Clinical Pathology Fecalysis and Urnalysis

Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test FECALYSIS Specimen collection  Clean dry container, leak-proof  Not contaminated by urine* Macroscopic screening 1. Color changes a) brown = normal b) gray=fecal obstruction or barium c) red=blood or food dyes d) black=blood form upper git, iron tx, charcoal tx e) green=vegetables/biliverdin 2.

Consistency a) formed=normal b) hard=constipated c) watery=diarrhea/steatorrhea (fat excretion >3g/day)

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Form a) b) c) d) e)

cylindrical=normal ribbon-like=intestinal strictures small, round=constipation bulky=steatorrhea mucus=colitis, constipation

Chemical examination  Test for blood:  Melena  large amounts of fecal blood  i.e. 50-100 ml/day  Occult blood  small amounts of fecal blood  i.e. 30-50ml/day  Testing principles for occult blood: 1) Hemoglobin reduction method  Based on the reaction of Hgb with H2O2 and an indicator  usually: blue green color (indicator)  usual indicators: a) benzidine (carcinogenic) b) Orthotoluidine c) Guaiac - most common  Interfering factors:  False (+) - turnips,brocolli, cantaloupes, banana, aspirin  False (-) - vitamin C 2) HemoQuant test  detects total fecal Hgb  higher specificity than #1 (not affected by interfering substances) 3) Immunodiffusion method  using antihuman Hb  most sensitive method MICROSCOPIC EXAMINATION  Fecal white blood cells a) Ulcerative colitis b) Dysentery (bacterial) 1. Salmonella 2. Shigella 3. Campylobacter 4. Yersinia 5. Enteroinvasive E. coli c) Ulcerative diverticulitis d) Intestinal TB e) Abscesses  NB:  Staph and Vibrio usually do not cause the appearance of fecal leukocytes  As few as 3 leukocytes per OIL immersion lens have 70% sensitivity for the presence of invasive bacteria.  other tests: 1) Lactoferrin* latex agglutination test By: Rem Alfelor 

A component of leukocytes’ secondary granules (a neutrophil protein*) which when detected can also a be an indicator of an invasive bacteria.  Major cause of fecal neutrophils in patients with chronic diarrhea is chronic inflammatory bowel disease of the colon. 2) Muscle fibers  pancreatic insufficiency (cystic fibrosis)  use of eosin to highlight the fibers  Only undigested fibers are counted and if more than 10, it is reported as increased.  Patients are instructed to eat red meat and specimens must be examined within 24 hours of collection. 3) Qualitative fecal fat  for suspected case of steatorrhea  Fats stains: Oil red O, Sudan III, IV  will appear as red orange droplets  confirmatory for steatorrhea  3 day specimen collection  Patient on a regular intake of 100g/day of fat during the collection period. 4) APT TEST (Fetal hemoglobin)  Grossly bloody stools and vomitus are sometimes seen in the neonates as the result of swallowing maternal blood.  the material to be tested is emulsified in water to release hemoglobin, and after centrifugation + 1% NaOH  Result: if maternal hemoglobin yellow brown supernatant  if fetal, supernatant remains pink (alkali-resistant fetal hemoglobin) Other chemical tests: 1) Carbohydrate detection 2) Enzyme tests 

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Parasites (Ameba)  E. histolytica/E. dispar Giardia  Giardia using Iodine as stain Direct fluorescent antibody assay  Giardia Others: Helminths…  Enterobius….UV light microscope…  Enterobius…adult worm

URINALYSIS History of urinalysis  Middle Ages  Johanes de Ketham  Fasciculus Medicinae” 1491  Urine color wheel  Based on four temperaments/humors: 1. Sanguineous-blood 2. Choleric-yellow bile 3. Phlegmatic-phlegm 4. Melancholic-black bile BASIC URINALYSIS  key points  Many different diseases can display abnormalities in the urine.  Quickly and economically  Therefore, examination of the urine is an important laboratory function.  Basic urinalysis consists of  gross examination of the urine  dipstick analysis for blood, white cells, sugar, and other substances  Dipstick may be read either manually or by an automated instrument.  Microscopic analysis of urine may be necessary in many cases.  This is to detect cellular elements, casts, and crystals.  Each of these items can be caused by several different disease states.

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test 

Although microscopic examination of the urine is usually performed manually, there are several automated instruments that can perform this analysis.

URINE FLOW CYTOMETRY  Cells are counted based on 1. light scattering 2. fluorescence 3. impedance properties  Considerations  Red blood cells within the urine can come from any point along the urinary tract.  Dysmorphic red blood cells are often a sign of glomerular disease.  The first voided morning urine, because it is the most concentrated, is often the best specimen for analysis.  Some procedure may require a 12- or 24-hour urine sample.  Specific gravity and osmolality measurements reflect the concentrating ability of the kidneys.  After a period of dehydration, the osmolality should be three to four times that of plasma.  Proteinuria of over 4 g/day is seen in the nephrotic syndrome.  Although nephrotic syndrome is usually seen in primary renal disease, it can occasionally be seen in a systemic disease which affects the kidneys.  Ketonuria can be seen in diabetics.  It can also be seen in other states, such as febrile illnesses and cachexia.  The dipstick nitrite and leukocyte esterase tests are used to help diagnose urinary tract infections.  Positive results should be confirmed by microscopic analysis of the urine.  Urinary calculi are most commonly formed from calcium.  Work-up of habitual stone formers should include both analysis of the urine and of the stone. THE NORMAL URINE  Water  Electrolytes  Bacterial toxins  Drugs  Vitamins  Pigments  Hormones  Urea  Uric acid  Ammonia  Creatinine  Trace of cells  Crystals URINE  The result of :  glomerular filtration  tubular reabsorption  selective secretion  Normal  Color--Pale yellow to amber  Appearance--Clear  Sp. Gravity---1.003-1.030  pH---4.5-8.0  Glucose, Protein, Nitrates, Blood, Crystals, Bilirubin, Leukocyte Esterase and Ketones---None  RBC’s---0-3/HPF  WBC’s---0-4/HPF  Urobilinogen---140/90mmHg) 3. Age (men over 45 years;F>55) 4. Obesity 5. DM 6. Sedentary lifestyle Key points: 1. Measurement of proteins present in cardiac myocytes indicates recent damage to cardiac muscle. 2. Measurement of substances that are damaging to the coronary arteries, or at least have been proven association with coronary heart disease, is used to assess risk and select appropriate preventive measures. The most important lab risk factors are LIPIDS. Serial sampling for cardiac markers  time window from death of muscle to release markers in blood  ESC & ACC>> 1. on admission 2. 6-9 hours 3. 12-24 hours (if earlier specimens were negative) and the clinical suspicion for MI is high  POC testing must be available ALL the time  Historically: LDH & CK  20+ years ago  Troponin antibodies  currently, the markers assay include:  Myoglobin  Troponins  CK isoenzyme (MB)

CARDIAC ENZYME MARKERS:  Historical development:  1950’s-La Due et al investigating transaminases (AST)  Very simple principle:  when heart muscle diesrelease proteins in blood (as markers)

By: Rem Alfelor 

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test 

Transaminases have not endured as cardiac markers because they are not specific to the heart. And soon afterLDH and CK.

CREATINE PHOSPHOKINASE

By: Rem Alfelor 

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

Newer tests:  Troponins…highly specific  A complex of three proteins that resides at regular intervals in the thin filament of striated muscle.  TnT, TnI, TnC  TnC=identical forms in Type 2 and cardiac muscle  TnI & TnT=cardio specific  In contrast with other markers, their levels are almost undetectable or zero in normal serum. (detection limits of 0.01ng/ml)  The 99th percentile of the healthy population is around 0.04ng/ml depending on the assay.  Levels above this threshold are almost certainly indicative of myocyte damage but possibly reveal a much lesser amount of damage than was detectable with earlier cardiac markers such as CK-MB.  Note that elevation maybe found in pericarditis, myocarditis, PE, renal failure, sepsis, and other critical illness.  Prolonged intense exercise, such as marathon can cause small elevations.  NB: in healthy newborns, levels as high as 3.0ng/ml has been found!

By: Rem Alfelor 

Other markers for coronary risk:  C-REACTIVE PROTEIN  First isolated in 1930 in patients with pneumococcal pneumonia  so named because it binds to the C-polysaccharide of the pneumococcus  Later it was found that it appeared in plasma during infectious or inflammatory conditions.  It is the original acute phase reactant protein.  Normal level=1mg/ml  in acute illness, levels may reach as high as 300mg/ml  Baseline average was 1.5mg/ml for those who developed MI.  It is pro inflammatory.  Still, CRP has an unclear role in the pathogenesis of vascular disease.  But the AHA suggested:  Asymptomatic men >50 years and women >60 years be screened even if LDL cholesterol is not elevated.  HOMOCYSTEINE  Hcy is sulfur containing amino acid and a metabolic intermediate.  HOMOCYSTINURIA:  homozygous defect in the enzyme cystathionine-Beta-synthase  lens dislocation, osteoporosis, MR, psychiatric disturbance, thromboembolic disease (e.g. CHD)  High levels of Hcy: Page 10 of 16

Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

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1. endothelial injury 2. platelet activation  To name a few damaging effects… as a CHD marker, it parallels that of a high cholesterol level Folate supplements can bring down elevated levels of Hcy.

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Summary: 1. The most important disease affecting heart is coronary heart disease, which is atherosclerosis affecting the coronary arteries.  CHD can lead to thrombotic occlusion of coronary blood flow, causing an acute coronary syndrome or ACS. ACS with frank necrosis of any amount is MI. 2. The primary tests for diagnosing ACS are ECG, lab measurements of cardiac markers which are proteins released in circulation from damaged heart muscle. The most important cardiac marker today is cardiac troponin (cTn). 3. Troponin is a complex of three proteins, two of which are suitable as specific cardiac marker tests: cTnI and ctnT 4. There is a delay of a few hours following MI before cTn is detected in the circulation. It peaks about 24 hours and then declines over several days 5. We can also measure risk factors associated with the development and progression of CHD: Lipids, homocysteine, CRP Assignment (got from Henry’s): Read on… 1. BRAIN NATRIURETIC PEPTIDE (BNP)  Circulating BNP derives from a 108 amino acid prohormone, proBNP, which is cleaved within the cardiac myocyte by the endoprotease furin to a 32 amino acid C-terminal fragment, the active BNP, and an inactive Nterminal fragment, N-BNP or NT-proBNP  Secretion of both fragments is enhanced by ventricular wall stretch and volume overload, as occur in HF  BNP is removed from the circulation by binding to a clearance receptor and also through the action of endopeptidases; its circulating half-life is approximately 22 minutes.  The circulating half-life of N-BNP is considerably longer (60–120 min), and its mechanism of clearance is not well understood.  BNP and the other natriuretic peptides exert their effects through two types of G-protein–coupled receptors, resulting in release of the second messenger cyclic guanosine monophosphate. They downregulate the renin-angiotensin-aldosterone system, decrease sympathetic nerve activity in the heart and kidney, increase renal blood flow, and increase sodium excretion via a direct effect on the renal collecting duct  Plasma levels of BNP are less than 100 pg/mL in most healthy individuals; reference ranges depend on age and gender.  The best established application of BNP measurement is for diagnosing acutely ill patients presenting to emergency service with shortness of breath.  Distinguishing HF from lung disease, such as emphysema, in these patients is occasionally difficult, and until now no laboratory test has been specifically applicable.  The multinational Breathing Not Properly study enrolled patients who presented to emergency centers with dyspnea and used a point-of-care method for measurement of BNP. At a decision point of 100 pg/mL, the BNP test had the following characteristics for diagnosis of HF: sensitivity 90%, specificity 76%, positive predictive value 79%, and negative predictive value 89%.  Among patients with a history of ventricular dysfunction, BNP was higher in those whose current symptoms were thought to be caused by HF; it was also higher in patients with more severe failure.  BNP levels decline when effective therapy for HF is instituted, and so the test may be used to monitor the course of treatment  Other proposed applications include risk stratification of patients with ACS; monitoring disease severity in patients with stable CHD; screening for ventricular dysfunction in selected populations; and testing for drug cardio toxicity  However, the test is relatively expensive, so routine application that leads to a large volume of testing needs to be carefully considered.  Some medical centers have introduced restrictions to limit the use of BNP for monitoring inpatients By: Rem Alfelor 

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A major limitation of BNP is that a wide range of values is observed in patients with and without HF, and all of the determinants of the circulating BNP level have not yet been well established.  BNP is increased in conditions of fluid imbalance other than HF, particularly renal insufficiency, which commonly coexists with HF.  In individuals without HF, higher levels are associated with female gender, advanced age, and lower body mass index  Also, patients with symptomatic HF, especially when it is chronic and stable, can have “normal” levels.  Intraindividual variability is relatively high; in a group of patients with stable, chronic HF, week-to-week variability for BNP and N-BNP was 30%–40%  At this time, it appears that the most appropriate use of the BNP test is as an adjunctive  test to rule out HF in the acute setting; it must not be used as a sole criterion for establishing the diagnosis of HF  it should be used judiciously as more information becomes Available.  The earliest assay for BNP commercially available in the United States was an immunoassay using an instrument most suitable for point-of-care measurement.  Recently, the test has become available on large, automated immunoassay platforms.  Assays for both BNP and N-BNP are available; a clear advantage of one biomarker over the other for any particular application has not been established  Besides being a biomarker for HF, BNP has natriuretic, vasodilatory, and other effects that are ameliorative for the syndrome, and in fact is available as the drug nesiritide (Natrecor) for the treatment of HF.  Because of the short half-life of BNP, measured levels several hours after its administration would reflect endogenous secretion. However, the utility of BNP measurement in the context of its therapeutic administration has not been established at this time. D-DIMER  Measures plasmin-cleaved, insoluble, cross-linked fibrin that originally arose from thrombin cleavage of fibrinogen  can be performed by immunonephelometric assay or by latex agglutination assay, and results can be obtained rapidly, facilitating the use of this test in clinical decision-making  As would be anticipated, the ddimer is elevated in a wide variety of thrombotic conditions, including DIC  By careful establishment of a cutoff value, the d-dimer may be judiciously used in concert with clinical findings as a negative predictive test to exclude deep vein thrombosis or pulmonary embolism  Level of d-dimer may decrease in response to anticoagulation.  In the past, fibrin degradation (split) products (FDPs) were used to recognize DIC (see Fig. 39-4, A). However, depending on the specificity of epitopes recognized by antibodies employed in the particular test systems, FDP may variably represent plasmin-cleaved fibrinogen, soluble fibrin, or insoluble fibrin.  The finding of increased FDPs accordingly does not necessarily translate into a measure of plasmin-cleaved, insoluble, cross-linked fibrin equivalent to that obtained with the d-dimer assay.  Fibrin monomer is the large molecular mass protein of fibrinogen that remains after fibrinopeptides A and B are liberated. It can be elevated in DIC, but if DIC is severe, it may be absent. Thus, it is an unreliable assay for use in recognizing DIC.  A scoring system that combines in a quantitative fashion the results of four commonly employed laboratory tests that may be helpful, in the appropriate clinical setting, to evaluate the likelihood of DIC

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test    

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From Book:

SEMEN ANALYSIS Dennis Macapagal 2014 Reasons for doing semen analysis 1. For fertility testing 2. To determine the need for IVF 3. As post vasectomy testing 4. Forensic analysis Outline I. Semen analysis II. Sperm preparation III. Quality assurance SEMEN ANALYSIS  Normal physiology:  During ejaculation, semen is produced from a concentrated suspension of spermatozoa, stored in the paired epididymides, mixed with, and diluted by, fluid secretions from the accessory sex organs.  It is emitted in several boluses.  During sexual intercourse, the initial, sperm-rich prostatic fraction of the ejaculated semen may come into contact with cervical mucus extending into the vagina with the rest of the fluid remaining as a pool in the vagina.  In contrast, in the laboratory setting, the entire ejaculate is collected in one container, where spermatozoa are trapped in a coagulum developed from proteins of seminal vesicular origin.  This coagulum is subsequently liquefied by the action of prostatic proteases By: Rem Alfelor 

There is some evidence that the quality of semen specimens varies depending on how the ejaculate is produced. Ejaculates produced by masturbation and collected into containers in a room near the laboratory can be of lower quality than those recovered from non-spermicidal condoms used during intercourse at home. This difference may reflect a different form of sexual arousal. Sperm concentrations in semen from young and old men may be the same, but total sperm numbers may differ, as both the volume of seminal fluid and total sperm output decrease with age, at least in some populations. In the absence of ejaculation, spermatozoa accumulate in the epididymides, then overflow into the urethra and are flushed out in urine. Steps: 1. In the first 5 minutes:  Placing the specimen container on the bench or in an incubator (37 °C) for liquefaction. 2. Between 30 and 60 minutes:  Assessing liquefaction and appearance of the semen.  Measuring semen volume.  Measuring semen pH (if required).  Preparing a wet preparation for assessing microscopic appearance, sperm motility and the dilution required for assessing sperm number.  Assessing sperm vitality (if the percentage of motile cells is low).  Making semen smears for assessing sperm morphology.  Making semen dilutions for assessing sperm concentration.  Assessing sperm number. 3. Within 3 hours:  Sending samples to the microbiology laboratory (if required). 4. After 4 hours:  Fixing, staining and assessing smears for sperm morphology. 5. Later on the same day (or on a subsequent day if samples are frozen):  Assaying accessory gland markers (if required).

SAMPLE COLLECTION 1. Dedicated private room for collection near the lab or within the lab. 2. Minimum of 2 days and max of 7 days abstinence 3. Verbal and or written instructions. (If a part is lost, it should be documented if possible).  Note: Collection at home is possible A. Submit within an hour B. 20-37 degrees C C. Condom collection:  nontoxic types  latex laden condoms  affect motility  Safe handling of specimens:  semen is a biohazard: 1. HIV 2. Hepatitis virus 3. Herpes simplex virus I.  

ANALYSIS Initial microscopic exam:  soon after liquefaction, around 30 mins but not >1 hr from ejaculation LIQUEFACTION:  initially as a coagulum  becomes thinner and watery thereafter  Usually, in 15 minutes, rarely in 1 hour  Condom specimens: If more than 1 hr, record.  This is the time when the sperms obtain the ability to move and is the best time to examine them microscopically.  It is helpful to shake gently the specimen during liquefaction to produce a homogenous sample  If there are no moving sperms, wait until full liquefaction.  Delayed liquefaction:  impossible to asses motility Page 12 of 16

Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

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 Dulbecco’s phosphate buffered saline  Proteolytic digestion by bromelain  Note: affects motility and morphology and use must be recorded SEMEN VISCOSITY:  after liquefaction:  use a 1.5mm bore disposable pipette  Normally, it leaves the tip of the pipette in small drops…if abnormal; they will form thread more than 2 cm long.  Alternatively, one may use a glass rod.  NB: high viscosity affects motility APPEARANCE OF THE EJACULATE:  after liquefaction, it is homogeneous, grey-opalescent  It may appear less opaque if the sperm concentration is very low.  The color may also be different, i.e. red-brown when red blood cells are present (haemospermia), or yellow in a man with jaundice or taking certain vitamins or drugs. VOLUME:  Collect the sample in a pre-weighed, clean, disposable container.  Weigh the vessel with semen in it.  Subtract the weight of the container.  Calculate the volume from the sample weight, assuming the density of semen to be 1 g/ml.  Note:  Empty specimen containers may have different weights, so each container should be individually pre-weighed.  The weight may be recorded on the container before it is given to the client.  Use a permanent marker pen on the vessel itself or on a label.  If a label is used for recording the weight, it should be attached before the empty container is weighed.  Alternatively:  Collect the sample directly into a modified graduated glass measuring cylinder with a wide mouth.  Read the volume directly from the graduations (0.1 ml accuracy).  Measuring volume by aspirating the sample from the specimen container into a pipette or syringe, or decanting it into a measuring cylinder, is not recommended, because not all the sample will be retrieved and the volume will therefore be underestimated.  The volume lost can be between 0.3 and 0.9 ml  Comments on volume: 1. Low semen volume is characteristic of obstruction of the ejaculatory duct or congenital bilateral absence of the vas deferens, a condition in which the seminal vesicles are also poorly developed. 2. Low semen volume can also be the result of collection problems (loss of a fraction of the ejaculate), partial retrograde ejaculation or androgen deficiency. 3. High semen volume may reflect active exudation in cases of active inflammation of the accessory organs.  Lower reference limit: 1.5 ml (5th centile, 95% confidence interval) pH  after liquefaction preferably after 30 minutes  use pH paper between 6-10  Spread a drop of semen evenly onto the pH paper.  Wait for the color of the impregnated zone to become uniform (