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Andrology Lab Booklet

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or Reprod er f uc nt ti e ve C M n a e c d i i r c e i Andrology Center and n m e A C e 3 9 n 9 tr 1 um t. E Es Reproductive Tissue Bank xcellentiae Who We Are What We Offer The Andrology Center and Reproductive Tissue Bank - The Andrology Center and Reproductive Tissue a section of the Glickman Urological & Kidney Institute Bank’s specialized laboratory offers a wide variety at Cleveland Clinic - provides specialized tests and of comprehensive tests and the latest technology services to evaluate male infertility. Our laboratory to meet patient needs. The Andrology Center offers offers referring physicians and patient’s quantifiable both research and clinical services. Our laboratory results using the latest state-of-the art technology. uses the latest World Health Organization (WHO, We are located in the Building X, which is part of the Fifth Edition, 2010) guidelines and reference ranges Downtown Main campus. in the evaluation of semen samples. Additionally, our laboratory’s Therapeutic Sperm Banking As part of the American Center for Reproductive program provides a complete fertility preservation Medicine, the Andrology Center and Reproductive service including a reliable system for the long-term Tissue Bank is staffed with highly qualified and preservation of human semen, epididymal aspirate experienced laboratory technologists who are well and testicular tissue. trained in fertility testing and certified by the American Society of Clinical Pathologists (ASCP). Our laboratory is certified by the Clinical Laboratory Improvement Table of Contents Amendments (CLIA) and the Department of Health and Human Services. Since 1994, the lab has been Who We Are And What We Offer 1 accredited by the College of American Pathologists Our Technology 2-6 (CAP). Our reproductive tissue bank is accredited by Sperm Processing Techniques 7-8 the JCAHO and FDA. Advanced Technology Brings Results 9-16 “Patients First” is the guiding principle of the Cleveland Therapeutic Sperm Banking 17-19 Clinic. To emulate this standard, our staff at the Dedicated Patient Service 20-21 Andrology Center and Reproductive Tissue Bank Make An Appointment 22 continually strives to provide compassionate care and Andrology Center Staff 24 outstanding service every step of the way. About ACRM 25 Get To Know Our Center 26 About Cleveland 27 1 February 2, 2018 Our Technology Semen Profile Clinical Utility of the A semen profile assesses semen quality using the most Semen Analysis comprehensive and advanced testing available. It is The results of both manual considered the cornerstone of infertility investigation semen analysis and CASA because it provides the physician with a clear understanding guide the clinician in Demonstration of a step in semen analysis. of the functional capacity of the sperm. A typical profile assessing semen quality assesses routine semen parameters and levels of reactive and creating an individualized treatment plan. Some of the oxygen species (ROS), oxidation reduction potential motion parameters are used to grade of sperm motility while (ORP) total antioxidant capacity (TAC) and sperm DNA others are used to assess the functional quality of the sperm fragmentation (TUNEL). such as the fertilization potential. Semen Analysis Turn-Around-Time: 1-3 days Semen analysis assesses a man’s fertility potential, sperm production, accessory gland function and Leukocytospermia Quantification/Endtz Test ejaculatory capability. It The presence of high numbers of white blood cells (WBCs) specifically examines in semen, especially the presence of granulocytes, is often sperm volume, pH, color, associated with an viscosity, sperm underlying infection Observe: 10X Brightfield concentration, total sperm and male infertility. Objective Lens Load Load N Leukocytes are powerful AME count and percent motility. Pt.# Microcentrifuge Da Makler te 10 µL tube Liquified Chamber generators of free Semen Count Cells in A computer assisted semen Figure 1: Microscopic view of the All 100 Sqaures 10 µL • All granulocytes radicals called reactive stain dark brown analyzer enhances this counting chamber. Tyrodes Buffer Incubate 5 min. oxygen species or ROS. ENDTZ analysis by measuring sperm motion characteristics not Calculation: 20 µL #WBC X 4 (dilution factor) = 105/mL semen The myeloperoxidase Endtz Reagent 105/mL semen 10 = 106/mL semen Endt z • Mix 4.0 mL stock solution with detected by the human eye such as velocity, linearity, Reagent or Endtz test 50 µL of 3% H2O2 progressive motility and amplitude of lateral sperm differentiates WBCs from head movement. It is also capable of determining sperm Figure 3: Quantification of white blood cells other immature germ morphology. Our CASA system is among the most by the Endtz test. cells in the semen. technologically advanced systems and yields far more accurate and quantifiable results than manual methods of Figure 4: Endtz semen analysis. positive cells - these are leukocytes that Computer Assisted Semen Analysis (CASA) stain brown. Our laboratory is equipped with a Computer Assisted Semen Analyzer and Integrated Visual Optical System (IVOS) software version 14 (Hamilton Thorne Research), which operates as a cell motion analyzer and computer. Computer Reference Range: assisted semen analysis has If the presence of WBCs is ≥ two advantages: it is precise 1X106 WBC/mL, and can quantitatively this is known as assess sperm kinematics. leukocytospermia. It is a semi-automated technique that provides Turn-Around-Time: 1-3 days data on sperm concentration, motility and special sperm Clinical Utility of the Test motion parameters. These Clinicians need to be aware that an Endtz value of ≥0.4 X include: average path 106 WBC/mL is indicative of a possible infection of the velocity, linearity, curvilinear male accessory glands. The patient should undergo further velocity, amplitude of investigation to identify the possible cause of infection. lateral head displacement, Antibiotics may be prescribed. Treating men with antibiotics flagellar beat frequency Figure 2: CASA screen shot may help reduce any infection by decreasing seminal and hyperactivation. The showing measurement of various leukocytes and ROS production. This often results in a percent progressive motility sperm motion parameters subsequent improvement in sperm motility and natural has been shown to relate with pregnancy rates. The CASA conception. The patient may be asked to repeat the Endtz results for motility and concentration are also verified test to confirm that the infection has cleared. 2 manually. Retrograde sperm count and preparation In some men, semen passes into the bladder instead of coming out through the urethra; this condition is known as retrograde ejaculation. Urine sample is centrifuged and the pellet re-suspended in culture media and examined for sperm count and motility. Processing of retrograde sample for IUI can be done using density gradient method. TAT: 3-5 Days Clinical Utility of test: Negative result indicates absence of retrograde ejaculation. Post-vasectomy screen Post vasectomy screen is ordered in patients who had a vasectomy done. The test involves examination of a semen sample for presence of sperm. If no sperm are seen then results pass the AUA criteria. TAT: 1-3 days Clinical utility of the test: Results AUA criteria No sperm seen on unspun wet prep Pass Sperm count between 0 to 100,000/mL Pass Sperm count ≥ 100,000/mL Fail Sperm motility ≥ 0% Fail 3 Sperm Morphology Sperm count and motility alone are not adequate predictors Reference Value: ≥4% normal sperm forms of fertility. These tests must be run in conjunction with Turn-Around-Time: 1-3 days sperm morphology in the evaluation of the infertile male. Clinical Utility of the Test The Cleveland Clinic Andrology Center uses the WHO Normal sperm morphology is a significant predictor of 2010 (5th edition) criteria for assessing sperm morphology. pregnancy in in-vivo and in-vitro fertilization. Smears are stained using the Diff-Quik method. Abnormal sperm forms are associated with lower fertilizing potential and increased DNA damage. Eosin-Nigrosin Test It is important to determine whether immotile sperm are alive or dead. This is accomplished using the Eosin-Nigrosin or vitality test. Spermatozoa that are alive have an intact sperm plasma membrane that does not allow the Eosin stain to penetrate. These sperm appear white when stained and viewed under a microscope. Dead spermatozoa have a compromised sperm membrane that absorbs some of the stain, which turns them pink. Figure 5: Reviewing sperm morphology slides on a monitor for various abnormalities. Figure 7: Eosin- Nigrosin stain and a microscopic view of viable (white) and non-viable or dead (pink) sperm. Vitality Staining: It is performed when the sperm motility is <25%. Viability test should be conducted within 30 but no more than 60 minutes after semen collection. Turn-Around-Time: 1-3 days Clinical Utility of the Test The presence of a high percentage of immotile and non- viable sperm is characteristic of epididymal pathology. Figure 6: Diff-Quik staining procedure for sperm morphology showing common sperm abnormalities. 4 Figure 8: Checking for the presence of spermatozoa using cytospin in azoospermic sample. Special Staining for Azoospermic Patients Special staining is performed when no sperm are seen Azoospermia is the absence of spermatozoa in the during a semen analysis. ejaculate. After analysis of the semen specimen, a special stain called the nuclear fast red picroindigocarmine, or Turn-Around-Time: 1-3 days NFPIC stain, is used. Clinical Utility of the Test This test may help the clinician determine whether to Sperm heads stain bright pink and tails green. This test perform a testicular biopsy or microscopic testicular sperm confirms the complete absence of spermatozoa or the extraction (MicroTESE). presence of rare spermatozoa in the ejaculate. Key Publication On Identification Of Sperm In An ‘Azoospermic’ Semen Sample By Andrology Staff Urology 1998; 51: 816‐819 Urology 1998; 51: 816‐819 5 Intrauterine Insemination of healthy spermatozoa available for fertilization during a Intrauterine insemination (IUI) is an effective method given cycle.
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