31

LABORATORY MANUAL

CLINICAL PATHOLOGY



BLOCK 2.1





Hemoglobin (Hb) Measurement
Erythrocytes Count
Hematocrit Measurement
Erythrocytes Indices
Reticulocyte Count
























FACULTY OF MEDICINE
UNIVERSITAS GADJAH MADA
2012

32

Learning Objectives:
At the end of the lab work, students are expected to be able to:
1. understand principle and procedure of hemoglobin test, Erythrocytes Count, Hematocrit
Measurement, Erythrocytes Indices, and Reticulocyte Count
2. perform hemoglobin examination, Erythrocytes Count, Hematocrit Measurement, Erythrocytes
Indices, and Reticulocyte Count
3. Give the normal values of Hb in different age and sex groups, Erythrocytes Count, Hematocrit
Measurement, Erythrocytes Indices, and Reticulocyte Count
4. interpret hemoglobin test, Erythrocytes Count, Hematocrit Measurement, Erythrocytes Indices,
and Reticulocyte Count results

1. HEMOGLOBIN (CYANMETHEMOGLOBIN METHOD)

Method
Cyanmethemoglobin method

Principle
Whole blood is added to cyanmethemoglobin (HiCN) reagent. The potassium ferricyanide in the
reagent converts the hemoglobin iron from the ferrous state (Fe
++
) to the ferric state (Fe
+++
) to form
methemoglobin (Hi) which then combines with potassium cyanide to form the stable pigment,
cyanmethemoglobin (HiCN). (Hi = hemoglobin = hemoglobin in which the iron has been oxidized to
the ferric state.HiCN = hemoglobin cyanide = hi which has been banded to the cyanide ions). The
nonionic detergent present in the reagent improves the lysis of the red blood cells and decreases
the amount of turbidity resulting from abnormal proteins, such as lipoprotein. The color intensity of
this mixture is measured in a spectrophotometer at a wavelength of 540 nm. The optical density of
the solution is proportional to the concentration of hemoglobin. All forms of hemoglobin are
measured with this method except sulfhemoglobin.

Specimen
Whole blood, using EDTA as the anticoagulant. Capillary blood may also be used.

Reagents
1. Cyanmethemoglobin (hemiglobincyanide) (HiCN) reagent contains potassium cyanide (50 mg),
potassium ferricyanide (200 mg), dihydrogen potassium phosphate (KH
2
PO
4
) (140 mg), and
nonionic detergent (1 mL) in 1 L of distilled water.

Equiment
1. Test tubes, 10 x 75 mm
2. Pipets, 0.02 mL.
3. Spectrophotometer (540 nm)

Procedure
1. Place exactly 5.0 mL of HiCN reagent into an appropriately labeled test tube. Place 5.0 mL of
the reagent into a test tube to be used as the blank.
2. Add 0.02 mL of well-mixed whole blood or control blood to the appropriately labeled tube. Rinse
the pipet 3 to 5 times with the HiCN reagent until all blood is removed from the pipet
3. Mix the preceding solutions well and allow to stand at room temperature for at last 3 minutes to
allow adequate time for the formation of HiCN
33

4. Transfer the mixture to a cuvette and read in a spectrophotometer at a wavelength of 540 nm
using the HiCN reagent in the blank tube to set the optical density (O.D.) at 0.0. Record the
readings for the patient and control samples from the O.D. scale and refer to the prepared chart
for the actual value of the hemoglobin in g/dL.

Clinical correlation
Estimation of Hb is usually done to detect anemia because it is convenient and less consuming
than the total RBC count. Anemia is said to be present when the Hb level in the blood is below the
lower limit of thye normal range for the age and sex of the individual.

2. HEMATOCRIT MEASUREMENT (MICROHEMATOCRIT METHOD)

Method
Microhematocrit

Principle
Whole blood is centrifuged for maximum red blood cell packing. The space occupied by the red
blood cells is measured and expressed as a percentage of the whole blood volume.

Specimen
Whole blood using dipotassium ethylene-diaminetetraacetic acid (EDTA) as the anticoagulant.
(The liquid EDTA is thought to cause a 2 to 3% decrease in the hematocrit due to slight shrinkage
of the red bllod cells)

Reagents and Equipment
1. Microhematocrit tube, approximately 75 mm long with an inner bore of approximately 1.2 mm.
Two types of microhematocrit tubes may be purchased: (1) those which contain heparin (color
coded with a red band) as the anticoagulant, for use with non-anticoagulated whole blood, and
(2) plain tubes (color coded with a blue band) for use with anticoagulated whole blood. The
microhematocrit tubes hold approximately 0.05 mL of whole blood.
2. Clay-like sealing blood.
3. Microhematocrit centrifuge capable of producing an RCF of 10000 to 15,000 g. The centrifuge
should be able to reach maximum speed within 30 seconds.
4. Microhematocrit tube reader.

Procedure
1. Allow the capillary or well-mixed anticoagulated whole blood to enter two microhematocrit tubes
until they are approximately two-thirds filled with blood. (Air bubbles denote poor technique but
do not affect the results of the test)
2. Seal one end of the microhematocrit tube with the clay material by placing the dry end of the
tube into the clay in a vertical position (the microhematocrit tube forms a 90
0
angle with the tray
of clay). The plug should be 4 to 6 mm long. Make certain blood is not forced out the top of the
microhematocrit tube during this process
3. Place the two microhematocrit tubes in the radial grooves of the centrifuge head exactly
opposite each other, with the sealed end away from the center of the centrifuge.
4. Centrifuge for 5 minutes
5. Remove the hematocrit tubes as soon as the centrifuge has stopped spinning. Determine the
results for both microhematocrits , using the microhematocrit tube reading device. Duplicate
34

results should agree within 1 unit (%). If they do not, repeat the procedure. The hematocrit may
be expressed in either of two ways: (1) as a percentage, e.g., 42%, or, (2) as a decimal fraction,
e.g., 0.42.

3. RED BLOOD CELL COUNT

Method
Haemocytometer

Principle
To facilitate counting and prevent lysis of the red blood cells, whole blood is diluted with an
isotonic diluting fluid.

Reagent and Equipment
1. Thoma red count pipet
2. Hayem Solution (Red count diluting fluid)
- Sodium sulphate 2,5g
- Sodium chloride 0,5g
- Mercury chloride 0,25g
- Aquades 100 ml
3. Microscope
4. Clean gauze or kimwipes
5. Hemocytometer and cover glass. The large middle square containing 25 smaller squares of
equal size is used for the RBC.
a. The five small squares labeled R are the areas to be counted the RBC (Fig. 1)
b. The large center square has a volume of 0.1 L. Therefore, the volume of each of the 25
smaller squares is 0.004 L for five small squares.



Figure 1.

Specimen
Whole blood, using EDTA or heparin as the anticoagulant. Capillary blood may also be used.




35

Procedure
1. Draw the blood up to exactly the 0.5 mark in the red count pipet and dilute to the 101 mark with
red count diluting fluids, thus making 1:200 dilution of blood.
2. Mix the diluted red blood cell counts for 3 minutes
3. Clean the counting chamber
4. Fill the counting chamber (one red count dilution filling each side of the hemocytometer). Once
the counting chamber is filled, allow approximately 3 minutes for the red blood cells to settle
prior to counting.
5. Count the red blood cells.
a. Carefully place the filled counting chamber on the microscope stage.
b. Using low power (10 x objective). place the large center square in then middle of the field of
vision. Examine the entire large square for even distribution of red blood cells
c. Carefully change to the high dry objective (40x)
d. Move the counting chamber so that the small upper left corner square is completely in then
field of vision. The square is further subdivided into 16 even smaller squares for ease of
counting.
e. Count all red cells in this square, remembering to count the cells on two of the outer margins
but excluding those lying on the other two outside edges.
f. Some of the red blood cells may be lying on their sides and, therefore, do not appear as
round. These cells are to be included in the count.
g. If there are any white blood cells in the area being counted, do not include these cells in your
count. (The white blood cell is usually much larger than the red blood cell and does not have
as smooth an appearance)
h. Count the red cells is each of the five small squares
i. Count the red blood cells on the opposite side of the counting chamber in the corresponding
five small squares.
6. Calculate the red blood cell count for each of the red counts performed and average the two
results for the final report

RBC/L = # Cells in five squares x Correction for Dilution x 10
6

Correction for Volume

RBC/L = # Cells in five squares x 200 x 10
6
5 x 0,2 x 0,2 x 0,1

For example :
# Cells in five small squares = 400
Dilution = 1: 200
Volume counted = five small squares
Conversion to liter = x 10
6

RBC/L = 400 x 1.0 x 200 x 10
6
02
= 4.0 x 10
12
The red blood cell count (RBC) is expressed as the number of red blood cells/liter (L) of whole
blood. The normal RBC is 3.6 to 5.6 x 10
12
/L for females and 4.2 to 6.0 x 10
12
/L for males. The
newborn shows an RBC of 5.0 to 6.5 x 10
12
/L at birth, which gradually decreases to 3.5 to 5.1 x 10
12
/L at 1 year of age.

36

4. RETICULOCYTE COUNT

Principle of Method
After the orthochromic normoblast loses its nucleus, a small amount of RNA remains in the red
blood cell, and the cell is known as a retticulocyte. To detect the presence of RNA, the red blood
cells must be stained while they are still living. This process is called supravital staining.

Reagents and Equipment
1. The reticulocyte stain can choosen one of the following:
a. New methylene blue stain solution :
New methylene blue (CI 52030) 1,0 g
Sodium chloride 0,89 g
Distilled water 100 mL
b. Brilliant Cresyl Blue:
Brilliant Cresyl Blue 1.0 g
Sodium chloride 0.85% 99 ml
Mix for at least 15 minutes, filter, and store at room temperature. Filter again on the day of
use.
2. Glass slide
3. Microhematocrit tubes
4. Small test tube
5. Microscope

Specimen
Whole blood (1 mL), using EDTA as the anticoagulant. Capillary blood may also be used.

Procedure
1. Place three drops of filtered reticulocyte stain in a small test tube
2. Add three drops of well-mixed whole blood to the tube containing the stain
3. Mix the tube and allow to stand at room temperature, or incubate at 37
0
C, for 15 minutes. This
allows the reticulocytes adequate time to take up the stain
4. At the end of 15 minutes, mix the contents of the tube well
5. Prepare several wedge or spun smears and allow to air dry.
6. Place the first slide on the microscope stage and, using the low power objective (10x), find an
area in the thin portion of the smear in which the red blood cells are evenly distributed and are
not touching each other. Carefully change to the oil immersion objective (100x) and further
locate an area in which there are approximately 100 to 200 red blood cells per oil immersion
field.
7. As soon as the proper area is selected, the reticulocytes may be counted. The red blood cells
will be a light to medium green in color. The RNA present in the reticulocytes stains a deep
blue. The reticulum may be abundant or sparse, depending on the cells stage of development,
The youngest reticulocyte shows a large amount of RNA whereas the more mature reticulocyte
shows only a small amount of RNA. Count all of the red blood cells in then first field on one cell
counter. At the same time, enumerate the reticulocytes in the same field with a second cell
counter. To be considered a reticulocyte the red cell must contain two or more blue-staining
particles. Move then slide as described in the differential Cell Count procedure, using the cross-
sectional method, until all reticulocytes in 1000 red blood cells have been counted

37

8. Average the two results and calculate the reticulocyte count as shown below.




Clinical correlation
The red blood cell goes through six stage of development: pronormoblast, basophilic normoblast,
orthochromic normoblast, reticulocyte, and mature red blood cell. The first four stages are normally
confined to the bone marrow. The reticulocyte, however, is found in both the bone marrow and
peripheral blood. The reticulocyte count is an important diagnostic tool. It is a reflection of the
amount of effective red blood cell production taking place in the bone marrow.

5. Erythrocytes Indices

Mean Corpuscular Volume (MCV)
The MCV is calculated from the RBC count and the hematocrit and indicates the average volume
of the RBC in femtoliters (fL).

The formula:
MCV = Hct x 10
3
fL
RBC/L

Normal range for the MCV: 80 to100 fL


Mean corpuscular haemoglobin (MCH)
The MCH is calculated from the HB and RBC count, indicates the average weight of Hb in the
RBC, and should always correlate with the MCV and MCHC.

The formula:
MCH = Hb (g/L)
--------------- pg
RBC/L
Normal range for the MCH: 27 to 31 pg


Mean corpuscular haemoglobin concentration (MCHC)
The MCH is calculated from the Hb and Hct and is an expression of the average concentration of
Hb in the RBC.

The formula:
MCHC = Hb (g/dL) x 100%
Hct
Or,

MCHC = Hb g/dL
Hct (expressed as a fraction)

Number of reticulocytes
% Reticulocytes = in 1000 red blood cells
10



38

Normal range for the MCHC: 31 to 36%, or 31 to 36 g/dL

Clinical correlation
The red blood cell (RBC) indices are used to define the size and hemoglobin content of RBC.
They concist of the mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), and
Mean corpuscular haemoglobin concentration (MCHC). With the widespread use of automated cell
counters trhat routinely determine the RBC indices on each blood sample tested, the indices are
commonly use as an aid in diagnosing and differentiating anemias based on themorphology.

References
1. Brown, B.A., 1993. Hematology: Principles and Procedures. 6
th
ed. Philadelphia: Lea & Febiger.
2. Stiene-Martin, E.A., Lotspeich-Steineger, C.A., and Koepke, J.A., 1998. Clinical Hematologi:
Principles, Procedures, Correlation. 2
nd
ed. Lippincott-Raven Publisher.


































39

SPERM ANALYSIS

Standard procedure for sample collection and delivery
1. Patient should do abstinence for 2-7 days
2. Semen collection is done by masturbation and ejaculated into a clean, wide-mouthed container
made of glass or plastic, from a batch that has been confirmed to be non-toxic for spermatozoa.
Dont spill out.
3. The semen must be kept at 20-37C and delivered to the laboratory as soon as possible after
its collection. The time must not exceed 1 hour after emission.
4. Container must be labeled with the patient identity, the date and time of collection
5. Patient should sign inform consent and report any spill out of sperm during collection

Semen analysis involves the following steps:
In the first 5 minutes:
1. Place the specimen container on the bench or in an incubator (37C) for liquefaction.
2. Between 30 and 60 minutes, the procedure below should be performed:
1. Asses macroscopic examination (liquefaction ,appearance of semen, semen volume, PH)
2. Preparing wet preparation and the dilution for sperm appearance, motility and number.
3. Asses sperm vitality, sperm concentration and number, round cell (leucocyte and immature
germ cell)
4. Preparing semen smear for sperm morphology
5. Preparing for biochemistry test by centrifugating semen.
Within 3 hours
Performing requested biochemistry test (fructose)
After 4 hours:
Fixing, staining and assesing smear for sperm morphology

MACROSCOPIC TEST
1. Likuefaksi
LO:
a. Mahasiswa mengetahui cara menghitung waktu likuikaksi
b. Mahasiswa dapat menghitung waktu terjadinya likuefaksi lengkap
c. Mahasiswa dapat menginterpretasi hasil likuifaksi semen

Prinsip:
Semen akan mengalami likuefaksi lengkap dalam waktu tertentu

Bahan:
Semen

Alat :
jam

Cara Kerja:
a. catat jam pengeluaransemen (sesuai keterangan pasien)
b. amati waktu semen mengalami likuefaksi lengkap catat waktunya

40

Perhitungan:
Saat likuefaksi lengkapsaat pengeluaran semen

Pelaporan:
Likuefaksi lengkap setelah . menit

Nilai normal:
15 menit - 60 menit

Kesalahan yang dapat terjadi :
a. Pasien tidak mencatat waktu/jam saat pengeluaran mani
b. Terlambat mencatat waktu terjadinya likuefaksi lengkap

2. Viskositas
LO:
a. Mahasiswa mengetahui cara menentukan viskositas semen
b. Mahasiswa dapat memeriksa viskositas semen
c. Mahasiswa dapat menginterpretasi hasil viskositas semen

Prinsip:
Semen mempunyaiviskositastertentu. Pada saat semen diaspirasi kemudian didorong keluar
karena adanya gaya gravitasi akan jatuh ke bawah membentuk benang.

Alat:
Disposable pipette with diameter 1,5 mm (pipet Pasteur)
Glass rod

Cara Kerja:
Pipet Pasteur
1. Semen diisap kedalam pipet Pasteur.
2. Teteskan mani, amati panjang benang yang terbentuk
Perhitungan : Catat panjang benang yang terbentuk pada waktu menetesnya mani
Pelaporan : . cm
Nilai normal : 2 cm

Glass rod
1. Masukkan batang pengaduk kaca ke dalam semen
2. Angkat batang pengaduk, amati panjang benang yang terbentuk saat penarikan batang
pengaduk.
Perhitungan : Catat panjang benang yang terbentuk pada saat penarikan batang kaca
pengaduk
Pelaporan : ....... cm
Nilai normal : 2 cm
41


Disposable pipette with diameter 1,5 mm Glass rod

Warna
Tujuan : Mengetahui warna semen
Prinsip : Semen mempunyai warna tertentu
Bahan : Semen
Alat : Indera penglihatan
Cara Kerja : Melihat semen secara langsung dengan latar belakang putih di tempat terang
Pelaporan : Warna putih seperti kanji, putih kekuningan, putih abu-abu, kemerahan,
kecoklatan
Nilai normal : putih seperti kanji

Bau
Tujuan : Mengetahui bau semen
Prinsip : Semen mempunyai bau tertentu
Bahan : Semen
Alat : indra penciuman
Cara Kerja : Mencium bau semen
Pelaporan : Bau khas (bunga akasia), busuk, amis
Nilai normal : Khas

Volume
LO : Mengetahui prinsip pengukuran volume semen
Mampu melakukan pemeriksaan volume semen
Mampu menginterpretasikan hasil laboratorium untuk tata laksana pasien

Prinsip : Semen dikeluarkan dalam volume tertentu
Alat : Modifed graduated glass measuring cylinder



Bahan : Semen
Cara kerja : - Tuangkan seluruh semen dari wadah kedalam gelas ukur
- Catat volumenya
Perhitungan : Volume semen dengan ketepatan 0,1 ml
Nilai normal : 1,5 ml
42

Kesalahan yang dapat terjadi :
- Salah membaca skala pada gelas ukur
- Mengambil semen/mani untuk pemeriksaan lain sebelum mengukur volumenya
Catatan : Volume semen diukur setelah 20 menit dari saat pengeluaran

pH
Tujuan : Mengetahui pH semen
Prinsip : Semen mempunyai pH tertentu
Bahan : Semen
Alat : Pipet Pasteur
Reagen : Kertaslakmus / indikator pH
Cara Kerja :
- Dengan pipet Pasteur teteskan 1 tetes semen pada kertas lakmus/indikator pH
- Perhatikan perubahan warna kertas lakmus/indikator pH
- Cocokkandenganstandar pH
- Mix the semen sample well
- Spread a drop of semen evenly onto the pH paper.
- Wait for the colour of the impregnated zone to become uniform (<30 seconds)
- Compare the colour with the calibration strip to read the pH.
Pelaporan : pH = .
Nilai normal : batas terendah 7,2
Jika pH < 7 pada zoospermia : disgenesis vas deferens, vesikula seminalis
atau epididymis


PEMERIKSAAN MIKROSKOPIK
Motilitas Sperma
Tujuan:
Mengetahui aktivitas gerak spermatozoa pada waktu tertentu

Prinsip:
Spermatozoa mempunyai gerak tertentu pada waktu tertentu

Alat:
- Kacaobyek
- Mikroskop
- Kaca penutup
- Pipet Pasteur
- Counter Tally

Bahan:
Semen

Procedure for sperm motility examination by wet preparation:
1. Mix the sample until homogenous.
2. Place 10 L of homogenous sample on an object glass.
3. Cover the sample by cover-glass 22 X 22 mm.
4. Incubate for one minute.
43

5. After one minute, observe the sperm under a microscope in a high power magnification.
6. First evaluation: observe the sperm motility. Count minimum 200 sperms on 8 different
observation fields as shown in the below figure. Asses sperm motility (while counting) as
progressive motility, non-progressive motility and immotile (based on WHO 2010 new category
for sperm motility). Calculate the percentage of sperm motility and also record any observed-
agglutination in the sample.
7. Below is the figure to aid assessing sperm motility:


Figure1. Aid to assessing sperm motility.

8. Second evaluation: repeat the sperm motility examination once again to asses the sample
homogeneity. Sample is considered as homogenous if the difference of sperms motility
between duplicate is less than 5%.

Category for sperm movement:
1. Progressive motility (PR): spermatozoa moving actively, either linearly or in a large circle,
regardless of speed
2. Non-progressive motility (NP): all other patterns of motility with an absence of progression, e.g.
swimming in small circles, the flagellar force hardly displacing the head, or when only a flagellar
beat can be observed.
3. Immotility (IM): no movement

Acceptable differences between two percentages for a given average, determined from replicate
counts of 200 spermatozoa (total 400 counted)
44


Nilai normal : The lower reference limit(PR + NP) is 40% (5th centile, 95% CI 3842).
The lower reference limit (PR) is 32% (5th centile, 95% CI 3134).

Viabilitas/Vitalitas
Tujuan:
Untuk melihat spermatozoa yang hidup dan mati

Alat:
1. Kacaobyek
2. Kacapenutup
3. Pipet Pasteur
4. Mikroskop

Reagen:
Eosin Y larutan eosin 0,5% (b/v) dalam garam faal

Procedure:
1. Wet preparation with Eosin dye
- Mix well the semen sample.
- Drop 5l of semen and 5l of eosin 0,5% onto an object glass.
- Mix with pippette tip, swirling the sample on the slide, cover with deck glass, leave for 30
seconds.
- Make the second replicate with same procedure as previous slide
- Examine under microscope at high power magnification (objective 100x) on 200
spermatozoa.
- Count the percentage of the live spermatozoa.
2. Smear preparation with Eosin dye
- Prepare the mixture of sample-dye as above. Make smear, dry it in room temperature
- Examine the smear under microscope in a high power magnification Assess the dead and live
sperm in 200 spermatozoa.
- Count the percentage of live spermatozoa.
45

Scoring to determine live or dead spermatozoa:
Live spermatozoa are characterized by:
- Spermatozoa with a white/colorless head or light pink color head
- Spermatozoa with limited staining on the neck region and white head that called as leaky-neck
membranes spermatozoa.

Pelaporan:
Hidup = %

Nilai normal:
The lower reference limit for vitality (membrane-intact spermatozoa) is 58% (5th centile, 95% CI
5563).

Jumlah Spermatozoa
LO:
1. Mahasiswa dapat mengetahui cara menghitung jumlah spermatozoa
2. Mahasiswa dapat menghitung jumlah spermatozoa
3. Mahasiswa dapat menginterpretasi hasil pemeriksaan jumlah spermatozoa

Prinsip:
Semen mengandung spermatozoa dalam jumlah tertentu

Alat:
1. Pipet mikro 50 ul dan 1000 ul
2. Pipet Pasteur
3. Mikroskop
4. KH Improved Neubauer

Larutan pengencer:
NaHCO3 : 50 g
Formalin : 10 ml
Gentian Violet Jenuh : 5 ml
Akuades : 1000 ml

Bahan:
Semen

Procedure for sperm count using wet preparation and haemocytometer chamber:
By wet preparation:
1. Examining a well-mixed, undiluted preparation of liquefied semen on a glass slide under a cover
slip, to determine the appropriate dilution and appropriate chambers to use. This is usually the
wet preparation used for evaluation of motility.
2. The dilution for counting by haemocytometer chamber depends on the average count of
spermatozoa in wet preparation. The dilution rules and ratio are as follows:




46

Table 1. Dilution Table of Sperms
/ HP DILUTION RATIO
CONVERSION FACTOR
ON SQUARE
25 10 5
15 1 : 5 ( 1 + 4 ) 20 8 4
> 15 40 1 : 10 ( 1 + 9 ) 10 4 2
> 40 200 1 : 20 (1 + 19 ) 5 2 1
> 200 1 : 50 (1 + 49 ) 2 0.8 0.4
Notes: / HP= average sum of spermatozoa per high power magnification

By haemocytometer chamber:
1. Mixing semen and preparing appropriate dilutions with fixative.
2. Loading the haemocytometer chamber and allowing spermatozoa to settle in a humid chamber.
3. Assessing the samples within 10-15 minutes (after which evaporation has noticeable effects on
sperm position within the chamber)
4. Counting at least 200 spermatozoa per replicate in middle square of erythrocyte chamber of
Improved Neubauerhaemocytometer
5. Comparing the replicate counts to see if they are acceptably close. If so, proceeding with
calculations; if not, preparing new dilutions. The difference of the counting must be <5% or
under the tolerance curve (Figure 2). If the difference > 5% and out of tolerance curve; repeat
the counting with homogenization sample.



Figure2. The Tolerance Curve.
6. Calculating the concentration of spermatozoa per ml.
Calculating formula:
The average of sperm x conversion factor (see dilution table)
7. Calculating the total number of spermatozoa per ejaculate.
Calculating formula:
The concentration of spermatozoa/ml x semen volume
Sum of the two counts
D
i
f
f
e
r
e
n
c
e

b
e
t
w
e
e
n

c
o
u
n
t
s

47


Figure3. Improved Neubauerhaemocytometer.


Figure4. Count Area in grid squares of Improved Neubaurer (in erythrocyte chamber).

Counting rules:
1. Counting only whole spermatozoa (with head and tails)
2. The counting of spermatozoa is determined by the location of spermatozoas head, not the tail.
The boundary of a square is indicated by the middle line of the three lines.
3. The examples of counted spermatozoa are shown in above figure (A, B, C): white circle =
counted spermatozoa, black circle= not counted spermatozoa.
It is recommended to calculate and report the total number of spermatozoa per ejaculate, as this
parameter provides a measure of the capability of the testes to produce spermatozoa and the
patency of the male tract. This is obtained by multiplying the sperm concentration by the volume of
the whole ejaculate

lower reference limit for sperm concentration is 15 106 spermatozoa per ml
(5th centile, 95% CI 1216 106).
The lower reference limit for total sperm number is 39 106 spermatozoa perejaculate
(5th centile, 95% CI 3346 106).

Morfologi Spermatozoa
Tujuan:
1. Mahasiswa dapat mengetahui pemeriksaan morfologi spermatozoa
48

2. Mahasiswa dapat melakukan pemeriksaan morfologi sperma
3. Mahasiswa dapat menginterpretasi hasil pemeriksaan morfologi sperma

Prinsip:
Spermatozoa mempunyai variasi bentuk dan ukuran dari bagianbagiannya (kepala, leher, ekor)

Alat:
1. Kacaobyek
2. Kaca pendorong (geser)
3. Pipet Pasteur
4. Mikroskop

Reagen:
Zat warna Giemsa

Bahan:
Semen

Procedure to check sperm morphology:
1. Put a drop of liquefied semen on an object glass. The drop volume is 5 l if the sperms number
by wet preparation is more than 20 million/ml, or 10 to 20 l sample if sperm number is less
than 20 million/ml.
2. Make two smear from the drops on object glass
3. Dry at room temperature
4. Fixate by methanol for 5 minutes
5. Stain with Giemsa for 30 minutes
6. Wash by phosphate buffer pH 6.9
7. Dry at room temperature
8. Examine by high power microscope 1000X on 200 or 100 of the sperms.
9. Count percentage of each type of the sperms.

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Figure5. Morphologically normal spermatozoa

Classification of abnormal sperm morphology
Head defects: large or small, tapered, pyriform, round, amorphous, vacuolated (more than
two vacuoles or >20% of the head area occupied by unstained vacuolar areas), vacuoles in the
post- acrosomal region, small or large acrosomal areas (<40% or >70% of the head area), double
heads, or any combination of these.
Neck and midpiece defects: asymmetrical insertion of the midpiece into the head, thick or
regular, sharply bent, abnormally thin, or any combination of these. Principal piece defects: short,
multiple, broken, smooth hairpin bends, sharply angulated bends, of irregular width, coiled, or any
combination of these.
Excess residual cytoplasm (ERC): this is associated with abnormal spermatozoa produced
from a defective spermatogenic process. Spermatozoa characterized by large amounts of irregular
stained cytoplasm, one third or more of the sperm head size, often associated with defective
midpieces (Mortimer & Menkveld, 2001) are abnormal. This abnormal excess cytoplasm should
not be called a cytoplasmic droplet (Cooper, 2005).
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Figure6. Schematic Drawings of Some Abnormal Form of Human Spermatozoa


Figure7. Sperm Morphology
The lower reference limit for normal forms is 4% (5th centile, 95% CI 3.04.0).

5. Round cell (leukocyte and immature germ cell) examination:
If during sperm morphology, the round cell are observed more than 10 cell/high power field, the
round cell examination should be performed.

Procedure to make smear for round cell examination by methylene blue staining:
1. Mix 2 drops of semen with 1 drop methylene blue 1%.
2. Mix on object glass, cover by deck glass, leave it for 30 minutes.
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3. Examine under microscope by high power magnification, find minimun 200 of sperms and count
the number of round cell among 200 of sperms.
4. Count percentage of leukocyte (blue) and germ cell (white)

Calculation:
C = NxS/200
Notes:
C = leukocyte or germ cell in million/ml
N = No. of leukocyte or germ cell in 200 sperms
S = sperms concentration in million/ml.
Normal value for round cell is less than 1x10
6
/ml.

Pemeriksaan Kimia
Pemeriksaan Fruktosa
Tujuan:
Menentukan kadar fruktosa dalam semen

Prinsip:
Fruktosa bereaksi dengan resorsinol membentuk senyawa berwarna merah dan resapannya
dibaca pada 490 nm

Pemeriksaan fruktosa kuantitatif
Alat:
1. Fotometer
2. Tabung reaksi
3. Pipetmikro 500 ul
4. Pipetukur 10 ml
5. Sentrifus
6. Tabung sentrifus
7. Pemanas air 90C

Reagen:
1. Lrtn Ba(OH)2 0,3 N
2. Lrtn ZnSO4 0,175 M
3. Lrtn Resorsinol 0,1 % dalam Alkohol 95%
4. HCl 10 N
5. Lrtn Standar fruktosa 200 mg/l

Cara Kerja :
1. Deproteinisasisemen
0,1 ml mani + 2,9 ml aq
+ 0,5 ml Ba(OH)2 homogenkan
0,5 ml Zn SO4 homogenkan
Sentrifus : 4000 rpm, 5 menit
2. Sediakan 3 tabung T (tes), S (standar), B (Blanko)
3. Isi dengan larutan yang tersebut dibawah ini
4. Homogenkan isi tabung masing-masingmasukkan kedalam penangas air 90C selama 10
menit
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5. Baca serapan T dan S terhadap Blanko pada 490 nm
Perhitungan:
Kadar fruktosa : Serapan T x 200 mg/dl
Serapan S

Pelaporan:
Kadar fruktosa = mg/dl

Nilai Normal:
120 450 mg/dl

Kesalahan yang dapat terjadi :
1. Reagen tidak baik
2. Pipet tidak akurat/tidak terkalibrasi
3. Fotometer tidak baik/tidak terkalibrasi

Catatan
Cara membuat larutan standar fruktosa 200 mg/dl
1. 50 mg fruktosa dilarutkan dalam 100 ml Asam Bensoat 0,2 %
2. 1 ml lrtn (a) diencerkan dengan akuades sampai 100ml

Pemeriksaan Imunologi
Aglutinasi:
Spermamotil saling melekat kepala dengan kepala,bagian tengah dengan bagian tengah, ekor
dengan ekor atau campuran seperti bagian tengah dengan ekor.
1. Spermamotil dengan tidak motil melekat pada benang mukus/selbukan sperma bukan
aglutinasi
2. Aglutinasi merupakan petunjuk (bukan bukti pasti) akan adanya factor imunologi sebagai
penyebab infertilitas

Lower reference limits (5th centiles and their 95% confidence intervals) for semen characteristics

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Distribution of values for semen parameters from men whose partners became pregnant within
12 months of discontinuing contraceptive use

Nomenclature related to semen quality
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