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CLS 1521 URINALSIS AND BODY FLUIDS
SEMINAL FLUID (SF)
All learning objectives are cognitive, unless identified as being psychomotor.
The student, at the end of each instructional component, whether in the
classroom or laboratory, is responsible for achieving these objectives.
Any student that achieves a cumulative score of 70% or better on all
problem sets, case studies, major exams, quizzes, and library assignments are
deemed to have met the objectives of this course.
Upon completion of each classroom and laboratory
component, the student will be able to demonstrate their responsibility and role
in learning by successfully completing the following learning objectives in this
study unit and textbook as follows:
01
LIST SYNONYMS FOR SEMINAL FLUID AND WRITE A
DEFINITION FOR SEMEN.
[1] Ejaculate
[3] sperm
[2] Semen
[4] s seed
Definition: “
It is a penile ejaculate consisting of a
thick, opaque, yellowish-white, viscid fluid containing spermatozoa; a mixture
produced by secretions of the testes, seminal vesicles, prostate, and
bulbourethral glands.
02
LIST AND DESCRIBE THE FOUR FRACTIONS OF SEMINAL FLUID.
•FRACTION ONE: Prostate portion.
Makes up about 20% -25% of the total volume, containing citric acid, enzymes
(for coagulation and liquefaction), zinc, and acid phosphatase.
•FRACTION TWO: Seminal vesicles portion.
Contributes 60% or more of the total volume and contains fructose, flavins
(enables the fluid to fluoresce, citric acid, and potassium. This portion is the
nutrient medium for spermatozoa.
•FRACTION THREE: Bulbourethral and urethral glands.
Make only a minor contribution.
•FRACTION FOUR: Testes and epididymis.
Contributes approximately 7% of the total volume. Spermatozoa are found in this
fraction.
03.
DESCRIBE IN GENERAL TERMS THE PROPER COLLECTION PROCEDURE FOR SEMINAL
FLUID.
The patient should appear at the physician's office/laboratory after three days
of sexual abstinence. A form should be completed that contains (1) the patient's
name, (2) days of abstinence, (3) time of collection, (4) how collected, and (5)
time the laboratory begin testing the seminal specimen. This form may also serve
as the report form, in which case, the analysis results will be included.
Ideally, the specimen should be collected at the testing site, with safeguards
for the patient's privacy. In the event, that this is not practical, the fluid
may be collected at home and delivered to the laboratory within one hour of
collection. The patient must be instructed on how to avoid temperature extremes
until the specimen is delivered to the laboratory. Ideal transport temperatures
are 20 0C - 40 0C. The advantage of collecting the
specimen on site lies in the laboratory accurately reporting the coagulation and
liquefaction events.
The specimen should be collected by masturbation into a chemically clean,
sterile, wide-mouth, warmed glass container. Ejaculation into a warm container
prevents temperature shock to the sperm. Plastic containers should be avoided
since some plastics have an inhibitory effect upon motility over a 60 minute
period. The use of most condoms is discouraged because of the use of spermicidal
agents. There are special, non-spermicidal condoms available if this type of
semen collection is required.
"Coitus interruptus" is generally not acceptable because the first portion of
the ejaculate is easily lost. The first part of the ejaculate contains the
largest concentration of sperm.
04.
LIST AT LEAST THREE REASONS FOR PERFORMING A SEMEN EVALUATION.
[1] sexual crimes (homicide, abuse, or rape), [2] infertility, [3]
post-vasectomy analyte, [4] biological testing (example: AIDS).
05
DISCUSS COAGULATION AND LIQUEFACTION OF SEMEN.
Once ejaculated, the semen will immediately form a coagulum and within
approximately 5 minutes, liquefaction begins. In most cases liquefaction will be
complete within 30 minutes. If the liquefaction process is not complete within
60 minutes, the semen specimen is considered to be abnormal. Until liquefication
is complete, the sperm are immobilized.
06
DISCUSS APPEARANCE AS A PART OF THE PHYSICAL ASSESSMENT OF SEMEN.
Normal semen is usually white and opaque. It also described as being light gray
or gray-white, opalescent, and opaque. If brown or red color is present, this
may indicate the presence of blood. Patients on medication may present a yellow
hued seminal fluid. Sperm content is not associated with appearance.
07
DEFINE THE FOLLOWING TERMS:
AZOOSPERMIA: absence of spermatozoa in semen.
CELLS OF LEYDIG: also "interstitial cells of Leydig". Located
between the interstitial spaces of the seminiferous tubules of the testes, these
cells produce and secrete testosterone.
COAGULUM: the formation of a semi-solid, gel-like clot of seminal
fluid.
EPIDIDYMIS: a complex, coiled tubular structure attached to the
testes and provided the final maturation site of the developing spermatozoa.
LIQUEFACTION: the conversion of the coagulum of semen to its liquid
form after ejaculation.
OLIGOSPERMIA: a decreased number of sperm, generally considered to
be fewer than 20 million/mL.
PROSTATE GLAND: a lobular gland that surrounds the male urethra and
is located in from of the urinary bladder. It is testosterone dependent.
SEMINAL FLUID: complex body fluid transport medium for
spermatozoa.
SEMINAL VESICLES: a pair of glands in the male reproductive tract
(lying close to the prostate gland) that produce the major volume of the seminal
ejaculate.
SEMINIFEROUS TUBULES: long, coiled, complex tubules located in the
testes and are the site of spermatogenesis.
SERTOLI CELLS: located interspersed among the germs cells of the seminiferous tubules, these cells limit the movement of chemicals from the blood
into the lumen of the tubules, provide a variety of nutrients to the developing
spermatozoa, and secrete a fluid for the lumen of the tubule. These cells have
been called "nurse" cells.
SPERMATID: an immature sperm cell.
SPERMATOZOA: also called sperm, it is a mature male sex germ cell
with the capacity to fertilize a mature ovum.
VIABILITY: in reference to spermatozoa, it is their ability to live in the
female reproductive tract and fertilize the ovum.
VISCOSITY: a measure of the ease of fluid flow or it resistance to
flow.
08
DISCUSS VISCOSITY AS IT RELATES TO EVALUATING SEMINAL FLUID.
This describes the expected consistency of the semen. If the viscosity is too
watery or too gel-like in nature, it is considered to be abnormal. Viscosity can
be evaluated on the way it pours from its container into a graduated cylinder
for measuring volume or the way droplets form from a Pasteur pipet or a 5 ml or
10 ml serological pipet. In either case, as the semen is poured from a container
or expelled from a pipet notice how the droplet free falls under the effects of
gravity. A thread or "tail" will form as the droplet falls and the length of the
droplet determines the viscosity. If there is no thread/tail, the viscosity is
low. The length of the tread/tail is a reflection of the degree of viscosity,
the longer the thread/tail the greater the viscosity. Try to measure the length
of the thread and droplet to the nearest 0.1 cm. If the semen is normal in
viscosity, distinct droplets form and fall with a thread/tail that will be less
than 2.0 cm (¾ inch). Normal semen will not appear stringy nor “clumpy”.
Abnormal semen forms threads that are >2.0 cm. Viscosity can be rated as
follows:
A. 0 = watery (near absence of the thread/tail) and is easy to
aspirate in
a pipet.
1 = somewhat watery and is reasonably
easy to aspirate into a pipet,
2 = normal viscosity and is somewhat
difficulty to pipet.
3 = moderate viscosity and is
difficult to pipet.
4 = viscid or gel-like and cannot be pipetted.
B. An alternate methods that have been used for reporting viscosity are:
a. low viscosity, normal viscosity,
more viscous than normal, or very
viscous.
b. slight viscosity, moderate
viscosity, or extreme viscosity.
In the semen sample that is normal in viscosity, it has been recommended that a 5 ml
or 10 ml pipet be used to pipet the sample. If the semen needs to be diluted,
use Delbecco’s fluid. If the semen is extremely viscous, used an enzyme
such as chymotrypsin, bromelin, or plasmin. Chymotrypsin is preferred by
some laboratories.
09
DISCUSS VOLUME AS IT RELATES TO EVALUATING SEMINAL FLUID.
Normal semen volume ranges for 2.0 mL to 5.0 mL. Use a chemically clean and
sanitized graduated cylinder or a wide-tipped pipet with a mechanical aspirating
devise. Simply measure the volume. The viscosity of the semen sample can be
determined at this time.
10
DISCUSS pH AS IT RELATES TO EVALUATING SEMINAL
FLUID.
pH testing measures the hydrogen ion concentration of semen. pH testing paper or
a pH meter may be used to measure the pH. Normal seminal fluid is slightly
alkaline with a pH range of 7.2 to 7.8. Textbooks give other ranges (examples:
7.3 - 8.2 and 7.2 to 8.0). The alkaline nature of the seminal fluid protects the
sperm from the acid environment of the vagina. Also the motility and metabolic
activity of the sperm declines as the pH drops below 7.2, in fact sperm will
either be of poor quality or will die. If there is an increase in prostatic
fluid, the pH of the semen will become more acidic.
11
DESCRIBE THE ODOR OF
SEMEN.
Odor of semen may help cue the technologist to something unusual about the
specimen. Normal odor is described as resembling sodium hypochlorite (bleach).
If it is suspected that something is wrong based upon the odor, first verify
that the collection process was properly conducted.
12
DESCRIBE HOW TO PREPARE A SEMINAL FLUID WET MOUNT AND WHAT INFORMATION CAN
BE OBTAINED.
Simply place a drop of well-mixed seminal fluid on a chemically clean slide and
overlay with a cover-slip. Ideally, 10.0 μL of semen should be deposited on a
slide and covered with a 22 mm
X 22 mm cover-slip. Some laboratories recommend
sealing the edges of the cover-slip with some type of sealant. This is not
necessary step to a successful motility study. Allow the preparation to "sit"
for one minute to stabilize and then perform the procedure.
The wet-mount analysis can provide information about sperm morphology,
viability, approximate count, motility, and any agglutination patterns.
If no sperm are noted in the wet mount, centrifuge the specimen at low speed
(1000 rpm) for a maximum of five minutes. Pour of the semen and resuspend the
button in the residue, make a new wet mount and observe for sperm. If no sperm
are found by this method, the patient may have azoospermia. When evaluating a
wet mount for sperm motility, be sure to note the presence of other cells. For
example, the presence of neutrophils may be an indicator of prostatitis.
13
DISCUSS MOTILITY AND THE GRADING CRITERIA AS IT RELATES TO EVALUATING
SEMINAL FLUID.
Determination of motility is an important characteristic as it reflects the
ability of the sperm to migrate to the ovum to fertilize it. Sperm motility is a
subjective and semi-quantitative evaluation. The motility of the sperm is
estimated by determining the numbers of motile and non-motile sperm in the
wet-mount. A minimum of 200 sperm could be counted and rated (under the 40X or
45X objective) in a minimum of 5 different fields. The percentage of motile
sperm may be determined as follows:
total sperm – non-motile sperm
% motility = -------------------------------------
× 100%
total sperm
An alternate method requires examining 25 fields and subjectively grading the
motility from zero to four. The following scale can be used for this subjective
evaluation:
0 = no motility observed
1 = motile, but no forward movement/progression noted
2 = motile, the movement/progression is slow and meandering
3 = motile, with moderate linear (forward) movement/progression
4 = motile, with strong linear (forward) movement/progression
Regardless of which grading system used to evaluate motility, evaluate the speed
of movement and the rate of linear (forward) movement in the specimen. Motility
should be determined within one hour after collection. Motility may be observed
over a period of hours. If this is the case, then keep the sperm in a moist
chamber (covered petri dish with moist toweling) at room temperature. Some
laboratory's stipulate the temperature should be at 37 0C. Sperm
motility at 60 minutes after collection should demonstrate 70% motile forms. If
motility is <70%, then some anomaly is present. The following table may be used
for normal motility values over time.
NORMAL VALUES
• up to 60 minutes 70% to 90% motility
• 60 to 120 minutes 60% to 69% motility
• third hour approximately 60% motility
• sixth hour 50% to 59% motility
• tenth hour 35% to 49% motility
• fifteenth hour 25% to 34% motility |
14
EXPLAIN WHAT A REVITALIZATION
CHECK IS IN THE SEMINAL FLUID EVALUATION.
If the spermatozoa are immotile or appear dead, add a drop of glucose-Ringer's
solution to the slide. If the sperm become motile, the procedure worked. If no
motility then the sperm may be dead. See Objective 15.
15
DESCRIBE HOW TO DIFFERENTIATE BETWEEN DEAD AND LIVING SPERMATOZOA IN A
MICROSCOPIC EXAMINATION.
Prepare a 0.5% solution of yellow eosin in saline. Add one drop of dye to one
drop of well mixed semen. If the sperm cell is alive, it will NOT take up the
stain, but retain a somewhat bluish-white color. If the sperm cell is dead, it
will absorb the stain and the head of the sperm will take on a yellow-pink
color. This test does not differentiate between motile and non-motile sperm. If
nigrosin is added to the stain, it will form a dark or purple-like background
that accentuates the sperm cells for easier viewing.
16
DISCUSS SPERMATOZOA
MORPHOLOGY IN THE MICROSCOPIC EXAMINATION AND ILLUSTRATE A NORMAL SPERM CELL AND
A MINIMUM OF 12 ABNORMAL SPERMATOZOA.
To evaluate sperm morphology, stained smears of semen are required. This slide
may be prepared as is the blood smear. After the smear has air dried, it may be
stained with crystal violet, hematoxylin-eosin, Papanicolaou stain, Wright's
stain, or Wright's-Giemsa stain. The slide should be observed under the oil
immersion lens and a minimum of 200 sperm cells counted and evaluated. Eighty to
eighty-five percent of the sperm examined should be normal. The following areas
of the sperm morphology are to be rated: head, neck, and tail.
The normal sperm cell (top view) has an oval head that ranges from 2 to 3 μM in
width to 3 to 5 μM in length. A side view shows an arrow shaped head. The
midpiece is from 7 to 8 μM long and the tail averages about 45 μM in length. See
Figure l for illustrations of normal and abnormal sperm morphology.
The categorizing the head of the sperm is the most important part of the
description.
[1] The head anomalies include vacuolation, acrosomal abnormality, bicephalic or
paired spermatozoa, malformation of the post-acrosomal (nuclear) region, and
size variation.
[2] Tailpiece anomalies include variation in length of tailpiece, coiled
tailpiece, and multi-tails.
[3] Mid-piece anomalies consist of length variations, absence, or other
distortions. Note that the mid-piece is barely discernable in the brightfield
microscope. Usually any abnormality other than shortening of length or absence,
is usually detected.
Immature forms may be observed on the slide. Spermatocytes and spermatids will
be present on the slide and will be characterized by variation in sizes. Late
spermatids will contain a flagellum but the head will not have a discernable
acrosome or nuclear area. Immature spermatozoa may have cytoplasmic residues
attached in the vicinity of the head.
17
IDENTIFY EIGHT SEDIMENT ELEMENTS THAT MAY BE OBSERVED IN SEMEN.
[1] Epithelial cells: These may be
cuboidal, columnar, or squamous
dependent upon their cell form.
[2] Sertoli cells: These are tall,
columnar epithelial cells that nourish the
developing sperm.
[3] Macrophages: Considered to be the
end phase of monocytes,
macrophages are a large, single nucleated, vacuolated
cell.
[4] Erythrocytes: Normal values are zero to
rare. Very few RBC’s should
be observed.
[5] Leukocytes: Normal values are none to
few. If in significant numbers,
then may be an indicator of an inflammatory
process.
[6] Lecithin granules: These are
starch-like granules containing lecithin.
[7] Corpora amylacea: These are small
degenerating cells. They will be
hyaline-like in appearance.
[8] Spermine hydrochloride crystals:
Generally are clear, rhomboid
crystals of variable shapes. These
crystals can
somewhat pleomorphic.
18
DESCRIBE HOW TO PERFORM A SPERM COUNT.
The sperm count is usually performed in the same manner as a cerebrospinal fluid
count. A well mixed semen specimen diluted 1:20 with an appropriate diluent
(consisting of 1.0 mL 40% formalin, 5.0 grams of sodium bicarbonate, and q.s. to
100 mLs with distilled water). Trypan blue or gentian violet stain may be added
to the diluent. The formalin immobilizes the sperm, the sodium bicarbonate
dissolves the mucus, and the stain can enhance the visibility of the sperm. Some
laboratories use distilled water or saline as a diluent. A drop of diluted semen
is added to the Neubauer hemocytometer. If the specimen contains 10 or less
sperm per sub-square in the WBC counting squares, then count two diagonal WBC
squares and multiple the count by the factor 100,000. If there are more than 10
sperm per sub-square, perform the count in the RBC counting area, using the four
corner sub-squares and central sub-square. Multiply the total by the factor one
million. Refer to the laboratory manual for additional information instructions
on the counting technique and strategy.
19
BRIEFLY DISCUSS THE VASECTOMY AND HOW TO CONFIRM AZOOSPERMIA.
A vasectomy is a surgical procedure that can be performed on an outpatient
basis. The surgeon excises a segment of the vas deferens and this brings about
sterility. It takes about 40 days to confirm azoospermia, in which no
spermatozoa can be found in the semen. Azoospermia is determined by a sperm
count. Documentation of azoospermia is required to confirm sterility. If the
male changes his mind after a vasectomy and requests reanastomosis of the vas
deferens, the procedure is called a vasovasostomy and is successful in up to 90%
of those requesting the procedure. Statistically up to 40% may achieve
fertility. The remainder who remain nonfertile is thought to be due to sperm
antibodies resulting from the vasectomy.
20
LIST THE NORMAL VALUES FOR A SPERM COUNT.
In one consensus the sperm count will ranges from a low of 20 million/mL to a high of 160 million/mL.
Another consensus recommends a range of 60 to 200 million/mL as a normal range. Some references
cite/recognize a high of 250 million/mL as the upper limits of normal.
There is a medical viewpoint that a sperm count of 20 to 40 million/mL is
generally considered to be low with a reduced chance for fertilization.
Sperm values below 20 million/mL are considered by many primary care
practitioners as being inadequate for fertilization.
21
BRIEFLY DISCUSS SPERM ANTIBODIES AND HOW THEIR PRESENCE CAN BE DETECTED IN
THE MICROSCOPE.
Sperm and semen proteins are antigenic and can cause the production of
antibodies. Both IgA and IgG antibodies have been identified. If a breakdown in
the blood-sperm barrier (through trauma, surgery, or infections) antibodies may
result. Men who undergo a vasectomy often (60%) develop agglutinating or
immobilizing antibodies against sperm. These antibodies can be detected in both
serum and semen. If you are evaluating a semen specimen and in the "wet mount",
you notice the presence of clumps of sperm, it is highly possible that such
antibodies are present. Specific tests should be performed to verify the
presence of the antibodies. See the following illustration for an example of agglutinating sperm
antibodies.
22
LIST THREE METHODS BY WHICH SPERM ANTIBODIES CAN BE ASSAYED.
(1) Immunobead assay, (2) Enzyme-lined ImmunoSorbant Assay (ELISA), (3) flow cytometry, (4) gelatin agglutination test, (5) sperm immobilization test, (6)
double-fluorochrome sperm-cytotoxic antibody assay.,
23
BRIEFLY DESCRIBE THE GELATIN AGGLUTINATION TEST.
This procedure requires preparing aliquots of semen. Each aliquot is mixed with
10% gelatin and patient's serum. If antibodies are present, the agglutinated
sperm will be observed as aggregates of white particles in a clear surrounding
medium.
24
LIST FOUR OR MORE BIOCHEMICAL PARAMETERS THAT CAN BE MEASURED IN SEMEN,
THE NORMAL RANGES, AND WHAT THEY CORRELATE TO.
FRUCTOSE: It is produced in the seminal vesicles. The normal
range 40 to 638 mg/dL (mean = 222 mg/dL). If this carbohydrate is absent, then
it may indicate the absence of the seminal vesicles, obstruction of the seminal
duct or some other abnormality. Note: If there is an obstruction involving the
vas deferens, azoospermia results.
CITRIC ACID: It is produced in the prostate and is considered
to be a biological marker for prostatic function. The normal values range from
96 to 1430 mg/dL (mean = 376 mg/dL). Decreased levels suggest a dysfunction of
the prostate gland.
SPERMINE: This is produced in the prostate gland. The normal
values range from 50 to 350 mg/dL with a mean value of 270 mg/dL. Abnormal
values would indicate a dysfunction.
ACID PHOSPHATASE: This is a proteolytic enzyme that is
commonly regarded as a biological marker for prostatic function. The normal
values range from 272-408 King-Armstrong (KA) units (mean = 340 KA units). This
enzyme has been used to diagnose prostatic cancer. This is a forensic marker to
determine if semen is present in the vagina in cases of alleged sexual assault.
ZINC: Most of the zinc found in seminal fluid comes from the
prostate gland. The normal values range from 5 to 23 mg/dL (mean = 14 mg/dL).
Zinc level are decreased in prostate infections.
GLYCERYLPHOSPHORYLCHOLINE: This biochemical marker can be
measured as an indicator of epididymis function. Mean values = 66 mg/dL. Note:
L-carnitine is a possible epididymis marker.
PHOSPHORYLCHOLINE:
This nitrogenous compound in found in the seminal fluid with a normal range of
250 to 380 mg/dL.
Figure 1. Normal and Abnormal Sperm.
| The sperm illustrations are derived from stained spermatozoa smears in
the teaching laboratory and from the information and
drawings/photos in the Atlas of Sperm Morphology by M. M. Adelman and E. M.
Cahill (ASCP Press) -- 1989. |
KEY FOR NORMAL AND ABNORMAL SPERM MORPHOLOGY
[1] Normal sperm, first variation.
[2] Normal sperm, second variation.
[3] Normal sperm, third variation.
[4] Normal sperm, fourth variation.
[5] Normal sperm, fifth variation.
[6] Normal sperm, sixth variation.
[7] Juvenile sperm with a cytoplasmic residue, first variation.
[8] Juvenile sperm with a cytoplasmic residue, second variation.
[9] Sperm with vacuolation.
[10] Sperm with acrosomal deficiency, first variation.
[11] Sperm with acrosomal absence.
[12] Sperm with acrosomal deficiency, second variation.
[13] Elongated post-acrosomal (nuclear) region, first variation.
[14] Elongated post-acrosomal (nuclear) region, second variation.
[15] Flattened post-acrosomal (nuclear) region.
[16] Bicephalic or double-headed sperm, first variation.
[17] Bicephalic or double-headed sperm, second variation
[18] Bicephalic or double-headed sperm, third variation
[19] Bicephalic or double-headed sperm, fourth variation
[20] Sperm with kinked tail.
[21] Sperm with a kinked mid-piece.
[22] Sperm with a coiled tail.
[23] Sperm with an aberrant implantation of the head.
[24] Sperm abnormal cytoplasmic extrusions of the head.
[25] Megalosperm or giant headed sperm.
[26] Sperm without a mid-piece (absence of mitochondria sheath).
[27] Sperm with coiled tail about the head.
[28] Sperm with an elongated head.
[29] Microsperm or miniature-headed sperm.
[30] Pin-headed sperm.
[31] Sperm with an amorphous head.
[32] Double-tailed sperm.
[33] Sperm with a roughened head, first variation.
[34] Sperm with a roughened head, second variation.
[35] Sperm with a roughened head, third variation.
[36] Sperm with post-acrosomal or nuclear deficiency.
[37] Immature germ cell, first variation.
[38] Immature germ cell, second variation.
[39] Spermatid with a short tail-piece. |
