Tri Ratnaningsih
Bagian Patologi Klinik Fak. Kedokteran UGM
Inst. Laboratorium Klinik RSUP Dr Sardjito
CELL COUNTING
Learning objectives
After following this lecture, the student will be able to:
1.
State the dimension of the counting area of a Neubauer ruled hemacytometer
2.
Described the parts and basic principle of the Neubauer chamber
3.
Perform a cell count step by step, in order to achieve reliable and reproducible
results
4.
Describes best practices and recommendations when performing a cell count
5.
Describe the performance of manual cell counts for leukocytes, erythrocytes,
and platelets, including types of diluting fluids, typical dilutions, and typical
areas counted in the hemacytometer
6.
Calculate dilutions for cell counts when given appropriate data
7.
Calculate hemacytometer cell counts when given numbers of cells, area
counted, and dilution.
8.
Describe the principle and procedure for performing a manual reticulocyte
count
9.
Calculate the relative, absolute and corrected reticulocyte values and
reticulocyte index
10. Correct WBC counts for the presence of nucleated RBCs
Materials
1. Cellular dilution to measure
2. Hemocytometer, or Neubauer chamber
3. Optical microscope
4. Cover glass
5. Pippette/micropippete with disposable tips
6. Dilution buffer
 The Neubabuer Chamber or hemocytometer
thick crystal slide, size (30 x 70 mm and 4 mm
thickness)
 Glass cover: squared glass of width 22 mm The
glass cover leaves room for the cell
concentration between the bottom of the
chamber and the cover itself.
 The pipette allows for the introduction of a
precise amount of liquid into the Neubauer
chamber. Take 10 μl of dilution ) In case of the
appearance of bubbles, or that the glass cover
has moved, repeat the operation.
Calculations
Total count= cells counted x dilution factor
or
Area (mm2) x depth (0,1
mm)
Total count= cells counted x dilution factor x 10
Area (mm2)
White Blood Cell count
 Number of WBC in 1 L of whole blood
 Sample: whole blood from capillary or +
EDTA
 Diluting fluid: weak acid (acetic or
hydrochloric) or a buffer amonium oxalate 
to lyse nonucleated RBC
 Dilution 1:20
Procedure
 Bersihkan dgn alkohol chamber dan cover




glass, dikeringkan dg tissue khusus
Dibuat pengenceran 1:20 (dalam tabung
kecil; 25 uL darah + 475 uL pengencer)
Tutup dan campur dg baik
Biarkan selama 10 menit (RBC lisis sempurna)
Penghitungan WBC dilakukan dalam 3 jam
setelah dilusi
 Isilah kedua sisi bilik
menggunakan pipet dgn
kemiringan 450
 Tempatkan bilik pada
piring petri utk
melembabkan selama 10
menit (hati2 posisi
coverslip)
 Bilik hitung harus selalu
dlm keadaan horizontal,
tempatkan pada
mikroskop
 Gunakan perbesaran 10x
untuk melihat distribusi sel
 Utk dilusi 1:20, hitung
sel pada seluruh 4
kotak besar di sudut,
 Ulangi penghitungan
pada sisi satunya
(perbedaan <10%)
 Rerata hasil kedua sisi
 masukkan dalam
rumus
First Chamber
Cells counted in each
square
Second Chamber
Cells counted in each
square
35
45
40
37
44
36
39
44
158 WBCs counted
162 WBCs counted
Sumber kesalahan
 Hindari: Debu, sidik
jari, kontaminasi diluen
 Bila hasil terlalu
rendah, hitung pada
area yg lbh luas (mis: 9
mm2)
 Pengisian yg
berlebih/kurang
 Inkubasi selama 10
menit supaya sel
“settle”
 Perlu koreksi hitung
WBC bila ada ≥5 NRBC
/100 WBC)
Rumus WBC terkoreksi=
WBC x100
NRBC(per 100 RBC)+100
 Akurasi WBC manual
bisa dinilai dg melakukan
estimasi WBC pada
apusan
sample
WBC count
(automated)
Number
WBC in 10 oil
field
Average
number
WBC/oil field
Auto WBC
count/average
number wbc/OF
1
30
∑ 1-30/30
Faktor estimaasi
sample
Number WBC in 10 Average number
area 40 or 50x
WBC/area
Average number
WBC/area x3000
1
Compare with WBC
count
Platelet Count
 Diluent: asam lemah atau larutan yg
mengandung BCB
 Blood diluted with solution containing
Brilliant Cresyl Blue so that the platelet seem
bright blue. The platelets then are counted
using hemocytometer. Results are doubled
checked by examination of the platelets on a
Wright/Giemsa stained smear
Prosedur
 Dibuat pengenceran 1:100 (dalam tabung kecil;
20 uL darah + 1980 uL pengencer)
 Tutup dan campur dg baik
 Biarkan selama 15 menit dalam petri disk yg
lembab
 Penghitungan WBC dilakukan dalam 3 jam
setelah dilusi
 Penghitungan dengan lensa objective 40x, pada
25 kotak kecil di bagian tengah
 Dihitung kedua sisi, hasil harus ≤10%
 Hitung jumlah trombosit sesuai rumus
Estimasi hitung trombosit
 Jumlah trombosit (dlm 10 lp 100x obyektif ) x
2000
 taksiran kasar: bila ditemukan 7 sd 25
trombosit/lp  jumlah trombosit adekuat
(utk jumlah eritrosit lk 200 per lp  Hb
normal)
 Utk pasien anemic/polisitemia:
Rerata jumlah trombosit/lpx total RBC count
200
Sumber kesalahan:
 Platelet clump  inadekuat mixing/sampling




yg tdk tepat  di redilusi masih menetap,
ulang sampling (jangan dari ujung jari)
Kotoran/kontaminasi pipet, bilik, dan diluent
Bila hasil hitung tiap sisi <50  ulang dg
pengenceran 1:20
Bila hasil hitung tiap sisi >50  ulang dg
pengenceran 1:200
Fenomena “platelet satellitosis” ulang
sampling dg sodium sitrat  hasil x 1,1
Erythrocyte Count
 Jarang dilakukan
karena hasil kurang
akurat dibanding Hb
atau Hmt
 Prinsip: diencerkan
dengan pelarut
isotonik
Hemocytometer (Levy chamber with improved Neubauer ruling)
Area= 0,04 mm2
21
White Blood Cell Differential
Counting Area
(ideal area)
Tail
Counting area
Sumber: Miwa (1998), Atlas of Blood Cells, Bunkodo Co.,Ltd, Japan
Head
 Untuk WBC> 40 ribu 
menghitung 200 sel
 Untuk lekopenia:
 Menghitung 50 selx2 (25 sel x4)
 Buffy coat
 Selalu menghitung daerah pinggir 
monosit, limfosit reaktif, blast
 Dilaporkan dalam persentase (%)
Reticulocyte count
Principle:
 After the orthochromic normoblast looses its
nucleus, a small amount of RNA remains in the red
blood cell, and the cell is known as a reticulocyte.
 The definition of a reticulocyte is any non-nucleated
erythroid cell containing two or more bluish–
stained material corresponding to ribosomal RNA.
 To detect the presence of RNA, the red blood cells
must be stained while they are still living. This
process is called supravital staining
 Reticulum is present in newly released blood
cells for 1-2 days before the cell reach its full
mature state.
RETICULOCYTE COUNT
Place 3 drops
Into tube
Filtered
reticulocyte
stain
+
Whole
blood
Allow to stand at RT
or
incubate at 37C (15 min)
At the end of 15 min.
mix the content
of the tube well
Make several
smears &
allow to dry
Mix well
 Untuk meningkatkan akurasi:
 count 500 cells on two slides each. They should
agree within ± 15% of each other. If they do not,
repeat the reticulocyte count on the third smear.
 Petugas lain utk menghitung slide yg lain, hasilnya
harus tidak lebih dari 20%
Miller disc
 Disc td 2 square: area yg kecil mengukur
1/9 dari area besar
 Disc ini diletakkan pada lensa okuler
 RBC dihitung pd area kecil, retik dihitung
pd area besar
 Minimal 112 sel dihitung pd area kecil 
ekuivalen dg 1008 RBC pd area besar
Sumber kesalahan
 perbandingan antara pewarna dan darah untuk
pasien yang sangat anemik dan polisitemik
 BJ retik<RBC matur, saat inkubasi berada di atas
 mix dgn baik saat akan membuat smear
 Kelembaban udara dan pengeringan yg tdk
sempurna  area refraktil  membingungkan
dlm identifikasi retik
 Inklusi lain (dg pengecatan suravital) (heinz,
howel jolly, pappenheimer)
Howell-Jolly bodies (small,
single, purple cytoplasmic
inclusions): These
represent nuclear remnant
DNA and are found after
splenectomy or with
functional asplenism.
Heinz bodies: (inclusions seen only
on staining with violet crystal):
Heinz bodies represent denatured
Hgb and are found in glucose-6phosphate dehydrogenase after
oxidative stress
On Wright stained smears, Pappenheimer bodies appear
as violet staining granules usually found along the
periphery of the red cells, often in clusters. They must be
confirmed with an iron stain. These iron-staining granules
are found in sideroblastic and megaloblastic anemias,
alcoholism, following splenectomy, and in some
hemoglobinopathies.
(A) basophilic stippling, (B)
Howell-Jolly bodies, (C) Cabot's
ring bodies and (D) Heinz's
bodies.
Reporting Results
Absolute Reticulocyte Count (ARC):
• Retic(%) x RBC count (1012 /L)/100
Corrected reticulocyte count
• Retic(%) x Hct pasien/45
Reticulocyte production index
• Corrected reticulocyte count (%)/maturation
time # Days
Maturation Hematocrit
Time
%
1 day
45
1.5 day
35
2 day
24
3 day
15
Alhamdulillaah
Referensi:
Rodak, et.al., 2012, edisi 4, hematology clinical principles and
applications, Elsevier Saunders
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