1. Urinalysis

							Clinical Microscopy
Department of Laboratory Medicine
 China Medical University Hospital
      Shih Mu-Chin (施木青)
Introduction
   Microscopy(鏡檢)：Examination of
    urine, stool, CSF and other body
    fluid under microscope.
       Gross observation
       Cell counts
       Morphology under microscope
       Rapid chemical tests
        1. Urinalysis
   1.1Purpose
     Estiment of renal funtion, urinary tract disorders.
     Metabolites in urine may reveal the systemic disease

      especially in liver disease, and toxicology, uremia.
    Eg. proteinuria－glomerulonephritis, nephrotic syndrome.
        glucosuria－diabetes mellitus.
        pyuria－urinary tract or kidney infection.
        hematuria－stone, T.B.kidney, tumor, acute
      glomerulonephritis.
Urine color
Multitest urine strip
Automatic urine chemistry analyzer
   1. Urinalysis
1.3. Summary of urine collection
   a.  First morning urine
   b.  Random urine
   c.  Postprandial urine
   d.  2 hr interval urine
   p.s. Decomposition of urine begins within 30
       min. at room temperature, and 4 hrs. at
       refrigeration. For long term storage, boric
       acid, Thymol can be used as preservative.
1. Urinalysis
1.4. Physical finding
   a.  Color
            light brown－Urochrome
            Gross red－hematuria, hemoglobinuria
            Deep brown－Bilirulin
            Dark brown－Delayed hemoglobinuria
            Black－Alkaptonuria (Homogentisic acid oxidase def.)
   b.   Turbidity：Normally clear and transparent
            Chyluria─Filariasis or lyphnode obst.
             Lipiduria─nephrotic sydrome
             Pyeuria─Infection
   c.   Volume：Normally 1000~1500 mL
        a.   Polyuria－more than 2000 mL
        b.   Oligouria－less than 500 mL
        c.   Anuria－less than 100 mL
        1. Urinalysis
1.5. Chemical testing
   a.    Protein: proteinuria, microalbuminuria, posture proteinuria,
         Bence Jones P.
   b.    Sugar: Glucosuria vs. mellituria
   c.    Ketone bodies: Diabetes and non diabetes ketonuria.
   d.    Bilirubin and urobilinogen: Differentiation of Jaundice
   e.    Porphobilnogen and Porphyrins: Porphyuria and lead poisoning.
   f.    Occult blood: Hematuria and Hemoglobinuria (Hb-uria),
         myglobin uria
   g.    Nitrite: Bacteria can reduce urinary nitrate to nitrite.
   h.    Leukocyte esterase: An indicator of increased WBC in urine.
   i.    Vit. C(Ascorbic acid): It inhibits reading of glucose, bilrubin,
         nitrite…etc.
   j.    pH: Normal 6.0, range 5.0~8.0
1.6. Normal protein in Urine
   Source：Plasma filtrate and nephron
    secretion (Tamm-Horsfall protein)
   Amount：150 mg/24hrs or 10 mg/dL
   Bromophenol blue used as indicator for
    albumin but not good for globulin and
    Bence-Jones protein.
   Heat denaturation or sulfosalicylic acid
    ppt. can be used.
1.7. Proteinuria (>150 mg/24hrs or 10 mg/dL)


                     Proteinuria


    Prerenal            Renal           Postrenal

    Hb-uria         Glomerular P.         Stones
   Myo-uria           Tubular P.         Cystitis
 Bence Jones P.                          Tumors
               Microalbuminuria:>50mg/24hr.
1.8. Glucosuria
   Normally less than 0.1%glucose or <130
    mg/24hrs in urine.
   Glucose oxidase reaction used for detection
    of glucose in urine.
   Mellituria indicates some reducing sugar
    ( galatose, fructose) in urine of newborn and
    infants.
   Clinitest can detect most reducing sugar
    include glucose.
1.9. Ketoacidosis
   Ketone bodies contains acetone, acetoacetic
    acid and beta-hydroxybutyric acid.
   Diabetes ketosis and starvation can produce
    ketone bodies.
   Ketone bodies are very acidic in plasma and
    in urine.
   Ketoacidosis makes blood pH<7.4
   Ketone-uria is a critical sign of ketoacidosis.
1.10. Bacteriuria
   E. coli, Proteus, Klebsiella, Pseudomonas…
    etc. are commonly found in urinary infection.
   Urinary infection resulting increased WBC in
    urine (Pyeuria)
   Lysed WBC releases leukocyte esterase.
   Prolong incubation (>4hrs), the bacteria can
    reduce urinary nitrate to nitrite which can be
    detected by Na-nitroprusside reaction.
   Nitrite(-) and WBC esterase (-) can rule out
    urinary infection.
1. Urinalysis
   1.11. Hematuria
    無痛血尿 painless hematuria
       腎癌
       多囊腎 Polycystic kidney
       外力傷害 Post-traumatic damage
       運動血尿 Postexercise hematuria
       腎結核 Kidney tuberculosis
       膀胱癌 Bladder tumor
    痛血尿 painful hematuria
       腎結石
1.12. Sediments in normal urine
Sediment                                   Quality
Blood cells:
  Red blood cells                          0-2/hpf
  White blood cells                        3-5/hpf
Casts:
  Hyaline                                  0-2/lpf
  Granular                                 0-1/lpf
Mucus                                      Variable
Epithelial cells
  Renal                                    0-1/hpf
  Transitional                             0-2/hpf
  Squamous                                 Variable/hpf
Crystal in acid, neutral, or basic urine   Variable
Microorgaisms:                             +
  Bacteria
  Fungi(yeasts)
Spermatozoa                                + present in both men and women
      1.13. Diseases State Related to Urinary Findings

Disease State        Urinary findings                      Clinical Features

Exercise             Microscopic hematuria, mild           Vigorous exercise
                     proteinuria cylindruria.

Trauma               Hematuria varing from                 History of trauma to region
                     microscopic to gross                  of kidney

Cystitis             White blood cells especially in       Urgency, frequency dysuria
                     clumps
                     Proteinuria (mild) Bacteriuria
                     No casts
Renal calculi        Ossasionally gross hematuria          Flank pain

Acute                Red blood cell casts, white blood     Smoky urine
Glomerulonephritis   cells, proteinuria, granular casts    Occasionally uremia

Chronic              Red blood cells, white blood cells,   Few symptoms
Glomerulonephritis   proteinuria, granular casts and
                     cellular casts.
RBC’s in urine

   Refractile disks
   Hypertonicity
Dysmorphic RBC’s
WBC’s in urine
   Lobe nuclei
   Refactile cytoplasmic
    granules
     Hyaline casts

   Very pale and
    slightly refractile,
    are common
    findings in urine
Granular casts
   There are granular
    casts with a roughly
    rectangular shape
Granular cast
   Casts which persist
    may break down, so
    that the cell forming
    it are degenerated
    into granular debris,
    as has occurred in
    this granular cast
Renal tubular cell cast
   This renal tubular
    cell cast suggests
    injury to the tubular
    epithelium
   Associated with
    interstitial tulular
    disaese .
Waxy cast
   This is a broad
   The edges are sharp
    and there are
    “Cracks” in this cast
   Tubular prolonged
    stasis
   Also called renal
    failure cast.
          Casts Formation
• Urinary casts are formed only in the
  distal convoluted tubule (DCT) or
  the collecting duct (distal nephron).
  The proximal convoluted tubule
  (PCT) and loop of Henle are not
  locations for cast formation.
• Hyaline casts are composed
  primarily of a mucoprotein (Tamm-
  Horsfall protein) secreted by tubule
  cells.
• The Tamm-Horsfall protein
  secretion (green dots) is illustrated
  in the diagram below, forming a
  hyaline cast in the collecting duct.
      Fat in urine
   Oval fat bodies
    consist of
    degenerated tubular
    cells containing
    abundant lipid
    Fat in urine (under polarized light)


   Oval fat bodies
    demostrate the
    “Maltese cross”
    appearance
Squamous epithelial cells
   Large polygonal
    Squamous epithelial
    cells with small
    nuclei are seen here
Oxalate crystals

   Look like little
    envelopes (or
    tetrahedrons)
   Can be found in
    some foods and
    Vit.C ingestion.
   Associated with
    renal stone
    Triple phosphate crystals
   Look like rectangles or
    coffin lids.
   Associated with renal
    stone,chronic pyelitis
Cystine crystals
 Shaped like “stop”
  signs
 Quite rare

 Inherited metabolic

  defect
Associated with
congenital cyctinuria
and renal stone.
   Diseases State Related to Urinary Findings

Disease State        Urinary findings                      Clinical Features

Exercise             Microscopic hematuria, mild           Vigorous exercise
                     proteinuria cylindruria.

Trauma               Hematuria varing from                 History of trauma to region
                     microscopic to gross                  of kidney

Cystitis             White blood cells especially in       Urgency, frequency dysuria
/pyelonephritis      clumps                                Cloudy urine and
                     Proteinuria (mild <0.5g/dL)           foul smelling
                     Nitrite and leucocyte esterase (+)    Bacteriuria No casts



Renal calculi        Ossasionally gross hematuria          Flank pain

Acute                Red blood cell casts, white blood     Smoky urine
Glomerulonephritis   cells, proteinuria (<1.0g/dL),        Edema,hypertention
                     granular casts

Chronic              Red blood cells, white blood cells,   Edema,hypertention,anemi
Glomerulonephritis   proteinuria(.2.5g/dL), granular       a,
Nephrotic syndrome   casts and cellular casts.             oligouria
2. Stool examination
2.1 Appearance
  a.   Tarry stool: upper GI bleeding.
  b.   Rice water stool: V. Cholera infection.
  c.   Steatorrhea: malabsorption or
       maldigestion.
  d.   Clay stool: barium sulfate or acholic stool
  e.   Bloody stool: Lower GI bleeding.
  f.   Bloody mucoid stool: Pyogenic colitis
S-Y® Feces test Bottle

   糞便檢查用傳送盒
   檢體量:花生米大小
2. Stool examination
2.2 Leucocyte in stool
  Neutrophil and monocyte are commonly
  found. Related to Shigellosis,
  salmonellosis, typhoid fiver and
  pyogenic colitis
   2. Stool examination
2.3 Parasite ova
  a.   Wet mount procedure.
  b.   Concentration procedure.
  c.   Cellophane (cellulose)tape procedure:
       for Enterobius Vermicularis(Pin-worm)
 2. Stool examination
2.4 Diarrhea
  a.   due to neruologic disease or trauma:
       soft to liquid stool.
  b.   Steatorrhea: oily stool due to
       maldigestion or malabsorption. It
       can be confirmed by Sudan III stain.
  c.   Watery diarrhea: Infection or
       intoxication. It can be confirmed by
       culture method or enterotoxin test.
Parasites of the Intestinal Tract
Amebiasis
   Causal Agent:
    Several species of the protozoan parasite
    genus Entamoeba infect humans, but
    Entamoeba histolytica is the only species
    known to cause disease. The other (non-
    pathogenic) species are important because
    they may be confused with E. histolytica in
    diagnostic investigations.
Laboratory Diagnosis:
   Fresh stool: wet mounts and permanently
    stained preparations (e.g. trichrome).

   Concentrates from fresh stool: wet
    mounts, with or without iodine stain, and
    permanently stained preparations (e.g.
    trichrome). Concentration procedures,
    however, are not useful for
    demonstrating trophozoites.
     Microscopy

   Trophozoites of Entamoeba
    histolytica/dispar. Reminder:
    in the absence of
    erythrophagocytosis, the
    pathogenic E. histolytica is
    morphologically
    indistinguishable from the
    nonpathogenic E. dispar!
Hookworm
[Ancylostoma duodenale] [Necator americanus]

   Causal Agents:
    The human hookworms include two nematode
    (roundworm) species, Ancylostoma duodenale and
    Necator americanus. (Adult females: 10 to 13 mm (A.
    duodenale), 9 to 11 mm (N. americanus); adult males:
    8 to 11 mm (A. duodenale), 7 to 9 mm (N.
    americanus). A smaller group of hookworms infecting
    animals can invade and parasitize humans (A.
    ceylanicum) or can penetrate the human skin (causing
    cutaneous larva migrans), but do not develop any
    further (A. braziliense, Uncinaria stenocephala).
   Microscopy
Hookworm eggs examined
   on wet mount (eggs of
   Ancylostoma duodenale
   and Necator americanus
   cannot be distinguished
   morphologically).
 Diagnostic characteristics:
  Size 57 to 76 µm by 35
   to 47 µm
  Oval or ellipsoidal shape
  Thin shell
      Ascaris lumbricoides

   Unfertilized and fertilized eggs
    (left and right, respectively).
   Three fertilized eggs (one
    decorticated, on the right)
    of Ascaris lumbricoides.
   An adult Ascaris worm.
   Diagnostic characteristics:
    tapered ends; length 15 to
    35 cm (the females tend to
    be the larger ones).
   This worm is a female, as
    evidenced by the size and
    genital girdle (the dark
    circular groove at bottom
    area of image).
      Enterobius vermicularis

   Microscopy
   A, B: Enterobius
    egg(s).
   C: Enterobius eggs       B
    on cellulose tape    A
    prep.




                                 C
Intestinal Parasites:
Comparative Morphology
   Protozoa Found in Stool Specimens of Humans:
    Amebae
   Nematode and Cestode Eggs Found in Stool
    Specimens of Humans
      Specimen Collection


   Collect the stool in a dry,
    clean, leakproof container.
   Make sure no urine, water,
    soil or other material gets in
    the container.
Relative Sizes of Helminth Eggs
3. CSF analysis
Indication for a spinal puncture：
 Diagnosis of meningitis

 Intercranial hemorrhage

 Neurologic disorders

 Malignancy
Formation of CSF
   70% CSF是由Chroid plexuses of
    Ventricle經血液ultrafiltration或secretion
    產生。
   30% CSF由腦室的ependymal cells及
    subarachnoid space產生。
The flow of CSF through the
brain and spinal column
Specimen collection
       Lumbar puncture
    3. CSF analysis
   Blood-brain barrier accounts for the
    filtration of blood components to CSF.
   Volume：140-170 mL in adult.
               10-60 mL in neonate.
   Complete analysis needs：
    4 tubes, 2mL CSF in each.
       Tube 1：microscopy study.
       Tube 2：Chemistry study.
       Tube 3：Cell count.
       Tube 4：Cytology
3.1 Macroscopic examination of CSF
  3.1.1 Color
      a.  Clear an colorless－normal
      b.  Dull red or brown－due to old (After 48 hrs),
          hemolysis of intracranial hemorrhage.
      c.  Yellow(Xanthochromic)－due to blood pigment,
          either conjugated or unconjugated bilirubin.
      d.  Greenish or grayish－due to pus cell in severe
          inflammatory reaction.
  3.1.2 Turbidity：different type of meningitis may
       exhibit varying degrees of cloudiness, from slight
       turbidity to opacity.
  3.1.3 pH： 7.24－7.40
  3.1.4 Specific gravity ：1.003－1.008
  3.1.5 Coagulation ：Normal CSF does not coagulate.
    3.2 Characteristics of CSF：

 Appearance           Causes            Important information

    Clear                                       Normal
   Cloudy         >200 WBC/μL                  Infection
                   >400 RBC/μL               Hemorrhage
                   Protein (+++)              Meningitis
    Oily        Radiographic media

   Bloody          >600 RBC/μL         Hemor. or traumatic tap

Xanthochromic    Elevated bilirubin            Jaundice
  (Yellow)
   Clotted      Increased fibrinogen    Demage to blood-CSF
                                       barrier (severe infection)
3.3 Chemical Tests of CSF
  a.   Glucose        50-80% of blood level
  b.   Protein        10-45 mg/dL
  c.   Alb/Glo ratio    8:1
  d.   Lactate        1.1—2.8 mg/dL
  e.   Enzyme         ALT, LDH, CPK
  f.   Immunoglobulins IgG, IgA, IgM, Ig-EP
3.4 Microbiologic tests of CSF
   Culture (bacteria, fungi, viruses, M. TB)
   Stain (Gram’s stain, Acid-fast stain)
   Bacterial and fungal antigens.
   Polymerase chain reaction (PCR)
   Lactate：Normally less than 25mg/dL
Microorganism in CSF
Streptococcus group B
Escherichia coli
Neisseria meningitidis
Haemophilus influenzae
3.4.1 Rapid test for Bacterial Ag of CSF

   Neisseria meningitis group A,C,Y, WB5
   Streptococcus pneumoniae
   Haemophilius influenza type B
   Streptococcus group B
   Escherichia coli
    3.4.2 Fungal Meningitis
   Cryptococcus is the most important
    pathogen.
   DM, cancer and AIDS patients are easily
    infected with Candia sp.
   India ink examination for cryptococcus
   Latex agglutination test.
Cryptococcus neoformans in CSF
Cryptococcus neoformans in CSF
       Eosinophilic meningitis
       (Causal agent:Clonorchis sinensis)

   Distomum spathulatum
   Chinese or oriental liver
    fluke
   Some fish and snails are
    intermediate host.
   mammals are definitive host
   Associated with
    Clonorchiasis and
    eosinophilic meningitis.
    First host of Clonorchis sinensis


   First host: vector snail
   Nine different species
    belonging to four
    families
3.5 Symptoms of Meningitis
   Fever
   Stiff neck
   Petechiae
   Head ache
   Unconscious
   convulsion
            3.5.1 Meningitis and CSF findings

Meningit    Cell count                      Protein   Glucose
                               Cell type                        Other tests
  -is         (μL)                         (mg/dL)    (mg/dL)

Normal      <50          mononuclear WBC   15-45      50-80
CSF

Acute       1,000-       Neutrophils       100-500    <40       Gram stain
bacterial   10,000                                              Lactate(+)


Viral       5-300        Lymphocytes       <100       Normal    Normal
            some                                                lactate
            1000
Leukocyte in CSF(with cytospin tech.)
WBC in CSF (without cytospin tech.)
   3.5.2 CSF pathogens found in different ages
Pathogen <1M 2M-4Y 5-29Y 30-59Y >60Y
Strepto      49    2      2       4      3
agalactiae
Listeria     9     0      2       6      14
monocytoge
n
Hemophilus   5    70      8       5      4
infla.
Strepto.     3    10      17     37      48
Pneumoniae
Neisseria    1    13      42     10      3
meningitis
     3.5.3 Comparison of 3 methods on detection
     of bacteria in 134 CSF specimen

     Method          Culture   Bacter. Ag   Gram’s stain
Positive cases no.     17          9             8
Positive rate        12.7%       6.7%           6%
Hemophilus influ.      3           4             4
Strepto.               4           5             4
Pneumonia
Cryptococcus           1           2            －
         Small lymphocyte


   直徑8um，大小似正常
    紅血球(7-8um)
   N/C >95%，細胞核呈
    圓型、卵形、腎形，細
    胞核染色質濃重粗短，
    有Clump
   細胞質:顏色呈向外漸
    進的深藍色，細胞質中
    偶見少數大顆的
    granules。
          Neutrophilic myelocyte


   此細胞尚未分化完全，
    細胞質中仍有核酸，因
    此其顏色紅中帶藍。
   細胞核：偏位，外形凹
    陷，染色質變粗，無核
    仁，因此期細胞已喪失
    分裂能力，細胞的chro-
    matin 已成群落狀的
    clump。
           Monocytes


   細胞核形狀多變，常以
    馬蹄型，M字型或扭轉
    的不定形狀出現，其
    Chromatin 細長，
    lymphocyte chromatin
    粗重。
   細胞質為灰藍色，常有
    大或小的空泡。
   細胞質中可能出現顆粒
    大多細小，如粉塵與
    lymphocyte出現的少量
    大型顆粒不同。
Atypical lymphocytes
Normal blood RBC vs. IDA RBC




               Iron deficiency anmia
              Leukemia
 Leukemia is a malignant proliferation of cells arising in a
single deranged stem cell and resulting in the overgrowth
   of certain cell lines. These malignant cells replace the
  bone marrow elements and collect in areas of the body
  such as lymph nodes, liver, and spleen. The disease is
    classified as either acute or chronic. Acute leukemia
progresses in a quick and severe manner. Acute leukemia
   presents microscopically with the majority of cellsas
       blasts, severe anemia, and markedly decreased
 platelets. Early detection and treatment is necessary in
      this disease. The term "chronic" is used for those
     disorders in which the leukemic cell is sufficiently
   differentiated to be identified with a certain cell type.
    Chronic leukemia presents microscopically with the
   majority of mature cells, a mild anemia, and variable
                            platelets.
Acute Lymphocytic Leukemia (ALL)
L1 - most frequently occurring form of ALL (85%)
     in children and adults.

L2 - occurs more frequently in adults.

L3 - Burkitt's lymphoma is more often observed
     in children and young adults than older
     persons but overall is an infrequently
     occurring form of ALL.
Acute Leukemia


  Acute Leukemia is classified by the
 French-America-British classification
   (FAB) . This classification breaks
  down the Acute Myelocytic into M1
 through M6. Acute Lymphocytic into
        groups L1 through L3.
CLL   vs   ALL
CLL




      CML
       Myeloblast/Promyelocyte with Auer rods



   Myeloblast細胞核呈
    圓形或橢圓形，佔據大
    部分細胞 N/C 6：1。
   具光滑薄核膜，並具微
    細網狀均勻分布紫色
    核染色質，核仁透亮。
   細胞質具淡藍或藍色，
    沒有顆粒或偶爾發現
    Auer rods。
AML in PB blood   AML in bone marrow