application manual - human-kabul.com · application manual!"#$%&"’(hematology...

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APPLICATION MANUAL

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Hematology Analyzer1.0 Release

Cat.-No.: 17401/4

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

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1 INTRODUCTION 32 BLOOD 4

2.1 Function of Blood 4

2.2 Composition of Blood 42.3 Blood Cell Parameters 5

2.3.1 Red Blood Cells, Hemoglobin 5

2.3.2 White Blood Cells 62.3.3 Platelets 7

2.4 Normal Hematology Ranges of Human Blood 7

3 MEASUREMENT METHODS 83.1 Volumetric Impedance Method 8

3.1.1 Diluting whole blood 9

3.1.2 Hemolysing blood cells, 3-part WBC differential method 93.2 Hemoglobin Determination by Photometry 11

4 ROUTINE UTILIZATION 12

4.1 Sample Handling 124.1.1 Anti-coagulant 124.1.2 Taking blood 12

4.1.3 Storage of Samples 124.1.4 Handling Blood Controls and Calibrators 13

4.2 Reagents and Environmental Conditions 13

4.3 Aperture Blockage - Clogging 144.4 Calibration 154.5 Quality Control 15

4.6 Measuring Patient Blood 154.7 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 164.7.3 Calibrating Pre-dilution Mode 16

5 MEASUREMENT RESULTS, HISTOGRAMS 17

5.1 Typical histogram 175.2 Warning Flags 18

6 HUMAN CASES 20

6.1 Neonate 206.2 Three year-old Child 216.3 WBC clogging 22

6.4 WBC histogram with Nucleated RBCs 236.5 Lymphocytosis: WBC out of linearity 246.6 Lymphocytosis: measured in 1:10 pre-diluted mode 25

6.7 High MID and MID% 266.8 Granulocytosis 276.9 Thrombocytopenia 28

6.10 Thrombocytosis, Anisocytosis 296.11 Microcytosis (Low MCV) 306.12 Macrocytosis (High MCV) 31

6.13 Under-lysed Sample 326.14 Over-lysed Sample 33


7 VETERINARY CASES 347.1 Normal Hematology Ranges of Animals 347.2 Effect of Lyse setting to 3-part WBC differential 35

7.3 Dog 367.3.1 Dog: Normal Sample 367.3.2 Dog: High LYM%, low GRA% 37

7.3.3 Dog: Flag „D”, high PLT 387.3.4 Dog: Flag „W” 39

7.4 Cat 40

7.4.1 Cat: Normal Sample 407.4.2 Cat: High LYM%, low GRA%, high RBC and HGB 417.4.3 Cat: Flag “M”, low RBC, HGB and very low PLT 42

7.4.4 Cat: Flag „R” 437.5 Horse 44

7.5.1 Horse: Normal Sample 44

7.5.2 Horse: Low LYM%, high GRA and GRA% 457.5.3 Horse: Low PLT 467.5.4 Horse: Low RBC and HGB 47

7.6 Rabbit 487.7 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;E0;=50, accept the results, and the overall responsibility is ours. If we know the ability of our analyzer, we willmake the necessary decisions easier, accept the good results – even though there are warning flags – or repeatmeasurement if necessary.

We tried to avoid complicated descriptions and definitions of the hematology terms. You will find several simplefigures and drafts describing the basic functionality, because our scope is to make you understand the basicseasily, 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 today’s technology.

%?4@912, I deals with the rules of routine utilisation of the analyzer from taking the blood to the results, andmeasurement 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. Everycase 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 informationnot detailed among the Veterinary cases.

The information in this !"#$"% is based on the knowledge of the "01230,#45647. So, please, keep it at hand ifyou need it for reference.

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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 toand remove waste products from all the cells of the body. Neurones, muscle cells, connective tissue cells andepithelial cells draw their nourishment from the interstitial space and respond to the glucose and oxygen contentof that environment. Fresh supplies of oxygen and glucose are obtained by exchange with the blood circulatingin 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 theinterstitial fluid environment. These subtonics are taken up by the plasma and 21:,87==:,B1770 and eliminated asthe blood passes trough the kidneys and lungs. Blood also delivers hormones, lipids, amino acids, salts andvitamins 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 hemostaticplugs.

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-##/ >#&'-#-'/

'=2C47,:150;9T 4.5 – 5.5 x1012 cells/l 5 – 10 x109 cells/l 150 – 450 x109 cells/l

'6B71491:V No* Yes No

*68E@=@6749;=5045:,9?1;2,W

RBC: 99.9%

*NRBC (Nucleated RBC)0.1%

GRAnulocytes: 65

NEUtrophil: 60%

EOSinophil: 4%

BASophil: 1%

LYMphocytes: 30

MONOcytes:

*?4@1L,0;R1Biconcave (donut) shapediameter: 7-8 µmthickness: 1.8-2 µm

GRAnulocytes: 13-16 µm

LYMphocytes: 8-15 µm

MONocytes: 15-25 µm

Fragments with a

diameter of 2-4 µm

X=76C1,=>,B177 80 – 100 fl 50 – 1500 fl (native) 5 – 15 fl

X=76C192;B,W,;5A?=71,87==:

45 % 0.1 % 0.3 %

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FOH,M7==:,%177,U424C19120FOHO+,.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 itsC145,B=2@60B6742,S=76C1 Y,#%X,Y is approx. 90 fl. RBCs are the most numerous cells in blood. There areapprox. J,Z+[+F,B1770\7 in the blood of a healthy person. RBC and MCV are measured primary parameters, sothey 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 mostaccurate 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 dependson 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).

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!1C=<7=8;5,Y,!]M,Y,is the main component of RBCs. It is conjugated protein (with Fe). Its main function is totransport 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 RBCand 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==:,%1770Q?;91,87==:,B1770,Y,QM%,Y,are formed in the bone marrow. During their maturation sequence they differentiatefrom the same parent-cell to 5 sub-populations. WBCs are nucleated and comprise granulocytes, lymphocytesand 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 WBCcount can increase dramatically, e.g. to 500,000/µl in extreme leukemia. In this case, pre-dilution of the sampleis 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,#-/WY 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 benecessary.

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 of3-part WBC differential is illustrated by the following figure.

To determine WBC sub-populations, first the softwareputs :;0B2;C;549=2, +O to the border of hemolysedRBCs + PLTs (on the left) and LYM population, thenfits normal distribution curves to the remaining WBChistogram (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 showswhere it found the MID population.

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You should remember this way of operation during the verification/validation process, when you accept the WBCand 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,U74917190U74917190,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 canvary from 0 to 1 million cells/µl in different conditions.

PLTs are relatively small compared to RBCs. The mean platelet volume Y,#UX,Y, is approx. 12 fl, so that theycan 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 SDparameters 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&#-

QM% 109 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

.M% 1012 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

UN( 109 cells/l 275 ± 125 275 ± 125

#UX fl 12.0 ± 2.0 12.0 ± 2.0

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H,#)$*".)#)'(,#)(!&/*In this chapter we make an overview of hematology measurement methods. It is important to know how yoursystem 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 ofblood cells, the claim arose to a more accurate and quicker method. The volumetric impedance method is anautomated electronic way of counting blood cells.

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The principle of this measurement is that the blood diluted with isotonic solution (diluent) can conduct current byionic conduction. A counting chamber made of insulating material (plastics) holds this dilution of the blood, whilethere 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 isotonicsolution 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 smallchange in electric impedance will occur, making the voltage a little bit higher, i.e. a small electric pulse will bepresent. The amplitude of the pulse will be proportional to the ratio of the cell volume (size) and the aperturevolume, so the bigger the cell is, the higher pulse we get. The typical aperture size diameter and length is 80 µm.

For proper counting – differentiation – of cells, we need high likelihood (more than 90%) of passing one cell theaperture 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 extremelyhigh concentration cases (e.g. leuke-mia) WBC density can be 100x higherthan in normal cases causing two ormore cells passing the aperture at thesame time, generating one pulse in-stead of two (or more). It is called coin-cidence, and it will result in non-linearcounting of cells. Flags C and # showthis case.

The WBC linearity range is:

KJZ+[^ cells/lL, +[[Z+[^ cells/l for theHUMACOUNT.

In this case you should pre-dilute thesample and repeat measurement.

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The applicable pre-dilution ratio depends on the setting of your analyzer (1:3 or 1:10). See section 4.7 for detailson usage.

HO+O+,/;769;5<,A?=71,87==:Automated analyzers make the following steps automatically, to reduce the cell concentration to the desiredlevel.

Typical sample preparation sequence:

1. The analyzer takes 25 µl of whole blood treatedwith EDTA. This will be diluted to MIX chamber with4ml of diluent to form +d+G[,#-e,:;769;=5 of wholeblood.

2. Another 25 µl of the MIX dilution is sampled, andput into the RBC chamber with 5ml of diluent toform an overall +dHF,[[[,.M%,:;769;=5. This is suit-able to measure RBC/PLT.

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3. Then 0.8ml of lyse is added to the MIX dilution (practically while it is moved to the WBC counting chamberto avoid contamination of MIX chamber with lyse) to form an overall +dF[[,QM%\!]M,:;769;=5. This liquid issuitable 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 RBCswould 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 fromthe 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.


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The membrane of WBCs become selectively permeable so in the slightly hypertonic lyse solution they begin toshrink to their nucleus. The properly hemolysed liquid contains WBC particles in the 30-400 fl region.

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Selective (3-part differential) hemolysis of WBCs depends onlyse reagent type, lysing time and the amount of lyse used.

If we add too much lyse or we wait too much, the result will notbe selective, all WBCs will be shrunk to their nucleus, i.e. weget a histogram with all WBCs in one population in 30-120 flregion (see the histogram on the left). We call this case 9=9477Tor =S12E7T01:,04C@71.

Using 3-part WBC differential lyse with and HUMACOUNT,lyse amount is the only parameter which can be modified.

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If the software gets over-lysed histogram, it will put flag ) – “No WBC 3-part differential” – or flag Q,Y, “3-partWBC 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-lysingmeans 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 dependingon the results. If the histogram shows over-lysed result, you can repeat the measurement after decreasing thelyse amount by pressing the down arrow (!"# $%&'(&#)*+,# -.#%./*&01+'*/#$2'*3#4&*''#%4#2&&(5# 67"# 8(# 9.$&*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 User’s 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 toget 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-/

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-9, ;0, ;C@=29459, 9=, D5=A that – independently of the manufacturer of the analyzer – volumetric impedancemethod (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;=5method for making qualitative hematology report.

HOF,!1C=<7=8;5,/1912C;549;=5,8T,U?=9=C192THGB 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 ispossible 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 nmwave-length through the WBC dilution, and measuringthe 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)

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The analyzer takes reference HGB measurements on clean diluent in each cycle during the cleaning phase toensure stability over time and temperature.


I,.&"(-'),"(-N-g$(-&'IO+,*4C@71,!45:7;5<IO+O+,$59;EB=4<67459Since some time will usually elapse between collection of samples and counting, it is necessary to preserve thesample with an anti-coagulant to prevent large groups of cells forming into clots or lumps of cell matter that willclog the cell counter. Choice of anti-coagulant is very important, as some anticoagulants will affect the shapeand size of blood cells. In general )/($, 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 usingmanufactured 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 asmall 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@710Anti-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 (25ºC).

This rule basically applies for the 3-part WBC differential. The granulocytes are destroyed first, after 12-16 hoursyou 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 thiscase, because the samples must warm up to room temperature before measuring them. Roll the sample tubesbetween 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=20M7==:,B=592=70 must be handled correctly to ensure their reliability. During transport and handling they shouldbe 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. +EFC;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 torefrigerator within H[,C;56910 of use. When opening a vial for the first time note the date on vial label as theopen 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;=50Always 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 bymodifying hemolysing characteristics.

The analyzer will automatically 265 8745D,C140621C159 to ensure instrument and reagent cleanliness. If blankvalues 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 intothe 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 aperturegenerates very small electrical signals in the several hundred micro-Volts (100 µV) region. If there is adevice generating high-energy signals near the analyzer, it can harm counting by generating electricalnoise.

The main protection against electromagnetic noise is <==:,<2=65:;5< and shielding. It is therefore veryimportant that the power supply has a solidly grounded earth lead. If this is not sufficient to overcome theproblem, 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 anotherroom, 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 theblank 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 thecross-section of the aperture.

Clogging can be detected by increased measuring time. The basic measuring time is 8 seconds for WBC andRBC/PLT. If there is a higher counting time, the corresponding aperture must have been blocked, repeating ofmeasurement 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 theHUMACOUNT analyzer. You can monitor the probe voltage range at RPrV and WPrV under the PLT histogramon 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 oflonger shut-down times. Run “Preparing for shipment” procedure to remove reagents and drain the system.


IOI,%47;8249;=5Calibration is used to set the absolute accuracy of the analyzer. For this sake, we measure control blood havingknown results. If there are differences between the reference values and the measured values, we modify thefactors to get the most corresponding results.

The HUMACOUNT offers automatic calibration where the user must enter the reference (or target) values, andthen 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 theageing of the control).

Be sure to run calibration only in perfect conditions, if there is no clogging, contamination, etc. otherwise you canhave much worse results. It is recommended to run a cleaning and a blank measurement before calibrating, toensure optimal conditions.

IOJ,k647;9T,%=592=7Quality 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 (suitablefor 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 resultsare 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 User’s 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 bebad). At least 30 minutes is required for a sample tube to reach the room temperature if it was in refrigeratorbefore.

It is recommended to use a 2=942T,C;Z12 to keep samples homogenous during the measuring session. If you donot have sample mixer, ;5S129,9?1,96810,6@0;:1,:=A5,01S1247, 9;C10 before sampling. '1S12,0?4D1 them toavoid formation of micro-bubbles inside, because 0C477, 8688710, @21S159, B=221B9, 04C@7;5< (air is sampledinstead of blood).

After measurement, B?1BD,9?1,2106790,45:,?;09=<24C0 to identify any sort of disorder. .1@149,9?1,C140621EC159 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 cliniciancan 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 youneed better WBC qualification.

IOK,"0;5<,U21E:;769;=5,#=:1Pre-dilution mode is used in two cases:

1. if B4@;7742T,9681 (e.g. 25 µl 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-dilutionmode 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 µl).

IOKO+,*4C@71,@21@4249;=5,>=2,+d+[,U21E:;769;=5,#=:11. 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,#=:11. 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,#=:11. %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 patientsamples 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/WBCdiscriminator (near 30 fl) arecounted as WBCs.

WBC histogram has threepopulations:

1. lymphocytes

2. MID population(monocytes and someeosinophils), and

3. granulocytes

Lymphocytes can be found approx.between 30-90 fl.

The two other discriminators showthe place of MID population (90-140 fl).

Granulocyte population is abovediscriminator 3 (140 fl)

All cells above the RBC/PLTdiscriminator (near 30 fl) arecounted as RBCs.

The RBC histogram follows normaldistribution.

The RDW value is within the normalrange, so the width of the RBChistogram is normal

The whole histogram is normalised(100% fit) to the RBC peak.

PLT histogram is a magnifiedportion of the beginning of theRBC histogram.

PLT value is near 250.

PLT cell population is approx.between 2-30 fl

PLT histogram follows a log-normal distribution, with a clearseparation from RBCs.


JOF,Q425;5<,P74<0In this section we summarise the error flags and we give an explanation of their possible cause and a few hintsto overcome the problem.

"@@12B401,7199120,21>12,9=,QM%,=2,!]M,@2=871C0d

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

Q

WBC three partwarning or WBC threepart diff. unsuccessful

0--*8&/-*OZ*@I*&):E(7*%&/-/

Possibly lyse problem. Try reducing the lyse amount and repeat themeasurement. In extreme Lymphocytosis case you can get this flag.

Check the discriminators in the WBC histogram. If the discriminators are in theproper place (the populations can be separated even by eye) then the resultsare correct and acceptable.

) No WBC three partPossibly lyse problem, but in some pathological samples (too highlymphocytes), it can happen.

! HGB blank is high, orno HGB blank

Repeat the blank measurement. If HGB blank is not stable there are probablybubbles in the WBC chamber. Run a cleaning and try blank again. Close theside door if open during measurement.

M WBC blank is high, orno WBC blank

Repeat blank measurement, or run prime lyse and try blank again.

Possibly lyse contamination, or noise problem.

N WBC/RBC limitwarning

Check the 1. RBC-LYM discriminator. If it is in the minimum point (or close toit), accept the results. Otherwise repeat the measurement.

If the repeated measurement gives similar results and discriminator 1. is inwrong place then the MID and GRA results are OK, but the WBC and LYMresults can be higher because of the RBCs.

.

Too many RBC cutfrom WBC

0--*8&/-*@I*&):*8&'%&/-/

Repeat the measurement with increased lyse amount.

If the WBC measuring time is too high (more than 8 sec. in ) it could beaperture clogging. In this case perform cleaning and repeat the measurement.

#

WBC coincidence istoo high. Linearity

error.

0--*8&/-*OZ*@I*&):8&'*%&/-/

The results are out of the linearity range. Make a dilution with an externaldilutor with the set-up (1:10 or 1:3) dilution rate.

Do not forget to calibrate pre-dilution mode before using it for measuringsamples.

/WBC data package

errors

0--*E(7*%&/-/

Perform cleaning and repeat the measurement (aperture clogging).

If it is a general problem, please call your Service Personnel.

* WBC time error Same action as in case of warning flag,/.

%WBC clogging

0--*8&/-*I

Aperture clogging. Same action as in case of warning flag,/.

Low temperature reagents can caused it as well (mainly diluent), in this caseyou will have to wait until they reach room temperature.

U PLT blank is high, orno PLT blank

Run cleaning and repeat the blank measurement.

Diluent or system cleanliness problem. If it is stable high, replace the diluentby opening a new tank.

M RBC blank is high, orno RBC blank

Same action as in case of warning flag,U.

?&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

7RBC/PLT limit flag

0--*8&/-*Y

RBC/PLT valley is too high, or the PLT peak is not high enough.

If the discriminator is in a wrong place (in the PLT or RBC histogram) thenrepeat the measurement for a correct PLT result.

D RBC peak warningPerform cleaning and repeat the measurement (probably clogging).

If it becomes a general problem, change the RBC aperture.

CRBC/PLT coincidence

is too high.Linearity error.

Same action as in case of warning flag,#.

: RBC/PLT datapackage errors

Same action as in case of warning flag,/.

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 tothe 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 themeasurement.

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 givesthe same result, you can accept the results, keeping in mind that parameters marked by asterisk (o) are not100% 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 istoo 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. Thereasons can be: a high PLT blank (PLT value will be marked), a case of indefinite discriminator setting (defaultlocation 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 itgets a (E) flag if under the range, or gets (f) if over the range. You can customise ranges for all kind of patientsby setting the corresponding lower and upper ranges. If you set 0 for a range limit, it will be not verified (seeUser’s 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 yousee 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 ingood position or not. Normally it should appear in the valley between the PLT and RBC populations.

GO+,'1=5491This 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

baby bloodlow RBC, anemia normal


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

! slightly high WBC

low HGB & MCH high PLT


GOH,QM%,B7=<<;5<In cases of aperture clogging we can see a histogram magnified to the right (the smaller aperture has highersensitivity, so we see the particles to be “bigger”). In case of RBC clogging MCV – and consequently – HCT willbe higher.

.1@149,C140621C159O

Evaluation:

QM% .M% UN(! flag C shows WBC aperture clogging, the

sensitivity of the aperture increases, speed offlow decreases

! we can see that WBC measuring time(WBCt) is high, the instrument does not giveany WBC values and the histogram is magni-fied to the right (all discriminators are puthigher)

low RBC normal


GOI,QM%,?;09=<24C,A;9?,'6B71491:,.M%0You can have some more information about the composition of the sample even though the analyzer cannot giveN-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).

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 tothe histogram area shown with the arrow, wecan be sure, this extra amount is approx. 0.5,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.

normal thrombocytopenia: low PLT

possibly N-RBCs


GOJ,NTC@?=BT9=0;0d,QM%,=69,=>,7;5142;9TDue to coincidence (see Section 3.1) WBC linearity is limited. This sample is a typical Lymphocytosis and itdemonstrates 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 onwhich 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 ofcoincidence pulses cause overlapping of populations,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.

normal normal


GOG,NTC@?=BT9=0;0d,C140621:,;5,+d+[,@21E:;7691:,C=:1In order to increase the linearity of the instrument, you should pre-dilute the sample in a clean tube for repeatedmeasurement. (Refer to section 4.7 and the User’s Manual for details, since operation can be slightly differentdepending on software versions).

The main conception of pre-dilution: by reducing the concentration of the cells we can put measurement back inthe 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 significantdifferences 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 usedfrom the previous measurement (case 5).


GOK,!;<?,#-/,45:,#-/WIn case of Monocytosis or Eosinophilia the MID population will have a significant peak in the WBC histogrambetween 100 and 180 fl.

$BB1@9,9?1,2106790O

Evaluation:

QM% .M% UN(! high MID and MID%

! MID and GRA populations areoverlapped

low RBC, HGB, HCT normal


GOh,]24567=BT9=0;0This case shows Granulocytosis. The peak of Granolucyte population is so high that the software is normalizingthe WBC histogram to it, so the LYM population looks much smaller.

$BB1@9,9?1,2106790O

Evaluation:

QM% .M% UN(! Leukocytosis: high WBC

! GRA is high, LYM is lowanemia: low RBC and HGB normal


GO^,(?2=C8=BT9=@15;4In case of Thrombocytopenia the PLT value is very low.

$BB1@9,9?101,2106790O

Evaluation:

QM% .M% UN(

! normal WBC

! high MID:suspect ofmonocytosis

microcytic RBC

! thrombocytopenia: low PLT

! warning flag 7: due to the relatively low peak of PLT,the analyzer is not sure about the correct placement ofPLT/RBC discriminator, consequently it will putasterisks near PLT related results

! if you can see that the PLT/RBC discriminator is inright place (like now) we can accept the marked results


GO+[,(?2=C8=BT9=0;0L,$5;0=BT9=0;0This 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

is wide, RDWc is high

thrombocytosis:

very high PLT


GO++,#;B2=BT9=0;0,`N=A,#%XcThis sample shows low MCV and GRA parameters.

$BB1@9,9?1,2106790O

Evaluation:

QM% .M% UN(lymphocytosis: high value of

LYM and LYM%low MCV value: microcytic

RBCsnormal


GO+F,#4B2=BT9=0;0,`!;<?,#%XcIf 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. Thebone-marrow tries to compensate low HGB (HCT) by releasing bigger RBCs.

QM% .M% UN(

MID% is high

! macrocytic RBC: MCV value is very high

! right-shifted wide RBC histogram

! anemia: very low RBC, low HGB

normal


GO+H,"5:12E7T01:,*4C@71If a sample is not hemolysed correctly (under-lysed) the RBC residuals will not disappear from the LYM region. Iftheir 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 arrowonce or twice before sampling).

If the result of a measurement with more lyse is similar, you can suspect the presence of N-RBCs, contaminationof reagents or noise (see Section 4.2).

Evaluation:

QM% .M% UN(! typically under-lysed blood

! we have flags #L,.,45:,Q, the instrument gives allWBC related results with asterisks

! flag #: linearity error, too many particles detected

! flag .: too many RBC was found in WBC

! flag Q: 3-part differential is unsuccessful

normal a little bit low


GO+I,&S12E7T01:,*4C@71If the lyse is too much for a sample, the resulting WBC histogram will be much narrower. The problem is thatGranulocytes 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 areoverlapped, therefore MID% is high (correct value is6%)

high MCV normal


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, andthey have >;S1,4::;9;=547,S1912;542T,C=:10 for other species you wish to measure. You can customise theseadditional modes for your needs by adjusting the normal ranges and lyse amount used (see under N;C;90 menupoint).

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 youneed 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 thesepredefined 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 controllyse volume and verify results. Some repeated measurement may be necessary after measuring with the defaultlyse amount. This is obvious, since the basic problem comes from the characteristics of animal blood (and theused lyse).

You have to know that measurement of other species (e.g. birds) can be limited, maybe a few parameters areapplicable only (for birds WBC measurement is impossible, because they have nucleated RBCs, so lysing ofthem is not possible, we measure only RBC/PLT).

Typically, animal blood histograms show narrower RBC and WBC curves (compared to human). This comesfrom 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 hairand 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

GHJ?0 @]Y[# @]@F[# 7[:# ^ B# "7 7[:# @]Y[#

?&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;47In the following histograms we try to demonstrate the importance of correct lyse settings, because the result ofWBC 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 onthe 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 bydiscriminators.

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 betterdifferential results.

KOHO+,/=<d,'=2C47,*4C@71This sample shows a normal dog histogram.

Evaluation:

QM% .M% UN(

! all cells larger than 35-40fl are counted as WBCs

! WBC histogram hasvisually rather twopopulations than three, sothey are different fromhuman samples

! all cells larger than 27 flare counted as RBCs

! RBC histogram followsnormal distribution

! expected MCV is near 60fl

! expected RBC is near 7.0x1012 cells/l

! PLT cell population is between 2 fland 30 fl

! PLT histogram follows log-normaldistribution like in human blood

! in most cases there is clearPLT/RBC valley, so the softwarecan put the discriminator easily inplace


KOHOF,/=<d,!;<?,N_#WL,7=A,].$WThis 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 theLYM/GRA valley, it is usually done likethis in DOG mode

normal slightly low PLT


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 calculatedWBC 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

! flag / means WBC data error.

! the reason of data error is similar toclogging

normal

high PLT

(see RBC histogram: the peak ofthe curve is higher than in normalcase)


KOHOI,/=<d,P74<,qQbThe 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 badpositions.

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: notraces of 3-part differential could be found

! this sample has mainly lymphocytes

! discriminator 2. and 3. are in bad position

normal low PLT


KOI,%49Important 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 havehigher variance (or even lower value).

It is typical for cat patients, that during taking blood, they suffer a kind of shock, which triggers a protectingmechanism, 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 oftheir RBCs will rarely happen resulting in a small peak at the beginning of their WBC histogram.

KOIO+,%49d,'=2C47,*4C@71This sample is not typical, rather ideal case of cat samples.

Evaluation:

QM% .M% UN(! slightly low WBC

! good 3-part WBC differentialnormal

ideal case: PLT is well separatedfrom RBC


KOIOF,%49d,!;<?,N_#WL,7=A,].$WL,?;<?,.M%,45:,!]MThis sample demonstrates some characteristics of cat blood. $BB1@9,9?101,2106790O

Evaluation:

QM% .M% UN(

! low WBC

! high LYM %

! low GRA %

! high RBC and HGB

! cats have smaller RBCs,so MCV value is normal

! low PLT

! typical case of cat: PLT histogram ispartly overlapped with RBC histogramin 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(

# flag shows that WBCcoincidence is too high, the re-sults are out of the linearityrange.

! low RBC and HGB

! high MCV, presence of macro-cytic RBCs, the curve is widerthan in normal case (RDWc ishigh)

very low PLT value


KOIOI,%49d,P74<,q.bA 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 ofWBC by interfering and increasing WBC in LYM section.

.1@149,C140621C159,A;9?,4,?;<?12,7T01,S=76C1O

Evaluation:

QM% .M% UN(

flag,.: too many RBC found inWBC

normal

! a little bit low PLT

! overlapped PLT/RBC, but the dis-criminator is correctly set


KOJ,!=201Horse 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

! 3-part parameters are correctnormal

PLT is correct, because there iscorrect separation at PLT/RBCdiscriminator


KOJOF,!=201d,N=A,N_#WL,?;<?,].$,45:,].$WThis is a typical horse sample with smaller MCV.

$BB1@9,9?101,2106790O

Evaluation:

QM% .M% UN(! slightly low LYM % and high

GRA %

! nice 3-part differential WBChistogram

low MCV: microcytic RBCs normal (MPV low)


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(

! perfect WBC separation fromRBC/PLT

! good 3-part differential results

normal

! low PLT value, as it is shown on RBChistogram as well

! the valley between the PLT and the RBC iswell defined, so PLT value is correct


KOJOI,!=201d,N=A,.M%,45:,!]MThis case shows normal sample.

$BB1@9,9?1,2106790O

Evaluation:

QM% .M% UN(

normal low RBC (HCT) and HGB show anemia normal


KOG,.488;9As the histograms show below, rabbit blood is very similar to the human blood, but generally with lower MCVand 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 thismode 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,.49Rat 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(

perfect separationNormal RBC histogram

MCV of rats generally smaller than that of dogshigh

***

HumanGesellschaft für Biochemicaund Diagnostica mbHMax-Planck-Ring 21 " D-65205 WiesbadenGermanyTelefon: +49 6122 9988 0Telefax: +49 6122 9988 100

eMail: [email protected]: http://www.human.de

01/2004-04

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