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Urinalysis and Body Fluids CRg

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1 Urinalysis and Body Fluids CRg
Welcome to the first session of Unit 5. Unit 5 Seminal Fluid

2 Seminal Fluids - objectives
Discuss the major components of seminal fluid with regard to source, function, normal and abnormal appearance. List three (3) reasons for semen analysis. Outline instructions to give to a patient for the correct method for collecting a semen specimen for laboratory analysis. List two (2) methods for identifying a questionable fluid as semen. State the significance of finding increased acid phosphatase in a suspicious fluid. Calculate a sperm count when provided with the number of sperm counted, the dilution factor and the area of the counting chamber used. List the normal values for: semen volume, viscosity, pH, sperm count, motility and morphology. The primary focus of this session will be the composition, formation, collection, and analysis of seminal fluid. Although objectives are listed on this slide, you should refer to the complete objective listing as posted at the course website – as there may be revisions or additions.

3 Seminal Fluids Composition of Semen Spermatozoa
Fluids to provide nutritional support and media or mechanism for delivery % of total Description / Purpose Spermatozoa 2-5% Formed in testes, stored in epididymis and vasa differentia Seminal Fluid 60-75% Alkaline fluid,; primarily responsible for nutritional support through: amino acids, enzymes, fructose. Also to suppress possible immune response by female Prostate Fluid 25-30% Acid phosphatase, citric acid, proteolytic enzymes and zinc Bulbourethral glands 1 – 5% Galactose, mucous The sperm cells needed to fertilize the female egg; producing a new life, actually make up only a small amount of the seminal fluid. The majority of the seminal fluid comes from the seminal vessicles ; Contributions to the fluid are made from the prostate gland and bulbourethral glands. The seminal fluid is composed of various enzymes, amino acids, citric acid, electrolytes, zinc and sugars to nourish and support the sperm; and aid them by providing the appropriate environment for their survival and transport through the female reproductive track. This slide and the next list the major components that make up the seminal fluid. >>>>>>>>>>>>>>>>>>> Semen consists of several fluids produced in various male reproductive organs. The slightly alkaline fluid from seminal vesicles comprises over half the volume of semen and contains citric acid, flavins, fructose, and potassium. These substances provide nutritional support for spermatozoa. Spermatozoa are formed in the testis and are stored in the epididymis and vasa deferentia. The prostate gland contributes a slightly acidic fluid containing acid phosphatase, citric acid, and proteolytic enzymes. These substances account for about 20% of the semen’s volume. The remaining reproductive organs, bulbourethral glands, epididymis, urethral glands, and vasa deferentia, contribute little additional volume to the semen. Upon ejaculation, the fluids from all of these sources form the mixture, semen.

4 Seminal Fluids Anatomy, composition and formation.
Testes – source of sperm (2-5%) Seminal vesicles – provides fructose & nutrients and is primary provider of fluid 60-75%) Prostate gland – Provides enzyme, acid phosphatase, citric acid, zinc, and proteolytic enzymes (for coagulation and liquification). 2nd source of fluid(25-30%) Bulbourethral glands 5%. Thick alkaline mucous-like fluid that neutralizes acids. Once again, this slide provides a breakdown of the major components of the seminal fluid. Anatomy, composition and formation. Testes – source of sperm (2-5%) Seminal vesicles – provides fructose & nutrients and is primary provider of fluid 60-75%) Prostate gland – Provides enzyme, acid phosphatase, citric acid, zinc, and proteolytic enzymes (for coagulation and liquification). 2nd source of fluid(25-30%) Bulbourethral glands 5%. Thick alkaline mucous-like fluid that neutralizes acids.

5 Seminal Fluids Spermatozoa - produced in the testes, mature in the epididymis. Spermatogenesis is the formation of spermatozoa. This process takes place in the seminiferous tubules of the testis under the control of numerous hormones, including testosterone. The sperm mature in the epididymis; a process that 74 days to complete. By the end of the final phase, the normal sperm cell will have a developed - head, mid-piece and a flagellum-like tail. Spermatogenesis is the formation of spermatozoa in the Sertoli cells of the seminiferous tubules of the testis and is under control of various hormones, testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Further maturation of sperm occurs in the epididymis. This approximately 74-day process involves several phases: spermatocytogenesis, meiosis, and spermiogenesis. Spermatocytogenesis is a two-step phase in which spermatogonia undergo mitotic division and maturation into spermatocytes. Meiosis is the specific type of cell division that results in haploid gamete cells. Spermiogenesis is the phase in which the gamete cell develops a flagellum and transforms from a spermatid into a spermatozoon

6 Seminal Fluids Reasons for Testing
Infertility issues – more often a problem with the woman, but easy to rule-out the male. With assisted reproductive technology, greater emphasis placed on sperm quality and quantity. Post- vasectomy – frequent reason for testing Test at one month intervals until 2 consecutive months are negative for sperm. When a man and a woman have difficulty conceiving a child, one or both of the pair may be infertile. More often than not, the infertility will be due to a problem with the woman; but it is technically easier to rule out the male partner, so the investigation begins there. The most frequent reason for seminal fluid evaluation – or more specifically – a sperm count – is to follow up on the effectiveness of a vasectomy. Microscopic evaluation of the seminal fluid for the presence of sperm cells is usually performed at one month intervals until there are 2 consecutive months that are negative for sperm.

7 Seminal Fluids Reasons for Testing
Forensic analysis of fluid as being semen as in alleged rape. Vaginal swab, washings, or scrapings microscopically evaluated for sperm chemical test for enzyme: acid phosphatase Contributed by prostate gland Present even in the absence of sperm cells Sperm donors - artificial insemination programs Additional reasons for testing include the Forensic analysis of suspect fluid – to determine if it is semen - as in alleged rape, - In these cases: A Vaginal swab, vaginal washings, or scrapings are microscopically evaluated for the presence of sperm cells. In addition, a chemical test for the enzyme: acid phosphatase may be performed on the specimen . As noted on the slide presented earlier, The prostate gland contributes acid phosphatase enzyme to the seminal fluid. No other fluid in the body will have such a high concentration of this enzyme as will the seminal fluid. Detecting and measuring very high levels of acid phosphatase will also confirm the presence of seminal fluid, even if sperm cells are absent as could be the case when the sexual perpetrator has had a vascectomy And lastly. Seminal fluid testing is also performed on samples from Sperm donors to determine the quality of the specimen.

8 Seminal Fluids Specimen Collection Sterile container
Direct deposit preferred no lubricants, spermacides, condoms, etc. Complete specimen Majority of sperm are in first part of ejaculate 3 day sexual abstinence required But not more than 5 days. Best if collected at laboratory site. If other, specimen must be kept warm and delivered to lab within 1 hour Time of collection important. Must be recorded! For the most accurate analysis, the entire ejaculate of seminal fluid should be collected directly in to a Sterile container – this will eliminate potential toxic effects of lubricants, and the spermicidal effect of condoms, etc. The patient should be advised against having any sexual activity for 3 days, but not more than 5 days prior to the collection – to produce and provide a specimen with the most accurate sperm count and viability. It is best to have the sample collected at the testing laboratory site – if this is not possible, the sample must be kept at near body temperature and delivered to the testing facility within 1 hour. Regardless of where it is collected, the time of the collection must be noted and recorded. The normal or expected values to which the sample’ s testing results are going to be compared – have been determined at specific time intervals. - Must be the complete specimen – as the majority of the sperm are in the first part of the ejaculate - no lubricants, spermacides, condoms, etc. 3 day sexual abstinence required (but not more than 5 days) Ideal collection site is at laboratory If other, specimen must be kept warm and delivered to lab within 1 hour Time of collection important. (& must be recorded) >>>>>>>>>>>>>>>>>>>>>>>>>> The preferred method of semen collection is by masturbation. This procedure ensures the opportunity to collect the entire ejaculate. Collection should be performed after a 48- to 72-hour continence (abstinence from sexual activity) to provide a specimen containing the most accurate sperm count and viability. A private, comfortable room should be provided for specimen collection that allows for quick delivery of the specimen to the laboratory. Written and verbal instructions for the procedure should be provided. Specimen collection containers should be clean glass or plastic and have a wide opening. Specimens should not be collected in a condom as these often contain spermicidal compounds and lubricants that may interfere with laboratory tests. If the specimen must be transported from a site distant to the laboratory, it must be kept near body temperature and extremes in temperature must be avoided. Shortly after ejaculation, the semen coagulates because of the action of a clotting enzyme, formed in the prostate, on a fibrinogen-like precursor substance that is produced by the seminal vesicles. Liquefaction occurs within 30–60 minutes. Ideally, the specimen should arrive in the laboratory as soon after collection as possible so that an accurate liquefaction time may be recorded. The specimen should be labeled with all patient information and time of collection. In addition, the patient should be asked whether any part of the specimen was lost during collection. This information is important to note because the highest concentration of sperm is usually found in the first part of the ejaculate.

9 Seminal Fluids Physical characteristics
Liquefaction – fresh specimen will clot, then liquefy within 30 – 60 minutes Persistence of clot is abnormal All further evaluation must wait until liquefaction is complete. The fresh seminal specimen will normally gel / clot – if it does not, a note should be made on the report. Within 30 – 60 minutes the specimen should again become liquid. The persistence of the clotted stage is an abnormal characteristic and should be noted on the report. No testing can be performed until the liquefaction is complete. If there is a large amount of mucous present, It may be necessary to treat the sample with amylase or bromelin to break it up in order to obtain accurate sperm counts.

10 Seminal Fluids Semen: Appearance Opaque Gray, white, light yellow
Normal Shades of yellow Correlate with flavin concentration Could also indicate contamination with urine. Deep yellow Associated with certain drugs Brown or red May contain blood Highly turbid Usually contains leukocytes indicating infection or inflammation >>>>>>>>>>>>>> The normal seminal fluid will appear opaque / translucent and can exhibit several normal colors. Typical colors include light gray, white, and light yellow. The higher the flavin concentration of semen, the darker the yellow color may be. A deep yellow color has been associated with certain drugs. Brown or red-colored semen may contain blood. A highly turbid semen specimen often will contain leukocytes and may indicate a reproductive tract infection or inflammation. The leukocyte esterase part of the urine dipstick may be a useful test.

11 Semen: Appearance, cont.
Seminal Fluids Semen: Appearance, cont. Volume 2.0 – 5.0 mL Measured in serological pipet Recorded to 1 decimal place pH 7.2 – 8.0 Measured with pH paper Alkaline to off-set acid vaginal environment Acid may indicate increased prostatic fluids pH > 8 may indicate infection >>>> The Semen volume is measured using a serological pipette, or perhaps in a small graduated cylinder. Volume is recorded in milliliters to one decimal place (0.1 mL). The Normal volume range is from 2.0 to 5.0 mL for a complete ejaculate. Volumes - both lower and higher - than this range have been associated with infertility. Seminal fluid pH should be in the range of 7.2 – 8.0 and is measured using a pH paper. This normally slightly alkaline pH is to offset / neutralize the normal acid environment of the female vagina. More acid pH may indicate that the prostate gland has contributed more fluid; while a stronger alkaline pH may indicate the presence of an infection.

12 Semen: Appearance, cont.
Seminal Fluids Semen: Appearance, cont. Viscosity Pours in droplets (as shown in picture) Rating: 0 = water-like 4 = gel-like Appearance cont. Viscosity - from a pipette tip, specimen should pour in droplets, and not be too stringy Using a semi-quantative rating scale: 0 = water-like through 4= gel-like >>>>The Viscosity of the seminal fluid may be assessed while measuring specimen volume or when pipetting the specimen for other tests. The viscosity is often evaluated and reported in semi-quantiatitve terms. Normal semen is slightly viscous and dispenses in droplets, as shown in the picture on this slide. Abnormally increased viscosity will result in the formation of a string of fluid and An abnormal decreased viscosity will result in the specimen’s drops falling like water.

13 Semen: Microscopic Analysis
Motility Concentration / cell count Morphology Agglutination Viability Penetration of cervical mucous Through research efforts, More and more fertility issues have been identified in recent years, - and as such, more testing procedures have been developed. Many of these procedures would be reserved or only performed in special cases or at laboratories that specialize in fertility testing. Of the tests listed on this slide, the evaluation of motility, the sperm cell count and sperm morphology would be those that are more commonly encountered and will be presented in more detail. Although there may be comments or explanation on the other tests presented, it is generally beyond the expectation for an entry level MLT to cover them in any great detail.

14 Semen: Microscopic Analysis
Microscopic examination Generally performed min after collection Must be after liquidification has occurred Motility Motility is a very necessary quality of sperm. Must propel through uterus & fallopian tubes which is quite a long distance. Must be evaluated within the 1st hour following collection Will decrease over time The microscopic examination part of semen analysis must begin as soon as the specimen is liquidified which is minutes after the collection. Again, it is important that the accurate time of collection has been recorded and the sample is watched carefully. The characteristic of Motility, which is a very important quality of sperm and critical to fertility, should be evaluated within 1 hour of collection, as it will decrease over time.

15 Semen: Sperm Motility Analysis to begin within 1 hour of specimen collection Evaluation times may vary between labs, but usually at set intervals Consistency in technique and procedure important Using hemocytometer & coverslip, examine a drop of undiluted specimen using high dry objective. Brightfield microscopy with light level reduced Some labs use phase microscopy Alternate method: High-resolution video photography / CASA (computer assisted semen analysis) Analysis of sperm motility must begin within 1 hour of specimen collection and is repeated / re-examined several times over specified intervals of time, according to the laboratory protocol with the Consistency in technique and procedure being very important. A drop of undiluted specimen is placed in a hemacytometer and observed using the high power dry objective on a Brightfield microscope. It is important that the light level is reduced, similar to what was required for urine microscopics. Some labs prefer to use a phase microscope in place the brightfield. More consistent and objective results have been obtained using a High-resolution video photography and computer assisted semen analysis or CASA procedures. These advanced techniques are going to be most likely found in laboratories that specialize in fertility issues. >>>>>>>>>>>>> New technology – CASA (computer assisted semen analysis) provides objective evaluation, but only found in specialized labs.

16 Semen: Sperm Motility Manual Subjective evaluation
Observe immediately following liquidfication; and within 1st hour. Place well mixed undiluted drop on pre-warmed hemacytometer slide Observe under high-dry objective; with reduced light. Rated from “0” to 4.0” 4.0 – rapid and straight line movement 3.0 – slower, and some lateral movement 2.0 – slow forward progression, noticeable lateral movement 1.0 – no forward progression 0 – no movement Other types of rating scales may be used Normal (authors vary greatly) but > 50-60% show 2.0 or greater at 1 hour. >>>>>>>>>>> Please take a moment to review the information presented on this slide. Once again, the sperm Motility should be evaluated within the first hour of specimen collection, because it will decrease over time. I have found that there are several approaches to determining sperm motility and that authors also vary on what is considered to be normal. I want to stress that following the specific protocol of the laboratory and using consistent technique will be critically important. For our purposes, the evaluation scale presented on this slide will be used. The expected or normal value is that at least 50% of the sperm evaluated at 1 hour post collection will be rated at 2.0 or higher; showing slow forward progression. >>>>>>>>>>>>>>>>> The movement of sperm is evaluated and may be subjectively estimated or counted into three categories. These categories may be called high-motile, low-motile, and nonmotile; or progressive, nonprogressive, and nonmotile. Some laboratories may use as many as five categories: nonmotile, nonprogressive, slow nonlinear progression, moderate linear progression, and strong linear progression. Some laboratories report the percent of sperm in each category, whereas others report only the percent of motile sperm. At least 80% of the sperm demonstrate some forward progress in a normal semen sample. An alternate method used by some fertility clinics is to evaluate sperm motility from a video recording that is played back with a grid overlaying the monitor. This method also provides for reevaluation should a motility result come into question. More recent use of technology for sperm evaluation includes the use of high-resolution video photography in combination with computer programs that can calculate velocity, linear progression, and motility efficiency and measure patterns of sperm motion. Motility can be effected by temperature and other factors, such as the presence of antisperm antibodies. Therefore, a viability test should be performed, especially if a high number of nonmotile sperm are present. When the manual method of determing inUnless the computerized Although there is some variation in exact procedure, In the more commonly found manual method of evaluation of sperm motility, the sperm movement of sperm is evaluated and may be subjectively estimated Evaluation times may vary between labs, but usually at set intervals Consistency in technique and procedure important NV = 50% showing 2.0 or greater motility at 1 hour after collection Using a hemocytometer, examine a drop of undiluted specimen using high dry objective Motility Subjective evaluation Rated from “0” to 4.0” 4.0 – rapid and straight line movement 3.0 – slower, and some lateral movement 2.0 – slow forward progression, noticeable lateral movement 1.0 – no forward progression 0 – no movement

17 Semen: Microscopic Analysis
Morphology May be performed in cytology, pathology, or hematology Oval/egg shaped head (3x5um) While oval from the front, appears flattened when viewed from the side appears flattened @ ½ covered with an enzyme laden acrosomal cap, which contains Middle piece provides energy Tail piece 45 – 55 um long Like all of the characteristics being presented, the Morphology of the sperm is critical to fertilization. Each component part of the sperm cell has a purpose and therefore its morphology evaluated. The sperm has three major component parts: A head, a middle piece and a tail. The head is Oval/egg shaped head and (3x5um) It is somewhat flattened, so when viewed from the top appears oval, but from the side is pyriform or flattened. The Papanicolaou stained microphotograph included on this slide shows 2 normal sperm cells: the one at the arrow is showing a side view. About half of the sperm head is covered with an acrosomal cap which contains enzymes that would aid it in penetrating the ovum. There is a neck piece that attaches the head to the middle piece. Middle piece of the sperm cell is surrounded by a sheathe of mitochrondia which provides the energy to propel the tail. Authors vary somewhat, but The tail piece 45 – 55 um long, - it serves as a flagellum providing movement.

18 Semen: Microscopic Analysis
Morphology At least 200 cells evaluated on smear (Wright’s, Giemsa or Papanicolaou) stained. Usually evaluated by pathologist, or cytologist Looking for double heads, pin heads, giant heads, or amorphous heads, double, coiled, or missing tails, etc. Many sources of good pictures available A smear of the seminal fluid is generally made on frosted slides and stained with Wright’s, Giemsa or Papanicolaou stain. These slides are usually evaluated by a cytologist or pathologist. At least 200 sperm cells should be evaluated for abnormalities of the head, including the acrosomal cap, the middle piece and the tailpiece. Drawings of Some example deformities are presented on the slide. There are many other good sources of example pictures available in the textbooks as well as on-line.

19 Semen: Microscopic Analysis
Morphology Normal = < 30% abnormal forms (NV varies considerably based on strictness of criteria. WBC, RBC, bacteria presence should be noted & may indicate infection Round cells (neutrophils and immature sperm) should be noted as well. A counting of the number and type / types of abnormal cells should be made. Normally there are fewer than 30% abnormal sperm cells seen, but, again the normal values vary, depending on the strictness of the criteria used. The presence of any WBC, RBC, or bacteria should be noted as they may indicate the presence of an infection The presence of ‘Round cells’ which could include neutrophils and immature sperm) should be noted as well.

20 Semen: Abnormal forms 2 headed sperm Flat-headed sperm
Sternheimer – Malbin stain X 320 Flat-headed sperm Here are a couple example microphotographs of abnormal sperm morphology. Again , Abnormalities can occur in any part of the sperm cell structure.

21 Semen: Microscopic Analysis
Sperm count NV= 20 – 160 million/mL Make 1 to 20 dilution with sodium bicarbonate and formalin, count 5 small squares (within the center large square) of the Neubauer hemacytometer. The normal Sperm count NV= 20 – 160 million/mL >>>>>>> Most laboratories use manual hemocytometer counting techniques as outlined previously. A manual dilution of 1:20 using distilled water OR sodium bicarbonate and formalin is used to immobilize the sperm. Professional judgment should be used when determining the area to count on the hemocytometer. The 5 small squares - shown in red - in the center square may be sufficient to obtain an accurate count when the sperm concentration is high. Otherwise, it may be necessary to count the four outside corner large squares or even one entire side of nine squares to obtain an accurate count when the sperm concentration is low. Following the rules for hemocytometer counting will require professional judgment as well, because sperm do not always lie entirely inside or outside the counting area. What usually works best is to determine the placement of the sperm heads on the hemocytometer grid rather than the tails. - counting sperm with heads that touch the upper and left borders of the counting grid and not counting those sperm whose heads are touching the bottom and right borders. The picture representation on this slide is from the course textbook and provides an example of what should and should not be counted.

22 Semen: Microscopic Analysis
Sperm cell count standard method to begin calculation of # cells (mature sperm) per microliter: Presented on this slide is the standard formula to calculate the number of cells seen per microliter – as used for manual cell counts. As for the other manual cell counts covered, the number of cells counted on each side of the hemacytometer are added and averaged. This average is multiplied by the dilution factor – which as just stated, is usually 20 The product is then divided by the number of squares that were counted times the volume of each square. As a reminder: if you count small squares located in the center square – their volume is 0.004; but if you count the large squares, the volume is 0.1 o obtain the Sperm cell count from the manual hemacytometer: - begin by calculation of # of mature sperm cells. per microliter:

23 Semen: Microscopic Analysis
Microscopic examination Example: 52 cells (mature sperm) x 20 5 (squares) x 0.004 This provides results as ___ cells / uL; Normal values are reported as ___ cells / mL Must multiply X to convert uL to mL = 52.0 x 106 / mL IN the example presented on this slide, The sample was diluted 1/ 20 And an average of 52 mature sperm were counted in 5 small squares. After performing the calculation the cell count is 52,000 cells / uL. If this was any of the other body fluid cell counts presented in this course, the answer would have been solved – as 52,000 cells / uL, however since the normal values for sperm cell counts is per milliter, we must multiply the per microliter result by 1000 to convert the results to per milliler to be able to compare the patient results to the range of expected or normal values.

24 Semen: Microscopic Analysis
Metric Internationalized system using decimals Common system of measuring units Length (M) , volume (L) , mass (G) , time (s), temperature (˚C) Prefixes allow for mL, uL, etc. International System of Units (SI) Modified / modern form of metric system Has 7 base units (but, unlike the original metric system does not include volume) Other units, such as volume are ‘derived’ Basic unit for volume is m3 mL = cubic centimeter (cumm), uL = cubic millimeter (mm3 ) Before leaving this area, a comment to hopefully clear up some confusion about the reporting units you may see being used. The normal values used in this course utilize the traditional metric system which has base units with prefixes. Included is the metric base unit for volume – as being the Liter. As a result, you will see the terms Millillter , microliter, etc. IN 1960, the metric system was modified and now is properly referred to as the SI / International System of Units . It retained many features of the original, but there were some changes in the base units - that included no longer using the liter - volume as a unit. In the SI system, volume is now a ‘derived unit. It is derived from length, width and height. When this system is followed: the basic unit for volume is cubic meter or m to the 3rd power. Examples of Smaller volumes would be cubic centi meter which is abbreviated as cumm And cubic millimeters which is mm to the 3rd power. Much of the laboratory and scientific world continues to use the traditional Metric system of measurement, where the liter is the standard unit of measurement of volume. For purposes of consistency, the metric system of liters, milliliters and microliters will be used.

25 Semen: Sperm Agglutination
Observed while performing motility evaluation. Few clumps are normal. Distinctly head-to-head or tail-to-tail clumping may indicate the presence of antisperm antibodies. IgG IgA Agglutination may be observed while evaluating a wet mount of semen for sperm motility. A few clumps of sperm or sperm sticking to mucus or other cells can normally be seen in a semen sample. However, true agglutination is present if sperm are distinctly clumped head to head or tail to tail. This may indicate the presence of antisperm antibodies. Both IgG and IgA antibodies have been found in the semen of some men with reduced fertility whose sperm demonstrate agglutination. Confirmation with immunologic tests can help determine the specific type of antibody present.

26 Semen: Sperm Viability
Eosin – Nigrosin stain supravital stain Add to drop of fresh sample Smear is made and allowed to dry Evaluated on oil immersion (1000x) Viable / live sperm do not take up the stain and remain colorless or blue-white Non-viable / dead sperm stain orange-red Reported as % viable Normal >75% Sperm viability test is to determine whether nonmotile sperm are viable & alive or nonviable The Eosin-Nigrosin stain is added to the fresh sample, after a short incubation, a slide is made and examined under oil immersion. If the sperm cell is alive, it will have resisted the stain and will appear colorless / blue white. Non-viable sperm have damaged membranes and the stain penetrates into the cell. They will have an orange to red appearance. At least 100 sperm heads need to be counted and evaluated as to being red & dead or white & viable. Normally, 75% or more of sperm are viable. It is important to note that Viability and motility do not always correlate. Sperm that are nonmotile may be alive - but may have defects of the tailpiece. One the other side, dead sperm will not be motile – so your proportion of motile sperm should not be higher than the proportion of viable sperm. Remember, Dead sperm cannot demonstrate motility. Examples of viable and non-viable Eosin Nigrosin stained sperm are provided on the slide;

27 Semen: Analysis Other tests Sperm penetration Microbial testing
Evaluates ability of sperm to make progressive movement through the cervical mucous. Microbial testing Increased WBC (>1 million/ mL) suggestive of infection Aerobic and anaerobic cultures Other testing of the semen sample include the sperm penetration test\ Sperm penetration testing is relatively new to the market. This test evaluates the ability of the sperm to move in a progressive fashion through the cervical mucous. Like other body fluids, the semen sample may be sent to the microbiology department to be cultured for microorganisms. Finding more than 1 million wbcs / mL) is strongly suggestive of infection. Normally both Aerobic and anaerobic cultures are performed.

28 Semen: Chemical Analysis
pH Measure within 1 hour of collection Normal Acid Phosphatase Used to evaluate the secretory function of the prostate Also used in forensic analysis – as prostatic fluid acid phosphatase is higher than other fluids. (>200 units) Fructose Provides energy / nutrition to sperm Indication of viability Presence of fructose – screen using resorcinol test Hormones Testosterone, LH, FSH Chemical analysis of semen routinely includes measuring pH. As stated previously, the pH is slightly alkaline. A higher ph may indicate an infection. Additional tests may include acid phophatase, which is produced by the prostate gland. Acid phosphatase level in seminal fluid is greater than in any other body fluid with levels being greater than 200 U/L. A fact has made this test useful in forensic evaluation of whether an unknown fluid is semen. Fructose is a nutrient providing energy to the sperm cells and its level an indication of viability. Low levels may indicate the presence of a blockage or a decrease in testosterone hormone levels. The screening test for the Presence of fructose is the resorcinol test Testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) levels are also sometimes analyzed in cases of infertility.

29 Post- Vasectomy Analysis
Post-vasectomy semen analysis Specimens tested at monthly intervals starting 2 months post-vas. 2 consecutive months of negative microscopic for sperm Wet prep with phase microscopy Examination of centrifuged specimen as well Post-vasectomy semen analysis has been addressed earlier in this presentation. This slide is a reminder to review the information in conjunction with the course / unit objectives. >>>

30 Semen: Forensic Analysis
Examination of fluid as to being semen (forensic) Acid phosphatase – highly sensitive, as no other body fluid contains as high level (2500 units compared 5 units) ABO, HLA typing DNA analysis UV light scan, semen fluoresces green/white The information on this slide is also a review. Again, use the course unit objectives to guide you as you study this material.

31 Semen: Analysis QC Quality control
Previously little or no QC materials available Commercial products now becoming available Proficiency testing now available CAP American Association of Bioanalysts (AAB) Quality control Commercial quality control and proficiency testing products are now becoming available. Laboratories that offer semen analysis must utilize QC materials to verify the accuracy of testing results. Proficiency testing now available CAP American Association of Bioanalysts (AAB

32 Reference Listing Please credit those whose work and pictures I have used throughout these prsentations. Lillian Mundt & Kristy Shanahan, Graff’s Textbook of Urinalysis and Body Fluids, 2nd Ed. Susan Strassinger & Marjorie Di Lorenzo, Urinalysis and Body Fluids, 5th Ed. Wikipedia, the free encyclopedia Please credit those whose work I have sited in this presentation. This concludes the first session of Unit 5

33 Urinalysis and Body Fluids CRg
Welcome to the second session of unit 5. Unit 5 2 Pregnancy & Amniotic Fluid Testing

34 Pregnancy Testing & Amniotic Fluid - objectives
Describe HCG, explain its role in pregnancy testing, and identify causes for false negative and false positive results. Analyze the formation, composition, and physiology of amniotic fluid. State three (3) reasons for amniotic fluid analysis. Describe tests performed on amniotic fluid to determine risk of Hemoyltic Disease of the Fetus and Newborn (HDFN) and fetal maturity. Define amniocentesis and list special precautions needed for this procedure. Describe the handling and processing procedures for testing amniotic fluid. Explain the principle of spectrophotometric analysis of amniotic fluid for bilirubin and the interpretation of results as to level of risk to the fetus. Evaluate the L/S ratio including its significance and normal value in a mature fetus. Identify the significance of phosphatidylglycerol and the “foam” or “shake” test. Explain the significance of alpha fetal protein and cytogenetic analysis of amniotic fluid. This session will begin with an overview of how pregnancy is detected. The unit’s primary focus will cover the formation and composition of amniotic fluid; the collection and laboratory testing procedures used to aid in the evaluation of the health and well being of the unborn child. Objectives are listed on this slide, however, you should refer to the complete objective listing as posted at the course website – as there may be revisions or additions.

35 Urine Pregnancy Testing
Pregnancy tests detect the hormone produced in pregnancy, beta-human chorionic gonadotropin hormone (β-hCG). A natural hormone produced in very small quantities in all persons, male / female. Following fertilization of an ovum, the special cells in the chorionic layer of the developing placenta produce increased amounts. Levels essentially double daily, until a peak level is normally reached near the end of the first trimester. The hormone begins showing up in the 8-10 days after fertilization (2-3 days after implantation of the embryo) Pregnancy tests detect the hormone , beta-human chorionic gonadotropin (β-hCG). This is A natural hormone produced in very small quantities in all persons, whether male / female . Following the fertilization of an ovum, specialized cells in the chorionic layer of the developing placenta begin to produce ever increasing amounts of HCG – with the production leveling off near the end of the first trimester. In most cases, the hormone becomes detectable in concentrated urine 8-10 days after fertilization. >>>>>>>>>>>>> a hormone that is secreted in urine within 2–3 days after implantation of the embryo (or approximately 8–10 days after fertilization). Levels of this hormone rise rapidly after conception and remain elevated in pregnancy, peaking in the first trimester of pregnancy. Some tests performed on serum can detect pregnancy much earlier, within days of conception. One reason that serum is able to detect pregnancy earlier is that the levels of the hormone -hCG vary a great deal due to the concentration of the urine, yet the levels are relatively stable in serum. Still, collecting a urine specimen is easier and urine pregnancy test kits are available over the counter. The best specimen for urine pregnancy testing is the first morning urine, which is the most concentrated specimen. For optimal results, the specific gravity should be or higher.

36 Urine Pregnancy Testing
Pregnancy tests detect the increased amount of, beta-human chorionic gonadotropin hormone (β-hCG). Pregnancy testing can be done on blood or urine. Blood levels detected earlier and are more constant Urine levels vary depending upon the state of hydration (first morning specimen or one that has SpGr > 1.015) Pregnancy testing may be performed on either blood or urine. Many authors promote the blood serum specimen testing as the HCG levels in the serum are more constant and a positive result can be detected much earlier, - within days of conception - however, the urine sample is still more frequently tested – as the sample is easier to obtain and urine pregnancy tests are available over the counter. Appropriate Accuracy and reliability of the urine pregnancy test results can be obtained, IF: the testing procedure is carefully followed, the test results properly evaluated, AND if the first morning’s urine sample, is used in the testing. The first morning urine specimen is most desirable because it is the most concentrated. Most urine pregnancy test manufactures state: that for optimal results, the specific gravity should be or higher.

37 Urine Pregnancy Testing (cont.)
Enzyme immunoassays (EIAs) are most popular methodology. Can show a positive in as little as 10 days after conception. Example of how results are reported: hCG negative or hCG positive False results can occur False negatives – generally due to testing too early False positives Misinterpretation of results , not following directions Contamination with large amounts of blood, protein, bacterial contamination True positive, but patient NOT pregnant Trophoblastic tumors, choriocarcinoma, germ cell tumors, etc. Enzyme immunoassays are the most popular type of test kit, but whatever the method, follow the product manufacturer’s guideline. You must also follow the laboratory’s protocol as to how the results are to be reported. An example is provided on this slide. False negative results – are seen if testing occurs too early in the pregnancy. In this case, the patient should re-collect a fresh sample the next morning - or better yet, the morning following that -for repeat testing -– as the hormone levels double daily in the early stages. False positives are most often associated with misinterpreting the results , or not following the manufacturer's directions. Testing a sample that contains large amounts of blood, or protein, or is heavily contaminated with bacteria can also give misleading results There are rare cases - where the patient is not pregnant, - but the test is positive. Increased HCG levels are seen in choriocarcinoma, trophoblastic and certain germ cell tumors. The test result is true positive, as an increased level of the HCG is found.

38 Amniotic Fluid Physiology, Composition and Formation
Contained within the amnion (The membranous sac surrounding the fetus). Function Provides protective cushion Allows exchange of water, nutrients, biochemical products Formed by Maternal circulation/plasma (early) Transfer of water across placental membrane Metabolism of fetal cells Fetal urine 36 weeks) Amniotic fluid is found around the developing fetus, inside a membranous sac, called the amnion. This fluid serves to cushion and protect the developing fetus and also serves a key role in the exchange of water and molecules between the fetus and the mother’s circulation. Initially the amniotic fluid is a strictly a product of the placenta and has a composition similar to that of the mother’s plasma . As time goes on, fetal metabolism by-products and the fetal urine adds to the composition of the fluid.

39 Amniotic Fluid Physiology, Composition and Formation
Volume weeks. Composition Similar to maternal plasma with sloughed fetal cells. Fetal urine increases creatinine, urea & uric acid Rise in creatinine levels after 36th week can be used to evaluate fetal age < 36 weeks = 1.5 – 2.0 mg/dL > 36 weeks = > 2.0 mg/dL Fetal lung secretions Lecithin & sphingomyelin surfactants. The volume of amniotic fluid increases steadily throughout the pregnancy , with the peak level of about 700 – 1200 mL occurring at around 34 weeks of gestation. The fetus begins producing / releasing around 36 weeks of gestation and increasing levels of urine creatinine can be detected in the fluid. After 36 weeks of age, the amniotic fluid creatinine level will be greater than 2.0 mg/dL. This information was once utilized to determine the fetal age – but has been replaced by ultrasound measurements of the unborn child’s skeletal development. Breathing movements of the fetus releases pulmonary substances into the amniotic fluid. Notable components within these fluids are the surfactants lecithin and spingomyelin. A ratio of the concentration of them, the L/S ratio, is a reference method used in the evaluation of lung maturity and will be presented in more detail on a later slide.

40 Amniotic Fluid Indications for analysis
Abnormal screening blood tests: maternal alpha fetal protein, human chorionic gonadotropin, unconjugated estriol Metabolic disorders, such as Tay Sachs Neural tube defects – such as spinal bifida or an encephalic Abnormal chromosome analysis and history of genetic disorders - such as Down’s syndrome Abnormal ultrasound In later pregnancy for possible early delivery Fetal lung maturity, hemolytic disease of the newborn (HDN), infection, confirmation of gestational age, fetal maturity, etc. The procedure for obtaining the amniotic fluid is called amniocentesis. Although the procedure is now considered ‘routine’, it is not routinely performed without cause – as there are numerous potential complications. Listed on this slide are some of the reasons amniocentesis is performed. Take a few moments to review and note these reasons. >>>>>>>>>>>>>>>>>> Amniocentesis is performed to 1. follow up on an abnormal blood screening test: 2. To look for a potential abnormal chromosome structure, if there is a family history of genetic disorders - such as Down’s syndrome 3. Amniocentesis may be performed to follow-up on an abnormal ultrasound 4. Or later pregnancy, amniocentesis may be performed to assess the unborn child’s condition and determine if an early delivery is both warranted and advisable. >>>>>>>>>>>>>> Fetal lung maturity, hemolytic disease of the newborn (HDN), infection, confirmation of gestational age, fetal maturity, etc.

41 Amniotic Fluid Specimen collection Amniocentesis (using ultrasound)
16-42 weeks gestation @ mL through fine needle collected in sterile syringes Immediately transfer into sterile tubes (brown colored for protection from light) Puncture heals and liquid replenished within 48 hrs. Before the start of the procedure, the mother may be given a local anesthetic that will relieve the pain felt during the insertion of the needle. After the anesthetic is in effect, the needle is inserted through the mother's abdominal wall, then through the wall of the uterus, and finally into the amniotic sac. The physician uses ultrasound to guide the placement of the needle in an area away from the fetus and then will extract approximately 20 – 30  ml of amniotic fluid. The amniotic fluid sample is then placed into one or more sterile tubes. If the sample is to be tested for bilirubin, it must be protected from light – in which case it should be placed into brown colored light - impermeable tube. >>> In regard to the fetus, the puncture heals and the amniotic sac replenishes the liquid over the next 24–48 hours.

42 Amniotic Fluid General Handling and processing
Special precautions Cytogenic study specimens at RT or 37 degrees Fetal lung maturity testing specimens must be kept cold until tested Specimens for bilirubin testing must be protected from light exposure and process immediately Other chemistry tests require separation of cells, etc. from the fluid to preserve constituents. Amniotic fluid samples require special Handling and processing procedures. Samples for cytogenic studies are generally held at room temperature or at 37 degrees; while fetal lung maturity testing specimens must be kept cold until tested. Samples that are to be tested for bilirubin, must be protected from light exposure, processed and tested immediately before a significant amount of deterioration occurs. Other chemistry tests require separation of cells, etc. from the fluid to preserve constituents. Be sure to consult with the lab’s protocol for the procedures and processing steps at your clinical facility.

43 Amniotic Fluid Color & Appearance Normally colorless – pale yellow
Some turbidity is normal - cellular debris, especially late in fetal development period. Normal amniotic fluid is colorless to pale yellow and may have some slight to moderate turbidity from the presence of cells.

44 Amniotic Fluid Color & Appearance cont.
Blood streaked - traumatic tap, abdominal hemorrage, intra-amniotic hemorrhage Fetal blood vs maternal blood: use Kleihauer-Betke Yellow – bilirubin Dark green- meconium Dark red-brown – probable fetal death has occurred. Amniotic fluid that is streaked with blood may indicate a traumatic tap, abdominal hemorrhage or an intra-amniotic hemorrhage. Blood can appear as pink or red. Determining the source of the blood, as to whether it is fetal or maternal in origin, can be distinguished by the Kleihauer- Betke test for fetal hemoglobin. A dark yellow or amber color to the amniotic fluid is associated with bilirubin, whereas a green color indicates meconium, the newborn’s first fecal bowel movements. A very dark red-brown amniotic fluid is associated with fetal death.

45 Amniotic Fluid testing
Cytogenic analysis Determination of chromosomal abnormalities and certain metabolic defects Picture by Clare O’Connor, PhD, Biology Dept., Boston College. Prenatal Screens Detects Fetal Abnormalities., Nature Education. Depending on a suspected diagnosis or condition, the amniotic fluid under goes any number of tests: that may include a cytogenic evaluation. Please take a few minutes to review the picture on this slide - which shows the basic steps in obtaining and processing the amniotic fluid for cytogenic analyses. The unit’s objectives don’t require extensive recall knowledge on this procedure, but I feel this picture by Dr O’Connor provides an excellent overview of the process and tests performed during a cytogenic evaluation of amniotic fluid. >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Scitable by nature education Prenatal screen detects fetal abnormalities by Prenatal Screen Detects Fetal Abnormalities By: Clare O'Connor, Ph.D. (Biology Department, Boston College) © 2008 Nature Education  Citation: O'Connor, C. (2008) Prenatal screen detects fetal abnormalities. Nature Education 1(1) A cytogenic evaluation will determining whether the infant has any chromosomal abnormalities or has inherited a metabolic defect, such as Tay Sachs. I have included a picture depicting this process. After the amniotic fluid is extracted, the fetal cells are separated from the sample. The cells are grown in a culture medium, then fixed and stained. Under a microscope the chromosomes are examined for abnormalities. The most common abnormalities detected are Down syndrome (trisomy 21), Edwards syndrome (trisomy 18) and Turner syndrome (monosomy X). Some cells are lysed and their contents analyzed for enzymes used to evaluate metabolic defects such as Tay Sachs. Cytogenic analysis is not performed on all specimens – as indicated on this slide. Cells cultured Chromosomess evaluated for appropriate number and completeness Some cells lysed and contents analyzed for enzymes to evaluate for metabolic defects, such as Tay Sachs. Not done on all patients >35, or history of problems Increased AFP Known carriers. >>>>>>>>>>>>> Th picture contact for permission information Depending on a suspected diagnosis or condition, the amniotic fluid may under go a number of tests.

46 Amniotic Fluid testing
Cytogenic analysis Determination of chromosomal abnormalities and certain metabolic defects Cells cultured Chromosomess evaluated for appropriate number and completeness Some cells lysed and contents analyzed for enzymes to evaluate for metabolic defects, such as Tay Sachs. Not done on all patients Patient is >35yrs. or has history of problems Increased AFP Known carriers. An extensive cytogenic evaluation of the amniotic fluid is not performed on all specimens. Mothers – to - be who are over 35 years of age or who have a family history of heritable conditions would be candidates. After the amniotic fluid is extracted, the fetal cells are separated and grown in a culture medium. Some cells will be processed, placed on a slide, fixed, stained and their chromosomes examined under a microscope. The most common abnormalities detected are Down syndrome (trisomy 21), Edwards syndrome (trisomy 18) and Turner syndrome (monosomy X) sometimes called XO, meaning there is only one sex chromosome – a single X. Some cells are lysed and their contents analyzed for enzymes used to evaluate metabolic defects such as Tay Sachs.

47 Amniotic Fluid testing in HDN
Hemolytic Disease of the Newborn (HDN) Also called Hemolytic Disease of the Fetus and Newborn (HDFN) or erythroblastosis fetalis. “classical” case: Rh-negative mothers with Rh+ infants Other red blood group discrepancies between Mom and Baby can also produce HDN Fetal cells with antigen foreign to the Mom enter her circulation and stimulate the production of antibodies. Danger increases with each exposure. In Hemolytic Disease of the Fetus and Newborn, also known as erythroblastosis fetalis, mother develops antibodies to an antigen on the fetal erythrocytes that the baby inherited from the father. The most severe form of HDN has typically resulted when Rh negative mothers deliver Rh positive infants, but anytime a baby inherits blood group antigens from its father – and the mother does not have the same antigens, - a potential blood group discrepancy or problem could be produced. Each time the Rh negative woman carries and delivers an Rh positive baby, her body gets re-introduced to the foreign antigens and she responds by making more and more anybodies which cross the placenta and attack the unborn child’s red cells. Modern preventive measures, such as prenatal screening and the administration of a commercially made immune globulin / antibody that targets the Rh positive baby cells that may enter the mother’s circulation during pregnancy, have dramatically lowered the incidence , but have not totally eliminated HDN.

48 Amniotic Fluid testing for Fetal Distress
Hemolytic Disease of the Newborn (HDN) Maternal antibodies cross the placenta and destroy fetal cells with the corresponding antigen Bilirubin from RBC destruction appears in the amniotic fluid Some level occurs naturally from normal RBC catabolism The amount of unconjugated bilirubin present correlates with the amount of RBC destruction As they age, red blood cells are removed from circulation to be catabolized and useful component parts are re-cycled. The heme-portion of the hemoblobin molecule, is degraded through a number of steps and the resulting product is generically referred to as: bilirubin. Through this normal process, some amniotic fluid bilirubin is expected due to normal amount of red cell catabolism. In HDN, however, the red cells are destroyed in greatly increased numbers resulting in appearance of an elevated unprocessed bilirubin, called unconjugated bilirubin in the amniotic fluid. The amount of unconjugated bilirubin present correlates with the amount of fetal red cells being destroyed. >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Further metabolism of the bilirubin results in conjugated bilirubin which is not cleared by the placenta and build up in the amniotic fluid. With this hemolytic disease process, the high unconjugated bilirubin triggers early production of fetal hepatic glucuronyl transferase activity and this unconjugated bilirubin is converted to conjugated bilirubin. The conjugated bilirubin is not cleared by the placenta, and variable amounts of the conjugated bilirubin are found in the amniotic fluid.

49 Amniotic Fluid testing for Fetal Distress
Purpose of bilirubin testing on amniotic fluid: Measurement of bilirubin is an indication of degree of hemolysis occurring in utero, therefore an indication of danger of anemia in the fetus. Again, amniotic bilirubin levels indicate the degree of fetal red cell hemolysis that is occurring in utero; therefore provides an indication of the level of danger to the fetus from anemia. In other words, The bilirubin concentration can be correlated to the severity of HDN.

50 Amniotic Fluid testing for Fetal Distress
Bilirubin by spectrophotometric analysis Scan fluid at increasing wavelengths Plot readings against a baseline Measure difference between baseline and peak bilirubin at 450 nn Measuring bilirubin in the amniotic sample is best performed by spectrophotometric analysis. A spectrophotometer is an instrument capable of passing light a specific or pure wavelength through the fluid and effectively determining how much of the light was absorbed by the fluid. When light of different wavelengths is passed through the fluid and absorbance readings taken an absorbance spectrum of the fluid is created. An example absorbance spectrum for amniotic fluid is shown in the graph on the left side of this slide. The absorbance spectrum of amniotic fluid is measured between the wavelengths of 365 and 550 nm. The amount that the curve deviates from a straight line at the 450 nm wavelength… is directly proportional to the amount of bilirubin in the amniotic fluid. It is important that the amniotic fluid specimen be thoroughly centrifuged before this analysis to remove any contaminate blood – which would absorb light and be measured as oxyhemoglobin. In addition to obtaining the measurement of the bilirubin, the absorbance difference can also be used on a Liley graph as shown on the right side of the slide. Plotting the delta 450 absorbance difference - against gestational age will place the fetus into one of three (3) zones based on hemolytic severity Zone I: mildly affected fetus Zone II: requires careful monitoring Zone III: severely affected fetus, may require induction of labor or intrauterine exchange transfusion This information along with other test results can be used by the physician to aid in deciding whether to induce labor or to perform an intrauterine blood transfusion exchanges in cases of HDN. Plot difference on Liley graph, against gestational age

51 Amniotic Fluid Testing for Neural Tube Defects
Anencephaly Spinal bifida Tests (these are usually tested together) Alpha fetal protein (AFP) Peaks at 16 weeks gestation Acetylcholinesterase **Test affected by presence of blood or hemolysis A neural tube defect is an opening in the spinal cord or brain cavity that occurs during the first couple weeks of development. Neural tube defects are one of the most common birth defects, occurring in approximately one in 1000 live births in the US. Fetal neural tube defects, such as anencephaly and spinal bifida cause elevated alpha fetoprotein or AFP in both the amniotic fluid and the mother’s circulation. In normal fetal development, AFP peaks at about 16 weeks of gestation and then declines gradually to term. With neural tube disorders, the neural tube is open and AFP is released from the cerebrospinal fluid that leaks directly into the amniotic fluid, and amniotic AFP levels are much higher. Testing for (amniotic acetylcholinesterase) is more specific than AFP and is sometimes used as a confirmatory test. Test results are affected by blood or hemolysis.

52 Amniotic Fluid Testing for Fetal Lung Maturity
-To determine whether fetus is capable of surviving an early delivery. Hyaline membrane disease Also called neonatal respiratory distress syndrome Major complication of early delivery & Most common cause for death of premature newborn. Immature lungs lack of lung surfactants, which allow lung alveoli to be able to open during when breathing. Surfactants decrease surface tension, permitting alveoli inflation. Fetal lung surfactants include three phospholipids: lecithin (also known as phosphatidylcholine), **major lung surfactant. sphingomyelin, phosphatidyl glycerol. Many tests developed to assess for fetal lung maturity. Tests for Fetal Lung Maturity are used to determine whether fetus is capable of surviving an early delivery. Lungs require a surfactant or soap-like substance, to reduce surface pressure and allow the lung tissue to inflate. Lungs in the premature baby have limited ability to fully inflate resulting in a neonatal respiratory distress syndrome, also called hyaline membrane disease. Lecithin (also known as phosphatidylcholine), is the major lung surfactant Other surfactants include sphingomyelin, and phosphatidyl glycerol. .

53 Amniotic Fluid Testing for Fetal Lung Maturity
Shake test & Foam stability Both tests utilize dilutions of amniotic fluid in 95% ethanol and look for formation of a persistent ring of foam / bubbles, an indication of total surfactant concentration. Shake test – crude, fast, cheap and can be performed at bedside. Physician can make an immediate decision regarding safety of early delivery of infant. 1:2 dilution (amniotic fluid : 95% ethyl alcohol), shake 15 seconds, If a complete ring of foam persists 15 minutes = positive test. Foam stability index – places fixed amount of amniotic fluid in series of tubes with increasing amounts of 95% ethanol (from 0.43 to 0.55) Dilutions are shaken vigorously and the higher concentration of 95% alcohol that is able to suppress a foam ring is known as the foam stability index Foam stability index >0.47 indicates fetal lung maturity Among the first tests for fetal lung surfactants were the very simple screening tests: The Shake and Foam stability tests. Both tests utilize dilutions of amniotic fluid in 95% ethanol and look for formation of a persistent ring of foam / bubbles, an indication of total surfactant concentration. All other information regarding these tests has been placed on the powerpoint slide. <>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Foam Stability This is a screening test for fetal lung surfactant in amniotic fluid. In this test, a fixed amount of amniotic fluid is mixed with an increasing volume of 95% ethanol in a series of tubes with alcohol concentrations ranging from 0.43 to The mixtures are shaken vigorously for 30 seconds, and the contents are allowed to settle for 15 seconds and the samples are examined for an uninterrupted ring of foam in the tube. The highest concentration of 95% ethanol that is able to support a ring of foam is known as the foam stability index. The principle of the test is that more surfactant is needed to maintain the foam in greater concentrations of ethanol and more fetal lung surfactant is needed to support fetal lung function at birth. An index of 0.47 or higher is considered to indicate enough fetal lung surfactant for fetal lung maturity.

54 Amniotic Fluid Testing for Fetal Lung Maturity
Lamellar bodies “packets” of surfactant lipids produced by pneumocytes Size 1-5 um (slightly smaller than platelets) Can run on automated cell counters (platelet mode) Lamellar body count > 30,000 / uL is highly predictive of pulmonary maturity Count < 10,000 suggest risk for RDS – respiratory distress syndrome Test not affected by hemolyzed blood or meconium Lamellar bodies are the storage form of surfactant. The detection of amniotic lamellar bodies is a rapid and simple way of assessing fetal lung maturity. The test requires <1 ml of amniotic fluid and takes 15 minutes to perform. The fluid is aspirated through a particle counter analyzer – similar to a hematology cell counter and the lamellar bodies counted. A lamellar body count >30,000/μl is highly predictive of pulmonary maturity, while a count <10,000/μl suggests a risk for RDS. Neither meconium nor lysed blood has an effect on the lamellar body count.

55 Amniotic Fluid Testing for Fetal Lung Maturity
Lecithin / Sphingomyelin ratio Lecithin is the major lung surfactant The role of sphingomyelin is not established Levels equal until 33 weeks of gestation @ 1/1 ratio After 34 the week of gestation, lecithin production greatly increases as compared to the sphingomyelin L/S ratio 2.0 or greater indicates lung maturity 2X as much lecithin / spingomyelin Lecithin, the major lung surfactant, and sphingomyelin are produced in relatively equal amounts during the pregnancy – up until the 33 week. After 34 weeks of gestation, the level of sphingomyelin decreases, while the level of lecithin increases significantly. A ratio of lecithin:sphingomyelin (L/S) of 2.0 or greater is an indication of fetal lung maturity.

56 Amniotic Fluid Testing for Fetal Lung Maturity
Phosphatidyl glycerol Not detected until 35 week of gestation Delayed in cases of maternal diabetes Amniostat FLM - PG Immunological test for phosphatidyl glycerol Uses antibodies for detection, thereby not affected by the presence of hemolysis or blood A measurement of phosphatidyl glycerol (PG) has also been found useful in evaluation of the fetal lung maturity. PG is not generally detectable until after the 35 week of gestation. The Amniostat-FLM –PG is a commercial product that uses antibodies to detect the phosphatidyl glycerol fetal lung surfactant. This is a rapid , qualitatitive slide agglutination assay test. A positive result occurs when the PG concentration reaches approximately 0.5 uG/mL or greater, which would indicate a sufficiently mature fetal lung. An advantage to this immunological test is that it is not affected by blood or meconium that might be present in the amniotic fluid.

57 Amniotic Fluid Testing for Fetal Lung Maturity
Microviscosity Fluoresent dye binds with surfactants and albumin. Test run on Abbot TDx and results correlate well with L / S ratio Microviscosity is another approach to evaluating fetal lung surfactants. All information is provided on the powerpoint slide.

58 Amniotic Fluid Testing
Creatinine Fetal age determination (at 36 weeks, fetal kidneys excrete >2.0 mg/dL creatinine) This test has been replaced by ultrasound measurements. Creatinine still used as: Measurement as means of determining a fluid to be amniotic or urine. Creatinine level up to 3.5 mg/dL & urea 30 mg/dL can be found in amniotic fluid Urine levels of mg/dL 300 mg/dL for urea. Creatinine levels in the amniotic fluid were at - one time - used to determine approximate fetal age. Today, determining the age of the fetus is routinely achieved using ultrasound measurements which are much more accurate and do not require the invasive amniocentesis procedure. Creatinine levels are still used to aid in the determination of whether a fluid from an unknown source is urine or amniotic fluid. As indicated on the slide, much higher creatinine levels are seen in urine.

59 Urinalysis and Body Fluids CRg
Welcome to the third session of Unit 5. Unit 5 3 Sweat Fluid Analysis

60 Sweat Fluid Analysis - objectives
Define cystic fibrosis (CF). Describe the methodology of the sweat chloride test. Analyze the sweat chloride values seen in patients suspected of having CF In this short session, the primary focus will be centered around Cystic Fibrosis and testing of sweat chloride levels to aid in its diagnosis. Although objectives are listed on this slide, you should refer to the complete objective listing as posted at the course website – as there may be revisions or additions.

61 Cystic Fibrosis (CF) Cystic fibrosis
1/25 white Americans are carrier of CF gene Autosomal recessive, 1/1500 – 1/2000 Caucasian births. Most common fatal inherited disease of Americans 1000 new cases diagnosed / year Most patients diagnosed before 2 years of age If diagnosed early, many will live to adulthood; ½ die before age of 30. Affects mucous secreting glands Very thick mucous produced Lungs, pancreas, GI tract, sweat glands According to the Cystic Fibrosis Foundation web page, Cystic fibrosis or CF, is an inherited chronic disease that affects the lungs and digestive system of about 30,000 children and adults in the United States (70,000 worldwide). A defective gene is inherited in an autosomal recessive pattern, meaning that that both males and females are affected , having inherited a defective gene from each parent. About 1 out of 31 Americans are carriers of a defective CF gene. CF is most common in Caucasians, but can affect all races. The defective gene and its protein product cause the patient’s body to produce unusually thick, sticky mucus that: clogs their lungs, leading to life-threatening lung infections The mucous also causes obstructions the pancreas and GI tract which stops natural enzymes from helping the body break down and absorb food. About 1,000 new cases of cystic fibrosis are diagnosed each year. More than 70% of patients are diagnosed by age two. More than 45% of the CF patient population is age 18 or older. The predicted median age of survival for a person with CF is in the mid-30s. >>>>>>>>>>>>>>>>>>> Approximately 30,000 children and adults in the United States have cystic fibrosis. An additional ten million more—or about one in every 31 Americans—are carriers of the defective CF gene, but do not have the disease. CF is most common in Caucasians, but it can affect all races.

62 CF – Symptoms vary: CF Symptoms
Coughing due to respiratory distress & frequent lung infections Viscous stools cause GI obstructions Pancreatic insufficiencies Leads to frequent greasy, bulky stools Failure to thrive Salty –tasting skin There are many Symptoms of Cystic Fibrosis and they vary from person to person. More common symptoms include persistent coughing , wheezing or shortness of breath and frequent lung infections. CF Patients produce frequent greasy and bulky stools and have difficulty in bowel movements. CF patients often have difficulty in gaining and maintaining much weight – a condition often summarized as: failure to thrive. Cystic Fibrosis patients have excessive sodium chloride in their sweat – making their skin taste salty

63 CF – Treatments vary: CF Treatments: Mycolytic agent Antibiotics
Bronchodilator to liquefy and expedite removal of mucous from lungs Antibiotics Anti-inflammatory medications such as ibuphrofen Physical therapy – breathing exercises People living with cystic fibrosis must follow a regular treatment routine to stay healthy and maintain optimal lung function. Most often, treatment for cystic fibrosis begins with techniques to trigger strong coughs that help loosen and clear thick mucus from the airways. Mucolytic agents are commonly used. These including medications and treatments such as bronchodiators are used to liquidfy the mucous and facilitate its removal from their lungs. Antibiotics are routinely used to combat frequent lung infections as well as Anti-inflammartory drugs such as ibuprofen . And physical therapy exercises to strengthen muscles and encourage proper breathing are also routinely used.

64 Sweat test - Laboratory Procedure
(Pilocarpine iontophoresis) Pilocarpine – chemical that will induce sweating Iontophoresis – mild electrical current The Sweat Test has been the “gold standard” for diagnosing cystic fibrosis (CF) for more than 50 years. When it is performed by properly trained technicians, and evaluated in an experienced, reliable laboratory, the sweat test is still the best test to diagnose CF. The cystic fibrosis foundation recommends that the sweat test be performed at a facility that follows strict guidelines to make sure the results are accurate. The technical name for the sweat test procedure is: (Pilocarpine iontophoresis) Pilocarpine – is a chemical that will enter the skin and increase the amount of sweat produces. Iontophoresis – is a process where a mild electrical current is used to speed up this process.

65 Sweat test - Laboratory Procedure
(Pilocarpine iontophoresis) Pilocarpine – chemical that will induce sweating Iontophoresis – mild electrical current Clean area Induce new sweat formation Collection on filter paper OR Collection in tube OR Directly measure on skin with chloride ion selective electrodes / ISEs Test sweat for chloride concentration in the sweat test or (Pilocarpine iontophoresis) procedure: Pre preparation of the skin must be carefully done - The test area is usually on the inside of the patient’s forearm. The skin must be carefully cleaned with a series of gauze pads that contain deionized water. To remove any residual soap, or creams, etc. and to remove any old dried sweat. After the skin is gently dried, a pad containing the pilocarpine is applied and secured in place. The pilocarpine along with a mild electrical current will Induce new sweat formation After the designated amount of time, the fresh sweat sample is collected . If it is collected in a tube or on a filter paper it can be returned to the lab for testing. Some instruments provide for an ion selective electrode or ISE for direct measurement. Either method, is designed to measure chloride ion concentration. The web site listed on the slide is at the Cystic Fibrosis Foundation. About half way down the page is a YouTube video of the sweat test procedure. I encourage you to take the short 4 minutes to view it.

66 Sweat Testing Results:
Normal Sweat chloride levels < 30 mEq/L (mmol/L) Sweat chloride levels of mEq/L are borderline and must be repeated. Sweat chloride (or sodium) levels greater than 50 mEq/L are consistently seen in 98% of CF patients. Normal or expected sweat chloride values vary slightly depending on the patient’s age. For the purposes of this course: values less than 30 mEq/L (which is the same as mmol/L) would not be seen in patients with cystic fibrosis Those above 30 or 40 mEq/L are considered borderline and the patient should be retested, usually on another day. @ 98% of the patients with CF will have sweat chloride levels above 50 mEq/L.

67 Reference Listing Please credit those whose work and pictures I have used throughout these prsentations. Lillian Mundt & Kristy Shanahan, Graff’s Textbook of Urinalysis and Body Fluids, 2nd Ed. Susan Strassinger & Marjorie Di Lorenzo, Urinalysis and Body Fluids, 5th Ed. Wikipedia, the free encyclopedia Cystic Fibrosis Foundation CF/ Please credit those whose work I have sited in this presentation. This concludes the session on Sweat Testing.

68 Urinalysis and Body Fluids CRg
Welcome to the 4th session of Unit 5. Unit 5 4 Gastric Fluids

69 Gastric Fluid Analysis - objectives
Describe the physiology and composition of gastric fluid; including the role of gastrin in its production. List two (2) reasons for gastric fluid analysis. Explain the special patient preparation that should occur before gastric fluid is analyzed. Define or describe the Zollinger-Ellison Syndrome, anacidity, hypochloryhydia, and achlorhydria. Describe the procedure for gastric acidity and state the clinical significance. Describe gastric fluid drug screening and its clinical significance. In this short session, the primary focus will be on the formation and composition of gastric fluid; its collection and laboratory testing procedures used to aid in the diagnosis of conditions of increased and decreased amounts of gastric acid. Although objectives are listed on this slide, you should refer to the complete objective listing as posted at the course website – as there may be revisions or additions.

70 Gastric Fluid - production
Physiology Full stomach, presence of amino acids, stimulation by vagus nerve, other factors Release of hormone Gastrin from stomach G cells Gastrin stimulates release of digestive gastric fluids from the stomach parietal cells. Inhibition / shut-down Negative feed-back system Presence of secreted HCl in stomach will inhibit gastrin release Hormones somatostatin, glucagon, calcitonin and others Gastric acid / fluid production is the result of a series of events that usually begin after a person has eaten. When the stomach is nicely full of proteins and amino acids, the G cells located primarily in the stomach, release – gastrin. Gastrin is a hormone that will, in turn, stimulate the parietal cells in the stomach to secrete gastric acid, which is primarily hydrochloric acid. Normally, the gastrin hormone production and release is restricted or shut down when there is excessive HCl in the stomach or as the result of other hormones, such as somatostatin, glucagon , calcitonin and others.

71 Gastric Fluid - composition
Composition and formation Produced by the parietal cells in the stomach under the hormonal influence of gastrin. Normal makeup of gastric fluid: * HCl, saliva, mucous, neutralizing chemicals, secretions from the intestines, bile & pancreas The Gastric acid and fluids are produced by parietal cells in the stomach under the direction of gastrin. Gastric fluids are composed of hydrochloric acid, swallowed saliva, mucous and neutralizing chemicals as well as secretions from the intestines, bile and pancreas. The hydrochloric acid component of the gastric fluids results in pH levels being as low as 1.0

72 Gastric Fluid Anacidity – lack of normal acidity
Sometimes called achlorhydria (hypochlorhydria) Absence (decrease) of HCl in gastric secretions Usually caused by Atrophy of gastric mucosa Gastric carcinoma Pernicious anemia Severe iron deficiency anemia The breakdown of the term Anacidity is - no- acid –meaning the lack of normal acidity A similar term sometimes used, Achlorhydria – more specifically means the lack of hydrochloric acid in the gastric fluid. - and hypochlorhydria – meaning decreased hydrochloric acid.. The reason for achlorydria of gastric fluids is usually due to atrophy of the gastric mucosa. Gastric carcinoma and pernicious anemia may also be reasons. In addition, Achlorhydria is also associated with severe iron deficiency anemia and decreased or lack of intrinsic factor production. Protein digestion is severely impaired in patients with achlorhydria. The combination of achlorhydria and peptic ulcers may be an indication of the presence of a gastric malignancy.

73 Gastric Fluid testing Indications for testing gastric fluids
Peptic ulcer evaluation Gastritis – inflammation of stomach wall Anacidity – inability to produce acid A gastric analysis is generally performed to evaluate gastric function by measuring the fluid contents of a fasting patient's stomach the its acidity, appearance, and volume. This test may be indicated for patients with obscure gastric pain, loss of appetite, and weight loss. It is also utilized for suspected peptic ulcer, severe gastritis, and suspected Zollinger-Ellison (Z-E) syndrome.

74 Gastric Fluid testing Indications for testing gastric fluids
Zollinger-Ellison (Z-E) syndrome Hypersecretion Gastrin secreting neoplasm usually located within the pancreatic islets Drug analysis Suspicion of recent overdose Examination of gastric fluid / aspirate for presence of pills, capsules, etc. generally performed by attending physician, or perhaps pathologist. Literature provides some information regarding quantatitive testing, ie. HPLC, GLC/Mass Spectroscopy. Detection of a hypersecretion activity is characteristic of Zollinger-Ellison syndrome. Patients with the Zollinger-Ellison syndrome have small tumors which are generally malignant. These tumors are usually found in the pancreas or perhaps the small intestine and produce gastrin hormone - which causes the stomach to hypersecrete the hydrochloric acid rich gastric fluids; as a 95% of the patients with Z-E syndrome have peptic ulcers. Another possible reason for gastric analysis is to detect the presence of drugs . Aspiration and analysis of gastric fluid may be ordered when there is suspicion of a recent overdose of a prescribed or illicit drug. In general the testing would include examination for signs of pills, tablets or capsules in the gastric fluid. This would generally be performed by the emergency room staff, or perhaps by a pathologist. The literature provides some information regarding quantatitave testing procedures utilizing high pressure liquid chromatography and gas chromatography. The literature continues on to indicate that analysis of urine for drugs would provide more valuable information. Any further discussion of this specific topic is beyond the scope of this course.

75 Gastric Fluid testing Normal Specimen Specimen collection
Nasal or oral intubation Fasting & avoid swallowing saliva Normal Specimen ≤ 75 mL Translucent, light gray Slightly viscous Laboratory procedures Gastric acidy (acid <4.0) pH paper Drug screening To obtain the gastric fluids for testing, A physician inserts a tube either nasally or orally into the stomach of the fasting patient. Prior to the procedure, the patient should be instructed to avoid swallowing saliva, which will have a pH neutralizing effect on the sample. Normal gastric fluid is translucent , pale gray and slightly viscous. It often will have a faint acrid / acid odor and its volume is generally less than 75mL. Generally, the specimen’s pH should be checked using a pH paper suitable for producing results in the acid range. In some cases, a patient may have a base line pH level checked, then be administered a medication to stimulate acid production. After a specified period of time, another fluid sample is aspirated and its pH tested to determine if the patient was able to produce acid.

76 Reference Listing Please credit those whose work and pictures I have used throughout these prsentations. Lillian Mundt & Kristy Shanahan, Graff’s Textbook of Urinalysis and Body Fluids, 2nd Ed. Susan Strassinger & Marjorie Di Lorenzo, Urinalysis and Body Fluids, 5th Ed. Wikipedia, the free encyclopedia Mosby’s Medical Dictionary, 8th 2009 Elsevier. Clinical Chemistry, Vol 22, by American Association of Clinical Chemistry. Pub Med.gov. U.S. National Library of Medicine, National Institutes of Health. Please credit those whose work I have sited in this presentation. This concludes the session on Gastric Fluid.

77 Urinalysis and Body Fluids CRg
Welcome to the 5th session of Unit 5. The primary focus of this session is the laboratory’s examination of fecal body fluid. Most laboratory technicians and technologist might prefer testing other specimens, however, testing feces can provide important information related to gastrointestinal disorders, infections, as well as several other medical conditions. The Macroscopic, microscopic, and chemical testing of feces is usually performed in the core lab. Tests are also performed in the microbiology department for various stool pathogens including bacterial, viral, and parasitic organisms as well as for the testing for their toxins. Unit 5 5 Feces & miscellaneous

78 Feces Feces Composition Bacteria,
Cellulose & other undigested foodstuffs GI secretions, enzymes, bile pigments Cells Electrolytes and water Feces are the end product of the digestive process. The majority of the digestion occurs in the small intestine and is aided by pancreatic enzymes and bile salt. Feces are largely composed of Bacteria, Cellulose & other undigested foodstuffs, but also contain gastrointestinal secretions, enzymes, bile pigments Cells, Electrolytes and water. The majority of the fluids that are consumed or are part of the digestive process are absorbed and on average only about 100 to 150 mL are excreted through the feces. If a large volume of water and fluids enters the colon and are processed through rapidly, a liquid diarrhea will result.

79 Overview of Indications for Fecal Testing
GI bleeding Occult blood Suspicion of pathogenic bacterial or parasitic infections Microscopic exam for leukocytes O&P Stool cultures Diagnosis / confirmation of a malabsorption syndrome or liver and biliary duct disorder Fecal fat & Fecal Carbohydrates Meat fibers Fetal hemoglobin Take a moment to review this slide that lists common reasons or tests for fecal testing . Most of the procedures and testing methods listed will be further outlined in this presentation. The most common detecting gastrointestinal bleeding using the ‘occult blood test’ . investigating the cause of a dysentery or unexplained diarrhea . During this process, a thin smear of Wright stained feces may be directly evaluated under the microscope for the presence of white blood cells; or the specimen may be filtered and chemically processed to eliminate debris before it is evaluated under the microscope for the presence of intestinal parasites. It is fairly common practice to culture the stool sample to detect the presence of pathogenic bacteria. Stool samples are also tested to aid in diagnosis's of various Malabsorption syndromes, Liver and biliary duct disorders, and to determine whether the blood in a newborn’s stool is its own or from its mother.

80 Diarrhea Diarrhea Common disorder of the GI tract
Increased frequency (> 3x / day) Increased amount (>200 gm stool wt / day) Associated with infections, toxins, malabsorption issues, etc. Any proper discussion of feces must include a few comments about Diarrhea ; as it is a very common disorder of the GI tract. In diarrhea the frequency and volume of bowel movements are increased and the bowel movements are more liquid. Diarrhea has been associated with infectious agents, toxins, malabsorption, and a variety of GI disturbances.

81 Diarrhea Diarrhea Mechanisms of diarrhea: 1. Secretory
Microbial infections Vibrio cholerae Some E. coli Clostridium Salmonella Shigella Staphylococcus Campylobacter Cryptosporidium Drugs, laxatives Inflammatory bowel disease / colitis Endocrine disorders, malignancy, others Diarrhea is basically the result of a large volume of fluid being presented and passed through the large intestine. There are three main mechanisms or categories of causes of diarrhea. Although various causes of diarrhea stated here are categorized, keep in mind that there is often an overlap, where a particular reason for the diarrhea may cause more than one mechanism or outcome. A diarrhea that is classified as ‘Secretory’ – is the result of either an active secretion of ions and fluid into the bowels - or an inhibition of the absorptive processes. One of the most classical examples of this type of diarrhea - occurs as a result of cholera toxin - which causes the secretion of ions, especially chloride ions into the bowel. When the negatively charged chloride ions are secreted, positively charged sodium ions are also eliminated. And where sodium goes, so too does water. In addition to Vibrio cholerae, there are a number of other Microbes as listed, that are known to cause a Secretory – type of diarrhea. There are other reasons for secretory diarrhea that include : Some Drugs, and laxatives; as well as inflammatory bowel disease/colitis, endocrine disorders, malignancy, and collagen vascular diseases.

82 Diarrhea Diarrhea Mechanisms of diarrhea: 2. Osmotic imbalance
Incomplete digestion / absorption Lactose intolerance Amebiasis, antibiotics, laxatives, antacids Irritable bowel syndrome, others Diarrhea can be the result of an osmotic imbalance ; due to increased amounts of ‘osmotically active’ substances remaining in the GI tract. Osmotically active substances - are substances that draw water toward them. A great example is the milk sugar - lactose. If a person is lactose intolerant, they do not produce much, if any, lactase enzyme. With out this enzyme, the lactose molecule is not broken down into the smaller molecule sugars, galactose and glucose which are more readably absorbed. Because the lactose is not digested in the stomach and small intestine, it enters the colon, - where enteric microbes quickly begin fermenting it. This fermentation process results in the production of gas which results in the classical symptoms associated with lactose intolerance. These include stomach cramps, bloating, acid reflux and flatulence - along with the diarrhea.

83 Diarrhea Diarrhea Mechanisms of diarrhea:
3. Altered motility Hypermotility with decreased absorption Irritable bowel syndrome, others Ramifications – diarrhea can easily result in dehydration and critical electrolyte imbalances. Diarrhea can also occur as a result of increased intestinal movement ( / hypermotility) – which results in decreased intestinal absorption. A number of conditions can result in hypermotility; including Irritable bowel syndrome - which can be brought on by a food , or stress, etc. Diarrhea is not an easy topic to discuss; but it is no joke either. What ever the cause, prolonged Diarrhea can quickly result in dehydration and critical electrolyte imbalances. – According to Wikipedia, Diarrhea is a common cause of death in developing countries and is the second most common cause of infant death world wide.

84 Other Disorders of the GI Tract
Malabsorption Abnormal digestion or absorption of one or more nutrients May lead to malnutrition or anemia Maldigestion Impaired digestion due to lack of digestive enzymes Malabsorption is a state of abnormal digestion or absorption of a single nutrient or of multiple nutrients through the GI tract that may lead to malnutrition or anemia. Fats, meat fibers, and carbohydrates all may be improperly digested and malabsorbed. Maldigestion is impaired digestion which is caused by lack of digestive enzymes. And can also lead to a malnutrition situation.

85 Other Disorders of the GI Tract
Colorectal cancer Relatively common cancer of GI tract Associated with ‘occult’ / hidden blood loss Pancreatic insufficiency and cystic fibrosis Decreased pancreatic digestive enzymes Trypsin Chymotrypsin Elastase I Results in maldigestion Other disorders of the digestive track that are of concern to us include: Colorectal cancer and cystic fibrosis. Colorectal cancer is a relatively common cancer. In colon cancer, there is an increased loss of blood through the GI tract – which can often be detected by occult blood testing. The term “occult blood” refers to hidden blood or small amounts of fecal blood that are often not visible to the naked eye. The “occult blood test’ is the most commonly performed test on fecal samples. This test will be presented in more detail later in this presentation. Cystic fibrosis, is a hereditary disease affecting mucous secretion in the pancreas and lungs. In cystic fibrosis, and other pancreatic insufficiency conditions, there are decreased levels of pancreatic digestive enzymes such as trypsin, chymotrypsin, and elastase I, - which lead to / resulting in maldigestion. Levels of These enzymes can be tested in feces.

86 Fecal Specimen Collection
Patient needs detained instructions Clean container, avoid contamination with urine or toilet water Qualitative tests require only small amount of random sample Quantitative / timed specimens, may require collection over several days. May require entire sample during the time period (72 hr fecal fat) or small amounts taken over several days (O& P) In some tests, timing of collection is important Some tests require restrictions of diet (occult blood) As for other body fluid specimen collections: proper collection of the fecal sample is important to obtain accurate and useful test results. To obtain the proper specimen, the patient must have detailed instructions. Clearly written instructions in the patient’s native language are always best. Patients must understand that the fecal sample should not be contaminated with urine or water. Toilet water often contains strong oxidizing cleaners that interfere with laboratory testing, and protozoa can be destroyed by urine contamination. The patient should be provided with the proper sample collection container. The type of container and the amount of specimen to be collected will depend upon the tests to be performed, but for most fecal specimens, the container simply needs to be clean, dry, sealable, and leakproof. A secure lid is very important. The use of enemas or barium sulfate - which is used in radiological examinations, will affect the stool sample often making them difficult to read microscopically. In these situations, it is best to wait until normal bowel movements have resumed before collecting the test specimen. Timing of specimen collection is important in some tests, such as for parasite examinations or quantitative fat testing. Most of the fecal tests require the patient adhere to a dietary plan for several days prior to the specimen collection time period.

87 Fecal Laboratory Procedures
Color / Appearance Possible Cause(s) Brown (normal) Normal – presence of urobilin (from bacterial breakdown of urobilinogen / stercobilinogen Black *Upper GI bleeding, Iron therapy, or some medications Red *Lower GI bleeding. Beets, food coloring & some meds. Pale yellow, white, gray Giardia infection. Bile – duct obstruction. Barium sulfate Green Strongly green vegetables. Some oral antibiotics. Biliverdin Bulky / frothy Bile-duct obstruction. Pancreatic disorders Ribbon-like Intestinal constriction Mucous /blood streaked Colitis, dysentery, malignancy, constipation The chart on this page is to assist in correlating the color & texture or appearance of stool samples with possible causes. The information was abstracted from the a table of Macroscopic Stool Characteristics’ in the “Urinalysis and Body Fluids” reference textbook by Susan Strasinger. The gross appearance including the color and appearance or consistency of the fecal sample can provide some clues to possible GI disorders. Review the chart in correlation to the information in your textbook. >>>>>>>>>>>>>>>>>>>>>>>>>>>>> The normal fecal specimen is dark brown; due to the oxidation of urobilinogen in the intestines, the color changes to orange brown urobilin. The presence of blood-streaked mucus or mucus with pus or eosinophils often accompanies bacterial or amebic dysentery. Dysentery is associated with damage to the intestinal wall due to invasion by these organisms Black color may indicate older blood from the upper GI tract, whereas bright red blood is more likely to be from the lower GI tract. Bright red blood in feces is known as hemachezia. A very pale stool (called an acholic stool) often indicates a biliary obstruction. Another common reason for a pale stool is the presence of barium sulfate from a barium enema performed for radiological testing. This barium sulfate will interfere with some fecal examinations, especially microscopic examinations for fat, fibers, or parasites. A ribbonlike fecal specimen could be associated with GI tract obstruction.. *Color varies shades of red to black depending upon where bleeding occurs in the GI tract.

88 Fecal Laboratory Procedures
Microscopic Examination for WBC Fecal leukocytes Mucous with blood / pus often seen in dysentery and damage to intestinal wall. Methylene blue, Wrights or Gram stain may be used to visualize WBCs. Wrights best for cell differentiation Can indicate pathogenic bacterial infection or ulcerative colitis Neutrophils associated with bacterial infection Eosinophils associated with amebic infestation. The first of the fecal laboratory procedures to discuss is the Microscopic exam for WBC. Mucous with blood or pus or is often seen with bacterial or amebic dysentary and damage to the intestinal wall. Fecal leukocytes, especially neutrophils, are commonly associated with dysentery or invasion of the intestinal wall. In amebic infections, eosinophils are also often present. Wet preparations of the loose stool are made, stained with methylene blue and examined microscopically for fecal leukocytes. Alternatively, dried smears of the stool sample can be stained with Wright’s stain – which greatly improves the ability to differentiate the cells. The Gram stain is also sometimes used, but Wright’s stain is best for differentiation. As noted on the slide, the type of WBC found can provide insight as to the type of infection occuring. There is also a latex agglutination test to detect fecal leukocytes. This test detects lactoferrin, an enzyme found in the granules of granulocytes. If positive, it strongly suggests the presence of these WBC.

89 Fecal Laboratory Procedures
Microbiology tests Gram stain – not much help. Stool full of gram negative rods (mostly E. coli.) Cultures – must use selective media which restricts or prohibits the growth of normal flora, and allows pathogens to grow. Salmonella, Shigella, Campylobacter, Yersinia, E. coli 0157, Clostridium difficile This slide points out a few discussion points regarding the fecal specimen testing that would be performed in the microbiology department. Performing a Gram stain for bacteria on the freshly collected stool sample is generally not performed as the Stool is overwhelmingly full of gram negative rods, most of which are part of the normal flora - E. coli. Even in infection / diarrhea caused by a pathogenic organism such as Salmonella, Shigella, Campylobacter, Yersinia, E. coli 0157, or Clostridium difficile, you would not be able to distinguish the pathogenic organism from the normal flora using the Gram stain of the specimen. The stool specimen must be placed on a selective microbiology media which restricts or prohibits the growth of the normal flora, while allowing and promoting the growth of the pathogenic organisms. The complete discussion of the Microbiology testing performed on stool samples is best covered within a clinical microbiology course.

90 Fecal Laboratory Procedures
Microbiology tests Ova & Parasites Giardia Enterobius vermicularis (pinworm) Taenia sagenata Taenia solis D. latum H. nana .. Tapeworms Ascaris Also in the microbiology department, the fecal specimen may be evaluated for the presence of intestinal parasites such as some of those listed here. Again, it is beyond the scope and objectives of this course to cover this in any more detail.

91 Fecal Laboratory Procedures
Blood in feces Melana A large amount of fecal blood May be black, tarry stool Lower GI tract bleeds usually bright red blood if not occult The excretion of large amounts of blood in the upper GI tract may cause the stool to be dark or black in color. A very large amount of fecal blood is called melena and the stool will have a black color and sticky or tarry appearance. Bleeds in the lower GI tract tend to have a brighter red color to the blood - which can be seen if in sufficient quantity. 91

92 Fecal Laboratory Procedures
Fecal Occult Blood Testing (FOBT) “Occult” = hidden Detection of occult blood may indicate Infection / inflammation / ulcers of GI tract Intestinal Trauma / hemorrhoids / Bleeding gums Colorectal cancer The American Cancer society recommends testing on all those over age 50 years. Occult blood is blood that is not easily identifiable by observation or is ‘hidden’. The Fecal Occult Blood Test / FOBT is designed to detect hidden blood. Occult blood is seen in Infection / inflammation or from an ulcer in the gastrointestinal tract. Occult blood can also be found as a result of intestinal trauma, in patients with bleeding hemorrhoids. The testing for occult blood has been most useful as a screening test for Colorectal cancer & the American Cancer society recommends annual screening testing for occult blood on all persons those over age of 50 as a means of early detection of colorectal cancer.

93 Fecal Occult Blood Testing (FOBT)
Two samplings from 3 consecutive stools for a negative Traditional screening tests based on detection of the *pseudoperoxidase activity of hemoglobin Different chromagens have been used Benzidine (most sensitive), ortho-toluidine, & *gum guaiac (least sensitive, but preferred as to limit false positives) Hydrogen peroxide oxidizes a colorless compound to for a blue color. View demonstration at: To appropriately screen for colorectal cancer using the Fecal Occult Blood Testing (FOBT), Two samplings from each of 3 consecutive stools should be submitted. When taking the ‘samplings’ the technician should obtain the specimen from different parts of the specimen with most of the samples being taken from within the stool body, rather than the surface area to avoid possible contamination from blood from hemorrhoids. Traditional screening tests are based on detection of the *pseudoperoxidase activity of hemoglobin 3 Different chromagens / color producing chemicals have been used: Benzidine (most sensitive), ortho-toluidine, & *gum guaiac (least sensitive, but preferred as to limit false positives) Hydrogen peroxide oxidizes a colorless compound to for a blue color. A demonstration of the testing procedure is at the website listed. >>>>>>>>>>>>>>>>>>>> . The most common method for detection of fecal blood involves a guaiac-impregnated filter paper in a cardboard holder. A thin layer of these stool portions is applied to the front of the guaiac card as directed. If hemoglobin or another peroxidase or pseudoperoxidase is present in the feces in sufficient amounts, the guaiac paper will develop (usually turns blue) when hydrogen peroxide developer is applied. Methods other than guaiac are available for detecting fecal blood including benzidine, orthotolidine, and even immunological methods for hemoglobin. A problem with these tests is a higher degree of sensitivity that leads to many false positives. The less sensitive guaiac seems to give the best results under most circumstances.

94 Fecal Laboratory Procedures
Stool guaiac test / gFOBT Diet restrictions No red meats, fish Turnips , Horseradish Melons, banannas, pears, plums Raw broccoli, & cauliflower Other restrictions No aspirin or other non-steroidal anti-inflammatory medications 7 days prior to collections to prevent GI irritation iron supplements avoided for 3 days High Vitamin C levels will reduce (False negative Rx) peroxidase activity The occult blood test may also be referred to as the ‘stool guaiac test’ or gFOBT. This fecal test requires proper patient dietary preparation. The foods listed on the slide have peroxidase-like activity that would react in the testing procedure. The aspirin and similar anti-inflammatory medications may cause micro-bleeds which like the restricted foods would produce a false positive test result. Recall how vitamin C or ascorbic acid suppressed many reactions in urinalysis testing? Well it also suppresses occult blood testing reaction. Therefore, the patient must discontinue vitamin C supplements 3 days prior to collecting the specimen to limit or prevent a false negative reaction. >>>>>>>>>>>>>>>>>>>> Diet restrictions No red meats, fish Turnips , Horseradish Melons, banannas, pears, plums Raw broccoli, & cauliflower Other restrictions No aspirin or other non-steroidal anti-inflammatory medications 7 days prior to collections to prevent GI irritation iron supplements avoided for 3 days Most of these foods and medications will cause false positive results. If the patient takes vitamins with High Vitamin C levels, the ascorbic acid will reduce peroxidase activity causing a false negative. Patients should be instructed to avoid red meat, fish, bananas, cantaloupe, pars, plums, turnips, horseradish, broccoli, cauliflower, aspirin, and vitamin C for the days preceding testing as these substances interfere with guaiac testing. The patients must be instructed to collect several portions of the same stool sample to maximize blood detection.

95 iFOBT Immunochemical fecal occult blood (iFOBT)
Fecal Immunochemical Testing (FIT) Hemoccult ICT(commercial product name) Specific for globin portion of human hemoglobin Uses anti-human hemoglobin antibodies No dietary or drug restrictions Most sensitive to lower GI bleeding (patients with upper GI bleeding, such as ulcer would not react as blood has been digested) Although many studies are in progress comparing the iFOBT and the traditional guiac , this test is quickly replacing the traditional fecal occult blood test! Because of the many dietary restrictions and the potential false positive reactions associated with the traditional occult blood screening tests that are based on the *pseudoperoxidase activity of hemoglobin ; a new methodology in testing for occult blood in stool has been developed. This testing is referred to as the Immunochemical fecal occult blood (iFOBT) or Fecal Immunochemical Testing (FIT) . Commercially prepared kits utilizing this methodology are quickly replacing the traditional guaiac tests. The testing utilizes antibodies that are specific to the globin portion of human hemoglobin – eliminating the false positive reaction that would occur due to red meat and other restricted foods presented in the previous slide. Another advantage is that the test is most sensitive to blood found as a result of lower GI bleeding – as would be expected in colorectal cancer. >>>>>>>>>>>>>>> Hemoccult ICT – distinct advantages Specific for globin portion of human hemoglobin Uses anti-human hemoglobin antibodies No dietary or drug restrictions Most sensitive to lower GI bleeding (patients with upper GI bleeding, such as ulcer would not react as blood has been digested)

96 Feces - fecal fat Fecal Fat – steatorrhea Notable characteristics
Floats in water Pale and greasy oily appearance Foul-smelling Causes Decreased production of pancreatic enzymes Absence of bile salts Malabsorption syndromes bacterial overgrowth, intestinal resection celiac disease, tropical sprue lymphoma, Crohn disease, Whipple disease, and giardiasis. Steatorrhea is the term used to describe the presence of excess fat in the feces. The excess lipids generally causes the stool sample to float in the toilet water; they also have an obvious oily appearance, are pale in color and exceptionally foul-smelling. There are a large number of conditions capable of producing steatorrhea: They include: Disorders such as cystic fibrosis and pancreatitis - which cause the decreased production of pancreatic enzymes, Disorders, such as obstructive jaundice, that restrict the flow of bile and bile salts into the intestines & reduce fat digestion bacterial overgrowth, and other conditions as listed on the slide can lead to fat malabsorption and subsequently steatorrhea. >>>>>>>>>> intestinal resection, celiac disease, tropical sprue, lymphoma, Crohn disease, Whipple disease, and giardiasis.

97 Feces - fecal fat testing
Patient preparation Normal diet with normal level of fats No contamination by oils, suppositories, or creams (could cause false positives) Qualitative method 2 procedures Neutral fats (triglycerides) Soaps and fatty acids Sudan III/IV or Oil red O stain Examine microscopically for large orange - red fat droplets. Both qualitative and quantitative fecal fat analyses are available. Regardless of which testing level is ordered the patient should remain on a normal diet – with a normal amount of fat intake prior to the testing period. Patients should also be advised that mineral oils, suppositories and many creams can cause false positive results and must be avoided. The Qualitative method uses 2 procedures used to detect the chemically different types of lipids – the neutral / triglyceride type and the soaps and fatty acid types that require additional processing before staining. In the final stage, the fats are stained with Sudan III or IV or Oil Red O and evaluated under the microscope for the number and size of orange-red stained fat globules. In general an increased amount of neutral fats are associated with impaired digestion leading to steatorrhea ; but as always, you should follow the specific protocol of the procedure and use the clinical site or manufacturer’s criteria when making any interpretation of the results. The picture on this slide is from the Graff Textbook of Urinalysis and Body Fluids by Mundt and Shanahan and shows a representative view from a Sudan III stained specimen with steatorrhea due to increased neutral fats. >>>>>> Lillian Mundt & Kristy Shanahan, Graff’s Textbook of Urinalysis and Body Fluids, 2nd Ed.

98 Feces - fecal fat testing
Quantitative method To follow up a positive quantatitive test Dietary requirements Requires adherence to a diet of 100g/ day fat intake before and during test collection. Chemistry dept – usually sent to reference lab 3day collection (72 hours) Test methods Van de Kamer – classical titration Use sodium hydroxide to chemically titrate the amount of fat. Acid steatocrit Near infra-red spectroscopy If qualitative fecal fat testing is positive, confirmatory quantitative fecal fat analysis is warranted. Patients must prepare by maintaining a diet that includes 100 grams of fat per day, before and during the specimen collection period. Generally, this procedure is performed in the chemistry department and it is often sent out to a reference laboratory. Forty-eight- to seventy-two-hour fecal collections are necessary for these tests. Fecal fat analysis can be performed by the classical Van de Kamer titration – in which sodium hydroxide is used to titrate the amount of fat present. Other methods include the acid steatocrit, and a method that requires near-infrared spectroscopy. It is beyond the scope of this course to include any more detail of this testing.

99 Feces APT - fetal hemoglobin
To determine whether blood found in newborn’s vomitus or stool is their own, or from the Mom. Testing makes use of fact that baby blood (Hgb F) cells are resistant to lysing with sodium hydroxide & remain pink, while mom adult (Hgb A) cells lyse changing from the pink to yellow - brown. The APT test was developed to identify the source of bloody stools in newborn infants. Determining whether the blood is that of the mother or is from the newborn is an important tool in determining the need and type of treatment necessary for the infant. This test determines whether the hemoglobin present is the adult-type hemoglobin A from the mother or fetal hemoglobin F. The test principle makes use of the fact that fetal cells containing fetal / Hemoglobin F are resistant to lysing by sodium hydroxide and will remain intact and dark pink when stained and viewed microscopically – while material cells with / hemoglobin A will lyse and be very pale pink to yellow in appearance. >>>>>>>>>>> The stool or vomitus is mixed with water to yield a pink supernatant. The supernatant is removed and then alkalinized with dilute sodium hydroxide. If the pink color remains after adding the alkali, the blood contains fetal hemoglobin. If the pink color changes to yellow or brown within 2 minutes, the hemoglobin in the sample is maternal hemoglobin.

100 Feces Fecal enzymes Pancreatic insufficiency & cystic fibrosis Pancreatic – associated enzymes Trypsin Classic trypsin test – series of diluted stool specimens are placed on x-ray paper (has a gelatin coating). After incubation, the stool is rinsed off and the paper evaluated to determine the dilution at which no gelatin has been digested by the protease trypsin . TEST NOT SENSITIVE Chymotrypsin – more sensitive and can be measured spectrophotometrically Elastase I pancreas specific enzyme not affected by motility or other mucosal issues Immunoassay procedure provides higher degree of specificity The measurement of Fecal enzymes is on occasion used to evaluate Pancreatic insufficiency & cystic fibrosis Three pancreatatic produced enzymes have been evaluated: Trypsin, chymotripsin and more recently, elastase I. The slide provides additional information. Please review course objectives to determine how much and at what depth this information should be studied. >>>>>>>>>>>>>>> Trypsin Classic trypsin test – series of diluted stool specimens are placed on x-ray paper (has a gelatin coating). After incubation, the stool is rinsed off and the paper evaluated to determine the dilution at which no gelatin has been digested by the protease trypsin . TEST NOT SENSITIVE Chymotrypsin – more sensitive and can be measured spectrophotometrically Elastase I pancreas specific enzyme not affected by motility or other mucosal issues Immunoassay procedure provides higher degree of specificity

101 Feces - Fecal Carbohydrates
Celiac disease – inability to absorb carbohydrates Lactose intolerance – lack enzymes to digest Inflammatory necrotizing entrocolitis – rare, but very serious condition, most often occurs in premature infants Increased carbohydrates in stool results in osmotic diarrhea. Disaccharides (lactose is example) in the large intestine and bowel are osmotically active and cause movement of a large amount of water into the intestine. Clinitest to detect the carbohydrate Fecal pH to determine increased acid level Stool pH 7-8 pH 5.5 – 6 indicates increased acid *Fecal carbohydrates are present in a number of conditions: Some individuals are unable to absorb carbohydrates – such as those with Celiac disease. While others may lack sugar digesting enzymes – such as seen in persons who are lactose intolerant and in congenital disaccharidase deficiencies. But the Fecal carbohydrate testing is most useful in infant diarrhea to assess fecal diarrhea and inflammatory necrotizing enterocolitis. The copper reduction test / Clinitest is used to detect the presence of significant reducing sugars. If this test is positive, the infant may be tested by other more specific serum tests for carbohydrate tolerance. If carbohydrates in the feces are not being absorbed, bacterial fermentation action causes a decrease in the pH. The pH of the feces will decrease from a fairly neutral pH of between 7 and 8 to levels below 5.5 – 6. Therefore when a test for stool reducing substances / Clinitest is ordered, there will often also be a request for Fecal pH which is usually determined using a pH paper.

102 Bronchial Washings & Bronchoalveolar Lavage
Fiberoptic bronchoscope placed in airway can be used to obtain specimen. Or sterile saline infused (lavage procedure) and retrieved for analysis. - Results may be as good as biopsy. Specimens usually sent to cytology / pathology to be examined for malignancy. Occasionally, they are examined for other cells: macrophages (60-80%), lymphs, up to 10% neutrophils up to 21% eosinophils < 1 % bronchial epithelial cells, squamous cells OR, more often cultured for microorganisms. This slide and the next contain information regarding testing of miscellaneous body fluids. Occasionally a specimen labeled bronchial washing / or bronchoalveolar lavage fluid will come to the clinical laboratory. The most commonly performed tests on this fluid include cytology evaluation for malignant cells, and culturing for microorganisms. Occasionally, they are examined for other cells, usually in the hematology department.: Again review the course objectives. macrophages (60-80%), lymphs, up to 10% neutrophils up to 21% eosinophils < 1 % bronchial epithelial cells, squamous cells

103 Other miscellaneous fluids.
Nasal smears Hansel stain for eosinophils Cyst fluids – cells, and organisms Tears – eosinophils Breast milk – eosinophils. always can culture them. And finally, there are additional miscellaneous fluids evaluated in the clinical laboratory. Review the information on this slide in context with the course objectives.

104 Reference Listing Please credit those whose work and pictures I have used throughout these prsentations. Lillian Mundt & Kristy Shanahan, Graff’s Textbook of Urinalysis and Body Fluids, 2nd Ed. Susan Strassinger & Marjorie Di Lorenzo, Urinalysis and Body Fluids, 5th Ed. Wikipedia, the free encyclopedia Please credit those whose work I have sited in this presentation. This concludes session 5 of Unit 5

105 Urinalysis and Body Fluids CRg
Welcome to the final session of unit 5. Unit 5 6 Vaginal Secretions

106 Vaginal Fluids - objectives
Define and list at least three (3) symptoms of vaginitis. Identify at least two (2) sources of error that can occur during the collection and processing of vaginal wet prep specimens. List three (3) common causes of infectious vaginitis. Describe "clue cells" and explain the significance of finding them in a vaginal wet prep. Evaluate the test for estrogenic activity including the appearance of positive and negative results. Although objectives are listed on this slide, you should refer to the complete objective listing as posted at the course website – as there may be revisions or additions. This session will focus on the causes and symptoms of vaginitis and the laboratory testing of vaginal fluids.

107 Vaginal Secretions Normal secretions Abnormal changes Clear mucus
May turn slightly white or pale yellow when exposed to air Healthy vagina - Lactobacillus species predominates pH< 4.5 ( ) Amount / volume varies through menstrual cycle Normal microscopic exam Abnormal changes Color Consistency Amount Glands in the cervix normally produce a clear mucus that may turn slightly white or pale yellow when exposed to air. The normal bacterial flora IN the Healthy vagina are members of the Lactobacillus species . This organism maintains the normal vaginal pH 4.5. While the amount of vaginal secretions normally vary somewhat throughout the menstrual cycle due to hormonal influence, noticeable changes in the color, consistency, or amount of vaginal secretions may be linked to various conditions and infections. ……………………… Examination of vaginal secretions may provide the caregiver with helpful information on the patient’s condition. Infections and sexually transmitted diseases can be detected via testing of vaginal secretions. Some testing may be performed in the microbiology department whereas other testing may be performed in the urinalysis department.

108 Normal Wet prep No symptoms Lactobacillus (normal) Normal discharge
The microphotograph on this slide is an example of what could be found in a microscopic examination of a vaginal fluid from an asymptomatic patient with a normal discharge. This microphotograph and several others throughout this presentation are from the Seattle STD & HIV Prevention Training Center sponsored by the Washington State Department of Health.

109 Vaginitis Vaginitis - inflammation or infection of the vulva and vagina NOT a specific disease, but is a very common reason women seek medical attention Estimated 1/3 to ½ outpatient visits by women Can occur in all age groups, sexually active as well as sexually non-active. Common symptoms Vaginal discharge Foul smell Itching Spotting Pain According to Wikipedia; vaginitis is an inflammation of the vagina that can result in a discharge, itching and pain,[2] and is often associated with an irritation or infection of the vulva. While NOT a specific disease, vaginitis is frequently the result of an infection and is a common reason women seek medical attention. Vaginitis can occur in all age groups. Both sexually active and sexually non-active women can have vaginitis. Common symptoms of vaginitis include a noticeable change from the normal vaginal discharge, often including the development of a foul smell, itching, spotting and pain.

110 Vaginitis - evaluation
Patient history Marital or relationship status Timeline of when symptoms began, etc. Symptoms / complaint(s) Physical examination Tests Physical properties Vaginal pH Microscopic exam / Wet Prep Amine (Whiff) test Cultures, if warranted The evaluation of vaginitis begins with a detailed patient history, including listing of the patient’s symptoms or complaints. The physician will then perform a physical examination, during which he / she will collect a sample of the cervical or vaginal discharge for testing. The physician will note the appearance and consistency of the vaginal discharge. A thick, white and clumpy discharge that resembles – cottage cheese, is associated with a yeast infection,; a green-yellow frothy discharge is seen in vaginitis cases caused by the one cell parasite - trichomonas vaginalis; and a thin grey discharge is most often seen in cases of a bacterial infection. Other testing includes determining the pH, a microscopic evaluation for the types of cells present and the Amine or Whiff test. If a bacterial infection is suspected, a culture may be also performed. There will be more details on these tests and a summary chart in slides upcoming.

111 Vaginitis Two (major) types Non- infectious
May be caused by soaps ( no bubble baths ladies !), chemicals, foreign objects, allergies to condoms / lubricants etc. Infectious (makes up 90% of all cases) Fungal / yeast Parasitic – Trichomonas vaginalis Bacterial An estimated 10 % of the vaginitis cases are non- infectious in nature and are the result of irritation of the vaginal tissues by soaps, chemicals, foreign objects, condoms / lubricants and so forth. Resolving these cases usually involves removing or discontinuing the irritant . These cases rarely involve the clinical laboratory. The majority of vaginitis cases are the result of an infection. From the laboratory’s perspective, there are 3 main causes for vaginosis. Specimens are sent to the laboratory to be examined for the presence of yeast, the parasite – Trichomonas vaginalis, and bacterial pathogens. Bacterial culturing is performed in the microbiology department and the microscopic examination of the wet prep sample is often performed in the urinalysis department.

112 Vaginitis Specimen – Vaginal Wet Prep
Sterile swab (moistened with normal saline) Must process these immediately, ie within 5 minutes Swab in tube ½ mL normal saline, or Ringer’s lactate Keeps organisms from drying out if delay is expected Special collection procedures: Microbiology cultures for gonorrhea (GC) must be placed in special transport media immediately. This microbiology testing being replaced by molecular diagnostics Collection / processing errors Insufficient specimen / poor collection Swabs / slide drying out The ‘vaginal wet prep’ is a sampling of the vaginal fluid collected on a sterile swab. It is critically important that the sample be kept wet with a sterile normal saline or Ringer’s lactate to maintain the viability of any organisms prior to culturing. To obtain maximum results, samples to be cultured for gonorrhea MUST be inoculated into a special transport media immediately after collection to preserve the organism. Please note: In modern reference laboratories, Testing for the gonorrhea organism is being performed using molecular diagnostic techniques which have the distinct advantages of sensitivity and specificity and do not require the special collection requirements of the traditional GC culture. Failure to collect an appropriate specimen or allowing it to dry out are the most common reasons for inaccurate testing results.

113 Vaginitis - testing vaginal pH Most important preliminary test
Normal (childbearing age) < 4.5 pH paper Evaluating the pH of a fresh sample of vaginal fluid is considered the most important preliminary test when differenting the cause of the vaginitis. The normal vaginal fluid pH for a woman of child bearing age is less than 4.5. The pH can be sufficiently determined using a pH paper, such as a litmus paper. The paper should provide results in-half units and be suitable for measuring in a pH range 3 to 5.

114 Vaginitis - testing Microscopic exam / Saline Wet Prep
Sample mixed with saline examined microscopically to look for Budding yeast with elongated pseudohyphae Motile trichomonads & increased segmented neutrophils PMNs & Clue cells The microscopic examination of the vaginal fluid is called the ‘saline wet prep’ or just’- ‘wet prep’. In this procedure, a drop of the fluid is mixed with saline, coverslipped and examined under the microscope. The specimen is initially evaluated on low power magnification to get an overview and then high power for the identification of specific structures. As with the urine microscopic, it is important to use sreduced light to improve contrast. The specimen should be evaluated for fungal yeast cells that demonstrate pseudohyphae, motile trichomonads, increased numbers of polymorphic nuclear neutrophils and the presence of ‘clue cells’. These structures will be discussed in further detail in the upcoming slides.

115 Vaginitis - testing Microscopic exam / Saline Wet Prep Limitations
Skill of collection Transport time Trichomonas organisms die / become immotile Skill of technician New wave in laboratory testing Immunologic Molecular diagnostic / pcr Although the wet prep is a vital part of the testing process, it has limitations. The specimen must be properly collected and transported to obtain and preserve the offending organisms. The graph located on the middle of the slide shows how quickly trichomonas organisms begin to die after the specimen is collected and removed from the body. The laboratory must efficiently process the sample and employ technologists and technicians that are skilled and knowledgeable. Testing in modern laboratories, may include immunologic and molecular diagnostic techniques to improve sensitivity. The second bar graph compares these technologies with the traditional wet prep analsis.

116 Vaginitis - testing Amine (Whiff) test
Also called potassium hydroxide or KOH preparation Vaginal fluid & 10% KOH placed on a slide Fumes from the slide are smelled to detect presence of ‘fishy odor’ (trimethylamine) . Presumptive for bacterial vaginosis, though can also be positive for trichomonal vaginosis The Amine test, also frequently called the Whiff test is to detect the presence of volatile amines which are not normally found in normal vaginal fluids. In this test, a small amount of the vaginal discharge is mixed with a similar sized drop of 10% potassium hydroxide and then carefully smelled or sniffed for the presence of a fishy odor. The test is reported as being positive or negative. According to eMedicine from WebMD, the Whiff test is one of the most specific tests for bacterial vaginosis, when used in conjunction with clinical symptoms, however, it can produce positive results due to other causative agents, most notably, the parasite, trichomonas vaginalis. >>>>> Potassium hydroxide (KOH) preparation: Vaginal discharge is placed on a slide with 10% KOH solution. Known as the whiff test, a positive finding is the release of a fishy odor after addition of 10% KOH to discharge. The odor is due to the release of amines such as putrescine, cadaverine, histamine, and trimethylamine.Bacterial vaginosis: Whiff test is one of the most specific tests for bacterial vaginosis and the least sensitive. Vaginal candidiasis: Negative whiff test is 65%-85% sensitive for candidal infection. Trichomonas vaginitis: Whiff test may be positive.

117 Vaginitis - suspect yeast (candidiasis)
Candida albicans Commonly causes a majority of cases Alteration of normal vaginal flora antibiotic regimens immunocompromised patients Thick white, clumpy or “curd-like” discharge. Laboratory findings Normal vaginal pH Identification of yeast cells and elongated pseudohyphae (mycelia forms) saline wet mounts 10% KOH wet preps Gram stain It is normal to find the yeast, Candida albicans in small numbers in the vagina, along with the normal bacterial flora. A candidiais infection can occur when there is a change in the normal vaginal environment, such as when the balance of the normal bacterial flora are altered – allowing the yeast to predominate. This can often occur as a side effect resulting from taking oral antibiotics, and can occur in celibate as well as sexually active women. Candidiasis is also more common in immunosuppressed patients. Women with candidiasis frequently complain of a whitish, curd-like vaginal discharge. Upon laboratory examination, the discharge fluid will generally have a normal acid pH of 4.5 or less . The microscopic examination – of a saline mount, KOH wet prep or gram stain - will reveal an increased number of yeast cells, many of which will be demonstrating mycelia forms, also called: pseudohyphae - which indicate active or invasive-type of growth, as well as an increased presence of WBCs. Included on this slide is a representative microphotograph - which can also be found in your textbook.

118 Vaginitis - suspect:Trichomonas vaginalis
Trichomonas vaginalis - Free- living organism Parasitic vaginitis Swimming / bathing in contaminated water Sexually transmitted Symptomatic - Yellow-Green frothy discharge; or may be asymptomatic Organism seen in urine or on wet-prep In males – sexually transmitted urogenital infection Usually asymptomatic Organism may be detected in urine microscopic Trichomonas vaginalis is a flagellated single cell parasite – that is similar in size to a white blood cell. This free-living organism can exist in warm pools of water and infect unprotected female swimmers / bathers – resulting in a vaginitis. IN men the infection occurs in the urogenital tract and is the result of having sex with an infected female. Like-wise, an infected man can transmit the parasite sexually to an uninfected woman. While Men are usually asymptomatic, Women usually complain of yellow green frothy or foamy appearing vaginal discharge. In pregnant women, Trichomonas is a risk factor for preterm rupture of membranes leading to preterm labor and delivery.

119 T. vaginalis - testing / detection
Laboratory findings Wet-prep microscopic Single celled flagellate demonstrating jerky movements @ size of WBC, but no nucleus and actively motile - unless specimen is old, dry or cold. May demonstrate WBCs DNA and immunological tests Elevated vaginal pH Positive amine / “whiff” test The wet mount is helpful to detect the majority of cases of Trichomonas. In a fresh sample the organism will demonstrate a characteristic jerky movement. The trichomonas organism can be confused with white blood cells – which are also often present, but the trichomonas lacks a discernable nucleus and is generally actively motile. If the sample is cold, has aged or if the patient is currently being treated, the movement of the organisms is much slower – with often only a twitching or occasional waiving of the flagella . Testing using a DNA probe or an immunological test may be useful when the wet preparation is negative, but trichomoniasis is strongly suspected. In vaginal trichomonas infections, the normal bacterial flora may become altered and the normal 4.5 pH will become elevated to 5.0 or The amine or “whiff” test may also be positive due to these changes. More information about the amine / whiff test will be presented in the upcoming slides. >>>>>>>>>>>>>>>>> A diagnosis can be made from the detection of the motile organism microscopically from vaginal or urine samples. There are also DNA and immunological tests on the market.

120 Vaginosis – suspect bacteria
Healthy vagina - Lactobacillus species predominates Bacterial vaginosis Gardnerella vaginalis Mobiluncus species Prevotella species (anaerobes) Characteristics (*Amsel criteria) *Homogenous vaginal discharge Amount & Color may vary, but often gray / off-white Usually thin in consistency and malodorous. Lack of WBCs, but increased epithelial cell exfoliation *“Clue cells” (make up 20+%) – most reliable finding *Vaginal pH > 4.5 *Positive amine test in the KOH prep A Bacterial infection is the most common cause of vaginosis in women. Normally the vagina is colonized predominantly by Lactobacillus species microorganisms, which maintain the normal acid environment. In bacterial vaginosis, the normal Lactobacillus flora is suppressed allowing other bacteria to predominate. Among these, Gardnerella vaginalis is frequently sited. Other bacterial species, as listed on the slide are also suspect. The Jan. 1983, the American Journal of Medicine included an article by R. Amsel and colleagues that resulted in the development of standardized criteria for the diagnosis of bacterial vaginosis. These findings, now referred to as Amsel’s Diagnostic Criteria for Bacterial Vaginosis are listed on the slide. Using Amsel’s Criteria - 90% of women with bacterial vaginosis will demonstrate at least three of the four characteristics listed. First on the list of characteristics: is the … Production of a homogenous vaginal discharge. The discharge will often be thin, light gray in color and usually have a foul smell. # 2 is observing 20 or more % ‘Clue cells’ during the microscopic evaluation of the fluid. Clue cells are squamous epithelial cells that have been completely covered with numerous small, thin curved gram-variable bacilli. Finding 20% plus Clue Cells is the most reliable and significant of the characteristics. # 3 on the list is obtaining a pH greater than 4.5 on the freshly collected vaginal fluid. Characteristic #4 is … a positive amine or “whiff ” test. >>>>>>>>>>>>>>>>>> The discharge will often be thin, light gray in color and usually have a foul smell. Because the infecting bacteria seem to only colonize the squamous cells lining the vagina – and not invade the subepithelial tissues – there is no or little immune response of white blood cells, but there is an increase in exfoliation – or sloughing off of the epithelial cells. Finding more than 20% of the sloughed off squamous epithelial cells to be completely covered with numerous small, thin curved gram-variable bacilli. These cells are called ‘clue cells’ & identifying them in the microscopic, wet prep examination of the vaginal fluid is the most reliable and significant finding. The next slide will have more information and pictures of normal vaginal epithelial cells and clue cells. number 3 is a vaginal pH greater than 4.5. & Is a positive amine or “whiff ” test.

121 “Clue cells” Normal examples Clue cells
As stated earlier, finding clue cells during the microscopic evaluation of the vaginal fluid is highly significant during the investigation of possible bacterial vaginosis. The left side of the chart on this slide has unstained and stained examples of normal vaginal cells. The arrow at the #1 on the top picture is pointing to the longer curved Lactobacillis that make up the normal vaginal flora. The arrow at the #2 is the normal vaginal epithelial cell. The pictures on the right side are examples of samples that would be expected when the lactobacilli have been depleted or suppressed by an overgrowth of Gardnerella vaginalis, or one of the other opportunistic bacterial organisms listed on the previous slide. The epithelial cells at the arrows are heavily colonized by the bacterial giving them a peppered appearance with ragged or chewed up edges to the cell wall. These are clue cells. Although identifying 20% or more clue cells is very significant, the authors note that as many as 30% of clinically normal women may have clue cells. Therefore it is important that at least 3 of the 4 Amsel criteria are positive before a definitive bacterial vaginosis diagnosis is made.

122 Vaginitis – testing summary
Observation / test Candidia vaginatis yeast Trichomonas vaginalis Bacterial Appearance Thick white, clumpy / curd-like Green-yellow & frothy Thin, gray homogenous pH < 4.5 >4.5 > 4.5 Wet Prep microscopic Budding yeast and pseudohyphae Motile trichomonads & PMNs > 20% Clue cells identified Amine (Whiff) test with 10% KOH Negative Negative, or Positive Positive: fishy odor Miscellaneous DNA & immunological tests available Amsel criteria: at least 3 of 4 must be positive. This is a summary the observations and basic tests differentiating types of vaginosis. Remember, according to the Amsel diagnostic criteria : at least three of the four criteria listed is required for the diagnosis of bacterial vaginosis. There will be additional examples of microscopic / wet prep slides at the end of this presentation for your review.

123 Summary From the lab’s perspective – 3 main causes for vaginitis
Yeast infection / candidiasis Candidia albicans / other species possible Trichomonas vaginalis Bacterial From disturbance of normal flora (ie decreased lactobacillus) that allows overgrowth of mixed flora, ie Gardnerella vaginalis and others Gardnerella – results in ‘clue cells’ Known pathogens, ie gonorrhea So in summary, from the laboratory’s perspective, there are 3 main causes of vaginitis Yeast, such as Candidia albicans The one celled parasite - Trichomonas vaginalis And bacterial causes that are due to a disruption or disturbance in the normal Lactobacillis flora. Or from a known pathogen, such as Neisseria gonorrhea. While Neisseria gonorrhea causes infections, -serious infections, … it rarely causes the classical symptoms of vaginosis that brings the female patient to her physician. …As a result, we will not devote additional time to it in this course - other than to say that Microbiology culturing for Neisseria requires special techniques and media – AND using molecular diagnostic / pcr tests to detect it is becoming increasingly popular. MORE…. information on infections caused by Neisseria will be provided in the Clinical microbiology course.

124 Fern test Test for estrogenic activity
Cervical mucous smeared on glass slide and allowed to dry Examine under the microscope – look for fern-like appearance / pattern Seen during times of increased estrogen – as occurs at time of ovulation. Also done to see if there has been pre-mature leakage of amniotic fluid - as it will also make a fern pattern due to its protein and sodium chloride content. There are other reasons for laboratory examination of vaginal secretions, other than determining the cause of a vaginosis. In the Fern test - A sample of cervical mucous is smeared onto a clean microscope slide, and allowed to dry. Under the microscope, the technician looks for a pattern similar to the leaf appearance of a fern plant. This ferning pattern begins to about 3 days prior to ovulation due to increased levels of estrogen. The test is a useful aid in determining the best time period for conception. The test is sometimes performed on the woman’s saliva. Additionally, The fern test has been used to determine whether a pregnant woman is leaking amniotic fluid. The protein and salt content of the amniotic fluid will cause the appearance of a fern pattern, indicating leakage of the fluid…. Premature rupturing of the membranes leading to the leakage could… lead to fetal infection ..and subsequent mortality; ..therefore if the test is positive for amniotic fluid, the physician would likely go ahead and rupture the membranes and induce labor. Great care must be taken during the collection of the vaginal fluids sample, as the presence of as cervical mucous would give a false positive.

125 Fern test – positive reactions
These microphotographs taken at differing levels of magnification, demonstrate positive fern test reactions.

126 Fern test – negative reaction
This microphotograph is an example of a negative fern test reaction.

127 Wet preps - 1 No symptoms Lactobacillus (normal) Normal discharge
The remaining slides in this presentation are for review purposes. All relevant information is provided on the slides themselves. The final slide is a listing of reference sources and sites used during the development of this session. This concludes the final session of unit 5

128 Wet preps - 2 pH <4.5 (normal)
KOH microscopic negative & Whiff test negative (no amine odor) Normal epithelial cells Predominately lactobacillus Rare WBC

129 Wet preps - 3 Positive clue cells pH > 4.5 Whiff test positive
KOH microscopic negative Normal lactobacilli have been overrun by Gardnerella vaginalis and other organisms

130 Wet preps - 4 Positive KOH microscopy Whiff test negative
No amine odor when mixed with the KOH Vaginal pH <4.5 Moderate – increased discharge White to light yellow, Etiology – Candida albicans / Candida species

131 Wet preps - 5 Microscopy – positive for motile ‘trich’
Whiff test often positive Vaginal pH >5.0 Discharge – greatly increased Green / yellow purulent, may appear foamy Etiology – Trichomonas vaginalis

132 Summary Yeast - Candidiasis Candida albicans / other species possible
Microscopic shows mycelia forms

133 Summary Trichomoniasis Trichomonas vaginallis
@ size of WBC (slide on Rt), but no nucleus and actively motile Unless specimen is old, dry or cold

134 Reference Listing Lillian Mundt & Kristy Shanahan, Graff’s Textbook of Urinalysis and Body Fluids, 2nd Ed. Susan Strassinger & Marjorie Di Lorenzo, Urinalysis and Body Fluids, 5th Ed. Wikipedia, the free encyclopedia eMedicine from Webb MD Family Practice notebook.com Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med Jan;74(1): [Medline] Seattle STD/HIV Prevention and Training Center, Washington State Dept. of Health Please credit those whose work I have sited in this presentation. This concludes session 6 of Unit 5 CRg


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