case of Thrombocytopenia the PLT value is very low.
$BB1@9,9?101,2106790O
Evaluation:
QM% .M% UN(

! thrombocytopenia: low PLT
! normal WBC ! warning flag 7: due to the relatively low peak of PLT,
the analyzer is not sure about the correct placement of
! high MID: microcytic RBC PLT/RBC discriminator, consequently it will put
suspect of asterisks near PLT related results
monocytosis
! if you can see that the PLT/RBC discriminator is in
right place (like now) we can accept the marked results

GO+[,(?2=C8=BT9=0;0L,$5;0=BT9=0;0
This case shows Thrombocytosis and Anisocytosis. The RBC histogram has a high PLT peak therefore the soft-
ware normalised the RBC histogram to the PLT peak.
$BB1@9,9?101,2106790O
Evaluation:
QM% .M% UN(

high WBC anisocytosis: the RBC curve thrombocytosis:
is wide, RDWc is high very high PLT

GO++,#;B2=BT9=0;0,`N=A,#%Xc
This sample shows low MCV and GRA parameters.
$BB1@9,9?1,2106790O
Evaluation:
QM% .M% UN(

lymphocytosis: high value of low MCV value: microcytic
normal
LYM and LYM% RBCs

GO+F,#4B2=BT9=0;0,`!;<?,#%Xc
If MCV is higher than in normal case, the RBC histogram will show a shift to the right (the analyzer detected big-
ger cells).
$BB1@9,9?1,2106790O
Evaluation:
High MCV usually incorporated with low RBC. This is typical in situations where the patient is bleeding. The
bone-marrow tries to compensate low HGB (HCT) by releasing bigger RBCs.
QM% .M% UN(

! macrocytic RBC: MCV value is very high
MID% is high ! right-shifted wide RBC histogram normal
! anemia: very low RBC, low HGB

GO+H,"5:12E7T01:,*4C@71
If a sample is not hemolysed correctly (under-lysed) the RBC residuals will not disappear from the LYM region. If
their number is much higher than the total number of WBCs the result is a WBC histogram like below.
%?1BD, 7T01L, @2;C1, ;>, 51B10042T, 45:, 21@149, C140621C159, A;9?, ;5B21401:, 7T01, p6459;9T (press up arrow
once or twice before sampling).
If the result of a measurement with more lyse is similar, you can suspect the presence of N-RBCs, contamination
of reagents or noise (see Section 4.2).
Evaluation:
QM% .M% UN(

! typically under-lysed blood
! we have flags #L, ., 45:, Q, the instrument gives all
WBC related results with asterisks
normal a little bit low
! flag #: linearity error, too many particles detected
! flag .: too many RBC was found in WBC
! flag Q: 3-part differential is unsuccessful

GO+I,&S12E7T01:,*4C@71
If the lyse is too much for a sample, the resulting WBC histogram will be much narrower. The problem is that
Granulocytes get overlapped with MID population making impossible correct estimation of MID region.
.1@149,C140621C159,A;9?,:1B21401:,7T01,p6459;9T (press down arrow once or twice before sampling again).
Evaluation:
QM% .M% UN(

! typically over-lysed blood: all discriminators are be-
low 100 fl
! although we do not have flags MID and GRA are high MCV normal
overlapped, therefore MID% is high (correct value is
6%)

K,X)().-'$._,%$*)*
In the following sections we are going to present you some typical histograms of different animals.
The HUMACOUNT analyzer provide measurement of animal blood in veterinary mode: :=<L,B49,45:,?=201, and
they have >;S1,4::;9;=547,S1912;542T,C=:10 for other species you wish to measure. You can customise these
additional modes for your needs by adjusting the normal ranges and lyse amount used (see under N;C;90 menu
point).
Setting the lyse amount (default is 0.5 ml) requires a few measurements and understanding of the histograms.
By going trough the following pages, and after having some experience, you will be able to judge whether you
need more lyse (i.e. you will have to repeat measurement of the same sample with an increased lyse amount),
or less.
Do not forget that the same animal blood measured in different modes will give you different values, since these
predefined modes (e.g. dog mode) are optimised to measure the corresponding sample the best way possible.
So, measure cat blood in cat mode for best results. In some extreme cases you can even try to measure e.g.
dog samples in other #1 mode.
The software will automatically set the discriminators in the other five modes as well, you will only have to control
lyse volume and verify results. Some repeated measurement may be necessary after measuring with the default
lyse amount. This is obvious, since the basic problem comes from the characteristics of animal blood (and the
used lyse).
You have to know that measurement of other species (e.g. birds) can be limited, maybe a few parameters are
applicable only (for birds WBC measurement is impossible, because they have nucleated RBCs, so lysing of
them is not possible, we measure only RBC/PLT).
Typically, animal blood histograms show narrower RBC and WBC curves (compared to human). This comes
from the characteristics of their blood compared to the human cases, since generally they have smaller cells.
]==:,@24B9;B1,A?15,94D;5<,87==:,>2=C,45;C470,;0,9=,21>601,9?1,>;209,>1A,:2=@0,=>,04C@71O It prevents hair
and skin pieces getting into the sample protecting better against clogging.
KO+,'=2C47,!1C49=7=<T,.45<10,=>,$5;C470
*@1B;10 QM% .M% !]M !%( #%X #%! #%!% UN(
/&] 6-17 5,5-8,5 12-18 37-55 60-77 19,5-24,5 32-36 200-500
%$( 5.5-19.5 5-10 8-15 24-45 39-55 13-17 31-35 300-800
!&.*) 5,5-14,3 6,8-12,9 11-19 32-52 37-58 10-20 31-38 100-600
M&X-') 4-12 5-10 8-15 24-46 40-60 11-17 30-36 100-800
#&"*) 9-21 4,8-6,3 8-15 30-44 50-90 12-13 30-36 200-900
.$( 6,4-26,2 7-10 10,8-17,5 35-51 57-65 15-22 30-35 190-900
.$MM-( 2-15 4-8,6 9-18 30-53 57-90 16-31 32-39 120-800
U-] 11-16 5-8 10-16 32-50 50-68 17-21 30-34 320-720
*!))U 4-12 9-15 9-15 25-50 28-40 8-12 31-34 110-800
]&$( 4-10 8-13 8-12 20-38 16-25 5-7 30-36 300-600
Y @F Y
GHJ?0 @] [# @] [# 7[:# ^ B# "7 7[:# @] [#
?&9#-*PA*0,22&.5*(B*H(.2&#*4-'-.$)&.5*;&)7-/)@O*$10(2)34B

KOF,)>>1B9,=>,NT01,0199;5<,9=,HE@429,QM%,:;>>12159;47
In the following histograms we try to demonstrate the importance of correct lyse settings, because the result of
WBC 3-part differential depends mainly on the correct lyse amount.
We measured the same :=<,87==:,9?211,9;C10 A;9?,:;>>12159,7T01,0199;5<0. The whole process depends on
the applied lyse type as well. This is just a demonstration of the effect of lyse setting.
M*P)E7?)'./-*P1"9)Q.-')I4R)9%)*+)%S/()@%.--%()T.-)$#,(1F%S/(,B
M*P)E7?)'./-*P1"9)Q.-')I4U)9%)*+)%S/()@%.--%()T.-)*V(1F%S/(,B
M*P)E7?)'./-*P1"9)Q.-')I4W)9%)*+)%S/()@*V(1F%S/(,B
If you compare the WBC histograms above, you can come to the conclusion that the amount of lyse greatly de-
termines the WBC 3-part differential parameters.
NT01d QM% N_#W #-/W ].$W
0.4 ml 11.4 23.6 2.3 74.2

0.6 ml 10.9 56.2 6.7 37.1
0.8 ml 10.3 53.8 1.8 44.3
Total WBC decreases as lyse goes higher, because lysing of RBCs is better with more lyse.
On the other hand, the GRA population gets overlapped with LYM making impossible their correct separation by
discriminators.
As a summary we can declare that the 8109,7T01,4C=659,?121,;0,4@@2=ZO,[OI,C7,(probably 0.45 ml),>=2,HE@429
:;>>12159;47,@424C19120, while WBC is acceptable in all cases. This is the reason why you should repeat meas-
urement in some cases with different lyse volume.

KOH,/=<
Dog samples show RBC peaks around 60 fl following good separation of PLTs from RBCs.
For 3-part WBC differential sometimes there are difficulties due to small Granulocytes and giant Lymphocytes
(this makes overlapping of these populations in some cases). A little under-lysing of the samples gives better
differential results.
KOHO+,/=<d,'=2C47,*4C@71
This sample shows a normal dog histogram.
Evaluation:
QM% .M% UN(

! all cells larger than 27 fl ! PLT cell population is between 2 fl
! all cells larger than 35-40 are counted as RBCs and 30 fl
fl are counted as WBCs
! RBC histogram follows ! PLT histogram follows log-normal
! WBC histogram has normal distribution distribution like in human blood
visually rather two
! expected MCV is near 60 ! in most cases there is clear
populations than three, so
fl PLT/RBC valley, so the software
they are different from
human samples ! expected RBC is near 7.0 can put the discriminator easily in
12
x10 cells/l place

KOHOF,/=<d,!;<?,N_#WL,7=A,].$W
This sample has high LYM%. Usually LYM% is near 25-30% for dogs.
$BB1@9,9?101,2106790O
Evaluation:
QM% .M% UN(

! high LYM%
! low GRA%
! MID population is estimated right to the normal slightly low PLT
LYM/GRA valley, it is usually done like
this in DOG mode

KOHOH,/=<d,P74<,q/bL,?;<?,UN(
This sample gave D warning flag. This case is similar to some kind of clogging, because there are no calculated
WBC parameters.
.1@149,C140621C159,A;9?,9?1,04C1,7T01,S=76C1O
Evaluation:
QM% .M% UN(

! WBC parameters are missing
although there is good histogram high PLT
! flag / means WBC data error. normal (see RBC histogram: the peak of
the curve is higher than in normal
! the reason of data error is similar to case)
clogging

KOHOI,/=<d,P74<,qQb
The warning flag Q may appear if there are no traces of different WBC populations (Granulocytes in this case),
so 3-part differential results are suspected to be bad. In this case the software may set the discriminators to bad
positions.
U21E:;7691,04C@71,9?15,C140621,4<4;5,;5,@21E:;769;=5,C=:1O
Evaluation:
QM% .M% UN(

! WBC is very high, other related values are all
marked with asterisks
! flag Q means WBC three part warning: no
traces of 3-part differential could be found normal low PLT
! this sample has mainly lymphocytes

! discriminator 2. and 3. are in bad position

KOI,%49
Important characteristic of cat blood is that the size of RBCs is much smaller (between 20 and 50 fl, MCV = 35-
40 fl) while their PLTs are normal in size (2-30 fl). The result is that the RBC and PLT histograms are over-
lapped. This overlapping makes sometimes very hard to separate PLTs from RBCs, so the PLT result may have
higher variance (or even lower value).
It is typical for cat patients, that during taking blood, they suffer a kind of shock, which triggers a protecting
mechanism, which has bad influence on the sample itself.
A relatively good practice during taking blood from cats to refuse the first few drops of the sample. This can pre-
vent even hair and skin pieces to get into the sample making it safer to avoid clogging.
It is general for cat samples that some resistive RBCs will remain in their blood, so normally total hemolysis of
their RBCs will rarely happen resulting in a small peak at the beginning of their WBC histogram.
KOIO+,%49d,'=2C47,*4C@71
This sample is not typical, rather ideal case of cat samples.
Evaluation:
QM% .M% UN(

! slightly low WBC ideal case: PLT is well separated
normal
! good 3-part WBC differential from RBC

KOIOF,%49d,!;<?,N_#WL,7=A,].$WL,?;<?,.M%,45:,!]M
This sample demonstrates some characteristics of cat blood. $BB1@9,9?101,2106790O
Evaluation:
QM% .M% UN(
! low WBC ! low PLT

! high RBC and HGB
! high LYM % ! typical case of cat: PLT histogram is
! cats have smaller RBCs,
partly overlapped with RBC histogram
! low GRA % so MCV value is normal
in most cases

KOIOH,%49d,P74<,a#bL,7=A,.M%L,!]M,45:,S12T,7=A,UN(
The following sample is the blood of a very sick cat.
U21E:;7691,9?1,04C@71,45: C140621,4<4;5,;5,@21E:;769;=5,C=:1
Evaluation:
QM% .M% UN(

! low RBC and HGB
# flag shows that WBC
coincidence is too high, the re- ! high MCV, presence of macro-
cytic RBCs, the curve is wider very low PLT value
sults are out of the linearity
range. than in normal case (RDWc is
high)

KOIOI,%49d,P74<,q.b
A typical problem for cat samples the presence of some RBC residuals (sometimes nucleated RBCs) at the be-
ginning of the WBC histogram. In extreme cases the amount of this residual influences correct measurement of
WBC by interfering and increasing WBC in LYM section.
.1@149,C140621C159,A;9?,4,?;<?12,7T01,S=76C1O
Evaluation:
QM% .M% UN(

! a little bit low PLT
flag,.: too many RBC found in
normal ! overlapped PLT/RBC, but the dis-
WBC
criminator is correctly set

KOJ,!=201
Horse blood samples are relatively easy to measure. The only problem might be caused by the very small PLTs.
Good separation of PLT/RBC and correct WBC histogram are the typical characteristics of horse samples.
The MCV is relatively low, while RBC is high to give a correct HCT near 40%.
For horse samples MID% is low in most of the cases, because there are few monocytes in their blood.
KOJO+,!=201d,'=2C47,*4C@71
Evaluation:
QM% .M% UN(
! WBC value is normal PLT is correct, because there is

normal correct separation at PLT/RBC
! 3-part parameters are correct discriminator

KOJOF,!=201d,N=A,N_#WL,?;<?,].$,45:,].$W
This is a typical horse sample with smaller MCV.
$BB1@9,9?101,2106790O
Evaluation:
QM% .M% UN(

! slightly low LYM % and high
GRA %
low MCV: microcytic RBCs normal (MPV low)
! nice 3-part differential WBC
histogram

KOJOH,!=201d,N=A,UN(
In some cases PLT is low. You can compare the height of the PLT peak to the RBC peak (on the RBC histo-
gram).
$BB1@9,9?101,2106790O
Evaluation:
QM% .M% UN(

! low PLT value, as it is shown on RBC
! perfect WBC separation from
histogram as well
RBC/PLT normal
! the valley between the PLT and the RBC is
! good 3-part differential results
well defined, so PLT value is correct

KOJOI,!=201d,N=A,.M%,45:,!]M
This case shows normal sample.
$BB1@9,9?1,2106790O
Evaluation:
QM% .M% UN(

normal low RBC (HCT) and HGB show anemia normal

KOG,.488;9
As the histograms show below, rabbit blood is very similar to the human blood, but generally with lower MCV
and higher RBC.
Separation between populations on all histograms is correct due to fine segregation by size.
Measurement of rabbit blood should happen in either of the additional (other) modes. Do not forget to set up this
mode for rabbit: name, lyse, and normal range limits.
Evaluation:
QM% .M% UN(

Correct WBC and 3-part parameters Like dog: MCV is near 60 fl Perfect PLT/RBC separation

KOK,.49
Rat blood is similar to human blood in measurement characteristics. Their normal ranges are still different (nor-
mally LYM is higher than GRA).
It is better to measure rat blood in one of the other modes after setting it up (name, lyse amount, limits, etc.).
Evaluation:
QM% .M% UN(

Normal RBC histogram
perfect separation high
MCV of rats generally smaller than that of dogs
***

Human
Gesellschaft fr Biochemica
und Diagnostica mbH
Max-Planck-Ring 21 " D-65205 Wiesbaden
Germany
Telefon: +49 6122 9988 0
Telefax: +49 6122 9988 100
eMail: human@human.de
Internet: http://www.human.de
01/2004-04

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APPLICATION MANUAL

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 1/49

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 2/49

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 3/49

;-:*<#((:*8-##/ =1$'-*<#((:*8-##/ >#&'-#-'/

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 4/49

*The HGB unit specifies MCH and MCHC units as well.

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 5/49

To determine WBC sub-populations, first the software

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 6/49

>&.&2-'-. G)$' +&#- D-2&#-

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 7/49

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 8/49

Typical sample preparation sequence:

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 9/49

Selective (3-part differential) hemolysis of WBCs depends on

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 10/49

U424C19120 '=,QM%,:;>>O HE@429,QM%,:;>>O JE@429,QM%,:;>>O

QM% V-/ V-/ V-/

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 11/49

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 12/49

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 13/49

If clogging is detected, B or % flag appears. You should repeat the measurement.

If the aperture is partially clogged, its volume (V) decreases.

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 14/49

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 15/49

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 16/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 17/49

P74< #145;5< .1B=CC15:1:,6012,4B9;=5

WBC data package

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 18/49

P74< #145;5< .1B=CC15:1:,6012,4B9;=5

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 19/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 20/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 21/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 22/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 23/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 24/49

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 25/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 26/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 27/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 28/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 29/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 30/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 31/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 32/49

QM% .M% UN(

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 33/49

Application !"#$"% HUMACOUNT Hematology Analyzer 1.0 release 34/49

NT01d QM% N_#W #-/W ].$W

0.4 ml 11.4 23.6 2.3 74.2

;-:<#((:8-##/ =1$'-<#((:8-##/ >#&'-#-'/