SYLLABUS BASED NOTES FOR THE POST OF JUNIOR LABORATORY ASSISTANT IN MEDICAL EDUCATION PART 1 CLINICAL PATHOLOGY (Module lll)

SYLLABUS BASED NOTES FOR THE POST OF JUNIOR LABORATORY ASSISTANT IN MEDICAL EDUCATION PART 1 CLINICAL PATHOLOGY (Module lll)

 MODULE III- (15 Marks )

CLINICAL PATHOLOGY - 5 Marks 

✅Analysis of Urine, 
✅Sputum,
✅ Stool, Semen, CSF -Types of sample, collection of sample, Physical, Chemical & Microscopic examination techniques & their Importance BIOCHEMISTRY Blood Glucose estimation Types of samples: FBS, PPBS,RBS Methods of estimation - enzymatic method and glucometer (dry chemistry)

Introduction 

Clinical pathology is the diagnosis of disease based on the laboratory analysis of body fluids. Blood, urine, stool, sputum, cerebrospinal fluid, semen, etc are specimens commonly submitted for analysis in a clinical pathology laboratory. Physical, Chemical, Serological and Microscopical examination of specimens are carried out in the laboratory 

General guidelines for sample collection 

Collect specimens in proper container ie wide mouthed, spill free, disposable containers Label the specimen such as name of patient, type of specimen, ID no., Date and Time of collection etc. Follow proper protocol for transportation of specimens Keep the samples ready for performing analysis. 

Urine Analysis 

Urine is the most common specimen examined in clinical pathology laboratory. Urine is formed by the kidneys. The body removes water and many harmful waste substances from the body through urine. This is a normal physiological process of the body

Urine Analysis

Urine analysis provides mainly diagnostic and prognostic information on conditions like kidney diseases, liver diseases, metabolic disorders, urinary tract infections and parasitic infestations. In addition pregnancy, endocrine disorders and drug overdose are also investigated with a specimen of urine. Simplicity in obtaining the specimen and lesser cost of test makes urine analysis an popular tool in laboratory investigations
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Types of urine specimen 


1.Random specimen 

2. 24 hour specimen. . 

3.Early morning specimen 

Methods of collection 

Random specimen is a sample collected without any priority to the time or diet and is collected by the patient in a wide mouth dry container, after cleaning the external genitalia with mild soap and water. Urine specimen from catheter can be obtained in necessary conditions.
 Urine collection bags are used in case of infants and in geriatrics. A clean, early morning, fasting specimen is generally the most concentrated specimen and is preferred for qualitative, quantitative and microscopic examination for the detection of abnormal constituents


24 hour urine specimen collection is done by collecting urine in a special container, 2.5 to 5 litre capacity with added preservative. Preferably morning 8 am to next day 8 am over a period of 24 hr. It helps to diagnose kidney problems. It is often collected to do tests like clearance tests and to measure protein, hormones, minerals, and other chemical compounds. The preservatives are added to prevent changes in the urine (pH, odour, bacterial growth etc) that may happen on standing longer periods. Preservatives commonly used are toluene, formaldehyde, chloroform,Boric acid,Hydrochloric acid .

Physical examination of urine 
All routine urine analysis should begin with a physical examination of the urine sample. This examination includes assessment of appearance (colour and transparency), specific gravity, volume, reaction and odour. For best results of the physical examination of urine the specimen should be evaluated immediately after collection. 

Colour 
Normally the urine is straw coloured (due to urochrome). Colour depends on the concentration of urine and varies with the presence of constituents. Normal colour of urine changes in different disease conditions or in persons taking medications. 
Deep Yellow : Mild to severe dehydration, Jaundice, B complex therapy 

Red to brown: Haematuria, haemoglobinuria, myoglobinuria, porphyria

Brown to black: Alkaptonuna, methaemoglobinuria 

Milky: due to the presence of lymph (Chyluria)


The colour of urine is usually described after visual inspection with common colour terms. 


Transparency

 Freshly voided urine is clear and transparent. It may become turbid if exposed for long time due to the urea being acted upon by bacteria or the presence of proteins, pus cells, blood, bacteria, urates, phosphates. 
Specific gravity 
It implies the capacity of kidney to concentrate urine. Normal specific gravity of urine is 1.010-1.025 

Values more than 1.025 indicate severe dehydration, DM (diabetes mellitus), Adrenal insufficiency. 

Values less than 1.010 indicates increased water output as in Diabetes insipidus.

 A low fixed specific gravity usually found is chronic renal failure.
 It is measured by urinometer or refractometer

Volume

Normal urinary output ranges between 1000-1500 ml/day. It depends upon fluid intake, solute load, loss of fluid by skin or otherwise, climatic condition etc 

A urine out put more than 3 litre/day is called Polyuria and is seen in- Diabetes Mellitus, Diabetes Insipidus, Recovery from acute renal failure (ARF), Diuretic therapy etc. 
A urine output Less than 500 ml/day is Oliguria and is seen in Acute renal failure, Vomiting, Fever, Burns. Anuria is the suppression of urine output (generally less than 50 ml per 12 hours is considered as anuria)
Urine volumes can be measured usualy with a volumetric cylinder 


Reaction (pH) 

Normal fresh urine is acidic approximately 6. Depending on the person's acid-base status, the pH of urine may range from 4.5 to 8. 
A Urine pH 8.5 or more may be found after heavy meals, Urinary tract obstruction, Chronic renal failure A pH 4.5 or less may be seen after heavy exercise, Metabolic Acidosis, Chronic Respiratory, Acidosis, Uncontrolled diabetes 
Extremely acidic or alkaline urine usually indicates a poorly collected specimen

Litmus Paper, Reagent strip testing(pH paper), are commonly employed for measurement of pH, 


Odour 

Freshly voided urine is slightly aromatic. Urine becomes more ammoniacal due to bacterial activity. A fruity odour is felt in case of severe diabetes due to presence of ketone bodies. A putrid or foul smell is felt in Urinary tract infections


Chemical Examination of Urine 


Chemical examination of urine is done to detect presence or absence of certain constituents which reflects disease state of body. The parameters include Protein, Sugar, Ketone bodies, Blood, Bile pigments, Bile salts and Urobilinogen. Conventional test methods are commonly employed.Reagent test strips (dip sticks) are commercially available and recently used.

 1. Proteins 

Normally very small amount of protein (100 mg/day) is excreted and is not detectable by routine qualitative test methods. Proteinuria is the term used for increased protein excretion in urine. Commonly found protein in urine is albumin so it is often termed as albuminuria. Proteinuria is seen in kidney diseases such as Nephrotic Syndrome, Multiple myeloma, chemical poisons etc


Methods of detection 

The methods are based on the principle of precipitation of protein by chemical agents or coagulation by heat 
  • Heat and Acetic acid method 
  • Sulphosalicylic acid method 
  • Reagent test strips (dip sticks) like albustix 

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2. Sugar 

Glucose is the predominant sugar and is normally absent in urine. The renal threshold for glucose is 180 mg%. If the blood sugar rises above renal threshold level, the glucose will appear in the urine and is called glycosuria
Major cause of Glycosuria isDiabetes mellitus. Transient glycosuria is seen in pregnancy, stress etc. 

Methods of detection


Benedicts test--When Benedicts qualitative reagent is heated with urine, glucose present in urine reduces cupric ions present in the reagent to cuprous ions. Alkaline medium is provided to the reaction by sodium
carbonate present in reagent. The color changes to green, yellow, orange or red according to the concentration of glucose in urine. Reagent test strips (dip sticks) like glucostix. 


3. Ketone Bodies 

Ketone bodies is a term used to describe three discrete but metabolically related chemicals-Acetone, Acetoacetic acid and ẞ-hydroxybutyric acid.Ketone bodies are spilled into urine and the presence of Ketone body in urine is called ketonuria. Ketone bodies are detected in conditions such as diabetic ketoacidosis and starvation. 

Methods of detection

•Rothera mest (Ketone bodies form purple coloured complexes with so- dium nitroprusside in alkaline medium) . 
•Reagent test strips (dip sticks) like ketostix A fresh urine specimen is always prefered for ketone body estimation. 


4. Blood 

Presence of blood in urine is called Haematuria, seen in cases of Nephrolithiasis (urinary calculi) and in malignancy. A microscopic exmination of urine helps to confirm the presence of intact erythrocytes. Healthy individuals normally will have no detectable blood in their

Methods of detection 


Benzidine test - It is the test for occult blood. It is highly sensitive but as benzidine is considered as a potent carcinogen, this test is no longer used now. Reagent test strips (Haem occult test) 

6. Bile Pigments


These are breakdown products of haemoglobin excreted in bile. The two most important bile pigments are bilirubin, which is orange or yellow, and its oxidized form biliverdin, which is green. Normally bile pigments are absent in urine. Presence of bile pigments in the urine indicates liver dysfunction such as obstructive jaundice. 
Fresh urine sample should be used for bilirubin determination because exposure of urine to light may cause loss of bile pigment by oxidation and leads to negative result


Methods of detection 

Fouchet's test. (Bilirubin is treated with ferric chloride in Tri chloro acetic acid to give greenish blue colour) Reagent test strips (dip sticks) 


7. Bile Salts 

Bile salts are made in the liver from cholesterol and these help in the fats absorption. Sodium taurocholate and sodium glycocholate are the major bile salts. Normally bile salts are negative in the urine. 
The presence of bile salt in the urine indicates disease like Obstructive Jaundice Hay's test. (Bile salts when present, lower the surface tension of urine. 
When sulphur powder is added to the urine, sulphur particles sink to the bottom of the tube indicates postive result.) 


8. Urobilinogen 

Majority of Bile pigment derived from breakdown of hemoglobin is excreted in the stool, but small amounts are reabsorbed into the blood from the intestines and then excreted into the urine.
 Urobilinogen is formed in the intestines by bacterial action on bilirubin. Urobilinogen is normally present in urine in low concentrations. 
A fresh specimen is essential for the detection of urobilinogen, as it is a light-sensitive compound. Positive test results help to detect liver diseases such as hepatitis, cirrhosis and haemolytic anaemia 


Methods of detection

Ehrlich's Test (Urobilinogen forms a cherry red complex with para dim- ethyl amino benzaldehyde in normal urine. On further dilution of sample (>1:20) a positive result is significant.)

Microscopic examination of Urine 


Microscopic Examination of urine sediments provides a direct sampling of urinary tract morphology, which helps the diagnosis very much. The specimen used for microscopic examination should be as fresh as possible. Red cells and many formed solids tend to disintegrate upon standing, particularly if the specimen is warm or alkaline.Concentrated first morning, mid-stream, clean catch urine specimen, is preferred for microscopy. Urine is collected without faecal & vaginal contamination


The urine sediments assessed under microscope can be of two types 

Organized - Leukocyte (Pus cells), Erythrocytes, Casts, Epithelial cells, Bacteria, Parasites and fungi

Unorganized - Crystals and amorphous sediments. 


Pus cells - They are round or oval in shape and present normally, 0 to 3 leukocytes per high-power field will be seen on microscopic examination. The pus cells are leucocytes. More than 5 cells per high-power field probably indicate urinary tract infection. 


Erythrocytes - Red cells are not usually present in normal urine and appear as refractile bodies, seen in acute glomerulo nephritis, stones in urinary tract and malignancy etc. Estimate their number per high-power field and report it. 


Epithelial cells - Normal urine shows a few epithelial cells. Marked increase in the number of epithelial cells signifies some pathological conditions of the site of their origin 


Bacteria - Presence of numerous bacteria in the centrifuged deposit of freshly voided urine signifies infection in the urinary tract. 


Casts-These are formed by coagulation of albaminous material in the kidney tubules. Casts are cylindrical and vary in diameter. The sides are parallel, and the ends are usually rounded. Casts in the urine always indicate some form of kidney disorder and should always be reported There are different types of casts. They are Hyaline casts, Red cell casts, Granular casts, Epithelial casts, Waxy casts and Fatty casts
Crystals Crystals of various substances can be seen according to the reactions (pH) of urine. 

Crystals found in normal acid urine- 

 Amorphous urates- yellow and granular precipitate Uric Acid Crystals appear in several forms, Multi colored when polarized most commonly Diamond shaped Calcium Oxalate Crystals is most frequently observed in urine and octahedryf in shapes, often referred to as an 'envelope' shape 


Crystals found in Alkaline urine 

 Amorphous phosphates-fine granular precipitates Calcium phosphate-stillete prism


Calcium carbonates-colourless spheres or dumbbell shaped Triple Phosphate Crystals are Colorless, 4-6 sided prisms Referred to as 'coffin lid crystals 


Crystal found in abnormal urine Cystine - colourless, retractile, hexagonal Tyrosine - Fine needle in clumps, yellow and silky Leucine-yellow oily spheres Sulphonamide - yellow brown striated sheaves or round with radial striations


Urine Pregnancy Test 


Most chemical tests for pregnancy look for the presence of the beta subunit of hCG or human chorionic gonadotropin, in the blood or urine. hCG can be detected in urine or blood after implantation, which occur six to twelve days after fertilization


The common test done for detection of hCG in Urine is Card Test. It qualitatively detects the presence of hCG It is a single step immunoassay and has high sensitivity. Paper strips coated with monoclonal anti-hCG antibody incorporated in a disposable card is used for the test. The urine is introduced in the sample window. Appearance of coloured bands in the control and test areas denote a positive test. 


Urine Analysis 

Automated machines are now available in the clinical laboratory to perform urine analysis called 'Urine Analyzer'. Using urine strip readers, the unit can detect and quantify a number of analytes including bilirubin, protien, glucose, red blood cells etc. The instrument works on the principle of Reflectance photometry and can process several hundred strips per hour. Different types of urine analysers include Cobas 6500, UF-1000-SIEMENS CLINITEK ANALYSER etc

Sputum examination


Sputum is mucus that is coughed up from the lower airways of the respiratory tract i.e lungs, trachea and bronchi. Saliva and nasopharyngeal secretions are not part of the sputum. 
Sputum samples are used for microbiological investigations of respiratory tract infections and cytological investigations of cancer. Purulent sputum contains pus, composed of white blood cells, cellular debris, dead tissue, serous fluid, and viscous liquid (mucus). 


Collection: 

An early morning sample obtained by deep cough is a preferred sample for analysis, For the diagnosis of Tuberculosis, sample may be collected on three consecutive days. Wide mouthed disposable containers are preferred for the collection of sputum sample. The patient should take a deep breath, and empty his lungs in one breath and at the same time cough as hard and deeply as he can. Whatever he brings up by coughing, should be spit into the container and collected


Physical Examination 

It involves measurement of colour and consistency or appearance. Normal sputum is pale yellow in colour and contains mucoid materials. It may be red in Pulmonary Tuberculsis due to the presence of blood (Haemoptysis), dark yellow in bronchitis and brown in pneumonia. Normal sputum is viscous due to the presence of mucus, purulent in cases of bronchitis or acute upper respiratory tract infection, frothy in pulmonary oedema. 


Microscopic examination 


It involves mainly the examination of unstained and stained smears. For preparing sputum smears for microscopic examination, the purulent portion of the sample must be included. While handling utmost care should be taken in suspected cases of TB. Examination of Ziehl Neelson stained smears are most routinely used method in the diagnosis of Pulmonary Tuberculosis 


Grams stained smears are commonly used for the detection of bacteria causing respiratory infections such as Pneumococci, Haemophilus and staphylococcus. Stained and unstained smears are used to detect the presence of puscells, epithelial cells, RBCs, malignant cells, Curshmann spirals, Charcot leydon crystals etc

Stool Analysis 


Stool is an important specimen for the diagnosis of diseases of gastrointestinal tract such as diarrhoea, dysentry, parasitic infection, gastrointestinal bleeding, peptic ulcer, carcinoma and malabsoption syndromes. 

Tests which reveal the presence of blood denotes an ulcerative lesion somewhere in the gastro intestinal tract, either inflammatory or as a result of cancer. Examination of stool shows the presence of parasites such as Entamoeba histolytica, various types of tape worms, pin worms, round worm and its egg. 

Collection of Specimen: 

A portion of fresh specimen is collected in a clean wide mouthed container with a spatula. Areas containing mucus, blood and pus should be incorporated in the specimen. 
Analysis includes Physical, Chemical and microscopic examinations


Physical examination


Macroscopic examination of the stool involves consistency, colour, blood odour.pH etc. The presence of adult worms in a freshly passed stool (adult stages of Ascaris lumbricoides and Enterobius vermicularis, Proglottids of Taenia species) is also reported. 


Chemical examination


 It includes the tests for occult blood and reducing substances
Blood may be present hidden in the stool called occult blood and is seen in malignancies, ulcers, haemorrhoids etc. Occult blood in stool were detected by Benzidine based tests, and is now not used due to the carcinogenicity of benzidine. It has been replaced by strip tests. 


The presence of reducing substances is usually done in children suspecting lactose intolerance. The unabsorbed sugars in stool are measured as reducing substances. Benedict's test is commonly employed for detecting reducing substances


Microscopic examination 

Microscopic examination of faeces can be done by saline preparation and iodine preparation. It helps detection of identification of cells, crystals, protozoa, ova of intestinal parasites 


Direct saline wet mount 


a. Place a drop of saline on the slide.

b. Pick up a small amount of faecal material on the end of an applicator stick 

c. Emulsify in the saline and cover with a cover slip. Examine on low and high power. The smear should be thin enough to read a printed page through it. 

d .The entire preparation must be examined for the presence of eggs, larvae and protozoa


Iodine wet mount 


Iodine preparation is useful for the identification and differentiation of protozoal eggs and cysts 

a. Place a drop of Lugol's iodine solution on a slide. 

b. Pick up a small amount of fecal material on an applicator stick 

c. Emulsify in the iodine solution and cover with a coverslip. 

d. Examine on low and high power as described in the previous procedure
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