Transcript Evaluation of the one-step eosin-nigrosin staining technique for

Evaluation of the one-step eosin-nigrosin
staining technique for human sperm
vitality assessment
การประเมินวิธีการย้ อมสี eosin-nigrosin แบบขั้นตอนเดียว
สาหรับการประเมินค่ าการอยู่รอดของตัวอสุ จิมนุษย์
L.Björndahl, I.Söderlund and U.Kvist
Human Reproduction Vol.18, No.4 pp. 813-816, 2003
อาจารย์ที่ปรึ กษา : อ.ดร.ศุภชัย ศุภลักษณ์นารี
นาเสนอโดย นางสาวอมรวดี จุแดง รหัส 51312335
Sperm
www.rothamsted.bbsrc.ac.uk
Semen Stains
• Eosin-haematoxylin
• Christmas tree stain
• Trypan blue stain
• Nigrosin- Eosin stain
Introduction
• The assessment of sperm vitality is one of the
basic elements of semen analysis.
• Motile sperm and Immotile sperm
• Immotile - immotile dead sperm
- immotile live sperm
• Eosin - mark dead cells
• Nigrosin - blackground
• The simplified one step technique :
Add sperm into the mixture of eosin and
nigrosin.
• Used on boar, bull and ram sperm(Campbell et al., 1956).
• Used on rabbit sperm(Beatty,1957) and various
mammalian sperm (Dott and Foster, 1972).
• Used on washed human sperm by Mortimer
(Mortimer et al., 1990).
• The one - step technique is now widely in use
for basic semen analysis.
Materials and methods
• Studied 1235 consecutive semen samples.
• The mainly men in couples consulting the
clinic due to infertility problems.
• Sample collected by masturbation and
complete vitality and motility assessments
performed.
The one step eosin-nigrosin staining
technique
• Contained 0.67% eosin Y and 10% nigrosin
according to Mortimer (Mortimer, 1985;1994)
• But dissolved in 0.9% sodium chloride in
distilled water.
0.67 g of eosin Y yellow
(CI 45380, VWR No 115935)
and
0.9 g sodium chloride
Dissolved in 100 ml distilled water under gentle heating
Add 10 g of nigrosin
(CI 50420, VWR No 115924; VWR™International)
Boil and allowed to cool to room temperature (20°C)
Filtered through quality filter paper
(Munktell, quality 3: 90 g x m-2 retention of coarse and gelatinous precipitates;
filtration speed 700 ml x min-1 x 100 cm-2; VWR No 108016-9; VWR™ International)
Stored in a dark and sealed glass bottle
Method
Semen samples were liquefied at 37°C for 30 min
One droplet of semen
+
One droplet of the eosin- nigrosin staining solution
Incubated for 30 s at room temperature (20°C )
12 l droplet was transferred to
a labelled microscope slide and smeared
The smears were air dried and examined directly
At least 200 sperm were assessed at a magnification
of 1000 x under oil immersion with a high-resolution 100 x
bright field objective
www.vivo.colostate.edu
• White sperm = Live sperm
• Red sperm or pink sperm = Dead sperm
• Slight pink or red appearance restricted to
the neck region (leaky necks) = Live sperm
• Reliability and repeatability of assessment :
IQC (inter-individual variation was <10%)
• Exposure time to eosin-nigrosin was varied
from 30-300 s.
Motility assessment
• Assess at least 400 sperm in each semen
sample.
• Four motility categories.
– Rapid progressive
– Slow progressive
– Non-progressive
– Immotile
• Using techniques for assessment and quality
control according to the World Health
Organization(WHO)
• The percentages of motile sperm (the sum of
the three categories of motile sperm)
Calculation and evaluation
• The sum of percentages motile and stained (dead)
sperm in a sample should be <100%
(some immotile but live sperm)
• If assessed without errors the sum of
percentages of motile and stained(dead) sperm
cannot be >100%
• The sum of dead and motile sperm is >100%
(overestimation of the percentage of motile sperm, or to
an error in the technique for vitality assessment
rendering too high a percentage of stained (dead) sperm)
results
• Technique 1 =1235 sample
• Technique 2-4 = 20 sample
• The mean for sum of stained (dead) and
motile sperm using the one-step eosinnigrosin technique was 91%(SD + 10%)
Figure 1. Proportion stained + motile sperm (mean, SD) distributed into classes according to
percentage of stained (dead) sperm. (A)The present study (n = 1235). (B) Graph drawn from
data in Eliasson and Treichl (Eliasson and Treichl, 1971), (n =724).
• 1 A :The distribution of sums for percentage
stained and percentage motile sperm was
similar regardless as to whether the samples
have many or few dead sperm.
• 1B : the original evaluation of the eosin alone
method (Eliasson and Treichl, 1971)
• The percentage of live sperm did not change
with increasing time of incubation (30-300 s)
in the eosin-nigrosin staining solution
• Mean percentages of live sperm were 81, 80,
81 and 80% for incubation times of 30, 60, 180
and 300 s respectively
Discussion
• the sum of percentages for dead and live
sperm is 100%.
• Live sperm are either motile or immotile.
• the sum of percentages for dead and motile
sperm should be somewhat <100%.
• The sum of percentages for motile and
stained (dead) sperm:
technique 1 - mean 91% (95%CI:72-110)
(the present 0.67% eosin-nigrosin technique)
technique 2 - mean 96% (95%CI:72-120)
(the 0.5%-eosin-alone technique)
technique 3 - mean 95% (95%CI:74-116)
(the two-step 1%-eosin and nigrosin technique)
technique 4 - mean 109% (95%CI:87-131)
(the two-step 5% eosin-nigrosin technique)
• The present technique gave the narrowest
95% confidence interval.
• The wider total range in the present results
(45-120%) : n=1235
• Technique 4 showed a systematic error
(sum> 100%)
• The increased proportion of dead sperm cause:
- Toxic effect of the 5% eosin concentration.
- Stored of stained.
- Unmounted smear in humid environment.
- Made and dry smear.
• The one-step eosin-nigrosin technique minimizes
the exposure of sperm to eosin (concentration
and duration of exposure)
• 30 s for mixing and incubation.
• Environmental temperature,mixing and
incubation, volume used for making the
smears and the size of the slides were kept
constant
• 30-300s in nigrosin mixture before smear :
do not affect the final percentage of dead
sperm in human semen.
• Comparison between the one step eosin
alone technique with the one-step eosinnigrosin technique : sum of percentage motile
and stained lower with few dead sperm.
• Run at 37 C, QC and training of
technicians.
• Change solvent, specific volume and
technique smear : the Nordic Association for
Andrology and the Special Interest Group in
Andrology of the European Society of Human
Reproduction and Embryology
conclusion
• Modified from Mortimer.
• Evaluated with sperm motility data : WHO
• Reliability in assessing sperm vitality in
samples.
• Using standard bright-field microscope.
• Few methodology steps to control
management.
• Recommended in the basic semen analysis
when sperm vitality is to be assessed.
Thank you