SESSION II URINALYSIS MICROSCOPICS COURSE INSTRUCTIONS

Transcription

1 STATE OF WEST VIRGINIA DEPARTMENT OF HEALTH AND HUMAN RESOURCES Je Manchin III Gvernr Patsy A. Hardy, FACHE, MSN, MBA Cabinet Secretary SESSION II URINALYSIS MICROSCOPICS COURSE INSTRUCTIONS 1. Write yur name n the designated area f each chapter exam. Unidentified exams will nt be graded. 2. The exams are generally made up f fur sectins. Fllw the instructins given in each sectin: A. Multiple Chice B. True r False C. Matching D. Shrt Answer 3. Cmplete all questins f the examinatin. Participants are reminded that this is an individual self-study curse. Taking the examinatin is nt meant t be a grup effrt. Credits are based n individual merit. Therefre, participants will be n the hnr system when cmpleting this examinatin. Grup study can be dne after the exams are graded and returned. 4. The Training and Evaluatin Sectin has n regulated deadline. In rder t avid urgent requests fr grading, we ask that yu return cmpleted exams in plenty f time t allw fr grading t use fr whatever ther prgram deadlines yu plan t use the credits. Return cmpleted exams t: West Virginia Office f Labratry Services Training & Evaluatin Sectin th Avenue Suth Charlestn WV A final exam that cvers the material f all three chapters is als enclsed and shuld be cmpleted and returned with the ther exams. Thank yu fr yur cperatin. BUREAU FOR PUBLIC HEALTH OFFICE OF LABORATORY SERVICES th Avenue Suth Charlestn, West Virginia Phne: (304) FAX: (304)

2 CORRESPONDENCE COURSE OUTLINE Chapter Tpic Page 1 Urinary System / Micrscpy 1 2 Micrscpic Examinatin f Urine Sediment 19 3 Quality Assurance Cnfirmatry Urine Screening Tests 39 Exams 61

3 URINALYSIS SESSION II URINE MICROSCOPICS Chapter 1 URINARY SYSTEM / MICROSCOPY Welcme t all participants in the Urinalysis crrespndence Curse. This is Sessin II f a twpart curse. This sessin is designed t meet the needs f individuals perfrming mderate cmplexity urinalysis. It als serves as a review fr all labratrians. COURSE GOAL The gal f bth curses is t prvide participant with the infrmatin needed t imprve the quality f urinalysis perfrmed at each testing site. COURSE OBJECTIVES At the cnclusin f Sessin II, participants can expect t knw r accmplish the fllwing: Knw the anatmy & physilgy f the kidney and relate t urinalysis Imprve micrscpy techniques Identify micrscpic elements in urine sediment Assess quality f urinalysis prcedures in yur labratry The kidneys perfrm fur majr functins. They: excrete mst f the unwanted end-prducts r wastes f bdy metablism; cntrl the cncentratin f mst f the cnstituents f the bdy fluids; regulate bld acid-base balance; and, regulate water and electrlyte balance. The mechanisms by which the kidney accmplishes these functins include: glmerular filtratin tubular reabsrptin tubular secretin renal bld flw Thrugh these mechanisms, the kidney selectively excretes r retains substances accrding t the specific bdy needs. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 1

4 RENAL ANATOMY & PHYSIOLOGY The purpse f this sectin is t discuss the basic principles f urine frmatin and the mechanisms by which metablic wastes are remved frm the bld. A basic knwledge f the anatmy f the urinary system and urine frmatin is an imprtant aid in understanding urinalysis. Medulla Crtex Renal Artery Renal Vein Renal Pelvis Ureters The urinary system includes tw kidneys, tw ureters, ne bladder and ne urethra. In general, kidneys frm the urine and the urine flws frm the kidneys int the ureters and is passed t the bladder fr strage. It is eliminated frm the bdy thrugh the urethra. Urethra Bladder Figure 1-1 Calyces Renal Artery Renal Vein Ureter Renal Pelvis Medulla Crtex The kidney has tw anatmical prtins: Crtex - The uter prtin f the kidney which is made up f the glmerular prtins f the nephrn and the prximal cnvluted tubules. Medulla - The Central prtin f the kidney cnsisting f the lp f Henle, distal cnvluted tubules and cllecting ducts. Figure 1-2 Figure Mdified frm A. Guyatn, Basic Human Physilgy, 2nd Editin, W. V. Saunders Cmpany Figure Mdified frm Ringsrud and Linne, Urinalysis and Bdy Fluids, Msby, 1995 WVOLS T&E 2010 URINALYSIS SESSION II P a g e 2

5 The nephrn is the functinal unit f the kidney. Urine is frmed in the nephrn. Each kidney cntains apprximately 1 t 1.5 millin nephrns. Each nephrn is capable f frming urine by itself. Therefre, it is nt necessary t discuss the entire kidney but merely the activities in the single nephrn t explain the functin f the kidney. Each nephrn is cmpsed f the: glmerulus prximal and distal cnvluted tubules ascending and descending lp f Henle Bwman s Capsule Glmerulus Prximal Tubule Afferent Arterile Distal Tubule Efferent Arterile Bwman s Space Cllecting Tubule Lp f Henle Figure 1-3: The structure f the nephrn alng with the renal circulatry system; mdified frm Mdern Urine Chemistry, Miles, Inc., WVOLS T&E 2010 URINALYSIS SESSION II P a g e 3

6 THE GLOMERULUS The glmerulus delivers bld t the nephrn and is essentially a filtering system. It is a netwrk f up t 50 parallel capillaries cvered by epithelial cells and enclsed in a capsule cmmnly referred t as Bwman s capsule. Bld enters the glmerulus frm the renal artery thrugh the afferent arterile and then leaves thrugh the efferent arterile. Pressure f the bld in the glmerulus causes fluid t filter int the Bwman s capsule. The Bwman s capsule frms a membrane that allws passage f substances with mlecular weights less than 70,000. This filtered fluid is called glmerular filtrate. Glmerular filtrate is basically bld plasma withut prteins and fats. It has a specific gravity f , an imprtant piece f infrmatin t knw if a urine specific gravity n a patient becmes fixed at (issthenuria) which indicates a lss f the cncentrating ability f the kidney. Apprximately 180 liters f glmerular filtrate is prduced daily, yet nly ne t tw liters f urine is eliminated frm the bdy daily. Therefre, much f the glmerular filtrate is reabsrbed int the bld. Frm here, the glmerular filtrate passes int the tubules where reabsrptin, secretin and urine cncentratin ccur. Bwman s (Glmerular) Capsule Glmerular Filtrate Efferent Arterile (nte directin f bld flw) Afferent Arterile (frm renal artery nte directin f bld flw) Glmerulus Figure 1 4: Functinal structure f the glmerulus. Mdified frm Ringsrud and Linne, Urinalysis and Bdy fluids, Msby, WVOLS T&E 2010 URINALYSIS SESSION II P a g e 4

7 TUBULES The lng tubule in which the glmerular filtrate is cnverted int urine is cmpsed f several distinct segments. The uppermst prtin, cntinuus with the glmerulus is the prximal cnvluted tubule, fllwed by the descending and ascending lp f Henle. This is fllwed by the distal cnvluted tubule. The distal cnvluted tubules frm several nephrns drain int the cllecting duct. It is in these tubules that reabsrptin, secretin and cncentratin ccur. See Figure 1-3 fr an illustratin f the tubules and there structure within the nephrn. PROXIMAL CONVOLUTED TUBULE Reabsrptin and Secretin The prximal cnvluted tubule is where 80% f the fluid and electrlytes filtered by the glmerulus are reabsrbed. Reabsrptin is accmplished thrugh tw transprt mechanisms called active and passive transprt. Active transprt prduces the energy necessary fr a substance t be absrbed against a gradient frm a regin f lwer cncentratin t ne f higher cncentratin. Active transprt is respnsible fr the reabsrptin f glucse, amin acids, uric acid, albumin, magnesium and phsphate ins. Passive transprt is the mvement f mlecules acrss a membrane caused by differences in their cncentratin r electrical ptential n ppsite sides f a membrane. Substances mve passively dwn a cncentratin gradient frm a regin f higher t a regin f lwer cncentratin. Mst f the water in the glmerular filtrate is passively reabsrbed. Chlride, bicarbnate and ptassium ins tgether with 40-50% f the urea present in the glmerular filtrate are passively reabsrbed with water at the prximal cnvluted tubule. The prximal tubule has a limit as t hw much substance will be reabsrbed frm the glmerular filtrate. This is referred t as the renal threshld. When the plasma cncentratin f a substance is greater than its renal threshld, it will remain in the glmerular filtrate and be excreted in the urine. Fr example, the renal threshld fr glucse is abut 160 t 180 mg/dl. When a patient with diabetes mellitus has a bld glucse cncentratin greater than 180 mg/dl, excess glucse will be eliminated in the urine. The renal threshld differs with each substance. Knwledge f the renal threshld and the plasma cncentratin can be used t distinguish between filtratin f excess slute and renal tubular damage. Fr example, glucse appearing in the urine f a persn with nrmal bld glucse is the result f tubular damage and nt diabetes mellitus. SECRETION In cntrast t tubular reabsrptin in which substances are remved frm the glmerular filtrate and returned t the bld, tubular secretin invlves the passage f substances frm the bld t the glmerular filtrate as shwn in the diagram belw. (Figure 1-5) WVOLS T&E 2010 URINALYSIS SESSION II P a g e 5

8 Glmerulus Prximal cnvluted tubule Bwman s capsule Reabsrptin (frm filtrate t bld) Secretin (frm bld t filtrate) Glmerular filtrate (nte directin f filtratin) Peritubular capillary Figure 1 5: Functinal diagram f the mechanisms invlved in kidney functin. Mdified frm S. K. Strasinger, Urinalysis and Bdy Fluids, 3 rd Ed., F.A. Davis, Tubular secretin serves tw functins: Eliminates waste prducts that are nt filtered by the glmerulus. Regulates acid-base balance thrugh the secretin f hydrgen ins. In the prximal cnvluted tubule, hydrgen ins, phsphate ins, rganic acids, and certain drugs are secreted. The regulatin f acid-base balance t maintain a nrmal bld ph f 7.4 is dependent n secretin f hydrgen (H+) ins. In general, the reactins amng hydrgen (H+), bicarbnate (HCO 3 - ) and phsphate (HPO 4-2 ) ins alng with ammnia (NH 3 ) maintain bld ph. The resulting ammnium in (NH 4 ) is excreted in the urine. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 6

9 LOOP OF HENLE The lp f Henle is the principal site where urine is cncentrated as a mechanism fr cnserving bdy water. The descending prtin f the lp is the cncentratin prtin. It is permeable t water but nt t slutes. As the lp passes farther int the medulla, the fluid sn t be urine cncentrates. The mechanism by which water mves dwn the descending lp and int the bldstream is called the cunter current mechanism. The ascending lp f Henle serves as a diluting segment because f its ability t actively secrete sdium and chlride, but prevent water lss. DISTAL CONVOLUTED TUBULE There are tw main functins f the distal cnvluted tubule: The final reabsrptin f sdium (maintaining water and electrlyte balance) Remval f excess acid frm the bdy (acid/base balance) Sdium is actively reabsrbed with sme bicarbnate. Hwever, the mechanism fr sdium reabsrptin is thrugh the sdium/ptassium pump under the cntrl f the hrmne aldsterne. The sdium ptassium pump is initiated by lw plasma sdium and/r lw bld pressure. Thrugh a cascade f hrmne stimulatin invlving renin and aldsterne, active absrptin f sdium ins are exchanged fr ptassium which is excreted by the tubular cells. Overall, there is an increase in plasma sdium and water with a decrease in bdy ptassium levels. The ph f the final urine is affected by the distal cnvluted tubules, especially by an excretin f hydrgen and ammnium ins in exchange fr sdium. The bld ph is maintained at abut 7.4 whereas urine cmmnly has a ph f 5 r 6. COLLECTING DUCT Finally, the fluid (sn t be urine) flws int the cllecting duct which cllects this fluid frm several nephrns. It is the prtin f the nephrn where the final cncentratin f urine is dne. Accrding t S. K. Strasinger, final cncentratin f the filtrate thrugh the reabsrptin f water begins in the late distal cnvluted tubule and cntinues in the cllecting duct. Reabsrptin is dependent n the smtic gradient in the medulla and a hrmne prduced in the pituitary gland called antidiuretic hrmne (ADH) r vaspressin. The prcess is cntrlled by the presence r absence f ADH which renders the walls f the distal cnvluted tubules and cllecting duct permeable r impermeable t water. A high level f ADH increases permeability, resulting in increased reabsrptin f water and a lw vlume, cncentrated urine. Likewise, absence f ADH renders the wall impermeable t water resulting in a large vlume f dilute urine. Prductin f ADH is determined by the state f bdy hydratin. ADH CONTROL SUMMARIZED Bdy hydratin = ADH = Urine vlume (diluted) Bdy hydratin = ADH = Urine vlume (cncentrated) URETERS AND BLADDER The fluid that leaves the cllecting ducts and enters the ureter is nw urine. The ureters cnnect the kidneys t the bladder. The urine is temprarily stred in the bladder and is eliminated frm the bdy thrugh the urethra. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 7

10 RENAL DISEASES Disease states as well as disrders directly affecting the kidney result in abnrmal renal functin and urinalysis tests. This sectin discusses the varius kidney diseases and the significance f these diseases n urinalysis results. ACUTE GLOMERULONEPHRITIS Glmerulnephritis is an inflammatry prcess that affects the glmerulus. Acute implies a rapid nset f symptms. It is frequently seen in children and yung adults fllwing respiratry infectins caused by strains f grup A streptccci. The streptccci are believed t frm immune cmplexes with circulating antibdies that becme depsited in the glmerular membrane which may result in glmerular membrane damage. Similar symptms may als be seen fllwing endcarditis, abscesses and pneumnia which als prduce circulating bacterial antigens. Eradicatin f the infectin in mst cases manages this disease. Primary urinalysis findings include hematuria, increased prtein; liguria, red bld cell casts, dysmrphic red bld cells, hyaline and granular casts, and while bld cells. RAPIDLY PROGRESSIVE GLOMERULONEPHRITIS This is a mre serius frm f glmerular disease with a pr prgnsis ften ending in renal failure. Symptms are initiated by dysfunctin f immune cmplexes in the glmerulus ften as a cmplicatin f anther frm f glmerular nephritis r a disrder such as lupus. Permanent damage may be caused t the glmerular capsule. Initial urinalysis findings are similar t acute glmerulnephritis but becme mre abnrmal with time. ACUTE INTERSTITIAL NEPHRITIS This is an inflammatry syndrme with a rapid nset f clinical symptms assciated with renal dysfunctin. Causes f the syndrme include acute pyelnephritis, drug txicity, graft rejectin and immune disrders. Symptms are usually reversed when the cause is remved. Urinalysis findings include red bld cells, white bld cells and white bld cell casts withut the presence f bacteria and mild t mderate prteinuria. The findings f esinphils in the urine sediment are als diagnstic. CHRONIC GLOMERULONEPHRITIS Chrnic glmerulnephritis is a variety f disrders that prduce cntinual r permanent damage t the glmerulus. The nset begins with mild symptms such as recurring hematuria (in yung adults) r hypertensin and gradually prgresses t end stage renal disease. The mst cmmn cause is an IgA nephrpathy in which immune cmplexes are depsited n the glmeruli basement membrane. Patients usually have with an episde f macrscpic hematuria assciated with strenuus exercise r an infectin. Recvery f the macrscpic frm is spntaneus; hwever, asymptmatic micrhematuria may remain elevated. Examinatin f the urine in symptmatic chrnic glmerulnephritis reveals the presence f bld, prtein, and a variety f casts, including brad casts and a fixed specific gravity f indicating lss f renal cncentratin ability. MEMBRANOUS GLOMERULONEPHRITIS This is a prnunced thickening f the glmerular capillaries with IgG depsits n the glmerular basement membrane. Autimmune diseases depsit the immune cmplexes n the membrane alng with increased prductin f epithelial cells n the membrane. Labratry findings include micrscpic hematuria and elevated urine prtein excretin. Demnstratin f systemic lupus erythematsus r hepatitis thrugh bld tests can aid in diagnsis. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 8

11 MESANGIOCAPILLARY GLOMERULONEPHRITIS This frm f glmerulnephritis is characterized by tw different alteratins in the cellularity f the glmerulus and peripheral capillaries. Type 1 displays increased cellularity in the subendthelial cells f Bwman s Capsule causing thickening f the capillary walls. Type II displays extremely dense depsits in the glmerular basement membrane. Many patients are children. The labratry findings include hematuria, prteinuria, and decreased serum cmplement levels. NEPHROTIC SYNDROME The nephritic syndrme is characterized by the appearance f massive prteinuria, edema, high levels f serum lipids, and lw levels f serum albumin. Circulatry disrders that affect the pressure and flw f bld t the kidney are ne f the mst frequent causes f the nephrtic syndrme, and it may ccur as a cmplicatin in cases f glmerulnephritis. Increased permeability f the glmerular membrane caused by changes in the electrical charges in the glmerular membrane permit the passage f high mlecular weight prteins and lipids int the glmerular filtrate. Absrptin f the lipid-cntaining prteins by the renal tubular cells fllwed by cellular slughing prduces the characteristic val fat bdies seen in the sediment examinatin. Fatty casts are als seen. MINIMAL CHANGE DISEASE Minimal change disease prduces little cellular change in the glmerulus. Patients are frequently children with edema, prteinuria, and transient hematuria. Althugh the etilgy is unknwn at this time, allergic reactins, recent immunizatins, and pssessin f the HLA-B12 antigen have been assciated. The prgnsis is generally gd. PYELONEPHRITIS Acute pyelnephritis is mst ften seen in wmen fllwing an untreated case f cystitis r lwer urinary tract infectin, and des nt cause permanent damage t the renal tubules. Recurrent infectins caused by structural abnrmality r bstructin f the urinary tract that allw bacterial t remain in the kidney, can result in tubular damage. Urinalysis findings in bth acute and chrnic pyelnephritis are similar and include white bld cells - ften in clumps - white bld cell casts, bacteria, psitive nitrite reactin, and pssible prteinuria r hematuria. With the exceptin f the presence f white bld cell casts that are indicative f tubular invlvement, similar results will be fund with infectins f the lwer urinary tract such as cystitis. CYSTITIS Cystitis is an inflammatry cnditin f the urinary bladder and ureters, characterized by pain, and frequency f urinatin. It may be caused by a bacterial infectin, calculus, r tumr. Urinalysis results shw macrscpic hematuria. Micrscpic findings include numerus white bld cells (predminantly neutrphilis), red bld cells, transitinal epithelial cells, and bacteria. There is a nted absence f casts. VIRAL URINARY TRACT INFECTIONS Rarely the urinary tract may have an infectin that is f viral rigin. The urinalysis reveals macrscpic hematuria and ccasinal prteinuria. The micrscpic urinalysis shws enlarged mnnuclear white bld cells (lymphcytes & mncytes) sme with intracellular and/r cytplasmic inclusins. Red bld cells are als present. As in bacterial cystitis, there is a nted absence f casts. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 9

12 THE MICROSCOPE The cncluding prtin f the urinalysis prcedure is the micrscpic examinatin f the urine sediment. Its purpse is t identify insluble materials that may be in the urine due t renal r urinary tract disease. The urine sediment cnsists f slid material which includes casts, cells, crystals and amrphus depsits f chemicals. The examinatin f urine sediment requires well-trained, skilled and knwledgeable persnnel. Accrding t the CLIA-88 federal labratry regulatins, the micrscpic analysis f urine sediment is a mderately cmplex test. Persnnel examining the urine sediment must fllw a prcedure that ensures cnsistency in all aspects f specimen preparatin, examinatin, identificatin and reprting. They must be skilled in the use f the micrscpe. This sectin is a general review f the micrscpe. It is nt intended t substitute fr the manufacturer s manual n the micrscpe(s) used at yur facility. During this curse, we challenge yu t read yur micrscpe manual and becme familiar with its cntents. Eyepieces Bincular Eyepiece Tube Arm Revlving Nsepiece Objectives Specimen Stage Cndenser (aperture iris diaphragm) Curse and Fine Fcusing Knbs Field Diaphragm Tungsten bulb r ther light surce Base The Cmpund Light Micrscpe WVOLS T&E 2010 URINALYSIS SESSION II P a g e 10

13 MICROSCOPE PARTS AND FUNCTIONS Arm Base Cnnects the tw lens systems t the base and supprts the stage, cndenser, and fcus adjustment knbs. The stand n which the micrscpe rests; huses the light surce and neutral density filter. Brightfield Cncentrates light n a specimen, s that specimen cntents alter the light, and prject their dark image against a light backgrund nt the bjective. Carse adjustment knb Cndenser Cndenser knb Eyepiece Field diaphragm Used fr rapid fcusing f the specimen. The lens system beneath the micrscpe state psitined t cncentrate light crrectly n the specimen and direct light rays int the bjective. When the cndenser is used at a lwered psitin, the reslving pwer is reduced. Lcated at the side f the micrscpe and belw the stage, it mves the cndenser up and dwn. During mst micrscpic examinatins the cndenser shuld be at its highest pint. Cntains the cular lenses. The interpupillary distance between the tw eyepieces f a bincular micrscpe is adjusted by mving the eyepieces tgether r apart. An aperture diaphragm which restricts the area f illuminatin. Fine adjustment knb Fcusing knbs Iris diaphragm Fcuses the lens in small increments. Used t change the distance between the specimen and the bjective lens. This is accmplished by mving the stage. The carse adjustment knb mves the stage quickly and fr a greater distance. The fine adjustment knb mves the state nly slightly fr a sharper fcus. An aperture lcated in the cndenser. It has a lever that cntrls the amunt f light passing thrugh it int the cndenser. Nsepiece Revlving plate that hlds the bjective lenses. The nsepiece easily rtates until the bjective lens is in line with the light path. Objective Stage The lens system nearest the specimen used t magnify the specimen. It directs the image frming rays f the specimen t the culars. Flat platfrm n which the slide is placed fr viewing. The slide is held in place by clips attached t the tp f the stage. The stage is mved frm side t side and frnt t back by tw cntrl knbs lcated. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 11

14 MICROSCOPE MAINTENANCE The micrscpic urinalysis begins with a prperly maintained micrscpe. In rder t keep yur micrscpe wrking prperly, it is imprtant t invest the time and mney in bth daily care and prfessinal servicing. Attentin t daily care and prmpt evaluatin f prblems can prlng the life f yur micrscpe and prevent expensive repairs. DAILY PERFORMANCE CHECK Each day, befre using yur micrscpe yu shuld: Check fr cleanliness, il residue, and scratches n the bjectives. Verify that bth curse and fine fcus adjustment knbs mve freely. Assure the bulb is functinal and that a spare bulb is available. Check the electrical crd t assure it is undamaged. Verify that the immersin il is clear (free f fungal grwth r debris). Assure the light surce is aligned t result in ptimal illuminatin. At the cmpletin f each day, yu shuld: Rtate the bjectives t assure that the lw pwer (10X bjective) is in the wrking psitin. Remve the slide frm the slide hlder and clean the stage using lens safe tissues and a mild detergent. Assure that the bjectives and the cndenser are clean by wiping with lens paper mistened in lens cleaning slutin. Turn ff the lamp. Cver yur micrscpe with dust cver. CLEANING THE MICROSCOPE In additin t the daily maintenance f the micrscpe, it is necessary t thrughly clean the micrscpe peridically. When cleaning the scpe, it is a gd time t examine it t ensure that prfessinal service and repairs are nt needed. In rder t thrughly clean the micrscpe yu will need: Cttn tipped swabs Lens safe paper Lens cleaner A mild detergent Clean each f the eye pieces f yur micrscpe using a cttn tipped swab that has been slightly mistened in lens cleaning slutin (D nt use Xylene r ther slvents.). Plish the lens with lens safe paper and hld it up t the light t assure the lens has nt becme scratched and that it is thrughly clean. Shake the eye piece slightly t assure that it des nt rattle. If rattling ccurs, the lens may need t be tightened. D nt attempt t clean the inside f the eyepiece r the eye tubes f yur micrscpe. Remve the bjectives ne at a time frm the micrscpe by unscrewing them frm the nsepiece. Clean the 10X and 40X bjectives using a cttn tipped swab which has been slightly mistened with lens cleaning slutin. Discard the swab as it cllects dirt. It may take a series f swabs t cmplete thrugh cleaning. Plish the bjective with lens paper. Remve ne eyepiece frm the micrscpe and invert it. Hlding the bjective 3 inches frm the lens f the bjective, use the eyepiece t magnify the bjective lens and check fr scratches and fingerprints. Blt excess il frm the il immersin bjective and examine the bjective using the eyepiece. It shuld be free f scratches and cracks. Wipe the micrscpe stage and bdy with a mild detergent n a sft clth. Assure that dust and il are remved. Clean the cndenser lens and any auxiliary lenses using cttn-tipped swabs which have been slightly mistened in lens cleaning slutin. Plish all lenses with lens paper. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 12

15 CHANGING THE BULB When changing the bulb n yur micrscpe, the prper bulb shuld be used. Check the mdel number fr an exact match r check with the manufacturer f yur micrscpe befre making any substitutin. It is pssible that bulbs which appear the same may differ in the placement f internal filaments which wuld affect the perfrmance f the light surce. When changing the bulb, always unplug the micrscpe. After the bulb has cled, use a clth r gauze t grasp the ld bulb and remve it frm the micrscpe. Use a clth r gauze t remve the new bulb frm the package and insert it int the micrscpe. It is imprtant that if yu d tuch the new bulb, use lens paper t carefully plish yur fingerprints frm the bulb befre installing it int the micrscpe. Oil frm yur fingers will ck nt the bulb resulting in a shrtened bulb life and pssibly in uneven illuminatin ver a perid f time. MICROSCOPIC TECHNIQUES Mst ften urine sediment is examined as a wet munt f a preparatin f cncentrated stained sediment using a brightfield micrscpe. Because it is a wet preparatin, il immersin (100X) is nt used. The preparatin is examined under lw pwer (10X bjective) and high dry pwer (40X bjective). 10X Objective The 10X bjective is used primarily t view casts and crystals, scan the slide, and lcate elements r areas fr mre detailed examinatin. It is imprtant t begin with the 10X bjective when a specimen is examined under the micrscpe. If yu d nt scan the sample n 10X, imprtant diagnstic infrmatin culd easily be verlked. 40X Objective The 40X bjective n mst micrscpes is a dry bjective. Immersin il is nt used t view the sample. Fr this reasn it is ften called the high-dry bjective. This bjective is used t examine smaller elements that have been lcated and placed int the center f the field using the 10X bjective. This bjective is mst useful fr identificatin f red and white bld cells, epithelial cells, bacteria, yeast and parasites. ADJUSTING THE LIGHT Adjustment f the brightfield micrscpe fr urine sediment can be difficult, but is essential. The light must be sufficiently reduced t give cntrast t varius unstained structures and the backgrund liquid. The light is adjusted by clsing the aperture iris diaphragm t increase cntrast and by lwering the cndenser slightly (1 t 2 mm). The cndenser shuld nt be lwered t much. Cntrast is achieved by pening r clsing the cndenser. Translucent elements are easily verlked using brightfield micrscpy. Especially difficult t see are hyaline casts. Brightfield micrscpy can be enhanced by the use f varius stains available fr urine sediment. Staining can be helpful in the identificatin f cells and casts. The mst cmmnly used urine sediment stains are supravital stains called Sternheimer - Malbin and Tluidine Blue (0.5%). Sudan stains are available t identify fat. There is a disadvantage t stains because they can precipitate causing fine crystals r granules that can be mistaken fr true crystals r amrphus materials. T stain r nt t stain depends upn the experience f the testing persnnel. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 13

16 SCANNING SPECIMENS Specimens shuld be examined carefully and systematically t avid verlking an imprtant element r detail. Always begin with the 10X bjective and scan the entire slide t lk fr elements which may require a clser lk using the 40X bjective. When scanning the slide, begin at ne edge f the cver-slip and scan using a back-and-frth mtin. Casts have a tendency t cllect near the edges f the cverslip. Therefre, lw pwer scanning f a minimum f 10 fields that include the perimeter is recmmended. Calculate the average number per field. Glass Slide Cver Glass POLARIZING MICROSCOPY The plarizing micrscpy technique is an imprtant tl fr identifying and differentiating urine sediment crystals, fat, starch and fibers. Plarized light is used t study structures that plarize light. Objects that plarize light are by definitin birefringent. When viewed with crssed plarizing filters, they appear white against a black backgrund. If a cmpensating filter is added, the birefringent bdy is seen against a magenta backgrund. Certain crystals are identified by their pattern. Birefringent bjects fund in the urine sediment include crystals, fat (as chlesterl drplets), starch and fibers (which may be cnfused with casts). Bth fat and starch shw a characteristic Maltese crss pattern (a lighted crss against a dark backgrund) when plarized. Prtein material such as casts, cells and bacteria is nt birefringent. Plarizing light can als differentiate amrphus materials. MICROSCOPY TROUBLESHOOTING When unable t btain full perfrmance frm the micrscpe, cnsult the charts belw fr trubleshting pints. PROBLEM CAUSE REMEDY I. Optical System With illuminatr n, the field f view is dark. Field iris diaphragm is nt pened sufficiently. Cndenser is lwered t much. Light path selectr lever is pulled ut t C psitin (tricular). Open diaphragm t prper diameter. Adjust cndenser height. Push in lever up t CV r V psitin. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 14

17 Field f view is cut ff r illuminated irregularly. Dust r dirt is visible in the field f view. Excessive image cntrast Reslutin prblems: image nt sharp; lack f cntrast; image details lack definitin. Field f view is partially ut f fcus, r image is partly ut f fcus. Specimen image is partially ut f fcus. Can fcus slide n lw pwer; unable t fcus n higher pwer bjectives Field f view becmes nly slightly brighter by increasing vltage. Light path selectr lever is stpped midway (tricular). Nsepiece is nt clicked int place. Nsepiece is nt crrectly munted. The pwer f the bjective used exceeds the illuminatin capacity f cndenser. Cndenser is nt centered. Field iris diaphragm is stpped dwn excessively. Dust, etc., n light exit lens. Dust n cndenser tp lens. Dirty specimen/slide/cver glass. Dust n eyepieces. Cndenser is lwered t much. Aperture iris diaphragm is stpped dwn t much. Imprper bjectives in use. Nsepiece is incrrectly munted. Objective incrrectly psitined in light path. Objective crrectin cllar nt adjusted right. Nsepiece is nt crrectly munted. Objective is nt crrectly psitined in light path. Specimen is nt crrectly psitined n stage. Nsepiece is nt crrectly munted. Cndenser is nt centered. Objective is nt crrectly psitined in light path. Specimen slide upside dwn. Objective nt screwed int nsepiece fully. Cndenser is nt centered. Cndenser is t lw. T many r wrng kind f filters in light path. Click it int prper psitin accrding t yur purpse. Slightly rtate nsepiece until it clicks int place. Insert nsepiece dvetail int micrscpe frame all the way, then lck. Chse crrect cndenser fr bjective and yur purpse. Center cndenser. Open diaphragm t prper diameter. Remve dust, and clean ptical glass parts. Adjust cndenser height. Open diaphragm t prper diameter. Use bjectives made fr the micrscpe & its cmpnent parts. Insert nsepiece dvetail int bdy all the way & lck int place. Click nsepiece int place. Rtate crrectin cllar, keeping specimen in fine fcus, until ptimum reslutin is btained. Insert nsepiece dvetail int micrscpe frame all the way & lck in place. Slightly rtate nsepiece until it clicks int place. Place specimen slide crrectly n stage, and secure with stage, and secure with stage clips. Insert nsepiece dvetail int micrscpe bdy all the way & lck in place. Center cndenser. Slightly rtate nsepiece until it clicks int place. Turn slide right side up. Securely tighten bjective int nsepiece. Adjust cndenser height. Adjust cndenser height. Remve filters frm light path. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 15

18 PROBLEM CAUSE REMEDY II. Electrical System Illuminatr is t bright (r t dark) even when adjusting cntrl lever. Illuminatr vltage cannt be raised. Lamp ges ff and n. Bulb burns ut frequently. Line vltage selectr switch nt matched with lcal facility vltage. Line vltage selectr switch nt matched with lcal facility vltage. Bulb filament is abut t burn ut. Lse electrical cnnectins. Line vltage selectr switch nt matched with lcal facility vltage. Bulb is nt the standard ne fr micrscpe. III. Carse and Fine Adjustments Match selectr switch t facility vltage. Match selectr switch t facility vltage. Replace bulb. Check all cnnectins. Match selectr switch t facility vltage. Use nly standard bulb. Carse adjustment knb is t tight. Stage drps r ges ut f fcus due t slipping fine adjustment knb. Stage cannt be raised t upper fcusing limit. Stage cannt be lwered t lwer limit. Objective frnt lens hits specimen befre achieving fcus. Incmplete bincular visin. Image easily ges ut f fcus when yu tuch the stage. Specimen stps midway n the side-t-side traverse. Tensin adjustment ring is t tight. User trying t raise stage abve fcusing limit set by prefcusing lever. Tensin adjustment ring is t lse. Prefcusing lever engaged lwer than fcusing limit. Stage munt t lw. Specimen placed n stage upside dwn. Stage munted t high bjectives ut f fcus. Objectives nt parfcal. Objective nt screwed in nsepiece securely. IV. Observatin tubes Interpupillary distance is nt crrectly adjusted. Dipter adjustment is incmplete. Right and left eyepieces are nt matched. User unaccustmed t bincular visin. V. Stage Stage is nt crrectly lcked in place. Specimen r slide is nt psitined crrectly. Lsen ring prperly. Unlck lever, and adjust stage fcusing level. Tighten ring prperly. Unlck lever and adjust. Raise sage munt. Reverse specimen slide. Lwer stage munt and realign fcus. Parfcal bjectives. Securely tighten bjective in nsepiece. Crrect the interpupillary distance. Cmplete the dipter adjustment. Use nly matched pair f eyepieces. Befre lking int bincular bservatin tube lk ff int distance. Clamp stage securely. Adjust specimen r slide psitin crrectly. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 16

19 GLOSSARY OF TERMS Bacillus Brightfield Brwnian Mtin Cccus/Ccci Cmpund micrscpe Cntrast Cver glass Definitin Depth f field Dry bjectives Fcal Length Field f view Filters Neutral Density Filters Ttal magnificatin Micrscpy Mtility Any rd shaped bacterium. A micrscpy technique in which light passes directly thrugh specimen and int the bjective, making specimen image appear dark against a bright backgrund. The randm, dancing, zigzag mvements f minute, micrscpic particles suspended in liquid. This mtin is due t cllisins f the particles with the individual randm-mving mlecules f the slvent. A bacterial cell with a spherical shape. A micrscpe made up f tw lens systems, eyepiece and bjective. A relative difference between the brightest and darkest parts f the specimen; crispness. It is cntrlled by the aperture diaphragm. T little cntrast results in lack f definitin; t much cntrast reduces reslutin. An ultra thin glass made t cver the specimen n the slide. It is a part f the image frming system. The cver glass has ptical prperties which are taken int accunt in cmputing and designing bjective. Manufacturers specify the thickness f the cver glass fr general use n the micrscpe. The brilliance, clarity, distinctness, and sharpness with which the micrscpe magnifies and reprduces specimen detail. Distance just abve and belw the fcal plane (area being examined) that can be fcused clearly. Micrscpe bjectives designed t be used dry, i.e. withut il. The distance f the fcus frm the surface f a lens. The visible area thrugh an in-fcus lens. Used t cntrl the intensity r clrs f illuminatin. A filter at the light surce t cntrl the intensity (brightness) f illuminatin. Magnificatin f eyepiece x magnificatin f bjective. Example: eyepiece = 10X and bjective = 40X Ttal magnificatin = (10X) x (40X) = 400X The science f the uses and applicatins f micrscpes. Tw bjectives f micrscpy are frming a magnified image with as few ptical defects as pssible, and achieving gd reslutin and cntrast. Cntrast is based n the differential absrptin f light between the specimen under study and its backgrund; reslutin is the ability t reveal and separate fine detail. Having spntaneus but nt cnscius mvement, cntractility, ability f an rganism t mve in the medium, usually assciated with the presence f flagella, cilia, r pseudpdia. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 17

20 Numerical aperture (NA) Parfcal Refractive index Reslutin Stage Fcal Plane Image Immersin il Interpupillary distance Iris diaphragm Kehler illuminatin Lens Magnificatin Wrking distance A number, usually engraved n the bjectives and cndensers, expressing the size f the cne f light delivered by the cndenser r cllected by the bjective. NA is defined by the frmula: NA = n sin θ where θ = the half-angle f the light cne entering the bjective; and, n = refractive index f medium between specimen and bjective: air: n = distilled water: n = il and glass: n = The higher the NA, the greater the reslving pwer; hwever, the NA f the cndenser must be the NA f the bjective t achieve the full reslving pwer f the bjective. The bjectives are cnstructed s that nly slight refcusing with the fine adjustment knb is needed after rtating t anther bjective. Rati f the speed f light in the first medium t the speed f light in the secnd medium. The ability f a micrscpe t reveal fine detail in a specimen. The better the reslving pwer f a micrscpe the clser tw bjects can be and still be distinguished as tw bjects. The platfrm n which the micrscpe slide is placed. The up/dwn level t which the specimen r bject may be clearly imaged and studied. A picture r cnceptin: Real - ne frmed by the cllectin rays in which the bject is pictured as being inverted. Its presence can be viewed nly by insertin f a receiving screen, etc. Virtual - frmed by a cnverging lens. The image seems t be situated n the same side f the lens as the bject. An il with the same refractive index as glass, 1.515; used between the cver glass and an il immersin bjective t prevent scattering f light in air. The distance between the eyes. The eyepieces f a bincular scpe must be adjusted s that left and right images merge int ne. Adjustable assembly f thin metal leaves fr varying the size f penings that determine the crss sectin f the light ray bundle entering the cndenser and the bjectives. Bth the field and aperture diaphragms are iris diaphragms. A methd f ptical illuminatin prviding bright, evenly dispersed, glarefree light with gd cntrast and reslutin. The light beam is fcused n the back fcal plane f the cndenser, the field diaphragm is fcused in the field f view, and then the light is fcused again at the backless f the bjective. A piece f glass r ther transparent substance shaped t gather r scatter light rays, and used in the micrscpe and ther instruments t magnify, increasing the visual acuity f the human eye. The number f times larger the image appears as seen thrugh the micrscpe, than it appears t the eye at a distance f 10 inches (25.3 cm). The rati, in diameters, usually is expressed as pwer, times, r X. Distance between cverslip f a slide and the tip f an bjective. The lw pwer bjective has the greatest wrking distance. The il immersin bjective has a very small wrking distance. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 18

21 MICROSCOPIC EXAMINATION OF URINE SEDIMENT CHAPTER 2 CHAPTER OBJECTIVES Recgnize the frmed elements in the urine sediment, which include: Red bld cells White bld cells Epithelial cells Casts Crystals Amrphus material Artifacts Understand the clinical significance and rigin f the varius frmed elements in urine sediment Describe the standardized examinatin f urine sediment URINE SEDIMENT STANDARDIZATION The urine sediment cnsists f any frmed elements suspended in the urine. These elements include cells, casts, crystals, amrphus materials, and artifacts. The physical appearance and reagent strip test results are clues in the identificatin f the sediment cnstituents. The visual, chemical, and micrscpic examinatin f sediment must be crrelated t ensure accuracy f the urinalysis results. Traditinally, a urine sediment examinatin has been part f rutine urinalysis. Hwever, recent studies have shwn that the micrscpic examinatin may nt be necessary n every urine specimen tested. NCCLS states that the decisin t perfrm a micrscpic examinatin must be made by each individual labratry based n its patient ppulatin. Urine micrscpics shuld be perfrmed when requested by the physician, when determined by labratry prtcl, r when any visual r chemical result is abnrmal. Befre adpting this plicy, the labratry must have gd standardized techniques and a quality cntrl prgram in place. Cnditins t skip the micrscpic examinatin: yellw r straw in clr, with a clear appearance reagent strip tests are negative r nrmal WVOLS T&E 2010 URINALYSIS SESSION II P a g e 19

22 Cnditins t perfrm the micrscpic examinatin: any clr ther than yellw hazy, cludy, r turbid prtein greater than trace glucse, bld, nitrite, r leukcyte is psitive if the clinician requests it SPECIMEN REQUIREMENTS The first mrning midstream clean catch urine specimen is the specimen f chice fr urine micrscpics. It is a gd representative sample f the patient s cnditin and is less likely t be cntaminated frm external surces. Randm specimens are acceptable but need t be interpreted carefully with respect t cntaminatin. Ideally, ml. shuld be cllected int a clean 100 ml. screw cap cntainer. At least 12 ml. is required fr urine sediment cncentratin. Specimens shuld be centrifuged and examined within 2 hurs f cllectin held at rm temperature. Refrigeratin f the urine retards urine decmpsitin but causes amrphus material t depsit which culd bscure pathlgic cnstituents. URINE SEDIMENTATION PROCEDURE T ensure cnsistent urine micrscpic results, it is necessary t standardize the prcedure. Standardized urine sediment systems with specially designed centrifuge tubes and pipettes are available. Systems differ in final urine sediment vlume, but are alike in prviding a capped centrifuge tube, transfer pipette, stain (ptinal), and specially designed slides. If the traditinal glass micrscpe slides and cver slips are used, a unifrm vlume f cncentrated sediment shuld be placed n the slide and cvered with a standard #1 cverslip (22 x 22 mm). Whatever system is used, the fllwing specific factrs must be standardized: Urine vlume. Twelve ml. is generally recmmended fr centrifugatin and is the vlume emplyed by standardized systems. If a different vlume is used, the final cncentratin f urine examined must be applied t numerical cunts. Time f centrifugatin. Five minutes is recmmended. Speed f centrifugatin. NCCLS recmmends a relative centrifugal frce (RCF) f 400 g. Other standard textbks recmmend 450 r g. Nmgram (a graphical plt used fr slving certain types f equatins) can be used t relate the revlutins per minute (RPM) t RCF by measuring the radius ( r ) f the centrifuge head in centimeters frm the center pin t the bttm f a hrizntal cup r frm the fllwing frmula: RCF(g) = (11.18 x 10-6 ) x ( r ) x RPM 2 Cncentratin factr f the sediment. This is based n the vlume f well-mixed urine centrifuged and the final vlume f sediment remaining after the supernatant urine is remved. A unifrm amunt f 0.5 t 1.0 ml. shuld remain in the tube after decantatin. Sediment drp unifrmity. The drp transferred t the slide shuld be f unifrm size, large enugh t fill the cverslip withut air bubbles, but small enugh nt t run ver the slide. An excessive amunt f fluid may cause casts t g ut f the visin area. Reprting Frmat. Every persn in an institutin wh perfrms a micrscpic examinatin f the urinary sediment shuld use the same terminlgy, reprting frmat, and reference ranges. Crrelate micrscpic results with physical and chemical findings. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 20

23 ROUTINE URINALYSIS CORRELATIONS Micrscpic Elements Physical Chemical Exceptins Red bld cells Turbidity, Red clr + Bld Number, Hemlysis White bld cells Turbidity + Prtein, + Nitrite, + Leukcytes Number, Lysis Epithelial cells Turbidity Number Casts + Prtein Number Bacteria Turbidity ph, + Nitrite + Leukcytes Number and Type Crystals Turbidity, Clr ph Number and Type Adapted frm S. K. Strasinger, Urinalysis and Bdy Fluids, Editin 3, F. A. Davis, 1994 NORMAL URINE SEDIMENT Nrmal urine cntains urinary sediment. It is the amunt and the type f cnstituent that determines abnrmality. The fllwing table is a reference guide fr distinguishing nrmal frm abnrmal. Keep in mind all labratries shuld establish their wn nrmal values. NORMAL REFERENCE VALUES FOR URINARY SEDIMENT SEDIMENT CONSTITUENT 12:1 CONCENTRATION Red bld cells White bld cells Hyaline casts Renal epithelial cells Transitinal epithelial cells Squamus epithelial cells Bacteria Abnrmal crystals 0 2 / hpf* 0 5 / hpf 0 2 / lpf few / hpf few / hpf few / lpf neg / hpf nne / lpf *hpf = high pwer (40x bjective) field.; **lpf = lw pwer (10x bjective) field. Adapted frm K. M. Ringsrud and J. J. Linne. Urinalysis and Bdy Fluids, Msby, 1995 WVOLS T&E 2010 URINALYSIS SESSION II P a g e 21

24 CELLS FOUND IN URINE SEDIMENTS 1. RED BLOOD CELLS (RBC) NORMAL CRENATED SWOLLEN Descriptin: Clrless discs which may appear bicncave, crenated (cncentrated urine) r swllen (dilute urine). Clinical Significance: Althugh the presence f 0-2 RBC s/hpf is cnsidered nrmal, the presence f RBC s in the urine (hematuria) is an early indicatr f renal disease. When bld is detected n the reagent strip, the micrscpic is an imprtant piece f infrmatin in distinguishing between hematuria, hemglbinuria, and myglbinuria, all cnditins f different rigin. Hematuria is the presence f intact RBC s. Hemglbinuria is the presence f free hemglbin in the urine. Myglbinuria is the presence f the muscle hemglbin called myglbin in the urine. When myglbin is fund in the urine, it is indicative f muscle wasting. In general, with hematuria, red bld cells are present in the urine sediment but absent with hemglbinuria and myglbinuria. Bleeding can ccur at any pint in the urinary system. The presence f RBC s nly indicates a prblem f the lwer urinary system. When RBC s are accmpanied by casts in the urine sediment and prtein n the reagent strip urinalysis, the bleeding may be f glmerular rigin within the kidney. The amunt and shape f the RBC s als crrelate with the severity f the prblem. Dysmrphic r irregularly shaped RBC s culd indicate glmerular rigin. Structures which can be misidentified as red bld cells: yeast, calcium xalate, bubbles, il drplets, lymphcytic white bld cells. 2. WHITE BLOOD CELLS (WBC) MONOCYTE LYMPHOCYTE NEUTROPHIL EOSINOPHIL MACROPHAGE Descriptin: Runded, glbular cells, mst having lbed nuclei; larger than RBC s. Supravital Stain may be helpful in identifying nuclear material. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 22

25 Clinical Significance: Nrmal cncentrated sediment may cntain up t 5 leukcytes per high pwer field. Thugh the presence f WBC s generally refers t neutrphils, the presence f lymphcytes and esinphils are f diagnstic significance. An increase in WBC s in the urine is called pyuria and indicates inflammatin at sme pint in the urinary system. Neutrphils plus bacteria are generally seen tgether. Clumps f WBC s indicate acute infectin. Glitter cells are a type f neutrphil seen in dilute urine that cntains granules which exhibit Brwnian mtin. This mtin results in a shiny r glittery appearance f the cell. Esinphils in urine sediment are identifiable nly with supravital stains. They indicate drug induced nephritis including hypersensitivity t antibitics. Lymphcytes can be seen with renal transplant rejectin. Lymphcytes will nt react with leukcyte esterase n the reagent strip. Structures which can be misidentified as white bld cells: red bld cells, renal epithelial cells 3. EPITHELIAL CELLS SQUAMOUS TRANSITIONAL RENAL TUBULAR Epithelial cells are cells that line all the urinary and genital tracts. They are f three different types: squamus, transitinal and renal. Identificatin is clinically significant. SQUAMOUS EPITHELIAL CELLS Descriptin: Squamus epithelial cells are the largest cells seen in nrmal urine specimens. They are thin, flat cells, usually with an angular r irregular cell membrane and granular cytplasm. The nucleus is small but prminent nucleus. They may be present as single cells r as variably-sized clusters. Clinical significance: Squamus epithelial cells line the lwer urethra and vaginal areas. Large numbers ften represent vaginal cntaminatin in females r freskin cntaminatin in males. A few squamus epithelial cells are seen in mst urine specimens. Their presence has little clinical significance. Clue cells are a special type f squamus cell seen in bacterial vaginitis caused by Gardnerella vaginalis, a rd-shaped bacterium. The bacteria encrust themselves t the surface f the epithelial cell. T be reprted as a clue cell, mst f the surfaces shuld be cvered with bacteria. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 23

26 TRANSITIONAL EPITHELIAL CELLS Descriptin - Transitinal epithelial cells are rund r pear shaped with a central nucleus. Mst ften they are rund r plygnal because they take in water and swell spherically. Sme transitinal cells have tails f cytplasm and are referred t as caudate cells. These riginate frm the renal pelvis r the bladder and are nt clinically significant. Clinical Significance - Transitinal epithelial cells frm the lining f the urinary tract frm the pelvis f the kidney t the base f the bladder in female and the prximal part f the male urethra. Thse cells clser t the kidney are deeper, thicker, and runder. Nrmal urine may cntain a few transitinal cells. Numbers are increased in infectins. Clusters r sheets may be seen after catheterizatin. Structures which can be misidentified as transitinal epithelial cells - white bld cells, renal epithelial cells. RENAL TUBULAR EPITHELIAL CELLS Descriptin: Renal tubular cells are riginally cubic in shape; but nce exfliated, they adpt a runded r elngated val shape with a granular cytplasm. They have a single rund eccentric nucleus. Renal tubular cells vary in size frm slightly larger t twice as large as a WBC. Renal cells d nt absrb water and swell as d transitinal cells; therefre they tend t retain an val rather than rund shape. Clinical significance - Renal epithelial cells riginate frm the prximal t the distal cnvluted tubules, as well as the cllecting ducts. Small numbers can be fund in nrmal urine. Increased numbers, e.g., 15 renal tubular epithelial cells per hpf, prvide evidence f active renal disease r tubular injury. Their clinical significance als includes tubular necrsis, viral kidney infectins, renal transplant rejectin and chemical txins. Oval fat bdies are degenerated r necrtic renal epithelial cells filled with fat drplets. They can be recgnized with brightfield micrscpy but may need special fat stains fr identificatin (Sudan stains). If chlesterl is present in the fat glbules, they may be visualized with plarized light. The characteristic Maltese crss (white crss n black backgrund) is seen when the specimen is viewed with crssed plarizing filters. Structures which can be misidentified as renal tubular epithelial cells: transitinal epithelial cells and white bld cells. 4. BACTERIA Descriptin - Bacteria are the smallest cellular elements f clinical imprtance fund in urine sediment. Mst bacteria present in urine sediment are rd-shaped (bacillus). Runded frms (ccci) can be seen mstly due t cntaminatin. Bacteria in a fresh specimen may be mtile. Clinical Significance - Bacteria are nt nrmal cnstituents f urine. Hwever, unless specimens are cllected under sterile cnditins, bacterial cntaminatin will ccur. Specimens that are stred at rm temperature may have a significant amunt f bacteria that represents nthing WVOLS T&E 2010 URINALYSIS SESSION II P a g e 24

27 diagnstically. Bacteria in a urine sediment that has been prperly cllected and stred indicates a urinary tract infectin. White bld cells are present and the reagent strip is psitive fr nitrite, leukcyte esterase, and prtein if a significant urinary tract infectin is present. Mst urinary tract infectins are due t bacteria f fecal rigin such as Escherichia cli r Prteus species, which are bth gram-negative rds. Structures which can be misidentified as bacteria - amrphus phsphates, amrphus urates. 6. YEAST Descriptin: Yeast can vary in appearance frm vid shape t vid with buds and elngated branches called pseudhyphae. They have a smth refractile appearance with n granules. Clinical Significance - Yeast in the urine are mst ften Candida species. They result frm urine cntaminatin frm a vaginal r skin yeast infectin. They can be assciated with the presence f glucse in urine, ften with diabetic patients. The presence f pseudhyphae (elngated cells resembling mycelia) may indicate a true kidney infectin caused by Candida species. Fr this reasn, pseudhyphae when present in the urine sediment shuld be nted n the reprt alng with yeast. Structures which can be misidentified as yeast - red bld cells 6. PARASITES Trichmnas vaginalis the mst cmmnly seen parasite. Descriptin - Trichmnas vaginalis is a flagellated prtzan that is pear shaped and abut the size f a large white bld cell. The rganism has a single nucleus, fur anterir flagella, an anterir undulating membrane, and a sharp prtruding axstyle. Their mst distinguishing feature is a rapid jerky mtility. Diagnstic Significance - Trichmnas vaginalis is a cntaminant f urine that is respnsible fr vaginal, urethral, bladder, periurethral, and prstate infectins. If present, it resides in the vagina in wmen and the prstrate in men. Structures which can be misidentified as Trichmnas - white bld cells, transitrial epithelial cells. Urine may rarely cntain a variety f parasites ther than Trichmnas. They are f fecal r vaginal rigin with exceptin f Schistsmes which can reside in the urinary tract. If a parasite is suspected, cnsult with yur micrbilgy persnnel. In mst cases, what lks like a parasite may be nthing mre than a pllen grain r starch granule. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 25

28 CASTS Casts are the result f precipitatin r clumping tgether f prtein material in the tubules f the kidney. This prduces slidified prtein that is mlded t the lumen f the distal cnvluted tubule, ascending lp f Henle, and/r cllecting ducts. Their shape generally cnsists f parallel sides and rund r blunt ends. The majr prtein in casts is Tamm-Hrsfall prtein which is excreted by the renal tubular cells. Casts prvide a micrscpic view f the nephrn. When the cast is frmed any material present in the tubule (e.g. cells, bacteria, fat) is trapped within the cast. This helps t identify the type f cast and the cnditin within the kidney that is represented. Cast frmatin is enhanced by decreased urine flw, an acid ph and high cncentratin f slutes (increased specific gravity). Casts are the nly element fund in the micrscpic examinatins that are unique t the kidney. STEPS IN THE FORMATION OF A CAST: Aggregatin f Tamm-Hrsfall prtein int individual prtein fibrils Attachment f the prtein fibrils t the surface f the tubular epithelial cells t prevent them frm being washed away Interweaving f prtein fibrils t frm a lse fibrillar netwrk (urinary cnstituents may becme enmeshed in the netwrk at this time) Further prtein fibril interweaving t frm a slid structure Pssible attachment f urinary cnstituents t the slid matrix Detachment f prtein fibrils frm the epithelial cells Excretin f the cast HYALINE CAST Descriptin: Hyaline casts are clrless with parallel sides, unifrm diameter, definite brders and runded ends. They are difficult t see with brightfield micrscpy and need careful light adjustment. The light must give cntrast by lwering the cndenser slightly. The use f supravital stains is helpful. The narrw diameter frm f this cast with ne end tapering ff int a tail is referred t as a cylindrid. Cylindrids are frmed at the lp f Henle and distal cnvluted tubules, which causes the characteristic shape. They are reprted as number f hyaline casts per lw pwer field. Clinical Significance - Hyaline casts are the least imprtant cast. Less than 2 hyaline casts per lpf may be seen in nrmal urine. They result frm Tamm-Hrsfall prtein secreted int the renal tubular epithelium that aggregates int fibrils. The reagent strip result fr prtein may be psitive r negative. Strenuus exercise will prduce an increased number f this type f cast but the urine sediment returns t nrmal after 48 hurs. Hyaline casts may be seen in large numbers in mderate t severe renal disease. Structures which can be misidentified as hyaline casts: mucus, epithelial cells rlled int a cigar shape, yeast with pseudhypae. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 26

29 WHITE BLOOD CELL (WBC) CASTS Descriptin: WBC casts have parallel sides, runded ends, and WBC s embedded within the prtein material. WBC casts are relatively easy t see with brightfield micrscpy r supravital stain. The number f WBC s within the cast varies frm packed t few. Clumps f WBC s adhering t the exterir f a mucus thread are called a pseudcyst which may be difficult t distinguish frm a WBC cast. Clinical Significance - WBC casts are nt a nrmal finding. They are mst ften made up f neutrphils. Pyelnephritis r acute interstitial nephritis will prduce WBC casts. When WBC casts are present, the rigin f the infectin is in the kidney rather than the bladder. Structures which can be misidentified as WBC casts - pseudcyst, epithelial cell casts EPITHELIAL CELL CAST (Renal tubular) Descriptin - Epithelial cell casts have parallel sides, runded ends and renal epithelial cells embedded within the cast. They vary in size and shape. The epithelial cell cast underges changes frm cellular, t granular, and finally waxy. Clinical Significance - Epithelial cell casts result frm destructin f the cells that line the renal tubules. Damage may be irreversible depending n the disease severity. Epithelial cell casts may be seen after expsure t nephrtxic substances such as mercury r antifreeze r assciated with viral infectins. Structures which can be misidentified as epithelial cell casts - epithelial cells rlled int a cigar shape. RED BLOOD CELL CAST (RBC AND HEMOGLOBIN) Descriptin - RBC casts have parallel sides, rund ends and red bld cells entrapped within the prtein matrix. White bld cells and epithelial cells als can be seen within the cast. They are refractile and have a characteristic clr ranging frm yellw t brwn frm released hemglbin under brightfield illuminatin. They are ften bserved as fragments r prtins because red bld cell casts are prbably the mst fragile cast. As the cast ages, cells lyse and the casts appear waxy but retain the characteristic yellw t brwn clr. The aged red bld cell cast is then called a hemglbin cast. Hemglbin casts shuld be reprted as RBC casts 1pf. Differentiatin between the hemglbin cast and red bld cell cast is nt necessary because diagnstically they represent the same disease states. Clinical Significance - The presence f red bld cell casts shw bleeding within the nephrn. Primarily they are assciated with glmerulnephritis r lupus nephritis; hwever anything that damages the glmerulus, tubules r renal capillaries may cause prductin f RBC casts. Very strenuus exercise can als cause RBC casts t be prduced but it is nly a temprary cnditin. Structures which can be misidentified as red bld cell casts: granular casts with bilirubin r pyridium in the urine sediment. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 27

30 GRANULAR CASTS Descriptin - Granular casts have parallel sides and runded ends with carse t fine granules. The size f the granules within the cast varies and becmes prgressively smaller with degeneratin. The number f granules als varies frm nly a few within a hyaline matrix t a cast that seems t be cmpletely filled. Granular casts, whether the granules are carse r fine, need nt be further classified. Althugh many labratries distinguish between carse and fine granular casts, it is nt necessary. Reprting the term granular is sufficient. If a cast has a definite hyaline matrix with nly a very few granules, it shuld be reprted as hyaline rather than granular. Clinical Significance - A few granular casts may be present in nrmal urines, especially during vigrus exercise. The granules may result frm disintegratin f tubular cells, cellular casts r prtein aggregates such as Tamm-Hrsfall prtein. Urinary stasis which allws the casts t remain in the tubules lnger must ccur t result in a granular cast. Granular casts may be seen in glmerular (glmerulnephritis) r tubular disease (interstitial nephritis). Renal graft rejectin may als prduce this cast. Structures which can be misidentified as granular casts: cellular casts, rlled epithelial cells, clthing fibers, scratches n lens. WAXY CASTS Descriptin - Waxy casts have a brittle texture that causes them t be fragmented, r cracked. They are hmgeneus, like hyaline casts, but are easier t see because they are refractile with a brader width and blunt ends. Clinical Significance - Waxy casts are the final stages f the degeneratin prcess frm cellular t granular t waxy. They indicate a state f extreme renal stasis and are assciated with chrnic renal disease. They may appear in telescped sediment which is urine sediment that cntains a range f casts in varius stages f degeneratin. This is seen during a perid when the kidney resumes functin after a perid f shutdwn. Structures which can be misidentified as waxy casts - diaper fibers (NOTE: differentiate frm casts fibers based n negative reagent strip results fr prtein and ther sediment findings. Fibers will plarize with plarizing filters and casts will nt plarize.) BROAD CASTS (RENAL FAILURE CASTS) Descriptin - Brad casts are f all cast types but appear wider. Many times they are waxy casts f greater diameter. Clinical Significance - Brad casts are frmed in the cllecting ducts when the flw frm the kidney tubules becmes severely cmprmised. The finding f this type f cast represents renal failure. CASTS: KEY POINTS & REVIEW Unique t the kidney, frmed in the tubules and cllecting ducts Acid ph, high specific gravity, increased prtein, decreased urine flw Parallel sides and runded r blunt ends Usually pathgenic D nt plarize as fibers d WVOLS T&E 2010 URINALYSIS SESSION II P a g e 28

31 SUMMARY OF URINE CASTS Type Origin & Key Pints Clinical Significance Hyaline Tubular secretin f Tamm-Hrsfall prtein that aggregates int fibrils. Cntain n granules r exclusins. Glmerulnephritis Pyelnephritis Chrnic renal disease Cngestive heart failure Stress and exercise 0-1/1pf nrmal Red bld cell Red bld cell enmeshed within the Tamm-Hrsfall prtein matrix. Cells are nt just stuck t the utside. Glmerulnephritis Strenuus exercise White bld cell White bld cells enmeshed within the Tamm-Hrsfall prtein matrix. Cells are nt just stuck t the utside. Pyelnephritis Acute interstitial nephritis Bacterial Epithelial cell Granular Bacteria attached t Tamm-Hrsfall prtein matrix. Renal Tubular cells enmeshed within Tamm-Hrsfall prtein fibrils. Disintegratin f cellular casts resulting in curse r fine granules. Pyelnephritis Renal tubular damage Glmerulnephritis Pyelnephritis Stress and exercise Waxy Fatty Brad casts Refractile fractures and very brittle r n granules exclusin. Urinary lipids Oval fat bdies Frmatin f all types f casts in the cllecting ducts r distended distal tubules. They are 2-6 times wider. Stasis f urine flw Nephrtic syndrme Extreme stasis f flw Serius Prgnsis Adapted frm S. K. Strasinger, Urinalysis and Bdy Fluids, 3rd Editin, F. A. Davis, 1994 WVOLS T&E 2010 URINALYSIS SESSION II P a g e 29

32 CRYSTALS Crystals are cmmnly present in urine sediment. They are seldm f clinical significance. In sme cases, they bscure mre imprtant pathlgical findings such as cells r casts. Sme can be beautiful, especially under plarized light with their birefringent prperties. Identificatin f crystals is necessary t ensure that they d nt represent an abnrmal cnditin. Abnrmal crystals are f metablic rigin. Crystals are frmed by the precipitatin f urine salts subjected t changes in ph, temperature, r cncentratin. The presence f crystals in the urine is called crystalluria. As urine cls t rm temperature r is refrigerated, specimens may becme cludy due t crystal precipitatin. Generally crystals are imprtant nly when present in freshly vided urine. Crystal frms fund in the urine sediment are classified as nrmal r abnrmal. These are further classified as acid crystals r alkaline crystals. Other crystals are f drug rigin. Crystals are identified by mrphlgy and urine ph. Nrmal crystals are generally reprted as few, mderate r many per high pwer field. Abnrmal crystals are reprted as average number per lw pwer field. Cnfirmatin f sme crystals may require a chemical test such as a diaz reactin fr the sulfnamides r the cyanide nitrprusside reactin fr cystine. Knwledge f medicatin r prcedures that invlve radigraphic cntrast media is als helpful. NORMAL CRYSTALS ACID URINE (ph f 6.5 r less) 1. URIC ACID Descriptin - Uric acid crystals are seen in a variety f shapes, including fur-sided and rhmbic plates, rsettes, wedges, and needles. They may be relatively thick r thin. Uric acid crystals are birefringent and give a beautiful display f clrs when viewed with plarized light. Uric acid becmes sluble at 60 C and in 10% sdium hydrxide. Clinical significance - Althugh f little clinical significance, the presence f uric acid may be assciated with gut, stne frmatin, guty nephritis (in large numbers), leukemia patients (particularly in chemtherapy), and Lesch-Nyln syndrme. Tgether with amrphus urates, uric acid crystals are the mst cmmn crystals seen in acid urine. The ph f the urine needs t be less than 6 fr the crystals t be present. They are dependent n dietary intake f purines and breakdwn f nucleic acids. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 30

33 2. AMORPHOUS URATES Descriptin - Amrphus means withut shape. Amrphus urates are yellw t red t brwn clred micrcrystals that plarize light. They precipitate in refrigerated r rm temperature urine. The physical appearance f urine cntaining amrphus urates resembles bldy urine. Amrphus urates are sluble at 60 C and in 10% sdium hydrxide. They will change t uric acid when acidified with acetic acid. Clinical significance - Amrphus urates can ften be seen in cncentrated urine assciated with dehydratin and fever. Generally, they are f n diagnstic significance and are ne f the mst cmmn crystals. Structures which can be misidentified as amrphus materials: amrphus appears in clumps r adhere t mucus). granular casts (when 3. CALCIUM OXALATE Descriptin - Calcium xalate is seen as a clrless ctahedrn - an eight-sided frm that may be thught f as tw fur-sided pyramids jined at far ends. They are ften described as envelpes r squares with a crss in the center. Large and small frms may be seen. Occasinally, calcium xalate is present in a small vid frm. The vid shape is trublesme t identify since the size and shape resembles a red bld cell. It may be distinguished frm RBC s by plarized light. Calcium xalate is birefringent when viewed with plarized light. Clinical Significance - The presence f calcium xalate withut symptms is f little clinical significance. Hwever, with symptms, the crystals may give evidence f urinary stnes, which can cntain calcium. Large calcium xalate crystals in clusters are assciated with stnes and ethylene glycl pisns. Excess xalate frm fd cntaining xalic acid (spinach, rhubarb and Vitamin C (ascrbic acid) may cause frmatin f calcium xalate crystals. Calcium xalate may rarely appear in neutral and alkaline urine. Structures which can be misidentified as calcium xalate: red bld cells. 4. CALCIUM CARBONATE Descriptin - Calcium carbnate crystals are small and clrless with a characteristic dumbbell shape. They may appear in clumps r singly. When acetic acid is added t urine sediment cntaining this crystal, calcium dixide gas is released which prduces bubbles. Calcium carbnate is strngly birefringent. Clinical significance - Calcium carbnate is f little clinical significance. It is an uncmmn crystal in nrmal urine. Structures which can be misidentified as calcium carbnate: amrphus phsphates. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 31

34 ALKALINE URINE 1. AMORPHOUS PHOSPHATES Descriptin - Amrphus phsphates are shapeless micrcrystals that lack clr and tend t be finer than amrphus urates. Unlike urates, phsphates d nt disslve when heated t 60 C and disslve when treated with acetic acid and dilute hydrchlric acid. Macrscpically, amrphus phsphates appear as a fluffy white precipitate that causes cludiness in alkaline urine. Diagnstic significance - Amrphus phsphates have little clinical significance. They are a nuisance because they bscure the presence f bacteria. Structures which can be misidentified as amrphus phsphates: bacteria. 2. CALCIUM PHOSPHATE Descriptin - Calcium phsphates are clrless prisms with a wedge-like end which can appear as plates r needles. They are weakly birefringent when viewed with plarized light. Calcium phsphate is nt sluble when heated t 60 C, but is sluble in dilute hydrchlric acid. Clinical Significance - Calcium phsphate is f little clinical significance. Structures which can be misidentified as calcium phsphate: sulfnamides. 3. TRIPLE PHOSPHATE - (AMMONIUM MAGNESIUM PHOSPHATE) Descriptin - Triple phsphate is a clrless crystal, characteristically a three-t-six-sided prism. They are ften referred t as a cffin lid shape. They can be small r large in size. Triple phsphate is birefringent when viewed with plarized light. Occasinally, triple phsphates will appear as a fern leaf when they start t disslve int slutin. Triple phsphates are sluble in acetic acid. Clinical Significance - Triple phsphate is f little clinical significance. Hwever, sme urinary stnes cntain triple phsphate. Structures which can be misidentified as triple phsphate: calcium xalate. 4. AMMONIUM BIURATE Descriptin - Ammnium biurate crystals are yellw-brwn clred spheres ften described as thrny apples. They are sluble at 60 C and cnvert t uric acid when treated with acetic acid r hydrchlric acid. Clinical Significance - Ammnium biurate is nt clinically significant. It precipitates as urine stands at rm temperature. Ammnium biurate is the alkaline cunterpart t uric acid. Structures which can be misidentified as ammnium biurate: sulfnmides. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 32

35 ABNORMAL URINE CRYSTALS In mst cases, abnrmal crystals are present in urine specimens with an acid ph r less. ABNORMAL CRYSTALS OF METABOLIC ORIGIN 1. CYSTINE Descriptin - Cystine crystals are clrless hexagnal plates which are refractile. Cystine crystals d nt plarize. Clinical Significance - Cystine crystals are fund in persns wh inherit a metablic defect that prevents the reabsrptin f cystine by the prximal cnvluted tubules. Patients with cystinuria frm cystine stnes which may cause kidney damage. Cystine stnes may fill the renal cllecting system, resulting in calculi. Cystine crystals are sluble in dilute hydrchlric acid and ammnia. They are insluble in acetic acid, alchl and biling water. Cnfirmatin f cystine may be dne with the cyanide-nitrprusside test. Structures which can be misidentified as cystine: uric acid. 2. CHOLESTEROL Descriptin - Chlesterl crystals are clrless rectangular plates with a characteristic ntched end in ne r mre crners. Their presence is smetimes assciated with prteinuria, free fat, val fat bdies and fatty casts. Clinical significance - Chlesterl crystals are rare in freshly vided urine sediment. They are seen mre ften in refrigerated urine. Drplets f chlesterl are present in nephrtic syndrme and lipid nephrsis. Structures which can be misidentified as chlesterl crystals: radigraphic media used in I.V. pyelgram. 3. LEUCINE Descriptin - Leucine crystals are yellw-brwn, ily lking spheres with radial and cncentric striatins and sheets f fine needles. Cnfirmatin f leucine is by liquid chrmatgraphy. They are birefringent and exhibit a pseud-maltese crss when plarized. Clinical Significance - Leucine is a rare crystal assciated with liver disease and disrders f amin acid metablism. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 33

36 4. TYROSINE Descriptin - Tyrsine crystals are clrless needles that appear black as the micrscpe is fcused. They are sluble in ammnia, dilute acids, and heat. Chemical cnfirmatin may be dne with the nitrsnaphthal test. Clinical significance - Tyrsine crystals are rare in the urine, but may be seen in patients with liver disease and hereditary tyrsinsis. Structures which can be misidentified as tyrsine: bilirubin crystals, sulfnamides, ampicillin. 5. BILIRUBIN CRYSTALS Descriptin - Bilirubin crystals appear as reddish brwn needles that cluster in clumps r as spheres. Clinical significance - Rarely d bilirubin crystals appear in urine sediment. It is present in patients with liver disease r hepatitis. The detectin f bilirubin n the dipstick helps in cnfirming identificatin. Structures which can be misidentified as bilirubin: tyrsine crystals. ABNORMAL CRYSTALS OF DRUG ORIGIN NOTE: Patient drug histry is imprtant in identifying all crystals f drug rigin. 1. SULFONAMIDES Descriptin - Sulfnamides are yellw t brwn (smetimes clrless) needles arranged in sheaves r rsettes. They smetimes appear as brwnish stcks f wheat with central binding. Greenish brwn fan shapes als are seen. Clinical significance - Sulfa drugs administered t patients may nt be sluble. Precipitated sulfnamides can damage the kidney. These crystals can be accmpanied by bld and liguria. It is imprtant t identify them s patients can be given sufficient fluid fr adequate hydratin and the urine made alkaline t disslve them. Structures which can be misidentified as sulfnamide: ampicillin, tyrsine. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 34

37 2. AMPICILLIN Descriptin - These clrless needles that appear in acidic urine are crystal clusters in refrigerated urine. Clinical significance - Ampicillin crystals may be fund in the urine after administratin f high dses f ampicillin. Structures which can be misidentified as ampicillin: tyrsine, sulfnamide. 3. RADIOGRAPHIC CONTRAST MEDIA (MEGLUMINE DIATRIZOATE) Descriptin - Crystals f radigraphic cntrast media are chemically meglumine diatrizate. They are flat rectangular plates ften ntched at the crner. Lng, thin prism frms als ccur. They are strngly birefringent when viewed with plarized light. Macrscpically, urine cntaining radigraphic cntrast media appears cludy with a very high specific gravity by refractmeter (greater than 1.035). If the specific gravity is measured by the reagent strip methd, it is nt high because radigraphic cntrast media des nt inize. Infrmatin prvided by the patient as t recent radigraphic imaging prcedures is an imprtant cnfirmatin f this crystal. Clinical significance - Radigraphic cntrast media is seen in the urine f patients fr a brief perid after injectin f these cmpunds fr diagnstic radigraphic prcedures. Their appearance can be f clinical significance in dehydrated patients with renal blckage frm crystals. Structures which can be misidentified as radigraphic cntrast media: chlesterl crystals. ARTIFACTS Cntaminants f all types can be fund in the urine, particularly when specimens are cllected imprperly r in dirty cntainers. Mst cnfusing are starch granules, il drplets, air bubbles, and fibers. 1. STARCH (TALC) Descriptin - Starch is generally a large rund granule that smetimes has an uninflated beach ball appearance. Starch is birefringent and exhibits a Maltase crss pattern under plarized light. Surces f cntaminatin are: (1) latex glves with pwder used in the cllectin and specimen prcessing and, (2) talc that may have been applied t the patient s hands r urgenital area. Structures which can be misidentified as starch: chlesterl crystals and drplets, urate crystals, parasites. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 35

38 2. FIBERS (INCLUDING DISPOSABLE DIAPER FIBERS) Descriptin - Fibers are large, lng cylindrical shapes that may have a twisted appearance. Fibers are birefringent and will plarize light. Surce f cntaminatin - cttn threads, synthetic fibers, and hair may enter the urine at the time f cllectin and at any time during the treatment prcess. In children and incntinent patients, the diaper is a majr surce. Structures which can be misidentified as fibers: casts - differentiate casts frm fibers with the plarizing prperties f fibers. 3. AIR BUBBLES Descriptin - Air bubbles are rund, clrless, and highly refractile. Surce - Air bubbles are intrduced as the cverslip is applied t the sediment slide. Care must be taken t apply the cverslip frm ne edge and gradually cvering the sediment drp s air is pushed ut rather than gathered under the cverslip. Structures which can be misidentified as air bubbles: red bld cells, parasites, fat glbules. 4. OIL DROPLETS Descriptin - Oil drplets are highly refractile and clrless and usually rund r val. Surces f cntaminatin - cntaminatin frm vaginal areas, catheter lubricant, micrscpic il immersin, vaginal creams. Structures which can be misidentified as misidentified as il drplets: red bld cells r fat glbules. 5. GLASS FRAGMENTS Descriptin - Glass fragments are clrless, highly refractile and take n different shapes and sizes. Surce f cntaminatin - slivers f glass frm the cverslip r micrscpe slide. Structures which can be misidentified as misidentified as glass: crystals. 6. POLLEN GRAINS Descriptin - Pllen grains have a very large raised shape with thick cell walls. Surce f cntaminatin - Pllen grains are mstly a seasnal cntaminant frm external surces. Structures which can be misidentified as misidentified as pllen: parasite eggs. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 36

39 MAJOR CHARACTERISTICS OF NORMAL URINARY CRYSTALS CRYSTAL ph COLOR SOLUBILITY GENERAL APPEARANCE uric acid acidic yellw-brwn alkali sluble amrphus urates acidic brick-dust r yellw-brwn alkali & heat calcium xalate acidic/neutral (alkaline) clrless (envelpes) dilute HCl acid amrphus phsphates alkaline neutral white-clrless dilute acetic acid calcium phsphate alkaline neutral clrless dilute acetic acid triple phsphate alkaline clrless (cffin lids) dilute acetic acid ammnium biurate alkaline yellw-brwn (thrny apples) acetic acid with heat calcium carbnate alkaline clrless (dumbbells) gas frm acetic acid Adapted frm S. K. Strasinger, Urinalysis and Bdy Fluids, 3rd Editin, F. A. Davis, 1994 WVOLS T&E 2010 URINALYSIS SESSION II P a g e 37

40 MAJOR CHARACTERISTICS OF ABNORMAL URINARY CRYSTALS CRYSTAL ph COLOR SOLUBILITY GENERAL APPEARANCE cystine acidic clrless ammnia, dilute HCl acid chlesterl acidic clrless (ntched plates) chlrfrm leucine acidic/neutral yellw ht alkali r alchl tyrsine acidic/neutral clrless-yellw alkali r heat bilirubin acidic yellw acetic acid, HCl, NaOH, ether, chlrfrm sulfnamides acidic/neutral green acetne radigraphic dye acidic clrless 10% NaOH ampicillin acidic/neutral clrless refrigeratin frms bundles f needles Adapted frm S. K. Strasinger, Urinalysis and Bdy Fluids, 3rd Editin, F. A. Davis, 1994 WVOLS T&E 2010 URINALYSIS SESSION II P a g e 38

41 QUALITY ASSURANCE CONFIRMATORY URINE SCREENING TESTS CHAPTER 3 CHAPTER OBJECTIVES At the cnclusin f Chapter 3, participants shuld be able t: Define quality assurance Describe the cmpnents f a quality assurance prgram Discuss causes f errrs: pre-analytical, analytical, pst-analytical Knw when t perfrm cnfirmatry urine screening tests Like ther labratry tests, urine results must be accurate if the physician is t treat the patient prperly. A quality assurance prgram is necessary t assure that errrs are prevented and urinalysis test results are accurate. This chapter will discuss the cmpnents f a quality assurance prgram. In additin, this chapter will discuss the imprtance f the three mst cmmnly perfrmed cnfirmatry urine screening tests, Clinitest, Icttest and Sulfsalicylic acid. QUALITY ASSURANCE IN URINALYSIS Quality Assurance (QA) is a cmprehensive set f plicies, prcedures, and practices necessary t make sure that the labratry s results are reliable. It is essential that all persns wrking in the labratry be cmmitted t the quality assurance cncepts with patient service being the tp pririty. Fr a QA prgram t be successful, interactin and cmmunicatin between the labratry staff and health care prviders is essential. Accrding t the federal labratry regulatins (CLIA-88), Each labratry perfrming mderate r high cmplexity testing must establish and fllw written plicies and prcedures fr a cmprehensive QA prgram. This prgram must evaluate the effectiveness f its plicies and prcedures identify and crrect prblems, assure accurate, reliable, and prmpt reprting f test results, and cmpetency f the staff. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 39

42 QUALITY ASSURANCE COMPONENTS Cmplaints and cmmunicatin Patient test management Persnnel cmpetency Prcedures Prficiency testing Quality cntrl Recrd keeping Test cmparisns I. QUALITY CONTROL Quality Cntrl (QC) is the mst familiar cmpnent f a quality assurance prgram. Often the terms quality cntrl and quality assurance are incrrectly interchanged. Quality cntrl is nly ne prtin f a quality assurance prgram and it is imprtant t make the distinctin. Quality cntrl validates and mnitrs the testing prcess which invlves checking the integrity f the micrscpe, urine reagent strip reading instrument, reagent strips, and the technique f testing persnnel. This is dne primarily thrugh the use f cntrl specimens which are similar in cmpsitin t patient specimens. The cntrls must be carried thrugh the entire test prcedure and treated in the same manner as patient specimens. CLIA-88 federal labratry regulatins states that at least tw levels f cntrl material must be run each day f testing and are tested in the same manner as patient specimens. Fr labratries perfrming urine micrscpics and/r using instruments fr reading reagent strips, a quality cntrl prgram is required. Fr labratries perfrming a manual reagent strip urinalysis nly, quality cntrl is recmmended but nt required. Waived labratries are t fllw manufacturer s quality cntrl recmmendatins (at a minimum). The Ames (Bayer) Multistix package inserts state: Fr best results, perfrmance f reagent strips shuld be cnfirmed by testing knwn negative and psitive specimens r cntrls whenever a new bttle is first pened, randmly, r if questinable results are btained. Each labratry shuld establish perfrmance standards and shuld questin handling and testing prcedures if these standards are nt met. Urinalysis cntrls are purchased cmmercially in ready-t-use r in dried frm. Care must be taken in recnstituting dried cntrl material. The crrect amunt f distilled r deinized water must be added t the cntrl bttle and disslved cmpletely. Labratries that d nt have pipettes r distilled water available shuld purchase the ready t-use cntrl material fr ptimal cntrl perfrmance. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 40

43 Cntrl material must have the fllwing prperties: Similar t patient samples Acceptable range - instrument specific Fcused at medical decisin pints At least 2 cncentratins Available in aliquts cnvenient fr use Validates the run Trubleshting tl DOCUMENT! DOCUMENT!! DOCUMENT!!! All QC results must be dcumented immediately n a lg r chart r in a cmputer. This dcumentatin prvides a link between cntrl and patient results, and serves as a permanent recrd fr the directr t review. The chart, whether it is manually recrded r cmputerized, shuld include the fllwing infrmatin: Name and lcatin f labratry site Test title (e.g. Urinalysis Cntrl Chart) Reagent Strip infrmatin: Name Manufacturer Lt Number Expiratin Date Cntrl Material Infrmatin: Prduct Cntrl level - Nrmal and Abnrmal Manufacturer Lt number Expiratin Date Cntrl Ranges Date tested Time cllected r (Time In) Time Out Initials f wh tested cntrl Cntrl Results Crrective Actin (may be separate frm) WVOLS T&E 2010 URINALYSIS SESSION II P a g e 41

44 Other elements f quality cntrl can include preventive maintenance and calibratin f micrscpe and/r reagent strip instruments, and reagent strip strage requirements. All must be dcumented. Quality cntrl des nt: Ensure the prper test is perfrmed Ensure the crrect patient is tested Ensure the urine specimen is prperly cllected Ensure the results are timely Ensure the results are prperly evaluated Hwever, with prper dcumentatin yu can clearly track when: Lts f quality cntrl reagent strips and cntrls are intrduced A different labratrian begins t perfrm the test Instrument maintenance is perfrmed Respnses are made t ut f cntrl results Prficiency testing is perfrmed II. PROFICIENCY TESTING Prficiency testing (PT) is an external frm f quality cntrl that cmpares test results frm each participating site t all participating labratries that use the same methdlgy. The practice f testing unknwn specimens frm an utside agency prvides additinal means t assure quality labratry test results. Every fur mnths the PT prvider sends unknwn specimens t their subscribers. These specimens are tested in the same manner as patient specimens. The test results are returned t the PT prvider. The results are reviewed t determine whether each participant passes r fails perfrmance levels as established by CLIA-88 and ther accrediting agencies. Under CLIA-88, labratries perfrming mderate r high cmplexity testing must be enrlled in an apprved PT prgram. Labratries that perfrm urine sediment examinatin, r use a reagent strip instrument, fall int the mderate cmplexity categry f testing. A few f the CLIA apprved PT prgrams available fr urinalysis is listed in the fllwing chart: WVOLS T&E 2010 URINALYSIS SESSION II P a g e 42

45 URINALYSIS PROFICIENCY TESTING PROVIDERS Prficiency Testing Prvider Prgram Names Descriptin Ordering Cde (If applicable) Cllege f American Pathlgists (CAP) Clinical Micrscpy Excell-Urinalysis Specimens fr reagent strip and analytes transparencies fr micrscpics CM XL-A American Assciatin f Bianalysts (AAB) Urinalysis Specimens fr reagent strip analytes and transparencies fr micrscpics N/A Wiscnsin State Labratry f Hygiene Urinalysis Urine Sediment Identificatin Specimens fr reagent strip nly Transparencies UR SU American Prficiency Institute (API) Urinalysis Urine sediment Specimens fr reagent strip nly Transparencies #232 #234 American Sciety f Internal Medicine (MLE) Urinalysis Transparencies American Academy f Family Physicians (AAFP) Urinalysis Transparencies Prficiency testing is nt cmplete withut a system f mnitring the results received frm the PT prvider. Prficiency testing assessment invlves: Regular mnitring f PT results Crrective actin taken and dcumented fr each unacceptable r unsatisfactry PT result Mnitring the effectiveness f crrective actin Verifying that PT is treated in the same manner as patient samples Checking that reagent strips used fr PT match thse used fr patient testing, and thse described the prcedure manual. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 43

46 EXAMPLE OF A PROFICIENCY TESTING REPORT (CAP) CONSTITUENT METHOD ph IN URINE AMES CLINITEK 10/100 SPECIMEN YOUR RESULT EVAL CODE GOOD PERFORMANCE CM CM ACCEPTABLE PERFORMANCE PROTEIN QUAL URINE AMES CLINITEK 10/100 GLUCOSE REDUC SUB-UR AMES CLINITEK 10/100 KETONES, URINE AMES CLINITEK 10/100 BILIRUIBIN, URINE AMES CLINITEK 10/100 BLOOD/HEMOGLOBIN, URINE AMES CLINITEK 10/100 LEUKOCYTE ESTERASE AMES CLINITEK 10/100 NITRITE, URINE AMES CLINITEK 10/100 CM MG/DL (1+) MG/DL (1+) 100 MG/DL (2+) CM MG/DL (3+) MG/DL (3+) CM-19 NEGATIVE 61 NEGATIVE CM MG/DL MG/DL 1000 MG/DL CM-19 NEGATIVE 61 NEGATIVE CM-20 MODERATE (2+, 40 MG/DL) 61 MODERATE (2+, 40 MG/DL) CM-19 POSITIVE (MOD OR 2+) 61 LARGE AMOUNT (3+) CM-20 POSITIVE (MOD OR 2+) 61 POSITIVE (MOD OR 2+) LARGE AMOUNT (3+) CM-19 POSITIVE (50 ERY/UL) 61 POSITIVE (50 ERY/UL) CM-20 NEGATIVE 61 NEGATIVE CM-19 TRACE 61 NEGATIVE CM-20 MODERATE (2+) 61 MODERATE (2+) LARGE (3+) CM-19 POSITIVE 61 POSITIVE CM-20 NEGATIVE 61 NEGATIVE TRACE MG/DL (4+) OR MORE 100 MG/DL (2+) 1000 MG/DL (4+) OR MORE 250 MG/DL 2000 MG/DL OR MORE SMALL (1+, 15 MG/DL) LARGE (3+, >80 MG/DL) POSITIVE (MOD OR 2+) TRACE (SMALL OR 1+) TRACE (SMALL OR 1+) TRACE (5-10 ERY/UL) MARKD POS (250 ERY/UL) SMALL (1+) URINE SEDIMENT IDENT CM-21 GRANULAR CAST 71 GRANULAR CAST CM-22 WAXY CAST 71 WAXY CAST CM-23 HYALINE CAST 71 HYALINE CAST WVOLS T&E 2010 URINALYSIS SESSION II P a g e 44

47 NAME: III. PROCEDURE MANUAL Anther essential cmpnent f a quality assurance prgram is the prcedure manual. The prcedure manual cntains the peratinal prtcls that standardize labratry testing. It must be accessible t all f the labratry staff. A gd prcedure manual shuld fllw NCCLS frmat. This sectin will review the NCCLS frmat with examples f urinalysis sediment examinatin infrmatin included under each heading. This is a guide fr each facility t develp its wn sitespecific prcedure manual. Example: I. Urinalysis - Sediment Examinatin Prcedure Manual II. Principle The kidneys are paired rgans which are lcated in the small f the back n each side f the spine. They functin in maintaining the fluid balance f the bdy and frming urine which is excreted. Urine can be an indicatr f health r disease especially in metablic r renal disrders. A micrscpic examinatin f urinary sediment is helpful in cnfirming the presence f red bld cells (RBC s), white bld cells (WBC s), bacteria, yeast, crystals, r casts. A qualitative r semi-quantitative evaluatin f urine sediment prvides adequate infrmatin fr the majrity f diagnstic and clinical needs. III. Specimen Cllectin and Handling Specify the cnditin fr patient preparatin, e.g., clean catch instructins. Indicate the preferred type f specimen and any ther acceptable specimen, e.g., first mrning, randm urine List the acceptable cllectin cntainers, e.g., sterile urine cntainer. Specificatins fr hme cllected specimens - clean leak prf cntainer Specify urine vlume requirements, ptimal (50 ml r mre) and minimal (12 ml) State the strage and handling requirements fr stability f the specimen, e.g., Urine specimens must be delivered t the labratry within 1 hur f cllectin and prcessed within 2 hurs held at rm temperature. Specify requirements fr specimen labeling, e.g., Urine specimen cntainer must be labeled immediately after cllectin by persn respnsible fr specimen cllectin. The specimen must be labeled with name, date, and time f cllectin. Include criteria fr unacceptable specimens and the actin taken by the labratry t crrect the prblem, e.g., unlabeled specimens and specimens ver 1 hur ld are rejected. Dcument n specimen rejectin lg. Include infrmatin fr ntifying the persn respnsible fr cllecting a rejected specimen. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 45

48 IV. Reagents, Supplies and Equipment Clean, dry cntainers fr cllecting the urine sample Antiseptic wipes fr patient preparatin befre cllecting urine Micrscpe slides 1" x 3" Cver slips 22 x 22 mm - #1 r #2 Centrifuge tube - 15 ml capacity with cnical r cnstricted bttm General purpse centrifuge Micrscpe with mechanical stage and lw pwer (10X) and high pwer (40X) bjectives. Include a brief descriptin f preventive maintenance f centrifuge, micrscpe, etc. V. Calibratin Step-by-step instructins n calibratin f micrscpes and centrifuges. Specify acceptable perfrmance and crrective actin t be taken when limits are exceeded. Indicate where t recrd calibratin results. VI. Quality Cntrl: List the manufacturer and prduct name f each level f cntrl. List cntrl strage requirements. Describe step-by-step instructins fr recnstituting lyphilized cntrl samples and testing cntrls. Indicate hw cntrl results are recrded (charts, cmputer, lgs). State limits f acceptability and crrective actin when cntrl results exceed these limits. VII. Test Prcedure: Give detailed instructins in a step-by-step manner fr preparatin f urine sediment, slides and micrscpic scanning technique. Keep instructins free f explanatin Include clean-up and dispsal f specimens and any ther cntaminated material. VIII. Results: Include criteria fr identificatin f specific elements in the urine sediment, e.g., red and white bld cells, epithelial cells, yeast, crystals, and casts. State the nrmal versus abnrmal elements fund in the urine sediment. Describe critical values which require immediate physician ntificatin. Describe site-specific reprting prtcl. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 46

49 GENERAL REPORTING SYSTEM FOR URINE SEDIMENT Average number per lw pwer field (X100) Casts Negative >50 Abnrmal crystals Negative >50 Squamus epithelial cells Rare Few Present Mderate Many Average number per lw pwer field (X400) Red bld cells >100 White bld cells >100 Nrmal crystals Rare Few Mderate Many Epithelial cells (renal tubular, val fat bdies, transitinal) Miscellaneus (bacteria, yeast, Trichmnas, fat glbules) Sperm (males nly) Rare Few Mderate Many Rare Few Mderate Many Present NOTE: Few means sme are present; mderate means easily seen; many means prminent. Adapted frm Linǹe JJ, Ringsrud KM: Basic Techniques in Clinical Labratry Science, 3 rd editin, St. Luis, 1992, Msby IX. Surces f Errr. List interferences and surces f errr. Sme examples are described later in this chapter. X. Ntes/Remarks Prvide helpful hints and miscellaneus infrmatin. XI. References Include surces such as: package inserts textbks NCCLS publicatins scientific jurnals XII. Review and Update Each prcedure shuld be signed by the directr, with date f apprval. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 47

50 XIII. Supplemental Materials: clr atlases, textbks, flwcharts reprting results prcedure fr ntificatin f errneus results panic values and reprting prtcls review and update references Keep in mind that each site must include site-specific infrmatin fr that site. Accrding t CLIA- 88 regulatins, manufacturer s instructins, and reference material can be used fr a prcedure but the fllwing infrmatin shuld be supplied by each labratry: IV. PERSONNEL COMPETENCY The cmpetence f persnnel is an imprtant determinant f the quality f any labratry including urinalysis. Only well trained persnnel wh are skilled in the prper use f the micrscpe shuld perfrm urine sediment examinatin. Plicies n hw emplyees are trained and assessed fr cmpetency must be develped and recrded. Activities t Assure Persnnel Cmpetency (Dcument all activities.) Develp standardized rientatin training plicies fr new emplyees. Cmpetence verificatin checklists prvide dcumentatin that a new emplyee is cmpetent t perfrm specific tasks r prcedures independently. Prvide peridic pprtunities fr upgrading the technical skills fr all persnnel. This can be accmplished thrugh in-service classes, cntinuing educatin curses, and by encuraging study f textbks and audivisuals. Make clr atlases f urine sediment and textbks readily available fr review. Instruct labratry persnnel t share with each ther all unusual r abnrmal urine sediment findings. Evaluate persnnel peridically; fr test result reprting, QC recrds, and preventive maintenance recrds. Make direct bservatin f persnnel perfrming patient preparatin, specimen handling, prcessing, and testing. Assess technical cmpetency thrugh prficiency testing. Each emplyee, smetime within the year, must cmplete a PT survey including any mnitring f unsatisfactry results. This prvides the emplyee with valuable prblem slving cmpetence. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 48

51 V. PATIENT TEST MANAGEMENT Patient test management is the prcess t assure that patient specimens and test results are nt cnfused with ther patient results. This cmpnent f quality assurance tracks patient infrmatin frm the time the specimen is cllected t the time the test result is reprted. The labratry must emply and maintain a system that prvides fr prper specimen submissin and handling by assessing the fllwing elements: patient preparatin specimen cllectin labeling specimen preservatin specimen transprtatin specimen prcessing reprting f crrect results Patient test management must be dcumented using the recrds described belw: 1. Test requisitin The test requisitin may have ne f several different frmats depending upn the testing site. Traditinally, it has been a preprinted frm printed with multiple carbn cpies. In cmputerized labratries, it may be cmputer generated frm with accmpanying specimen labels. At small vlume testing sites, the patient chart may serve as the test requisitin. Regardless f frmat, the test requisitin must include the fllwing infrmatin: patient name r unique identifier patient sex authrized persn requesting test test rdered date and time f specimen cllectin relevant medical infrmatin 2. Labeling Patients must be identified befre the specimen is cllected. Once the patient is identified, the specimen can be cllected and prperly labeled. All cntainers must be labeled by the persn ding the cllectin t make certain that the specimen has been cllected frm the patient whse identificatin is nted n the label. The label must be placed n the cntainer and must adhere during refrigeratin. Never label the lid because nce the lid is remved, identity is lst. The label shuld include the patient s full name, unique identificatin number, date and time f cllectin. The cmplete request frm must accmpany the specimen t the labratry. The request frm must be clean and legible. The infrmatin n the specimen cntainer must match exactly the patient identificatin n the request slip. All specimens received by the labratry withut prper identificatin must be rejected. It is better t recllect the specimen than reprt false results. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 49

52 3. Test Recrds Devise a specimen lg-in bk t recrd: patient name identificatin number accessin number date and time f receipt int labratry test results initials f persn perfrming the test time the analysis is finished (time ut) explanatin fr rejected specimens When the specimen is received by the labratry, the infrmatin listed abve shuld be cmpleted. These recrds can be handwritten r cmputer generated. 4. Test Reprt The test reprt may be dne n the same frm as the test requisitin r patient chart. Duplicate cpies f the test reprt shuld be kept, ne in the labratry and ne n the patient chart. The test reprt must cntain the fllwing infrmatin: identity f the lcatin f labratry perfrming testing test results units f measurements infrmatin regarding specimen cllectin reference r nrmal ranges All test reprts shuld be accurately reprted in a timely manner t the health care prvider wh rdered the test. Once the test reprt reaches the patient chart, the health care prvider review shuld be dcumented either by initials r cmments t cnfirm that the results were nted n the crrect chart. Peridic meetings with labratry staff and health care prviders are necessary t discuss test reprting prblems. 5. Specimen Referral Lg Any urine specimen that is referred t anther labratry fr further study must be dcumented n a reference labratry lg. The reference lg shuld have the fllwing infrmatin: date cllected patient name r unique identifier test requested date mailed date results were received back in the labratry name and address f reference labratry WVOLS T&E 2010 URINALYSIS SESSION II P a g e 50

53 VI. COMMUNICATION AND COMPLAINT INVESTIGATION In rder fr labratries t survive managed care cmpetitin, cnsumers (patients, physicians) must be pleased with the service. Cmmunicatin f prblems is essential. The labratry must have a system in place that dcuments and reslves cmplaints that ccur as the result f cmmunicatin breakdwns, e.g., incrrect test, patient name, test results and unacceptable specimens. After cmmunicatin prblems are detected and dcumented, the labratry must investigate the cmplaints t determine a curse f actin. All cmplaints shuld be taken seriusly. N matter hw insignificant the cmplaint may seem at the time, it may be a symptm f a bigger prblem. The cmmunicatin r cmplaint lg dcuments the cmmunicatin prcess. This lg shuld answer the fllwing questins: Was the prblem dcumented? Was the prblem crrected? Has a plicy r prcedure changed? If a plicy change ccurred, has the change been evaluated t determine that the actin taken was effective? Were the apprpriate peple ntified? Was each step f the crrectin prcessed initialed (if mre than ne persn was invlved)? VII. COMPARISON OF TEST RESULTS An imprtant quality assurance cmpnent is cmparisn f test results. The infrmatin generated fr each patient shuld be crrelated with ther results btained by the labratry. In urinalysis, it is imprtant t crrelate the physical appearance, chemical (reagent strip) screen and micrscpic results. Inspect the test results fr incnsistencies and investigate the pssible cause befre releasing the results t the physician. Incnsistencies may r may nt be errneus and may lead t clues fr determining specific clinical prblems. Althugh there are exceptins, the physical, chemical and micrscpic prtins f a urinalysis shuld crrelate. The chart belw summarizes sme cmmn urinalysis test cmparisns. COMMON URINALYSES TEST COMPARISONS Appearance Reagent Strip Screen Sediment examinatin Hazy t cludy Prtein - trace t large amunt Casts Pink t red clr Bld - trace t large amunt Red bld cells Hazy t cludy Leukcyte esterase - trace t large amunt White bld cells Hazy t cludy Nitrite - Psitive Bacteria WVOLS T&E 2010 URINALYSIS SESSION II P a g e 51

54 RELATIONSHIP OF PATIENT INFORMATION TO PATIENT TEST RESULTS Labratries shuld develp a system t identify patient test results that appear incnsistent with patient status r medical infrmatin. EXAMPLES OF RELATING PATIENT CHART INFORMATION TO PATIENT URINALYSIS TEST RESULTS EXAMPLE URINALYSIS RESULTS PATIENT CHART INFORMATION INVESTIGATION Clr - Yellw Appearance - cludy Clues: -Cludy appearance #1 Reagent strip reactins - negative Specific gravity: By refractmeter By reagent Strip Urine Sediment Exam: Crystals resembling chlesterl Patient had a radigraphic prcedure perfrmed ne day prir t the urinalysis -Specific gravity incnsistency -Crystals Withut knwing the radigraphic prcedure infrmatin, the crystals may have been reprted as chlesterl. Clr - yellw Clues: Appearance - hazy Hazy appearance Nitrite - psitive #2 Reagent strip - Nitrite - psitive Prtein - trace All ther reactins - negative r nrmal Specific gravity: By refractmeter Rutine examinatin with n symptms f a urinary tract infectin Leukcyte esterase - negative Large amunt f bacteria Patient asymptmatic - questin urinalysis review prcedure By reagent strip Suspect: Cntaminatin Urine sediment examinatin: Large amunt f bacteria present WVOLS T&E 2010 URINALYSIS SESSION II P a g e 52

55 In each f the abve examples, investigatin is needed t determine whether r nt the urinalysis results are clinically crrect r are errneus. All actin must be dcumented. In small testing sites, the patient charts are available fr investigatin. In larger institutins, patient medical infrmatin is limited. In this case, pen cmmunicatin with nurses and physicians is essential. VII. RECORD KEEPING Thrugh recrd keeping is essential t quality assurance. The phrase If it isn t dcumented, it isn t dne, is reiterated by inspectrs and supervisrs in every labratry. This last sectin lists sme f the mst cmmn urinalysis recrds. Retain these recrds fr a minimum f tw years. Types f Labratry Recrds Cntrl lgs - tw levels f cntrl alng with strip infrmatin Temperature charts: refrigeratrs rm temperature freezers Instrument preventive maintenance Centrifuge calibratin recrds and preventive maintenance Patient lgs Test requisitins Refractmeter: preventive maintenance calibratin cntrls Prficiency testing t include: results submitted summary reprts crrective actin n deficiencies. Crrective active lgs fr cntrls and instruments Review f test results Persnnel training Unsatisfactry specimens/cllectin f additinal specimens Instrument service & repair Micrscpe preventive maintenance Cmplaint lgs Cmmunicatin reviews with staff and clients (mems, meeting agenda) Quality Assurance Mnitrs and Review Retain package insert infrmatin n cntrls and reagent strips (ne per lt r revisin) WVOLS T&E 2010 URINALYSIS SESSION II P a g e 53

56 SCOPE OF QUALITY ASSURANCE A gd quality assurance prgram incrprates all f the cmpnents discussed in this chapter int a prgram t prevent errrs and assure accuracy. Errrs can ccur in any phase f the testing prcess pre-analytic, analytic, pst-analytic. Accrding t a survey cnducted by Q prbe, the least amunt f errrs ccurs in the analytic phase: 7.3%. The pre-analytic phase has an errr distributin f 45.5%, and the pst-analytic phase accunted fr 47.2% f labratry errrs. This data indicates that labratries d very well with their accuracy and precisin during the actual testing phase but may be faulty in specimen cllectin r reprting f results. The lists belw summarize the cmmn errrs assciated with urinalysis in each phase f testing. PRE-ANALYTIC ERRORS Patient physilgy factrs: Fds which prduce abnrmal urine clr Medicatins Strenuus exercise which results in excretin f cells and prtein Failure t use a clean cllectin cntainer Failure t deliver specimen t the labratry prmptly (within 1 hur) and failure t perfrm test prmptly Cntaminatin f urine with skin cleansers Failure t prvide patients with instructins n hw t prperly cllect specimen (e.g. cleancatch methd) Failure t wash hands befre cllecting specimen Patient misidentified r nt identified Specimen cup nt labeled r incrrectly labeled The wrng type f urine specimen cllected, fr example, randm urine specimen cllected fr a urine culture. Randm r shrt vlume urine specimens (may nt truly represent the patient s cnditin) ANALYTICAL ERRORS Imprper dipping f a reagent strip. Prper timing f the reactins ignred. Tuching the reagent strip test pads. Failure t prtect the reagent strips frm light and humidity (leaving the cap ff the bttle) Failure t test reagent strips with cntrls Failure t thrughly mix the urine specimen immediately befre testing r befre puring urine specimen int a centrifuge tube fr sedimentatin Cntrl specimens imprperly recnstituted r prly mixed Failure t maintain and perfrm functin checks n refractmeter Refrigerated specimen tested befre allwing it t reach rm temperature Using expired reagent strips and cntrls Failure t test specimen prmptly Technical errrs in using the micrscpe Failure t QC and prperly maintain micrscpe Urine centrifuge RPM s and time nt checked. Lack f a written and updated prcedure manual Clerical errrs, e.g., results recrded fr incrrect patient WVOLS T&E 2010 URINALYSIS SESSION II P a g e 54

57 Cnfirmatry test nt prefrmed fr glucse, bilirubin, and prtein Technical prblems with urine strip instrument The human eye s perceptin f clr Failure t reject an unsatisfactry specimen Pr quality water supply Unstable pwer supply and temperature Failure t crrelate the visual, chemical, and micrscpic results POST ANALYTICAL ERRORS Failure t reprt results in a timely manner Nt identifying critical results (panic values) Transcriptin errrs Failure t reprt result t health care prvider Results placed n incrrect patient chart Misfiling r disrganized patient recrds Failure t prvide labratry interpretive infrmatin t health care prvider Failure t refer specimens Illegible results Lack f cmmunicatin and interactin between the labratry and thse directly invlved with patient care (physicians, nurses). CONFIRMATORY URINE TESTS Errrs can ccur if cnfirmatry urine tests are nt perfrmed as part f a chemical screen. In general, prblems arise with the human eye s perceptin f clr and the clr f the urine specimen masking the clr interpretatin f the reagent strip. Cnfirmatry urinalysis tests detect the same substance with greater sensitivity r specificity. They may use a different chemical reactin t detect the analyte. Repeating a reagent strip reactin is nt a cnfirmatry test. Beynd reagent strip urinalysis, micrscpic sediment examinatins represent cnfirmatry tests fr bld, leukcyte esterase, and nitrite detected by reagent strips. The three mst cmmn cnfirmatry tests invlve reducing sugars, prtein and bilirubin. These tests are described in this sectin. REDUCING SUGARS (CLINITEST COPPER REDUCTION TEST) Principle: The cpper reductin test is a nnspecific test fr reducing sugars in the urine which include: glucse, galactse, fructse, lactse, and pentse. The methd is based n the ability f sugar t reduce cpper sulfate t cuprus xide in the presence f alkali and heat (Benedict s Principle). The clr change which crrelates with the amunt f sugar present in the urine prgresses frm blue (negative) t yellw and then t red. It is imprtant t bserve this reactin clsely because high levels f glucse may cause a pass thrugh phenmenn t ccur. When the pass thrugh phenmenn ccurs, the clr prduced by the Clinitest reactin passes thrugh the red-clr and returns t a blue clr. In this situatin, a high glucse level may be reprted as negative. Using the 2-drp Clinitest methd instead f the traditinal 5-drp methd can prevent the pass thrugh phenmenn frm ccurring. Hwever, the ability t detect lw levels f reducing sugars may be cmprmised by the 2-drp Clinitest methd. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 55

58 Sensitivity: Be aware that the reagent strip screen fr glucse is mre sensitive than the Clinitest methd. The reagent strip will detect glucse levels as lw as 40 mg/dl while the Clinitest glucse sensitivity is 250 mg/dl. Clinical Reasns fr Perfrming Clinitest Galactsuria - The mst imprtant reasn t perfrm the Clinitest methd is t detect galactsuria in pediatric patients. All children under 2 years f age (especially newbrns) shuld be screened using the reagent strip and Clinitest methd. The presence f galactse in the urine results frm a metablic errr in which the enzyme galactse-1-phsphate uridyl transferase is lacking. This results in a failure t metablize galactse, with increased levels in the bld (galactsemia) and urine. Galactsemia will result in permanent physical and mental deteriratin. This deteriratin can be prevented by early detectin and dietary restrictin f galactse. Lactse Intlerance - Lactse intlerance can be detected by the Clinitest methd. Lactse intlerance will cause gastrintestinal distress in adults. In infancy, a failure t gain weight may be because f intestinal lactse deficiency. Lactse may als be seen in the urine in late pregnancy r early lactatin. BILIRUBIN (ICTOTEST ) Principle: The Icttest is the cnfirmatry test fr urine bilirubin which is based n a diaz reactin. The test is perfrmed by placing 10 drps f urine n an absrbent mat. The bilirubin (if present) will remain n the mat surface as the urine filters int the mat. A reagent tablet is then placed n the mat and ne drp f water is added t the tablet. After 5 secnds, a secnd drp is added and allwed t run dwn the side f the tablet. After 60 secnds, the reactin between the tablet and bilirubin prduces a blue r purple clr appearing n the mat. Clrs ther than blue r purple appearing n the mat are interpreted as negative. Clinical Reasns fr Perfrming Icttest Sensitivity - The Icttest is mre sensitive fr bilirubin than the reagent strip methd. Urine suspected f cntaining bilirubin due t the urine appearance r clinical histry shuld be cnfirmed by the Icttest methd. Clr interpretatin - The clr changes in the reagent strip bilirubin test pad can be very subtle. The purple clr reactin f Icttest is easier t interpret than the brwn clred reactin f the bilirubin reagent strip test pad. When in dubt abut bilirubin clr interpretatin, perfrm an Icttest. PROTEIN (SULFOSALICYLIC ACID TEST) The sulfsalicylic acid test (SSA) is a cld precipitatin test f prtein. Varius cncentratins f sulfsalicylic acid have been described. In general, equal amunts f urine and SSA are placed in a test tube and prtein (if present) precipitates. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 56

59 PROCEDURE: Sulfsalicylic Acid Test fr Prtein 1. Centrifuge a ml aliqut f well mixed urine. 2. Decant 4-5 ml f the urine supernatant int a 16 x 125 mm test tube. Nte clarity f the supernatant. 3. Add equal parts 3% sulfsalicylic acid reagent (SSA). 4. Stpper the tube and mix by inverting twice. 5. Let stand exactly 10 minutes. 6. Invert tube nce again. 7. Observe the degree f precipitatin and grade. GRADE PRECIPITATED PROTEIN AS FOLLOWS: SSA result Negative Trace Descriptin N turbidity, r n increase in turbidity Clear ring is visible at bttm f tube when viewed frm abve (tp t bttm) Barely perceptible turbidity in rdinary rm light Printed material distrted but readable thrugh the tube Cannt see a ring at bttm f tube when viewed frm abve. Apprximate prtein Cncentratin mg/dl by reagent strip* <5 mg/dl 5-20 mg/dl 1+ Distinct turbidity but n distinct granulatin 30 mg/dl 2+ Turbidity with granulatin but n flcculatin 100 mg/dl 3+ Turbidity with granulatin and flcculatin mg/dl 4+ Clumps f precipitated prtein r slid precipitate >500 mg/dl *Based n a cmparisn f SSA values by reagent strip (Multistix) and Cbas prtein results UMHC Jan Unpublished. Clinical Reasns fr Perfrming SSA Difficulty in clr interpretatin - The prtein area f the reagent strip is ne f the mst difficult t interpret, particularly in relatin t the trace reading. Questinable results shuld be cnfirmed using the SSA methd. Specificity f the reagent strips - Reagent strips measure primarily albumin and may nt detect tubular prteins and Bence Jnes prtein which are pathlgical. The SSA methd will detect albumin, glycprtein glbulins, hemglbin and light chain immunglbulin. Reactin Interference Any substance precipitated by acid will prduce false turbidity in the SSA methd. The mst highly encuntered substances are radigraphic dyes, cephalsprins, penicillin and sulfnamide. The patient histry will prvide the necessary infrmatin t rule ut these interferences. This is an example f why cmmunicatin amng labratries, nurses and physicians is crucial t quality patient care. Imprtant pieces f infrmatin prevent misinterpretatin. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 57

60 GLOSSARY OF QUALITY ASSURANCE TERMS acceptable cntrl range accuracy analysis analyte assayed values bias (inaccuracy) calibratin calibratr centrifuge clean-catch urine CLIA 88 carse adjustment cefficient f variatin cntrl crrective actin (labratry) critical limits critical results (critical values, panic values) deficiency distilled water (dist. H 2) equivcal expiratin date The range f results that indicate adequate perfrmance when analyzing a cntrl sample. The range is shwn in the cntrl s prduct insert. Crrectness; freedm frm errr. The extent t which measurements agree with the true value f the quality being measured. The labratry prcedure that enables yu t measure the amunt f an analyte in a specimen. The substance r cnstituent being measured, e.g., glucse. The measurement f the amunt f a cnstituent in a specimen; a test. A measure f the departure frm accuracy. A numerical difference between the mean f a set f replicate measurements and the true value f the sample. The prcess by which readings btained frm an instrument r ther measuring device are related t knwn cncentratins f an analyte. A material, slutin, r freeze-dried preparatin used in calibratin. The cncentratin f the analyte in a calibratr is knwn t be within a particular range. A machine using centrifugal frce (prceeding in a directin away frm the center) fr separating substances f different densities. A urine sample cllected after the urethral pening and surrunding tissues have been cleansed. Clinical Labratry Imprvement Amendments. Laws passed by federal gvernment in 1988 t imprve the quality f labratry testing. Adjusts psitin f micrscpe bjectives; used t bring bjects int fcus. An index used t describe the precisin f a labratry methd. It is calculated with this frmula: CV = standard deviatin/mean. Expressed as a percentage. A material, slutin, r lyphilized preparatin used in quality cntrl. The sme value. Actin taken when quality cntrl tests r instrument calibratin are ut f cntrl; must be dcumented. Test results f a patient s specimen that is dangerusly lw r high; used fr emergency ntificatin f clinicians. Test results that fall utside f the lw r high critical limit fr the particular test in questin. URGENT ntificatin f the apprpriate medical persnnel must be made if critical results are btained. Nncmpliance t a standard. The cndensate cllected when water has been biled (distilled) t remve impurities. A labratry result f a dubtful r uncertain nature which requires retesting. The last day a reagent, kit, cntrl, etc. can be used; the unpened expiratin date is printed n prduct by manufacturer. If the expiratin date changes when the prduct is pened, the labratrian must recrd the new expiratin date. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 58

61 false negative (result) false psitive (result) in cntrl Levy-Jennings chart linearity lt number (cntrl number) lyphilized maintenance (preventive maintenance) mean methd NBS thermmeter A negative test result fr a patient wh is psitive fr the cnditin r cnstituent being tested fr. A psitive test result fr a patient wh is negative fr the cnditin r cnstituent being tested fr. Term used t describe the testing prcedure when the results frm a cntrl sample r series f cntrl samples are within the acceptable cntrl range. Quality cntrl chart; a graph r table that shws results f cntrl results ver a perid f time; used in a quality cntrl prgram. The measure f the range (the linear range) f cncentratin f an analyte ver which a methd r test prduces cnsistent (i.e., linear, straight line) and accurate results. The number given t a batch f a prduct by the manufacturer. Lt number must be recrded when prduct is used in testing. Lt number is imprtant if prduct fails t perfrm adequately. Freeze-dried; a lyphilized calibratr, cntrl, r reagent has been specially dried t make its analytes mre stable. It must be refrigerated t maintain its stability and is recnstituted by adding an apprpriate diluent. Steps fllwed t keep instruments in gd state f repair; must be dcumented. The average f the numerical results btained frm a series f analyses. Analytical methd; the instructins including prcedures, material, equipment, and everything else needed fr an analyst t perfrm an analysis. Natinal Bureau f Standards thermmeter; readings f thermmeters used in labratry instruments must be cmpared t reading with NBS thermmeter. NCCLS ncturia nrmal values (expected values, reference values) ut f cntrl panic values parallel testing Natinal Cmmittee fr Clinical Labratry Standards - cmmittee f experts that develps standards fr labratry prcedures. Standards are published t be fllwed by labratries. Excessive urinatin at night A range f values established fr each analyte which includes results expected when perfrming a test n a healthy persn. Term used t describe the testing prcedure when the results frm a cntrl sample are utside the acceptable cntrl range. Test results that fall utside f the lw and high critical limit. URGENT ntificatin f the apprpriate persnnel must be made if a panic value is btained. Cmparisn testing f a new prduct with prduct currently being used. percentage Parts per 100. precisin (reprducibility) prcedure manual prduct insert The measure f the clseness f the results btained when analyzing the same sample mre than nce; the measure f agreement between replicate measurements. The smaller the variatin between results, the better the precisin. A labratry manual that cntains the methds, materials, and ther infrmatin needed t perfrm a test. Infrmatinal material that cmes with instrument, reagents, and ther labratry prducts giving instructins fr the use f prduct and ther infrmatin required f the manufacturer by the U.S. Fd and Drug Administratin WVOLS T&E 2010 URINALYSIS SESSION II P a g e 59

62 prficiency testing quality assurance quality cntrl qualitative quantitative reactivity reagent recnstitute reliability replicate reprducibility result run (analytical run) saline sample sediment sensitivity specific gravity specificity split-sample testing A prgram in which samples are sent t a grup f labratries fr analysis. The results are tabulated by the prgram spnsr, and a participating labratry can cmpare its results with thse f ther labratries that use the same methd. A cmprehensive set f plicies, prcedures, practices necessary t make sure that the labratry s results are reliable. QA includes recrd-keeping, calibratin and maintenance f equipment, quality cntrl, prficiency testing, and training. The set f labratry prcedures designed t ensure that the test methd is wrking prperly and that the results meet the diagnstic needs f the physician. QC includes testing cntrl samples, charting results, and analyzing the results statistically. This term is applied t tests that detect whether a particular analyte, cnstituent, r cnditin is present. This term is applied t tests that give results expressing the numerical amunt f an analyte in a specimen. The ability f a reagent t prduce its prper chemical reactin. Reagents can lse their reactivity if they are misused, mishandled, r t ld. A substance that prduces a chemical reactin in a sample that allws an analyte t be detected and measured. T add a diluent t a freeze-dried calibratr, cntrl, reagent. The methd s capacity t maintain bth accuracy and precisin. T repeat an experiment r an analysis in rder t check fr accuracy f the results. Each repeat is a replicate test r measurement. See precisin. The value btained by analysis fr a particular analyte in a particular sample. A grup f measurements by a particular methd ver a given perid f time during which the accuracy and precisin f the methd are expected t be stable. An istnic slutin f sdium chlride and distilled water; nrmal saline; physilgical saline; usually made in 0.85 r 0.9% cncentratin fr use in medical labratry prcedures. The part f a specimen that is used fr an analysis. Slid substances which settle t the bttm f a liquid. The ability f a test t give a psitive result fr patients that have the disease r cnditin being are tested fr; measured as the rati f psitive tests t the ttal number f tests in thse that have the disease; expressed as percentage. Rati f weight f a given vlume f a slutin t the weight f the same vlume f water; a measurement f density. The ability f a test t give a negative result fr patients that d nt have the disease r cnditin they are tested fr; measured as the rati f negative tests t the ttal number f tests in thse that d nt have the disease r cnditin; expressed as a percentage. Dividing a sample in half, and testing half in yur labratry and having the ther half tested in anther labratry, and then cmparing the results. This is a technique fr testing accuracy. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 60

63 NAME: URINARY SYSTEM / MICROSCOPY CHAPTER 1 MULTIPLE CHOICE: CIRCLE THE BEST ANSWER 1. The nrmal kidney perfrms all f the fllwing functins except: a. remves metablic waste prducts frm the bld b. regulates acid-base balance in the bdy c. remves excess prtein frm the bld d. regulates the water and electrlyte cntent in the bdy 2. Each kidney is cmpsed f apprximately: a. 100 nephrns b nephrns c. 15,000 nephrns d. 1,500,000 nephrns 3. The essential functin(s) f the prximal cnvluted tubules include: a. secretin b. reabsrptin c. renal bld flw d. secretin and reabsrptin 4. Maintaining a bld ph f apprximately 7.4 is dependent n secretin f: a. hydrgen ins b. ptassium ins c. ammnium ins d. water WVOLS T&E 2010 URINALYSIS SESSION II P a g e 61

64 5. Name the hrmne which is prduced by the pituitary that helps t cntrl the reabsrptin prcess. a. renin b. vaspressin c. angitensin II d. angitensin I 6. The mechanism by which water mves dwn the descending lp and int the bld stream is called: a. active transprt b. cunter - current c. passive transprt d. renal threshld 7. The amunt f glmerular filtrate prduced daily is apprximately: a. 1-2 liters b. 180 liters c. 600 ml/min d. 70 liters 8. The glmerular filtrate is described as: a. a prtein filtrate f plasma b. a glucse and prtein-cntaining filtrate f plasma c. a plasma filtrate withut glucse and prtein d. an ultra filtrate f plasma that des nt cntain prtein and fats 9. The renal threshld fr glucse is: a mg/dl b mg/dl c mg/dl d. ver 240 mg/dl WVOLS T&E 2010 URINALYSIS SESSION II P a g e 62

65 10. Active transprt is respnsible fr the reabsrptin f all f the fllwing except: a. glucse b. albumin c. bicarbnate d. amin acids 11. A wman is suffering frm recurrent urinary tract infectins. The fllwing results were btained frm her urinalysis: REAGENT STRIP MICROSCOPIC clr yellw WBC s 30-40/hpf appearance cludy WBC clumps ccasinal ph 8.0 bacteria large prtein 2+ WBC casts 5-10/hpf ccult bld glucse ketnes trace negative negative specific gravity nitrite leukcyte esterase psitive psitive Based n the abve results, what renal disease is mst likely? a. acute glmerulnephritis b. nephrtic syndrme c. pyelnephritis d. minimal change disease 12. The inflammatry kidney disease in which circulatry antibdies frm grup A streptccci are depsited n the glmerular membrane is called: a. glmerulnephritis b. acute interstitial nephritis c. pyelnephritis d. nephrtic syndrme WVOLS T&E 2010 URINALYSIS SESSION II P a g e 63

66 13. An image that is in fcus under the lw pwer becmes ut f fcus under the high pwer bjective. What culd be the cause f the prblem? a. cndenser is nt lwered b. bjective nt screwed int nse piece fully c. bulb filament is abut t burn ut d. dirty specimen SHORT ANSWERS: FILL IN THE BLANK 14. List the fur main parts f the urinary system. 15. List the tw functins f tubular secretin. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 64

67 16. Label the varius parts f the micrscpe that are indicated. WVOLS T&E 2010 URINALYSIS SESSION II P a g e 65