PRINCIPLE :

The HOS test is based on the ability of live spermatozoa to withstand moderate hypo-osmotic stress. Dead spermatozoa whose plasma membranes are no longer intact do not show swelling. Also the dead spermatozoa with intact plasma membranes have no osmo-regulatory mechanism active and show uncontrolled swelling resulting in the rupture of their over distended plasma membranes.
The spermatozoa that show controlled swelling under test conditions is considered the good fraction with low osmotic fragility.

Reagents : 

1. Cryo-HOS reagent – 1.0 ml in ampoule (150 mOsm solution of Sodium Citrate and Fructose)

Storage and Stability : 

Store at 2-8 C till expiry date. Expiry indicated on label

Method :

1. Transfer the number of ampoules required to run the batch of tests to incubator at 37 C. Allow to stabilize temperature for 10-15 minutes
2. Mix 0.2 ml of liquefied semen with 1.0 ml reagent in ampoule and cover with parafilm

3. Incubate for 30 minutes at 37 C. Remix the solution and transfer one drop of sperm suspension to clean microscope slide and mount using 22×22 mm coverslip.
4. Examine using preferably phase contrast. (If phase contrast microscope is not available normal binocular
microscope with reduced lighting can be used equally) Each spermatozoon is scored as either normal or showing swelling of the tail region.

Result :

The result of the test is expressed as the percent swollen cells, equivalent to the proportion of osmotically competent spermatozoa. Osmotically incompetent and dead spermatozoa burst open and show straight tails. Report as percentage normal vitality.

SPERM VITALITYSTAINING KITS :

The sperm vitality is reflected in the proportion of spermatozoa that are “alive”. It is measured by assessing the ability of sperm plasma membrane to exclude extracellular substances like dyes. Sperm vitality should be determined in semen samples with less than fifty percent motile spermatozoa. Vitality assessment also provides check on the accuracy of motility assessments; as the percentage of live spermatozoa should be slightly exceed the total percentage of motile spermatozoa. Eosin staining is used for assessing vitality. The technique is based on the principle of dead cells will take up the Eosin, and as a result stain pink. The assessment can be carried out immediately. Plain Eosin staining also can be used to assess vitality in wet smears. This provides quick assessment at the same time of count and motility assessment.

MATERIAL INCLUDED IN THE KIT :

Cryo-Vitality E 1 ml (Replace the cap and inner plug with dropper cap before first use.)

STORAGE : 

Store reagents at room temperature.

EQUIPMENT REQUIRED : 

Light microscope (40X objective)
Microscope slides pre-cleaned
Cover slips
Pipettes
Test tubes

METHOD :
Cryo-Vitality E :

1 Mix equal volumes of well-liquefied semen and CryoVitality E (small drops) on a pre-cleaned microscopic slide. Cover with a 22×22 mm cover-slip If there is excess mixture of semen and stain seen, blot lightly with tissue paper. Allow 5 minutes for settling and staining of the sperms.
2 Examine under high power (40X magnification) using high quality non-phase-contrast objective and correctly adjusted bright-field optics.

INTERPRETATION :

Spermatozoa that are white (unstained) are counted as live and those showing any degree of pink or red are dead.

LIMITATIONS OFTHE METHOD :

Spermatozoa stained with this kit can not be used for any further procedures. To differentiate live spermatozoa from dead spermatozoa for use in ICSI (low viability samples), the HOS (hypo-osmotic swelling) test should be used. All human, organic material should be considered potentially hazardous. Handle all specimens as if capable of transmitting HIV or hepatitis. Always wear protective clothing when handling specimens.

WARNINGS AND PRECAUTIONS :

All human, organic material should be considered potentially hazardous. Handle all specimens as if capable of transmitting HIV or hepatitis. Always wear protective clothing when handling specimens.

SPERM VITALITY STAINING TEST

The sperm vitality is reflected in the proportion of spermatozoa that are “alive”. It is measured by assessing the ability of sperm plasma membrane to exclude extra-cellular substances like dyes. Sperm vitality should be determined in semen samples with less than fifty percent motile spermatozoa. Vitality assessment also provides check on the accuracy of motility assessments; as the percentage of live spermatozoa should be slightly exceed the total percentage of motile spermatozoa.

Eosin-Nigrosin staining is used for assessing vitality. The technique is based on the principle of dead cells will take up the Eosin, and as a result stain pink. The Nigrosin provides a dark background, which makes it easier to assess the slides. The assessment can be carried out at any time and slides also can be preserved for future assessment and record.

Plain Eosin staining also can be used to assess vitality in wet smears. This provides quick assessment at the same time of count and motility assessment.

MATERIAL INCLUDED IN THE TEST

Cryo Vitality – EN 1 ml (Replace the cap and inner plug with dropper cap before first use.)

STORAGE

Store reagents at room temperature

EQUIPMENT REQUIRED

Light microscope (40X objective)

Microscope slides pre-cleaned

Cover slips

DPX

Pipettes

Test tubes

METHOD

Cryo Vitality – EN

1. Mix equal volumes of the well-liquefied semen sample and Cryo Vitality – EN (small drop) on a pre-cleaned microscopic slide. Allow about 30 seconds for staining.

• Make a smear on the slide using other slides as spreader. Smear should be thin enough for allowing proper visualization. Thick smears are useless for evaluation.
• Allow air-drying and preferably mounting with DPX or equivalent mountant as soon as possible. These slides can be stored at ambient temperature indefinitely.
• At least 200 spermatozoa are examined at a magnification of 40X under oil immersion using high quality non-phase-contrast objective and correctly adjusted bright-field optics.

INTERPRETATION

Spermatozoa that are white (unstained) are counted as live and those showing any degree of pink or red are dead.

Please Count a Minimum of 200 Spermatozoa for Accurate Results

LIMITATIONS OF THE METHOD

Spermatozoa stained with this kit can not be used for any further procedures. To differentiate live spermatozoa from dead spermatozoa for use in ICSI (low viability samples), the cryo HOS (hypo-osmotic swelling) test should be used.

WARNINGS AND PRECAUTIONS

All human, organic materials should be considered potentially hazardous.

Handle all specimens as if capable of transmitting HIV or hepatitis. Always wear protective clothing when handling specimens.

Intended use :

Cryocell Sperm Morphology staining  kit, is a diagnostic kit for staining human spermatozoa. The purpose of staining spermatozoa is to be able to differentiate morphologically normal from abnormal sperm cells.

General information :

The definition and criteria for normality have been largely based on studies done on sperm recovered from the female reproductive tract (especially in post coital cervical mucus) and also from the surface of zona pellucida, which are considered normal. Now WHO also has accepted strict morphological criteria for classification of sperms and normal or abnormal. Cryocell Sperm Morphology Stain Kit is a rapid aid in evaluating morphology in a way that it helps distinguish the different parts of the sperm cell (Head, Acrosome, Equatorial Region, Midpiece, Tail), making it easier to differentiate between a normal and an abnormal spermatozoa.

Material included with the Kit :

Reagent A: Red stain – 50ml in coplin jar

Reagent B: Blue stain – 50ml in coplin jar

Fixative 10 ml in dropper bottle (Replace the cap and inner plug with dropper cap before first use.)

Material not included with the Kit :

Glassware

Microscope

Immersion oil

Warm plate at 37°C

Tap or distilled water

Storage and stability :

Cryocell Sperm Morphology stain should be stored in closed Coplin jars (the original bottles), at 15-25°C. The reagents are stable for 36 months after date of manufacture if unused. However, staining removes constituents and introduces contaminants, and thus stains should be replaced when adequate staining is no longer achieved. Filter stains if deposit is noted.

Preparation

Ensure the fluid level is high enough to cover the area that is to be stained. Fill two Coplin jar, or any other recipient that can hold the slides, with tap water (for washing the slides between the different dyes). If the tap water is alkaline (pH > 7), then use distilled water for washing.

Use washed clean slides with frosted end for making smears.

Method

1. Allow a thin feathered-edge smear of fresh, undiluted semen to air dry for about 5 minutes on a warm plate at 37°C.
2. Fix the smear by putting few drops of fixative on horizontal slides so that the fixative covers all the areas of smear. Allow the fixative to evaporate and let the slides dry completely.
3. Stain in stain ‘A’ using 20 dips of 1 second each. Wash by dipping 7-10 times in fresh tap water. Briefly drain excess water onto absorbent paper.
4. Stain in stain ‘B’ using 20 dips of 1 second each. Wash by dipping 7-10 times in fresh tap water. Briefly drain excess water onto absorbent paper.
5. Allow smear to air dry.
6. Observe staining under a light microscope (100x) using oil immersion:

• Acrosome = bluish pink
• Nucleus = stained dark blue
• Equatorial region = pale pink
• Midpiece and tail = pink

Interpretation of the results :

• Count at least 200 spermatozoa and classify them as either normal or abnormal, specifying which defects are most common.
• Only include identifiable sperm cells in the count.
• The criteria for classifying sperm cells as either normal or abnormal depends on the classification method used in the lab.

Storage :

Cryocell Sperm Morphology Stain should be stored at room temperature (15-25°C).

Mounting slides :

If slides are mounted staining will fade under mounting medium (after a few weeks). So do not mount slides if you want to refer back later. Gently blot off immersion oil, which also fades the staining. It is preferable to make duplicate slides for future reference if necessary, or photographic or video records.

Warnings and Precautions:

• All semen samples should be considered potentially infectious. Handle all specimens as if capable of transmitting HIV or Hepatitis.

PrincipleUnder                                                      

The chromatin in spermatozoa is in a highly condensed state prior to fertilization. The appropriate nuclear chromatin de-condensation and subsequent male pronucleus formation is essential for fertilization and normal zygote development.Highly condensed nuclear chromatin in spermatozoa is because of the formation of S-S cross links between its histone units. The cleavage of S-S link can be induced in vitro by Sodium Dodecyl Sulphate and EDTA. Such induced decondensation is predictor of good fertilizing ability of spermatozoa.

Material included with the Kit

1. NCD Incubation reagent 0.5 ml x 10 ampoules
2. NCD Dry reagent disks 10 nos in a vial
3. Fixative and stain reagent  1 ml
4. Parafilm sheets

Material not included with the Kit

Incubator for 37^oC

Pipettes

Microscope slides and cover slips

Microscope

Storage and Stability

Store at room temperature till expiry date. Expiry indicated on label.

Method:

1. Open one ampoule of NCD Incubation reagent. Add one disk of NCD Dry reagent to the pipette reagent, cover with piece of parafilm and shake to let the disk elute.
2. Incubate the ampoule at 37^oC for 10 minutes to achieve equilibration at this temperature.
3. Add 100 / 200 microlitre of well mixed semen sample to the incubation mixture and incubate further for 10 minutes exactly.
4. At the end of incubation time add one drop of fixative and stain reagent to the incubation mixture.
5. Mix well and take a small drop on slide and place a 22×22 mm cover-slip.
6. Examine under low power (20X) and then high power (40X) of microscope. The cells showing swelling of nucleus with uniform chromatin are considered positive (intact DNA) and the cells showing intact nucleus are considered negative NCD (Fragmented DNA). Fragmented DNA  Good DNA

Please Count a Minimum of 200 Spermatozoa for Accurate Results

Results

The result of the test is expressed as the percent swollen cells or NCD reacted cells to total sperms. More than 70 % sperms in normal samples show NCD positivity. Less than 70 % NCD positivity is associated with reduced fertility of the sperm.

Warnings and Precautions

All semen samples should be considered potentially infectious. Handle all specimens as if capable of transmitting HIV or Hepatitis.After taking out the disk of the dry reagent the cap on the vial containing the rest of the disks should be immediately replaced as the chemical on disk is highly hydrophillic

The sperm acrosome is located on the top of the sperm head. During acrosome reaction, the outer acrosomal membrane and the plasma membrane fuse and vesisculate to discharge the acrosomal contents. Multiple enzymes are present in the sperm acrosome including several acid hydrolases commonly found in lysosomes. Acrosomal enzymes help the sperm to penetrate the cumulus mass and the zona pellucida A sperm with defective acrosome may not have the sufficient stock of these proteolytic enzymes and would eventually fail to penetrate the zona, a prerequisite for successful fertilization.

Sperm acrosome status and function may be assessed by various methods including the measurement of direct proteolytic activity present in the acrosome.

Acrosome integrity test (Gelatin film lysis) is one such method which detects acrosome proteolytic activity in terms of gelatin digestion in the area surrounding the sperm head. The pre-coated gelatin film on the glass slide is lysed coming in contact with proteolytic enzymes

leaching out of acrosome and allow light to pass through creating a halo around the sperm head which can be examined using a microscope.

Material included with the Kit

1. Incubation reagent 0.5 ml x 10 ampoules
2. Pre-coated Gelatin Film Slides 10 no.

Material not included with the Kit

1. Pipettes
2. Incubator at 50^o
3. Petri dish with moist filter paper

Storage and Stability :

Store at room temperature till expiry indicated on label.

Method

1. Open the diluents ampoule (Incubation Reagent).
2. Add 100 µl of well mixed semen sample in the diluent ampoule. Shake a little cover using parafilm and incubate the mixture for 5 minutes at room temperature.
3. Apply one small drop of mixture to the pre-coated gelatin film slide and spread smoothly and evenly.
4. Allow the fluid to dry partially and then transfer to a moistened petri dish (not touching the moistened tissue) and incubate at 50⁰ C for 30 minutes.
5. Examine halos around the sperm head under microscope using high magnification ( x40).

Sperm with halos  Sperm without halos

Interpretation:

Under microscope halos will be seen around the sperm heads. The sperm heads showing no halos are considered negative for acrosomal status and function.

WARNINGS AND PRECAUTIONS

All human, organic material should be considered potentially hazardous. Handle all specimens as if capable of transmitting HIV or hepatitis. Always wear protective clothing when handling specimens.

Fructose is a marker for seminal vesicle function. Its function in the seminal plasma is as a substrate for the glycolytic (Anaerobic) metabolism of the spermatozoa. This is an important energy source for the sperm and exclusion of the seminal vesicular component from the ejaculate will result in almost completely immotile sperm.

Fructose levels in semen are androgen dependent. Fructose is secreted by seminal vesicles. Fructose levels should be determined in any patient with azoospermia and especially in those whose ejaculate volume is less than 1 ml, suggesting seminal obstruction or atresia or ejaculatory tract duct obstruction. Absence of fructose, low semen volume, and failure of the semen to coagulate indicate either congenital absence of the vas deference and seminal vesicles or obstruction of the ejaculatory duct.

Disorders of the seminal vesicles and a subsequent reduction in the fructose concentration in semen will also result in a reduced motility of sperm. As the seminal vesicles contribute more than half of the total volume of semen, absence of seminal vesicular secretions will reduce semen volume. Thus the measurement of fructose concentration in men with low volume ejaculates may be of great value diagnostically.

Another situation where fructose estimates are helpful is in men with polyzoospermia and low motility. Occasionally in men with very high sperm concentrations (more than 350 million sperm per ml), the sperm are immotile due to relative deficiency of fructose. If this semen picture is present, the diagnosis can be confirmed by finding very low concentrations or even absence of fructose in semen.

Storage and Stability : Store at 2-8^O C till expiry date indicated on label.

Method :

1. Take 2 ml Cryo-Fructo-Qual reagent in 2 test tubes (2 ml in each tube). Label one as test (T) and other and standard (S).

• Add 0.2 ml standard in S tube and 0.2 ml semen sample in T Mix well.
• Keep both the tubes in boiling water bath which is already boiling for one minute. Cool by keeping in cold water.
• Examine the color of both the tubes. Standard will show pink-red color.

Interpretation :

Samples showing similar or more color than the Standard are interpreted as positive for fructose, Colorless or very faint are interpreted as negative for fructose.

WARNINGS AND PRECAUTIONS

All human, organic material should be considered potentially hazardous.

Handle all specimens as if capable of transmitting HIV or hepatitis. Always wear protective clothing when handling specimens.

Fructose is a marker for seminal vesicle function. Its function in the seminal plasma is as a substrate for the glycolytic (Anaerobic) metabolism of the spermatozoa. This is an important energy source for the sperm and exclusion of the seminal vesicular component from the ejaculate will result in almost completely immotile sperm.

Fructose levels in semen are androgen dependent. Fructose is secreted by seminal vesicles. Fructose levels should be determined in any patient with azoospermia and especially in those whose ejaculate volume is less than 1 ml, suggesting seminal obstruction or atresia or ejaculatory tract duct obstruction. Absence of fructose, low semen volume, and failure of the semen to coagulate indicate either congenital absence of the vas deference and seminal vesicles or obstruction of the ejaculatory duct.

Disorders of the seminal vesicles and a subsequent reduction in the fructose concentration in semen will also result in a reduced motility of sperm. As the seminal vesicles contribute more than half of the total volume of semen, absence of seminal vesicular secretions will reduce semen volume. Thus the measurement of fructose concentration in men with low volume ejaculates may be of great value diagnostically.

Another situation where fructose estimates are helpful is in men with polyzoospermia and low motility. Occasionally in men with very high sperm concentrations (more than 350 million sperm per ml), the sperm are immotile due to relative deficiency of fructose. If this semen picture is present, the diagnosis can be confirmed by finding very low concentrations or even absence of fructose in semen.

CRYO-FRUCTO~QUANT Reagents :

1. Precipitating Reagent, 10 % TCA for precipitation of semen proteins – 0.9 ml x20 microcaps.
2. Color Reagent, Indole-3-acetic acid reagent 5 ml.
3. Calibrator, Fructose Standard solution 500 mg/dl.
4. Empty microcaps – 20

EQUIPMENT REQUIRED

1. Test tubes
2. Test Tube Holder
3. Droppers
4. Pipettes
5. HCL.
6. Incubator at 37^o
7. Colorimeter

Storage and Stability :

Store at 2-8^O C till expiry date indicated on label.

CRYO-FRUCTO~QUANT Method :

1. Mark one micrcap for calibrator and one for each sample.
2. Pipette 0.1 ml of calibrator and 0.1 ml well mixed semen sample in each labeled microcap. Mix well and allow 10 minutes for precipitation.
3. Centrifuge all sample microcaps at 1500 RPM for 5 minutes to settle the precipitated proteins and obtain clear supernatant.
4. Label another set of empty microcaps and transfer 20 micro-liters of diluted calibrator and sample supernatant to appropriately labeled microcaps. Add 100 micro-liters color reagent in all the tubes.
5. Add 1 ml concentrated Hydrochloric acid to each tube and mix well.
6. Incubate at 37⁰C for 75 minutes. Keep all the microcaps closed.
7. Read absorbance at 520 nanometer or equivalent filter on colorimeter.
8. Calculate using following formula Concentration of test = mg/dl

Assay levels and interpretation:

Concentration of fructose in semen ranges from 63 to 500 mg/dl (3.5 to 28 mmol/l). It must be remembered that as sample ages; the fructose level will fall due to utilization of fructose by spermatozoa. The more sperm that is present in the ejaculate, the greater the fall in fructose concentration.

WARNINGS AND PRECAUTIONS

All human, organic material should be considered potentially hazardous.

Handle all specimens as if capable of transmitting HIV or hepatitis. Always wear protective clothing when handling specimens.

The reagent contains Hydrochloric Acid which is highly corrosive and irritant. Proper safety gear should be used while performing the test. The reagent should be properly disposed.