Актуальная Медицина - Laboratory Diagnosis

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Laboratory Diagnosis 
Laboratories are potentially dangerous places to work and it is essential that in planning a laboratory great attention is paid to safety.
Technical staff should be well trained or supervised. They should be aware of the risks of infection in the laboratory and know how to handle the specimens they are examining.
• Protective clothing should be supplied and hand-washing facilities should be available 
• Apparatus used in the laboratory should have written instructions for its proper use and maintenance.
• Provision for disinfection and disposal of specimens by incineration is necessary.
• A procedure for disposing of needles and other "sharps” into a suitable pot should be implemented.
Parasitic organisms are recognized by their size, colour and morphological appearance, often with the assistance of stains and after concentration to increase the numbers of parasite stages present in the case of scanty infections.
In chronic infections, active stages of a parasite may not be easily found and diagnosis may be more appropriately made by serological means.
The parasitological laboratory differs from a microbiology laboratory and certain additional pieces of equipment are essential for differentiation and speciation:
• A microscope, equipped with a binocular head and 10x eyepieces is preferred. The microscope should have a good quality sub stage Abbe condenser with a diaphragm to control the entry of light.
• A calibrated graticule should be available for one of the eyepieces; the graticule can be left in site or inserted as required. It should be calibrated, using a slide micrometer, for each power objective.
• A vortex mixer and electric or battery powered centrifuge capable of receiving 10cm centrifuge tubes and operating at 2.500g is also required for the faecal concentration method.
• Eyepiece graticule: a circular piece of glass on which is etched a like of variable length with 100 divisions.
Calibration:
• Place the calibrated slide on to the stage of the microscope.
• Using the 10x objective initially, bring the calibration line into the focus.
Example:
• If each slide calibration = 10 μm then if 10 slide divisions = 10 graticule divisions, 100 μm = 10graticule divisions and each graticule division will equal 10 μm.
• 40x: if 10 slide divisions = 40 graticule divisions, 100μm = 40 graticule divisions and each graticular division will equal 2.5 μm;
• 100x: if 10 slide division = 100 graticule divisions then 1 graticule division will equal 1μm.
Measurement enables separation of morphologically similar parasites to be made.

Investigation of the Causes of Diarrhoea

Direct examination of feces:
Direct examination of a suspension of faeces in warm saline enables the presence of motile trophozoites of protozoa to be seen. It will also show cysts and helminth ova present in sufficient numbers. Concentration methods are necessary when cysts and ova are present in low numbers.
Preparation of Direct Faecal Smear
Materials: 
• Physiological saline (0,9% sodium chloride)
• Lugol’s iodine.

Method: 
1. Take a microscope slide and label one end with the patient’s name 
2. Place a drop of saline at each end of the slide.
3. Using an applicator stick, select a piece of faecal material approximately the size of a small pea and emulsify the faeces into the two drops of saline on the slide. If the faeces contain any blood or mucus, prepare a separate slide for this. Add a drop of iodine solution to the right hand suspension.
4. Place a 22mm cover slip on to each preparation and view the slide using the 10x and 40x objectives.
Faecal concentration method: Formol-ether concentration method for ova and cysts.
Materials: 
• 10% formalin (100ml formaldehyde+900 ml distilled water; 
• Ether or ethyl acetate;
• 40 mesh (425μm) brass wire filter, 3 inch (7.5cm) in diameter, or a nylon coffee stainer;
• Small 3 inch (7,5 cm) dish or breaker
Investigations are mandatory for causes of diarrhoea, OCP, ova, cysts parasites.

Method:
1. Using applicator sticks, select a quantity of faeces (approximately 1g) to include external and internal portions
2. Place in a centrifuge tube containing 7ml of 10% formalin.
3. Emulsify the faeces in the formalin and filter through the brass/plastic filter.
4. Wash the filter and discard any lumpy residue.
5. Transfer the filtrate to a boiling tube; add 3ml of the ether and mix well on a vortex mixer for 15 seconds or by hand for one minute.
6. Transfer back to the centrifuge tube and centrifuge at 3000 r. p. m. for one minute.
7. Loosen the fatty plug with an orange stick and pour the supernatant away by quickly inverting the tube.
8. Allow the fluid on the side of the tube to drain on to the deposit; mix well and transfer a drop to a slide for examination under a cover slip.
9. Use the 10x and 40x objectives to examine the whole of the deposit for ova and cysts
Saturation Salt Flotation Method:
1. Boil coarse granular NaCl in excess in water to produce a saturated salt solution.
2. Half fill a wide-mouthed flat-bottomed container with the saturated salt solution.
3. Emulsify 1g of faeces in the solution and remove the larger debris from the surface.
4. Fill the container to the top with saturated salt solution until a convex meniscus forms.
5. Lay a glass slide or cover slip over the top, making sure that no bubbles are trapped. 
6. Leave for 20 minutes before quickly inverting the slide.
7. Cover with a cover slip or slide and scan for ova using the 10x objective.
This is a cheap preparation using simple apparatus. It concentrates nematode ova well but it does not concentrate cysts.

The String Test "Enterotest”:
The procedure utilizes a length of thread in a weighted gelatine capsule. The patient lies on the right side so that the capsule will travel to the duodenum. The string will be stained yellow with bile and mucus where it has been in the duodenum. If no part of the string is yellow the test should be repeated. The string is placed in a pot and covered with 5 ml of saline.

Method:
• Agitate the string and saline, preferably using a whirl mixer, to remove jejunal mucus from the string.
• Wind the string around an applicator stick, pressing it against the side of the container to remove jejunal mucus and excess saline, and then discard it.
• Centrifuge the saline at 2000 r. p. m. for 2 minutes.
• Discard the supernatant and transfer the deposit to a microscope slide and examine using the 10x and 40x objectives for trophozoites of Giardia and active larvae of Strongyloides. Ova from trematodes of the biliary tract may also be found.

Straining Methods for Intestinal Protozoa:
Temporary stains for wet preparations following concentration by the formol-ether method.
Lugol’s iodine solution: 
Reagent 1
• Potassium iodide 20g. 
• Iodine 10g.
• Distilled water 100ml.
Add potassium iodide to the distilled water; when dissolved, add the iodine crystals store in a brown bottle. It remains stable for many weeks.
Reagent 2 
It can be 25% glacial acetic acid (Mix equal parts of reagents 1 and 2 for use) results. Iodine stains glycogen brown and the nuclear chromatin of amoebic cysts brown /black.
Burrows stain:
• Thionin -20 mg.
• Acetic acid -3ml. 
• Ethanol-3ml.
• Distilled water -94ml.
Add an equal volume of the stain to the faecal concentrate and allow standing for 12-18 hours. Results: Chromidial bars stain deep blue.

Permanent Stains:
The trichrome method for staining protozoa is especially recommended for identifying features of amoebic cysts and trophozoites.
Solution A: Schaudinn’s fixative
• 95% Ethanol 33.3 ml.;
• Mercuric chloride 66.6 ml. (saturated aqueous solution approximately 14g./100ml.;
• Just prior to use, add 5 ml glacial acetic acid /100ml. of solution.

Horen’s fixative (alternative to Schaudinn’s fixative)
• Cupric sulphade 2% - 100ml.
• Ethanol - 50ml.
• Glacial acetic acid - 7, 5 ml.
Mix all ingredients together. Fix prepared slides for 30 min before they are completely dry. This fixative does not use the potentially harmful mercuric chloride.
Solution B: 
Iodine alcohol
• To prepare stock solution add enough iodine crystals to 70% ethanol to make a dark concentrated solution.
• To prepare working solution dilute stock solution by adding 70% ethanol. The exact concentration is unimportant.
Solution C: 
Acid ethanol
• Glacial acetic acid - 0, 5 ml.
• 90% Ethanol - 100ml.
Method:
• Make a thin smear of the faecal material on a glass slide.
• While the smear is still wet, immediately place the slide in a Coplin jar containing Schaudinn’s fixative (solution A) for 5 minutes at 50˚C (or 1 hour at room temperature).
Results:
Nuclei, chromidial bars, chromatin, red cells and bacteria stain red. Cytoplasm stains blue-green. Background and yeasts stain green.

Investigation of Blood/ Tissue Parasites
• Palsmodium spp.
• Trypanosoma spp.
• Spirochetes
• Babesia spp.
• Leishmania complex
Blood parasites can be identified from peripheral blood, bone marrow aspirate, splenic aspirate, gland aspirate or cerebrospinal fluid or from biopsy material.
Procedures:
• Direct staining of blood or impression smears;
• Culture of appropriate sample;
• Concentration methods; 
• Detecting by using DNA recognition methods.

Staining Blood Films
Giemsa stain is a Romanovsky stain and will stain chromatin material red and cytoplasm blue. Inclusion bodies within the cell will stain red or blue according to their origin.
Materials:
• Giemsa stain; a good quality stain is necessary
• Solvent methanol
• Buffered water pH 6.8 and pH 7.2; tablets are available for the preparation of 1-litre volumes.
Allow smears prepared from blood, aspirates or CSF deposits to dry thoroughly before staining.
• Flood the smear with solvent methanol and allow to fix for 1 minute
• Tip off the alcohol
• Prepare a 1 in 10 dilution of Giemsa stain using 5 drops of stain and 45 drops of buffered water (pH 7.2 for malaria, pH 6.8 for the other blood parasites)
• Flood the fixed slide with diluted stain and allow staining for 30 minutes.
Results:
 Parasite chromatin stains red, cytoplasm stains blue, cellular inclusions red. Red cell nuclear remnants (Howell-Jolly bodies) stain deep blue.
Parasites that are scanty can be concentrated before staining by several methods:
• The thick blood film is suitable for concentrating malaria parasites, trypomastigotes and spirochetes.
• "Buffy coat” (malaria parasites, trypomastigotes, spirochetes)
• Mini anion exchange column: (trypomastigotes)
Thick Blood Film:
Using an applicator stick or directly from a finger prick, place 2 or 3 drops of blood on to one end of a slide. Spread the blood over an area of 1 cm² and allow drying thoroughly.
Field’s satin consists of the two components of the Romanovsky stains in separate solutions.
It is a useful method for the concentration of malaria parasites, Trypanosoma and Borrelia spp.
Materials: 
• Field’s stain A solution (purchased as a prepared satin or prepared as 2,5g % in distilled or filtered water from the powder form)
• Field’s satin B solution (prepared as for Field’s stain A)
Method:
1. Dip the unfixed, dried, thick blood film in Field’s stain A for 3 seconds.
2. Carefully rinse the slide in tap water for 3 seconds.
3. Dip the slide in Field’s stain B for 3 seconds
4. Rinse the slide in water again and then stand vertically to dry.
Results: 
Parasite chromatin will stain red and cytoplasm blue; inclusion dots, if seen, stain red.
The Buffy coat will contain malaria parasites, spirochetes and trypanosomes and may demonstrate parasites when none can be seen in the thick or thin film.
Mini Anion Exchange Column Technique is a useful concentration technique where other investigations for trypanosomes in the blood have proved negative.
Blood is passed through a column of cellulose. Erythrocytes are retained in the column and trypanosomes pass out into a collecting tube.

Laboratory Diagnosis of the Filaria Parasites
Filariasis is diagnosed either serologically or by finding the L3 larvae, or microfilaria, in peripheral blood, urine, hydrocele fluid or skin snips.
Occasionally adult worms can be removed as they cross the eye (Loa-Loa) or from a subcutaneous nodule (Onchocerca volvulus)
Because of the periodic appearance of microfilaria, the peripheral blood sample is collected between 10.00 and 14.00 (day blood) and between 22.00 and 02.00 (night blood). An early morning sample of urine is most suitable.
Urine may show a milky appearance, called chyluria, if filariasis is present.

Modification of Knott’s method for examining blood for microfilaria:
• Collect 20 ml. of blood into sodium citrate anticoagulant, as described above
• Add the blood to an equal volume of 1% saponin in sline (or 2% formalin if saponin is not available)
• Mix well and allow standing for 15 minutes before transferring to centrifuge tubes and centrifuging for 20 minutes at 2000 r. p. m.
• Pour off the supernatant into a container of disinfectant and mix the deposit well before transferring a drop to a slide, covering with a cover slip, and scanning for microfilaria. The saponin preparation will show actively moving larvae; those in the formalin preparation will not be moving.
Urine and hydrocele fluid can be filtrated in a similar manner to blood to show any microfilaria present. The supernatant discarded and a drop of the deposit transferred to a slide.
Reagents:
• Giemsa stain
• Delafield’s haematoxylin 
If sufficient microfilarias are present, films prepared directly from peripheral blood can be used to stain them. If not, the blood can be prepared as a thick film or microfilaria can be washed from the membrane filter by placing it into a small pot of saline. Giemsa stain will stain the nuclei of the microfilaria and the haematoxylin will stain the sheath, if present.
Filarial worms of Onchocerca volvulus live in subcutaneous tissue nodules, and discharge their microfilaria into the tissue. They are identified by examining small pieces of skin taken from various parts of the body.
The leishmanial organisms are a broad complex of species responsible for a wide variety of clinical responses. Culture is an important diagnostic aid; culture media are varied but NNN media, a rabbit blood-agar base using a salt-based overlay and a liquid tissue culture medium. Cultures are incubated at 20ºC for up to 28 days. On examination they will show a conversion from amastigote stage to motile promastigote stage.
Detection of specific antibodies is only really useful in visceral leishmaniasis when high titres of antibody can be detected using the direct agglutination test. 

Cutaneous Leishmaniasis
• Slit-skin smears from an ulcer edge. The edge of the ulcer is compressed to provide a blood-free area, then, using a fine point scalpel blade, a slit is made into the subcutaneous tissue and the base is gently scraped. The tissue juice and cells are transferred to a slide and smeared over an area of 1 cm².
• Needle aspiration using a small syringe to inject saline around the lesion, and then re-aspiration to prepare slides and cultures, can also be used.
• Biopsies of the ulcer edge are taken and divided into two parts. One part is fixed in buffered formalin for histopathology. The second piece is aseptically cut into small pieces for culture and to make impression smears. Impression smears are made by dabbing the tissue several times on to a slide to deposit the cells. These are then dried and fixed in methyl alcohol before staining with Giemsa stain.

Visceral Leishmaniasis
• Bone marrow, splenic aspirate and lymph node aspiration are the tissues used to demonstrate Leishmania spp. in this clinical form. Peripheral blood Buffy coat may yield parasites in the immunocompromised host.
• Smears are made and stained with Giemsa stain.
• Cultures are also made into NNN and Schneider’s media.
Identification of specific species of Leishmania can be made using two principal techniques: 
• Zymodeme: using the isoenzyme pattern
• Polumerase chain reaction (PCR)

Examination of Cerebrospinal Fluid (CSF)
CSF can be used to search for parasites causing cerebral malaria or trypanosomiasis or to demonstrate bacteria causing meningitis.
One sample is sent for bacterial culture. The second is used for:
a) Cell count;
b) Examination of stained deposit after centrifugation; 
c) Biochemical tests in the supernatant.
Stain one slide with Giemsa stain in order to differentiate any blood cells present. Stain the second with Gram stain to detect any micro-organisms present. Stain the third with Ziehi-Neelsen stain for Mycobacterium tuberculosis.
• Normally the CSF has no more than five blood cells per micro litre and these are usually lymphocytes.
• Increased white blood cells are seen in bacterial infections. Pyogenic meningitis will give an increase in polymorph nuclear cells, and tubercular meningitis gives an increase in lymphocytes. Red blood cells are not a normal finding and indicate an accidental tonumatic tap or bleeding into the sub arachnoid’s space.
Grams stain materials:
• Gram stain: 1g. gentian or crystal violet dissolves in 100 ml. distilled water; 
• Lugol’s iodine – Reagent1:
  Potassium iodine (-20g).
  Iodide (-10g).
  Distilled water (-100ml).
Add potassium iodide to the distilled water; when dissolved add the iodine crystals. Store it in a brown bottle.
• Acetone or methylated spirit
• Neutral red: 0, 5 g. neutral red dissolved in 100ml. distilled water. 
Results:
• Pneumococci – Gram-positive cocci in pairs are seen more commonly;
• Haemophilus influenzae – small slender Gram-negative bacilli;
• Meningococci – Gram-negative intracellular diplococci seen inside the polymorphonuclear cells
• Crystococcus neoformans may be visualized.

Ziehi-Neelsen Stain
Materials:
• Carbol fuchsin stain;
• 1% Acid alcohol (1ml. concentrated hydrochloric acid) 99ml. methylated spirit.
• 0, 5% Malachite green (0,5g. malachite green dissolved in 100ml. distilled water. 
Results: Mycobacterium is acid-fast bacilli; and will stain as red bacilli against a green background.

Examination of Sputum
Sputum is commonly examined for parasites of the respiratory tract and for bacteria causing pulmonary infections such as tuberculosis or pneumonia. Sputum is usually described by its appearance as:
• Salivary-frothy, white and watery.
• Purulent-thicker consistency, often with a greenish color.
• Mucopurulent-thick, sticky consistency, containing pus, may be blood-stained.

Examination for Parasites
 
Paragonimus spp. Discharge ova with the sputum and usually cause "rusty”, blood-stained sputum.
Ova can be recovered after dissolving mucus in the sputum by mixing a portion with an equal portion of 10% potassium hydroxide.
The deposit is examined for ova using the 10x objective.
The sputum can also be concentrated, after dissolving the mucus, using the formol-ether method.
Pneumocystis Carini:
Demonstration of this parasitic organism of the immunocompromised host requires specialized laboratory techniques. In sputum the method used is a fluorescent monoclonal antibody stain on a specially prepared concentrate.

Examination for Bacteria 
Two thin smears of sputum are made using a wire loop that can be sterilized in a flame afterwards, or an applicator stick that can be burned.
The smears can then be fixed when dry by passing the back of the slide through a flame twice.
• One stained for acid-fast bacilli using the Ziehi-Neelson method.
• The second smear is stained for other bacteria using the Gram stain.
Sputum should be sent for routine culture if available, and for culture for Mycrobacterium tuberculosis if required.

Hematological Assessment
Hematological values are necessary in the diagnosis of infection or anemia. The most useful criteria include:
• Total white cell count;
• Differential white blood cell count; 
• Hemoglobin; 
• Haematocrit;
• Mean corpuscular hemoglobin;
• Platelet count;
• Examination of the blood picture.

Observations:
Red blood cells:
• The cell may be enlarged (macrocyte), or appear smaller (microcyte).
• An enlarged area of central pallor (hypochromia) indicates an iron deficiency.
• Target cells or sickle cells may indicate an abnormal hemoglobin type. 
• Basophilic stippling and nucleated cells or spherocytes may indicate a hemolytic process.
• Note any intracellular or extra cellular parasites which may be present (malaria, Trypanosome, Babesia, Borrelia, micrafilariae)
White blood cells:
• An average of 1-2 cells in each field is normally seen.
• A differential count will indicate the types of cells present.
• Note the morphological appearance of the cells.
• Neutrophils may show a shift to the left or to the right.
• Mononuclear cells may be "atypical”.

Platelets:
Those are seen in every field, either singly or in small clumps.
A decrease in platelets is noted when they are scanty, seen in every 3-5 fields only.


Vocabulary
Specimens [`spesǐmǐnz] Зразки
Disposal [dǐs`pəυzl] Розпорядження, видалення
Be implemented [bi:ǐmplǐ`mentǐd] Бути втіленим у життя
Pieces [pi:sǐz] Частини
Graticule [`grætǐkјυl] Поділ
Ova [`əυvə] Яйця
To emulsify [ǐ`mјυlsǐ`fa ǐ] Емульсувати
Lumpy residue [`lǎmpǐ`rǐzǐdјυ] Грудкоподібні залишки
Bubbles [`bǎblz] Кульки
Convex [`konvǐks] Випуклий, вигнутий
Rusty [`rǎstǐ] Іржавілий
To dilute [dǐ`lu:t] Розводити, розбавляти
Remnants [`remnənts] Залишки
Scanty [`skæntǐ] Рідкісний
Snips [`snǐps] Обрізки
Subcutaneous [səbkјυ`tænǐəs] Підшкірний
Nodule [`no:dјυl] Вузол
Overlay [əυə`leǐ] Покриття, покривати
Mobile [məυ`ba ǐl ] Рухливий
Iodine [`a ǐədi:n] Йодид, сіль йодистоводневої кислоти
Dissolved [dǐ`zolvd] Розчинений
Flame [`fleǐm] Полум’я, запалення

Questions
1. What are the essentials of laboratory diagnosis in the tropics?
2. How can we investigate the causes of diarrhoea in the developing countries?
3. What do you know about temporary and the permanent stains in the diagnosis of infections in the tropics?
4. What are the procedures of blood investigation and tissue parasites?
5. How can we diagnose in laboratory common filarial parasites?
6. What do you know about coetaneous and visceral leishmaniasis?
7. How can organize the examination of cerebrospinal fluid?
8. What are the essentials of sputum examination, examination for bacteria in the tropical environment?
9. What are the main laboratory observations in case of hematological assessment?




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