1. MANUAL LEUKOCYTE COUNT (Microscopic Hemocytometer Method)

* Principle:

A suitably diluted specimen is loaded onto a hemocytometer and the cellular elements are identified and counted microscopically and reported as number of cells per liter.

* Specimen Requirement:

. Whole blood (must not have any clots) EDTA, heparin or ammonium potassium oxalate may be used.

*' Reagents and Equipments:

a. Hemocytometer

b. Cover slip, optically flat, designed for


c. Thoma diluting pipet or unopette

d. Pipet suction apparatus

e. Diluents for leukocyte counting:

::::: must be capable of lysing erythrocytes without destroying erythrocytes

e.l. Glacial Acetic Acid, 2% (v/v)

e.Z. Hydrochloric Acid (Hf:l), 1 % (v/v)

e.3. Turk's Solution: enhances leukocyte

nuclear definition

3 ml. glacial acetic acid 1ml. gentian violet

96 ml.distilled water

* General Procedure for Specimen Preparation:

Recommended Dilutions for Manual WBCCount Specimen Preparation Based on Anticipated WBC Count

Anticipated Recommende Type of
WBC Count dDilution Thoma Pipet.
(x 10 91L)
0.1 - 3.0 1 :10 WBC
3.1 - 30.0 1:20 WBC
> 30.0 1:100 RBC
> 100.0 1:200 RBC * Calculation of Dilution Using WBe pipet:

= blood up to 0.5 mark and diluent to 11 mark, dilution is 0.5:10 or 1 :20

= blood up to 1.01hark 1: 10

* Sources of Error:

a. failure to mix blood specimen prior to taking a sample

b. failure to immediately mix blood and diluent in Thoma pipet produces clumps of erythrocyte stroma, which entrap leukocytes and make accurate counting impossible

c. using diluent contaminated with blood

d. bubbles in Thomapipet

e. drawing blood too far past the appropriate dilution mark and then drawing it out




The Unopette systenl provides a senes of reservoirs containing pre-measured diluent and pipets that automatically measure the amount of sample required for diluting blood specimens in preparation for manual counting.

Various reservoirs are available with appropriate diluting fluids for leukocyte and eosinophil counts.· The standard TTnAnpttp reservoir used -FAt" leukocyte count

~'" J. ........ ..1.""- \......I.1...l.VPVl,..-I,..V ~Vi.JV..l. 't' . .L \..f..IrJV .L..VL u.. r.,v . -

contains 0.475 ml of a 3% aqueous solution of glacial HAc.

The technique for Unopette is universal, no matter what laboratory procedure is to be performed. To ensure accurate results, follow manufacturer's instruction carefully .

.. Calculation ofWBC Count

= Total "ells counted X Sneci rYlpn dilution factor

.I_ L ~.J.. V . _ .J....l.L "'-f.. ~ '_'J::'vv . ..l..L.~'-".I....l. .I..I...UL..l.\.J..l. .J... . L ..l.

mnr' (area) counted x 0.1 mm (depth) = Total cells counted x 20

4 mnr' x (ll mm

= Total cells counted x SO/mm3

cells/L = Total cells counted x Specimen dilution factor x 106 area counted x depth

= Total cells counted x 50 x 106/L

Correction Factor

Area x Dilution x Depth

= 4 mnr' x 1/10 rnm x 1/20 = 4/200 or 1/50


Reciprocal of factor x no. of cells counted - no. WBC/cumm

* Quality Control

* Leukocyte Reference Range

Each laboratory should determine each own population reference range.

Leukocyte counts are age dependent. Based on automated cell counters:

Leukocyte values in man:

Age Total WBe x 109/L

12 hr 13.0-38.0

1 wk 5.0-12.0

12 rno 6.0-17.5

6yr 5.0-14.5

14yr 4.5-13.0

21 yr 4.5-11.0

Our Laboratory: '5, 000 - 10, OOO/cumru or 5.0-10 x 109/L


* Definitions

Leukocytosis ,;, an increase above normal in the number

of leukocytes in the peripheral blood .

• :. Physiologic Leukocytosis

caused by conditions that are physiologic such as exercise, time of day, stress

.:. Pathologic Leukocytosis

caused by a disease; always accompanied by absolute increase in one or another type of WBe

Leukopenia - a decrease below normal in the number of leukocytes in the peripheral blood

Leukemia ., a malignant increase in the number of leukocytes in the peripheral blood as well as in leukocyte producing organs.

• High WBC Count • Low WBC Count
appendicitis . measles sandfly fever
leukemia smallpox influenza psittacosis
tonsilitis ulcers brucellosis myxedema
diptheria . Typhoidfever paratyphoid
meningitis peritonitis agranulocytosis dengue
abscesses chickenpox Infectious hepatitis radiation
rheumatic fever lupus erythematosus
erythrblastosis fetalis cirrhosis of liver
newborn protein therapy
pregnancy rheumatoid arthritis
menstruation scrub typhus 5

2. Automated Method

2.1. Optical- Automated

Principle: Blood cells are counted as deflections of light beams as they pass through the dark field area of the machine.


Fisher Autocytometer

Frommer - Sanborn Autocytometer Technicon Autoanalyzer

2.2. Electrical Automated

Principle: Blood cells are counted as changes in voltage pulse.


Coulter Counter Celloscope




Principle: the same as WBC Count

Specimen: Peripheral blood.

Anticoagulated venous blood (with dry heparin or EDTA - must be used within 4 hours)

Rea.gent: Diluting Fluids

~:·Phloxine· Diluting Fluid 50 ml propylene glycol

IOml of t%water solution of phloxine

l m[ of lOG~ water solution of sodium carbonate 4ttO) mJ1 of distilled water


[p>IhllQ)ooinei's p Ius 100 unirs of heparin

.:. iOltlblter· Diluting Fluid

'falllUIDlleltil's Manner's

~alm)j(lltO[ph"s Dunger's

DO b'-

tscomo s


.:. Thoma WBC Pipeae or UilIDopette . ·:·lHiemocytometer

• Improve Neulbal!-ulJer Ruling

. area: 9 mnr' dlepth: 0.1 mm

section: 2




• Fuchs ~ Rosenthal

area: 16 mnr' depth: 0.2 ntnlsection: 2

I • Spiers ... Levy area: 10 mm~

depth: 0.2 mm section: 4


Procedure: $l,IlI111Dlea:s ~C IO(mmt Note: itllrue ~lJl'8n.IDaln ;diIilllllllti~Q)J]l

[ : [jQlWlilOm WlmlC 1I1lru(l)ma pipette [ : J2 'Vvimln. Ullli<DI:P>;erune

- :eedfhs atve ·atI~hQ)\"\Wiedl 1U0 Qimlkie up the stain for 15 - 20 min. , ,eiltJln!er rum.ltlIruc' IP'.Cluue or in the, counting chamber

- ,oeUsClJl1e·Q@1ffilIDtiedl liIDlmJl11alll!eas of the counting chamber

Calculation: use rttlThe~e1llieratll m0IDlIDJJula

Eosinophils/mm" = i1l0IDatll mo. ofeosinophils x dilution area x depth


The Absolute Eosinophil Count & Thorn Test (to check adrenal cortex function)

blood is drawn for eosinophil count

. . ... . d1. h d .'. h .

. patient IS mjecte WIt a renocorticotropic ormone



after four (4) hours another blood sample is drawn for eosinophil count


ilRl(i)it1I.1lDJal[tl, second count is 51Q)1% fue'$\s1tlhlaD. initial

11ypok3l!drenalism . ieO'sliIDllolPlhil count stays the .. _ ....

Si(lUJilllte (adrenal cortex unable tUo respond to ACTH)



= occasionally requested for diagnostic purposes as the indirect absolute count is not very reliable

, 1 d t f b' ik.;r •

" = may oe iUlrSle! I \ 0 con lrm,aSOip.!l--uua

B!all$\0@lhliHa is associated W[WN chronic myelogenous ~eil.lllklemiapolycythemfua Viera, myelofibrosis, mYXiedlema, colitis antGisometimes chronic hemolytic

= MleltllruQ)c[ fUlsedt:

ffi'.A!ieiftmID.dl ;Q)[ Cooper '

UsriJlll!g cooper aad (Cruruclksllruaumlk SlTIalQ\ll


= ,cOtiltll,ltJedli@ltil ,at Fil\l!CIlil!S - lIitol$\eiIDJ11hllatll ,(~if S!P1ie[tfS - Levy hemocytetaeter

== Reference Range

O 0", "0' 2' x l'09/L (0' 2'00'./ ,3)

...', - , , • "- , !.y illl1lID.lJl .

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