MICROSCOPIC EXAMINATION OF URINE

Published in Clinical Pathology
Thursday, 10 August 2017 00:57
Microscopic examination of urine is also called as the “liquid biopsy of the urinary tract”.
 
Urine consists of various microscopic, insoluble, solid elements in suspension. These elements are classified as organized or unorganized. Organized substances include red blood cells, white blood cells, epithelial cells, casts, bacteria, and parasites. The unorganized substances are crystalline and amorphous material. These elements are suspended in urine and on standing they settle down and sediment at the bottom of the container; therefore they are known as urinary deposits or urinary sediments. Examination of urinary deposit is helpful in diagnosis of urinary tract diseases as shown in Table 825.1.
 
Table 825.1 Urinary findings in renal diseases
Condition Albumin RBCs/HPF WBCs/HPF Casts/LPF Others
1. Normal 0-trace 0-2 0-2 Occasional (Hyaline)
2. Acute glomerulonephritis 1-2+ Numerous;dysmorphic 0-few Red cell, granular Smoky urine or hematuria
3. Nephrotic syndrome > 4+ 0-few 0-few Fatty, hyaline, Waxy, epithelial Oval fat bodies, lipiduria
4. Acute pyelonephritis 0-1+ 0-few Numerous WBC, granular WBC clumps, bacteria, nitrite test
HPF: High power field; LPF: Low power field; RBCs: Red blood cells; WBCs: White blood cells.
 
Different types of urinary sediments are shown in Figure 825.1. The major aim of microscopic examination of urine is to identify different types of cellular elements and casts. Most crystals have little clinical significance.
 
Figure 825.1 Different types of urinary sediment
Figure 825.1 Different types of urinary sediment
 
Specimen: The cellular elements are best preserved in acid, hypertonic urine; they deteriorate rapidly in alkaline, hypotonic solution. A mid-stream, freshly voided, first morning specimen is preferred since it is the most concentrated. The specimen should be examined within 2 hours of voiding because cells and casts degenerate upon standing at room temperature. If preservative is required, then 1 crystal of thymol or 1 drop of formalin (40%) is added to about 10 ml of urine.
 
Method: A well-mixed sample of urine (12 ml) is centrifuged in a centrifuge tube for 5 minutes at 1500 rpm and supernatant is poured off. The tube is tapped at the bottom to resuspend the sediment (in 0.5 ml of urine). A drop of this is placed on a glass slide and covered with a cover slip (Figure 825.2). The slide is examined immediately under the microscope using first the low power and then the high power objective. The condenser should be lowered to better visualize the elements by reducing the illumination.
 
Figure 825.2 Preparation of urine sediment for microscopic examination
Figure 825.2 Preparation of urine sediment for microscopic examination
 
CELLS
 
Cellular elements in urine are shown in Figure 825.3.
 
Figure 825.3 Cells in urine
Figure 825.3 Cells in urine (1) Isomorphic red blood cells, (2) Crenated red cells, (3) Swollen red cells, (4) Dysmorphic red cells, (5) White blood cells (pus cells), (6) Squamous epithelial cell, (7) Transitional epithelial cells, (8) Renal tubular epithelial cells, (9) Oval fat bodies, (10) Maltese cross pattern of oval fat bodies, and (11) spermatozoa
 
Red Blood Cells
 
Normally there are no or an occasional red blood cell in urine. In a fresh urine sample, red cells appear as small, smooth, yellowish, anucleate biconcave disks about 7 μ in diameter (called as isomorphic red cells). However, red cells may appear swollen (thin discs of greater diameter, 9-10 μ) in dilute or hypotonic urine, or may appear crenated (smaller diameter with spikey surface) in hypertonic urine. In glomerulonephritis, red cells are typically described as being dysmorphic (i.e. markedly variable in size and shape). They result from passage of red cells through the damaged glomeruli. Presence of > 80% of dysmorphic red cells is strongly suggestive of glomerular pathology.
 
The quantity of red cells can be reported as number of red cells per high power field.
 
Causes of hematuria have been listed earlier.
 
White Blood Cells (Pus Cells)
 
White blood cells are spherical, 10-15 μ in size, granular in appearance in which nuclei may be visible. Degenerated white cells are distorted, smaller, and have fewer granules. Clumps of numerous white cells are seen in infections. Presence of many white cells in urine is called as pyuria. In hypotonic urine white cells are swollen and the granules are highly refractile and show Brownian movement; such cells are called as glitter cells; large numbers are indicative of injury to urinary tract.
 
Normally 0-2 white cells may be seen per high power field. Pus cells greater than 10/HPF or presence of clumps is suggestive of urinary tract infection.
 
Increased numbers of white cells occur in fever, pyelonephritis, lower urinary tract infection, tubulointerstitial nephritis, and renal transplant rejection.
 
In urinary tract infection, following are usually seen in combination:
 
  • Clumps of pus cells or pus cells >10/HPF
  • Bacteria
  • Albuminuria
  • Positive nitrite test
 
Simultaneous presence of white cells and white cell casts indicates presence of renal infection (pyelonephritis).
 
Eosinophils (>1% of urinary leucocytes) are a characteristic feature of acute interstitial nephritis due to drug reaction (better appreciated with a Wright’s stain).
 
Renal Tubular Epithelial Cells
 
Presence of renal tubular epithelial cells is a significant finding. Increased numbers are found in conditions causing tubular damage like acute tubular necrosis, pyelonephritis, viral infection of kidney, allograft rejection, and salicylate or heavy metal poisoning.
 
These cells are small (about the same size or slightly larger than white blood cell), polyhedral, columnar, or oval, and have granular cytoplasm. A single, large, refractile, eccentric nucleus is often seen.
 
Renal tubular epithelial cells are difficult to distinguish from pus cells in unstained preparations.
 
Squamous Epithelial Cells
 
Squamous epithelial cells line the lower urethra and vagina. They are best seen under low power objective (×10). Presence of large numbers of squamous cells in urine indicates contamination of urine with vaginal fluid. These are large cells, rectangular in shape, flat with abundant cytoplasm and a small, central nucleus.
 
Transitional Epithelial Cells
 
Transitional cells line renal pelvis, ureters, urinary bladder, and upper urethra. These cells are large, and diamond- or pear-shaped (caudate cells). Large numbers or sheets of these cells in urine occur after catheterization and in transitional cell carcinoma.
 
Oval Fat Bodies
 
These are degenerated renal tubular epithelial cells filled with highly refractile lipid (cholesterol) droplets. Under polarized light, they show a characteristic “Maltese cross” pattern. They can be stained with a fat stain such as Sudan III or Oil Red O. They are seen in nephrotic syndrome in which there is lipiduria.
 
Spermatozoa
 
They may sometimes be seen in urine of men.
 
Telescoped urinary sediment: This refers to urinary sediment consisting of red blood cells, white blood cells, oval fat bodies, and all types of casts in roughly equal proportion. It occurs in lupus nephritis, malignant hypertension, rapidly proliferative glomerulonephritis, and diabetic glomerulosclerosis.
 
ORGANISMS
 
Organisms detectable in urine are shown in Figure 825.4.
 
Figure 825.4 Organisms in urine
Figure 825.4 Organisms in urine: (A) Bacteria, (B) Yeasts, (C) Trichomonas, and (D) Egg of Schistosoma haematobium
 
Bacteria
 
Bacteria in urine can be detected by microscopic examination, reagent strip tests for significant bacteriuria (nitrite test, leucocyte esterase test), and culture
 
Significant bacteriuria exists when there are >105 bacterial colony forming units/ml of urine in a cleancatch midstream sample, >104 colony forming units/ml of urine in catheterized sample, and >103 colonyforming units/ml of urine in a suprapubic aspiration sample.
 
  1. Microscopic examination: In a wet preparation, presence of bacteria should be reported only when urine is fresh. Bacteria occur in combination with pus cells. Gram’s-stained smear of uncentrifuged urine showing 1 or more bacteria per oil-immersion field suggests presence of > 105 bacterial colony forming units/ml of urine. If many squamous cells are present, then urine is probably contaminated with vaginal flora. Also, presence of only bacteria without pus cells indicates contamination with vaginal or skin flora.
  2. Chemical or reagent strip tests for significant bacteriuria: These are given earlier.
  3. Culture: On culture, a colony count of >105/ml is strongly suggestive of urinary tract infection, even in asymptomatic females. Positive culture is followed by sensitivity test. Most infections are due to Gram-negative enteric bacteria, particularly Escherichia coli.
 
If three or more species of bacteria are identified on culture, it almost always indicates contamination by vaginal flora.
 
Negative culture in the presence of pyuria (‘sterile’ pyuria) occurs with prior antibiotic therapy, renal tuberculosis, prostatitis, renal calculi, catheterization, fever in children (irrespective of cause), female genital tract infection, and non-specific urethritis in males.
 
Yeast Cells (Candida)
 
These are round or oval structures of approximately the same size as red blood cells. In contrast to red cells, they show budding, are oval and more refractile, and are not soluble in 2% acetic acid.
 
Presence of Candida in urine may suggest immunocompromised state, vaginal candidiasis, or diabetes mellitus. Usually pyuria is present if there is infection by Candida. Candida may also be a contaminant in the sample and therefore urine sample must be examined in a fresh state.
 
Trichomonas vaginalis
 
These are motile organisms with pear shape, undulating membrane on one side, and four flagellae. They cause vaginitis in females and are thus contaminants in urine. They are easily detected in fresh urine due to their motility.
 
Eggs of Schistosoma haematobium
 
Infection by this organism is prevalent in Egypt.
 
Microfilariae
 
They may be seen in urine in chyluria due to rupture of a urogenital lymphatic vessel.
 
CASTS
 
Urinary casts are cylindrical, cigar-shaped microscopic structures that form in distal renal tubules and collecting ducts. They take the shape and diameter of the lumina (molds or ‘casts’) of the renal tubules. They have parallel sides and rounded ends. Their length and width may be variable. Casts are basically composed of a precipitate of a protein that is secreted by tubules (Tamm-Horsfall protein). Since casts form only in renal tubules their presence is indicative of disease of the renal parenchyma. Although there are several types of casts, all urine casts are basically hyaline; various types of casts are formed when different elements get deposited on the hyaline material (Figure 825.5). Casts are best seen under low power objective (×10) with condenser lowered down to reduce the illumination.
 
Figure 825.5 Genesis of casts in urine
 Figure 825.5 Genesis of casts in urine. All cellular casts degenerate to granular and waxy casts
 
Casts are the only elements in the urinary sediment that are specifically of renal origin.
 
Casts (Figure 825.6) are of two main types:
 
  1. Noncellular: Hyaline, granular, waxy, fatty
  2. Cellular: Red blood cell, white blood cell, renal tubular epithelial cell.
 
Hyaline and granular casts may appear in normal or diseased states. All other casts are found in kidney diseases.
 
Figure 825.6 Urinary casts
Figure 825.6 Urinary casts: (A) Hyaline cast, (B) Granular cast, (C) Waxy cast, (D) Fatty cast, (E) Red cell cast, (F) White cell cast, and (G) Epithelial cast
 
Non-cellular Casts
 
Hyaline casts: These are the most common type of casts in urine and are homogenous, colorless, transparent, and refractile. They are cylindrical with parallel sides and blunt, rounded ends and low refractive index. Presence of occasional hyaline cast is considered as normal. Their presence in increased numbers (“cylinduria”) is abnormal. They are composed primarily of Tamm-Horsfall protein. They occur transiently after strenuous muscle exercise in healthy persons and during fever. Increased numbers are found in conditions causing glomerular proteinuria.
 
Granular casts: Presence of degenerated cellular debris in a cast makes it granular in appearance. These are cylindrical structures with coarse or fine granules (which represent degenerated renal tubular epithelial cells) embedded in Tamm-Horsfall protein matrix. They are seen after strenuous muscle exercise and in fever, acute glomerulonephritis, and pyelonephritis.
 
Waxy cast: These are the most easily recognized of all casts. They form when hyaline casts remain in renal tubules for long time (prolonged stasis). They have homogenous, smooth glassy appearance, cracked or serrated margins and irregular broken-off ends. The ends are straight and sharp and not rounded as in other casts. They are light yellow in color. They are most commonly seen in end-stage renal failure.
 
Fatty casts: These are cylindrical structures filled with highly refractile fat globules (triglycerides and cholesterol esters) in Tamm-Horsfall protein matrix. They are seen in nephrotic syndrome.
 
Broad casts: Broad casts form in dilated distal tubules and are seen in chronic renal failure and severe renal tubular obstruction. Both waxy and broad casts are associated with poor prognosis.
 
Cellular Casts
 
To be called as cellular, casts should contain at least three cells in the matrix. Cellular casts are named according to the type of cells entrapped in the matrix.
 
Red cell casts: These are cylindrical structures with red cells in Tamm-Horsfall protein matrix. They may appear brown in color due to hemoglobin pigmentation. These have greater diagnostic importance than any other cast. If present, they help to differentiate hematuria due to glomerular disease from hematuria due to other causes. RBC casts usually denote glomerular pathology e.g. acute glomerulonephritis.
 
White cell casts: These are cylindrical structures with white blood cells embedded in Tamm-Horsfall protein matrix. Leucocytes usually enter into tubules from the interstitium and therefore presence of leucocyte casts indicates tubulointerstitial disease like pyelonephritis.
 
Renal tubular epithelial cell casts: These are composed of renal tubular epithelial cells that have been sloughed off. They are seen in acute tubular necrosis, viral renal disease, heavy metal poisoning, and acute allograft rejection. Even an occasional renal tubular cast is a significant finding.
 
CRYSTALS
 
Crystals are refractile structures with a definite geometric shape due to orderly 3-dimensional arrangement of its atoms and molecules. Amorphous material (or deposit) has no definite shape and is commonly seen in the form of granular aggregates or clumps.
 
Crystals in urine (Figure 825.7) can be divided into two main types: (1) Normal (seen in normal urinary sediment), and (2) Abnormal (seen in diseased states).
 
Figure 825.7 Crystals in urine
Figure 825.7 Crystals in urine. (A) Normal crystals: (1) Calcium oxalate, (2) Triple phosphates, (3) Uric acid, (4) Amorphous phosphates, (5) Amorphous urates, (6) Ammonium urate. (B) Abnormal crystals: (1) Cysteine, (2) Cholesterol, (3) Bilirubin, (4) Tyrosine, (5) Sulfonamide, and (6) Leucine
 
However, crystals found in normal urine can also be seen in some diseases in increased numbers.
 
Most crystals have no clinical importance (particularly phosphates, urates, and oxalates). Crystals can be identified in urine by their morphology. However, before reporting presence of any abnormal crystals, it is necessary to confirm them by chemical tests.
 
Normal Crystals
 
Crystals present in acid urine:
 
  1. Uric acid crystals: These are variable in shape (diamond, rosette, plates), and yellow or red-brown in color (due to urinary pigment). They are soluble in alkali, and insoluble in acid. Increased numbers are found in gout and leukemia. Flat hexagonal uric acid crystals may be mistaken for cysteine crystals that also form in acid urine.
  2. Calcium oxalate crystals: These are colorless, refractile, and envelope-shaped. Sometimes dumbbell-shaped or peanut-like forms are seen. They are soluble in dilute hydrochloric acid. Ingestion of certain foods like tomatoes, spinach, cabbage, asparagus, and rhubarb causes increase in their numbers. Their increased number in fresh urine (oxaluria) may also suggest oxalate stones. A large number are seen in ethylene glycol poisoning.
  3. Amorphous urates: These are urate salts of potassium, magnesium, or calcium in acid urine. They are usually yellow, fine granules in compact masses. They are soluble in alkali or saline at 60°C.
 
Crystals present in alkaline urine:
 
  1. Calcium carbonate crystals: These are small, colorless, and grouped in pairs. They are soluble in acetic acid and give off bubbles of gas when they dissolve.
  2. Phosphates: Phosphates may occur as crystals (triple phosphates, calcium hydrogen phosphate), or as amorphous deposits.
    Phosphate crystals
    Triple phosphates (ammonium magnesium phosphate): They are colorless, shiny, 3-6 sided prisms with oblique surfaces at the ends (“coffinlids”), or may have a feathery fern-like appearance.
    Calcium hydrogen phosphate (stellar phosphate): These are colorless, and of variable shape (starshaped, plates or prisms).
    Amorphous phosphates: These occur as colorless small granules, often dispersed.
    All phosphates are soluble in dilute acetic acid.
  3. Ammonium urate crystals: These occur as cactus-like (covered with spines) and called as ‘thornapple’ crystals. They are yellow-brown and soluble in acetic acid at 60°C.
 
Abnormal Crystals
 
They are rare, but result from a pathological process.
 
These occur in acid pH, often in large amounts. Abnormal crystals should not be reported on microscopy alone; additional chemical tests are done for confirmation.
 
  1. Cysteine crystals: These are colorless, clear, hexagonal (having 6 sides), very refractile plates in acid urine. They often occur in layers. They are soluble in 30% hydrochloric acid. They are seen in cysteinuria, an inborn error of metabolism. Cysteine crystals are often associated with formation of cysteine stones.
  2. Cholesterol crystals: These are colorless, refractile, flat rectangular plates with notched (missing) corners, and appear stacked in a stair-step arrangement. They are soluble in ether, chloroform, or alcohol. They are seen in lipiduria e.g. nephrotic syndrome and hypercholesterolemia. They can be positively identified by polarizing microscope.
  3. Bilirubin crystals: These are small (5 μ), brown crystals of variable shape (square, bead-like, or fine needles). Their presence can be confirmed by doing reagent strip or chemical test for bilirubin. These crystals are soluble in strong acid or alkali. They are seen in severe obstructive liver disease.
  4. Leucine crystals: These are refractile, yellow or brown, spheres with radial or concentric striations. They are soluble in alkali. They are usually found in urine along with tyrosine in severe liver disease (cirrhosis).
  5. Tyrosine crystals: They appear as clusters of fine, delicate, colorless or yellow needles and are seen in liver disease and tyrosinemia (an inborn error of metabolism). They dissolve in alkali.
  6. Sulfonamide crystals: They are variably shaped crystals, but usually appear as sheaves of needles. They occur following sulfonamide therapy. They are soluble in acetone.

CARE AND MAINTENANCE OF MICROSCOPE

Published in Microbiology
Monday, 31 July 2017 18:06
Routine care and proper maintenance of microscope will ensure good performance over the years. In addition to this, a properly maintained and clean microscope will always be ready for use at any time. Professional cleaning and maintenance should be considered when routine techniques fail to produce optimal performance of the microscope.
 
Cleaning and maintenance supplies
 
Dust cover: When not in use, a microscope should be covered to protect it from dust, hair, and any other possible sources of dirt. It is important to note that a dust cover should never be placed over a microscope while the illuminator is still on.
 
Lens tissue: Lint-free lens tissues are delicate wipes that would not scratch the surface of the oculars or objective. Always ensure that you are using these types of tissues. Never substitute facial tissue or paper towel, as they are too abrasive.
 
Lens cleaner: Lens cleaning solution assists in removing fingerprints and smudges on lenses and objectives. Apply the lens cleaner to the lens tissue paper and clean/polish the surface.
 
Compressed air duster: Using compressed air to rid the microscope of dust particles is far superior to using your own breath and blowing onto the microscope. Compressed air is clean, and avoids possible contamination of saliva particles.
 
Maintenance tips
 
  1. Whenever the microscope is not in use, turn off the illuminator. This will greatly extend the life of the bulb, as well as keep the temperature down during extended periods of laboratory work.
  2. When cleaning the microscope, use distilled water or lens cleaner. Avoid using other chemicals or solvents, as they may be corrosive to the rubber or lens mounts.
  3. After using immersion oil, clean off any residue immediately. Avoid rotating the 40× objective through immersion oil. If this should occur, immediately clean the 40× objective with lens cleaner before the oil has a chance to dry.
  4. Do not be afraid to use many sheets of lens tissue when cleaning. Use a fresh piece (or a clean area of the same piece) when moving to a different part of the microscope. This avoids tracking dirt/oil/residue to other areas of the microscope.
  5. Store the microscope safely with the stage lowered and the smallest objective in position (4× or 10×). This placement allows for the greatest distance between the stage and the objective. If the microscope is bumped, the likelihood of an objective becoming damaged by the stage surface will be greatly minimized.

EXAMINATION OF BLOOD SMEAR

Published in Hemotology
Thursday, 27 July 2017 11:22
A blood smear is examined for:
 
 
A peripheral blood smear has three parts: Head, body, and tail. Also read: PREPARATION OF BLOOD SMEAR BY WEDGE METHOD.
 
A blood smear should be examined in an orderly manner. Initially, blood smear should be observed under low power objective (10×) to assess whether the film is properly spread and stained, to assess cell distribution, and to select an area for examination of blood cells. Best morphologic details are seen in the area where red cells are just touching one another. Low power view is also helpful for the identification of Rouleaux formation, autoagglutination of red cells, and microfilaria. High power objective (45×) is suitable for examination of red cell morphology and for differential leukocyte count. A rough estimate of total leukocyte count can be obtained which also serves to crosscheck the total leukocyte count done by manual counting or automated method. Oil-immersion objective (100×) is used for more detailed examination of any abnormal cells.
 
Further Reading:
 

MORPHOLOGY OF PLATELETS

Published in Hemotology
Wednesday, 26 July 2017 17:45
Box 802.1 Role of blood smear in thrombocytopeniaPlatelets are small, 1-3 μm in diameter, purple structures with tiny irregular projections on surface. In blood films prepared from non-anticoagulated blood (i.e. direct fingerstick), they occur in clumps. If platelet count is done on automated blood cell counters using EDTA-anticoagulated blood sample, about 1% of persons show falsely low count due to the presence in them of EDTA dependent antiplatelet antibody. Examination of a parallel blood film is useful in avoiding the false diagnosis of thrombocytopenia in such cases. Occasionally, platelets show rosetting around neutrophils (platelet satellitism) (see Figure 802.1). This is seen in patients with platelet antibodies and in apparently normal persons. Blood smear examination can be helpful in determining underlying cause of thrombocytopenia such as leukemia, lymphoma, or microangiopathic hemolytic anemia (Box 802.1).
 
Also Read:
 

NUMERICAL ABNORMALITIES OF LEUKOCYTES

Published in Hemotology
Wednesday, 26 July 2017 16:28
For meaningful interpretation, absolute count of leukocytes should be reported. These are obtained as follows:
 
Absolute Leukocyte Count = Leukocyte% × Total Leukocyte Count/ml
 
 
Neutrophilia:
 
An absolute neutrophil count greater than 7500/μl is termed as neutrophilia or neutrophilic leukocytosis.
 
Causes
 
  1. Acute bacterial infections: Abscess, pneumonia, meningitis, septicemia, acute rheumatic fever, urinary tract infection.
  2. Tissue necrosis: Burns, injury, myocardial infarction.
  3. Acute blood loss
  4. Acute hemorrhage
  5. Myeloproliferative disorders
  6. Metabolic disorders: Uremia, acidosis, gout
  7. Poisoning
  8. Malignant tumors
  9. Physiologic causes: Exercise, labor, pregnancy, emotional stress.
 
Leukemoid reaction: This refers to the presence of markedly increased total leukocyte count (>50,000/cmm) with immature cells in peripheral blood resembling leukaemia but occurring in non-leukemic disorders (see Figure 801.2). Its causes are:
 
  • Severe bacterial infections, e.g. septicemia, pneumonia
  • Severe hemorrhage
  • Severe acute hemolysis
  • Poisoning
  • Burns
  • Carcinoma metastatic to bone marrow Leukemoid reaction should be differentiated from chronic myeloid leukemia (Table 801.1).
 
Table 801.1 Differences between leukemoid reaction and leukemia
Table 801.1 Differences between leukemoid reaction and leukemia
 
Figure 801.2 Leukemoid reaction in blood smear
Figure 801.2 Leukemoid reaction in blood smear
 
 
Absolute neutrophil count less than 2000/μl is neutropenia. It is graded as mild (2000-1000/μl), moderate (1000-500/μl), and severe (< 500/μl).
 
Causes
 
I. Decreased or ineffective production in bone marrow:
 
  1. Infections 
    (a) Bacterial: typhoid, paratyphoid, miliary tuberculosis, septicemia
    (b) Viral: influenza, measles, rubella, infectious mononucleosis, infective hepatitis.
    (c) Protozoal: malaria, kala azar
    (d) Overwhelming infection by any organism
  2. Hematologic disorders: megaloblastic anemia, aplastic anemia, aleukemic leukemia, myelophthisis.
  3. Drugs:
    (a) Idiosyncratic action: Analgesics, antibiotics, sulfonamides, phenothiazines, antithyroid drugs, anticonvulsants.
    (b) Dose-related: Anticancer drugs
  4. Ionizing radiation
  5. Congenital disorders: Kostman's syndrome, cyclic neutropenia, reticular dysgenesis.
 
II. Increased destruction in peripheral blood:
 
  1. Neonatal isoimmune neutropaenia
  2. Systemic lupus erythematosus
  3. Felty's syndrome
 
III. Increased sequestration in spleen:
 
  1. Hypersplenism
 
Eosinophilia:
 
This refers to absolute eosinophil count greater than 600/μl.
 
Causes
 
  1. Allergic diseases: Bronchial asthma, rhinitis, urticaria, drugs.
  2. Skin diseases: Eczema, pemphigus, dermatitis herpetiformis.
  3. Parasitic infection with tissue invasion: Filariasis, trichinosis, echinococcosis.
  4. Hematologic disorders: Chronic Myeloproliferative disorders, Hodgkin's disease, peripheral T cell lymphoma.
  5. Carcinoma with necrosis.
  6. Radiation therapy.
  7. Lung diseases: Loeffler's syndrome, tropical eosinophilia
  8. Hypereosinophilic syndrome.
 
Basophilia:
 
Increased numbers of basophils in blood (>100/μl) occurs in chronic myeloid leukemia, polycythemia vera, idiopathic myelofibrosis, basophilic leukemia, myxedema, and hypersensitivity to food or drugs.
 
Monocytosis:
 
This is an increase in the absolute monocyte count above 1000/μl.
 
Causes
 
  1. Infections: Tuberculosis, subacute bacterial endocarditis, malaria, kala azar.
  2. Recovery from neutropenia.
  3. Autoimmune disorders.
  4. Hematologic diseases: Myeloproliferative disorders, monocytic leukemia, Hodgkin's disease.
  5. Others: Chronic ulcerative colitis, Crohn's disease, sarcoidosis.
 
Lymphocytosis:
 
Box 801.1 Differential diagnosis of LymphocytosisThis is an increase in absolute lymphocyte count above upper limit of normal for age (4000/μl in adults, >7200/μl in adolescents, >9000/μl in children and infants) (Box 801.1).
 
Causes
 
  1. Infections: 
    (a) Viral: Acute infectious lymphocytosis, infective hepatitis, cytomegalovirus, mumps, rubella, varicella
    (b) Bacterial: Pertussis, tuberculosis
    (c) Protozoal: Toxoplasmosis
  2. Hematological disorders: Acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma.
  3. Other: Serum sickness, post-vaccination, drug reactions.

WHITE BLOOD CELLS MORPHOLOGY

Published in Hemotology
Wednesday, 26 July 2017 13:33
Approximate idea about total leukocyte count can be gained from the examination of the smear under high power objective (40× or 45×). A differential leukocyte count should be carried out. Abnormal appearing white cells are evaluated under oil-immersion objective.
 
Morphology of normal leukocytes (see Figure 800.1):
 
  1. Polymorphonuclear neutrophil: Neutrophil measures 14-15 μm in size. Its cytoplasm is colorless or lightly eosinophilic and contains multiple, small, fine, mauve granules. Nucleus has 2-5 lobes that are connected by fine chromatin strands. Nuclear chromatin is condensed and stains deep purple in color. A segmented neutrophil has at least 2 lobes connected by a chromatin strand. A band neutrophil shows non-segmented U-shaped nucleus of even width. Normally band neutrophils comprise less than 3% of all leukocytes. Majority of neutrophils have 3 lobes, while less than 5% have 5 lobes. In females, 2-3% of neutrophils show a small projection (called drumstick) on the nuclear lobe. It represents one inactivated X chromosome.
  2. Eosinophil: Eosinophils are slightly larger than neutrophils (15-16 μm). The nucleus is often bilobed and the cytoplasm is packed with numerous, large, bright orange-red granules. On blood smears, some of the eosinophils are often ruptured.
  3. Basophils: Basophils are seen rarely on normal smears. They are small (9-12 μm), round to oval cells, which contain very large, coarse, deep purple granules. It is difficult to make out the nucleus since granules cover it.
  4. Monocytes: Monocyte is the largest of the leukocytes (15-20 μm). It is irregular in shape, with oval or clefted (kidney-shaped) nucleus and fine, delicate chromatin. Cytoplasm is abundant, bluegray with ground glass appearance and often contains fine azurophil granules and vacuoles. After migration to the tissues from blood, they are called as macrophages.
  5. Lymphocytes: On peripheral blood smear, two types of lymphocytes are distinguished: small and large. The majority of lymphocytes are small (7-8 μm). These cells have a high nuclearcytoplasmic ratio with a thin rim of deep blue cytoplasm. The nucleus is round or slightly clefted with coarsely clumped chromatin. Large lymphocytes (10-15 μm) have a more abundant, pale blue cytoplasm, which may contain a few azurophil granules. Nucleus is oval or round and often placed on one side of the cell.
 
Figure 800.1 Normal mature white blood cells in peripheral blood
Figure 800.1 Normal mature white blood cells in peripheral blood
 
Morphology of abnormal leukocytes:
 
  1. Box 800.1 Role of blood smear in leukemiaToxic granules: These are darkly staining, bluepurple, coarse granules in the cytoplasm of neutrophils. They are commonly seen in severe bacterial infections.
  2. Döhle inclusion bodies: These are small, oval, pale blue cytoplasmic inclusions in the periphery of neutrophils. They represent remnants of ribosomes and rough endoplasmic reticulum. They are often associated with toxic granules and are seen in bacterial infections.
  3. Cytoplasmic vacuoles: Vacuoles in neutrophils are indicative of phagocytosis and are seen in bacterial infections.
  4. Shift to left of neutrophils: This refers to presence of immature cells of neutrophil series (band forms and metamyelocytes) in peripheral blood and occurs in infections and inflammatory disorders.
  5. Hypersegmented neutrophils: Hypersegmentation of neutrophils is said to be present when >5% of neutrophils have 5 or more lobes. They are large in size and are also called as macropolycytes. They are seen in folate or vitamin B12 deficiency and represent one of the earliest signs.
  6. Pelger-Huet cells: In Pelger-Huet anomaly (a benign autosomal dominant condition), there is failure of nuclear segmentation of granulocytes so that nuclei are rod-like, round, or have two segments. Such granulocytes are also observed in myeloproliferative disorders (pseudo-Pelger-Huet cells).
  7. Atypical lymphocytes: These are seen in viral infections, especially infectious mononucleosis. Atypical lymphocytes are large, irregularly shaped lymphocytes with abundant cytoplasm and irregular nuclei. Cytoplasm shows deep basophilia at the edges and scalloping of borders. Nuclear chromatin is less dense and occasional nucleolus may be present.
  8. Blast cells: These are most premature of the leukocytes. They are large (15-25 μm), round to oval cells, with high nuclear cytoplasmic ratio. Nucleus shows one or more nucleoli and nuclear chromatin is immature. These cells are seen in severe infections, infiltrative disorders, and leukemia. In leukemia and lymphoma, blood smear suggests the diagnosis or differential diagnosis and helps in ordering further tests (see Figure 800.2 and Box 800.1).
 
Figure 800.2 Morphological abnormalities of white blood cells
Figure 800.2 Morphological abnormalities of white blood cells: (A) Toxic granules; (B) Döhle inclusion body; (C) Shift to left in neutrophil series; (D) Hypersegmented neutrophil in megaloblastic anemia; (E) Atypical lymphocyte in infectious mononucleosis; (F) Blast cell in acute leukemia
 
Further Reading:
 

PARTS AND FUNCTIONS OF A COMPOUND MICROSCOPE

Published in Microbiology
Tuesday, 25 July 2017 15:42
The microscope is the most important piece of equipment in the clinic laboratory. The microscope is used to review fecal, urine, blood, and cytology samples on a daily basis (see Figure). Understanding how the microscope functions, how it operates, and how to care for it will improve the reliability of your results and prolong the life of this valuable piece of equipment.

Parts and functions of a compound microscope

Compound Microscope(A) Arm: Used to carry the microscope.
 
(B) Base: Supports the microscope and houses the light source.
 
(C) Oculars (or eyepieces): The lens of the microscope you look through. The ocular also magnifies the image. The total magnification can be calculated by multiplying the objective power by the ocular power. Oculars come in different magnifications, but 10× magnification is common.
 
(D) Diopter adjustment: The purpose of the diopter adjustment is to correct the differences in vision an individual may have between their left and right eyes.
 
(E) Interpupillary adjustment: This allows the oculars to move closer or further away from one another to match the width of an individual’s eyes. When looking through the microscope, one should see only a single field of view. When viewing a sample, always use both eyes. Using one eye can cause eye strain over a period of time.
 
(F) Nosepiece: The nosepiece holds the objective lenses. The objectives are mounted on a rotating turret so they can be moved into place as needed. Most nosepieces can hold up to five objectives.
 
(G) Objective lenses: The objective lens is the lens closest to the object being viewed, and its function is to magnify it. Objective lenses are available in many powers, but 4×, 10×, 40×, and 100× are standard. 4× objective is used mainly for scanning. 10× objective is considered “low power,” 40× is “high power” and 100× objective is referred to as “oil immersion.” Once magnified by the objective lens, the image is viewed through the oculars, which magnify it further. Total magnification can be calculated by multiplying the objective power by the ocular lens power.
 
For example: 100× objective lens with 10× oculars = 1000× total magnification.
 
(H) Stage: The platform on which the slide or object is placed for viewing.
 
(I) Stage brackets: Spring-loaded brackets, or clips, hold the slide or specimen in place on the stage.
 
(J) Stage control knobs: Located just below the stage are the stage control knobs. These knobs move the slide or specimen either horizontally (x-axis) or vertically (y-axis) when it is being viewed.
 
(K) Condenser: The condenser is located under the stage. As light travels from the illuminator, it passes through the condenser, where it is focused and directed at the specimen.
 
(L) Condenser control knob: Allows the condenser to be raised or lowered.
 
(M) Condenser centering screws: These crews center the condenser, and therefore the beam of light. Generally, they do not need much adjustment unless the microscope is moved or transported frequently.
 
(N) Iris diaphragm: This structure controls the amount of light that reaches the specimen. Opening and closing the iris diaphragm adjusts the diameter of the light beam.
 
(O) Coarse and fine focus adjustment knobs: These knobs bring the object into focus by raising and lowering the stage. Care should be taken when adjusting the stage height. When a higher power objective is in place (100× objective for example), there is a risk of raising the stage and slide and hitting the objective lens. This can break the slide and scratch the lens surface. Coarse adjustment is used for finding focus under low power and adjusting the stage height. Fine adjustment is used for more delicate, high power adjustment that would require fine tuning.
 
(P) Illuminator: The illuminator is the light source for the microscope, usually situated in the base. The brightness of the light from the illuminator can be adjusted to suit your preference and the object you are viewing.

KOHLER ILLUMINATION

Published in Microbiology
Tuesday, 25 July 2017 13:43
What is Kohler illumination?

Kohler illumination is a method of adjusting a microscope in order to provide optimal illumination by focusing the light on the specimen. When a microscope is in Kohler, specimens will appear clearer, and in more detail.

Process of setting Kohler
 
Materials required
 
  • Specimen slide (will need tofocus under 10× power)
  • Compound microscope.
 
Kohler illumination
 
  1. Mount the specimen slide onthe stage and focus under 10×.
  2. Close the iris diaphragm completely.
  3. If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
  4. Using the condenser adjustment knobs, raise or lower the condenser until the edges of the field becomes sharp (see Figure 797.1 and Figure 797.2).
  5. Open the iris diaphragm until the entire field is illuminated.
 
Note the blurry edges of the unfocused light
Figure 797.1 Note the blurry edges of the unfocused light
 
Adjusting the condenser height sharpens the edges of the ball of light
Figure 797.2 Adjusting the condenser height sharpens the edges of the “ball of light.”
 
When should you set/check Kohler?
 
  • During regular microscope maintenance
  • After the microscope is moved/transported
  • Whenever you suspect objects do not appear as sharp as they could be.
 
Further Reading:
 

BIOGENESIS THEORY IN MICROBIOLOGY

Published in Molecular Biology
Tuesday, 27 June 2017 00:44

The theory of biogenesis states that living things can only arise from living things and cannot be spontaneously generated. Learn more about this popular microbiology theory to better understand what it means.

The Spontaneous generation hypothesis proposed by scientists to explain the origin of the “animalcules" observed by Antoni van Leeuwenhoek in his magnifying lenses had received wide acceptance all over Europe from Antoni’s time until the time of Louis Pasteur. Erroneous experimental set up, results, and conclusions of some scientists had supported and strengthened the hypothesis.

For example, the Englishman John Needham claimed that vital life is needed for the spontaneous generation of microbes. He added that the reason why no living organisms emerged from heated and sealed solutions in containers is that the “vital life" was destroyed by the heat and new “vital life" was not supplied to the solutions because they cannot enter the sealed containers.

Fortunately, there were scientists skeptical about the hypothesis, so they designed their own experimental set up and from the results they gathered, they drew the most feasible explanation on the origin of the “animalcules".Among the scientists was the Italian Lazzaro Spallanzani who opposed Needham’s idea of the “vital life".

Proponents and opponents of spontaneous generation hypothesis debated a lot starting from the time Leeuwenhoek presented his discoveries (1670s) to the public until the time of Rudolf Virchow, who in 1858 challenged the spontaneous generation with his concept and definition of biogenesis.

This concept claims that living cells can arise only from preexisting living cells. Virchow defended this concept to the scientific community but he did not come up with a convincing experiment to back up his idea. In 1861, the French scientist Louis Pasteur resolved the issue on the origin of microbes (“animalcules") through a series of ingenious and persuasive experiments.

Pasteur showed that microorganisms exist in the air and can contaminate sterile solutions, but he emphasized that air itself does not produce microbes. He filled a number of short-necked flasks with beef broth and then boiled their contents. He immediately sealed the mouths of some of the flasks while he left the others open and allowed to cool.

After few days, the contents of the unsealed flasks were found to be contaminated with microorganisms. No evidences of growing microorganisms were found on the sealed flasks. Pasteur concluded that the microorganisms in the air were responsible for contaminating non-living matter like the broths in John Needham’s flask.

Pasteur performed another experiment but this time he put beef broth in open-ended long-necked flasks. He bent the necks of the flasks into S-shaped curves and boiled the contents of the flasks. Amazingly, the contents of the flasks were not contaminated even after several months.

The unique S-shaped design of Pasteur’s flasks allowed air to pass but trap microorganisms that may contaminate the broths. Do you know that some of the original vessels used by Pasteur in his experiments are still displayed in the Pasteur Institute, Paris today? A few of the flasks contain broths that remain uncontaminated for more than 100 years!

Pasteur demonstrated the presence of microbes in non-living materials whether they are solid, liquid, or air. In addition, he laid the foundation of aseptic techniques, techniques that prevent contamination by unwanted microbes.

These techniques are based on Pasteur’s idea that microbes can be killed by heat and that procedures can be designed to inhibit the access of airborne microbes to nutrient environment. Application of aseptic techniques is now the standard practice in medical and laboratory procedures.

Disproving the idea that microorganisms spontaneously generated from non-living matter through mystical forces is one of the greatest contributions of Pasteur in science. He provided the evidence that any appearance of “spontaneous" life in nonliving solutions can be attributed to microbes that already exist in the air or in the fluids themselves.

Wallach’s Interpretation of Diagnostic Tests, 9th Edition (Interpretation of Diagnostric Tests)

Published in Downloads
Tuesday, 11 April 2017 00:42

Description: Wallach’s Interpretation of Diagnostic Tests, now in its Ninth Edition, has been completely revised and updated by a new author team from the Department of Hospital Laboratories, UMass Memorial Medical Center faculty, who are carrying on the tradition of Jacques Wallach’s teachings. This text serves as a practical guide to the use of laboratory tests which aids physicians in using tests more effectively and efficiently by offering test outcomes, possible meanings, differential diagnosis, and summaries of tests available.

The book has been reorganized into 2 sections. The first section is devoted to an alphabetical listing of laboratory tests while stressing the integration of the clinical laboratory in the clinical decision making process. Test sensitivity, specificity, and positive and negative infectious disease probabilities are included whenever appropriate. Microbiology tests are listed in a separate chapter. The second section is devoted to disease states. Where appropriate, a patient’s chief complaint and/or physical findings are initially presented with subsequent discussions focused on discrete disease states as they relate to a patient’s chief complaint. Current molecular diagnostic testing, cytogenetics, common pitfalls, test limitations, and identification of appropriate tests for specific clinical presentations are also addressed.

Ninth Edition highlights include:
Detailed listing and description of routine and esoteric tests listed alphabetically, with information on when to order and how to interpret the test results based on evidence-based laboratory medicine.
Information on how to work up patients with specific symptoms and the appropriate lab tests to order
Up-to-date test procedures including molecular diagnostic tests
Detailed microbiology chapter of infectious diseases

Page 1 of 7

Useful Sites

  • NCBI

    National Center for Biotechnology Information
  • LTO

    Lab Tests Online® by AACC
  • ASCP

    American Society for Clinical Pathology
  • ASM

    American Society for Microbiology
  • The Medical Library®

    Project of BioScience.pk
Advertisement

Connect With Us

Contact Us

All comments and suggestions about this web site are very welcome and a valuable source of information for us. Thanks!

Tel: +(92) 302 970 8985-6

Email: This email address is being protected from spambots. You need JavaScript enabled to view it.

Website: https://www.bioscience.pk

Our Sponsors

The Physio ClubB2BPakistan.com

By using BioScience.pk you agree to our use of cookies to enhance your experience on this website.