USING 10% KOH FOR FUNGAL DETECTION

If the specimen is skin, hair or nail, the background cellular material may mask fungal elements. Potassium hydroxide will dissolve the keratin in these specimens, thus making any fungi more visible.

If there are a lot of cellular material in sputum or vaginal secretions, KOH mount may be prepared to dissolve the background thus making any yeast more visible.

However a saline mount must be additionally done to quantitate epithelial cells and WBCs and note presence of Trichomonas.

How to prepare 10% KOH

Potassium hydroxide……….…………10 g
Glycerol…………………………….20 mls
Distilled water………………………80 mls

Dissolve KOH in water, and then add glycerol. Glycerol prevents crystallization of the reagent and allows KOH preparations to be maintained for two days before drying up.

The procedure of using 10% KOH


1. Place specimen on clean grease free slide.

2. Add a drop of 10% KOH.

3. Apply coverslip and gently warm the slide over a flame.

4. Gently press the coverslip to spread the already softened tissue evenly on the slide.

5. Examine under a microscope X10 then X40.

6. Record the findings

Potassium hydroxide is a highly corrosive deliquescent chemical, therefore handle it with great care and make sure the stock bottle of chemical is tightly stoppered after use’.

THROMBOCYTOSIS

Occurs when platelet count rises above the normal range of 150–400 10^9 per litre of blood

What causes Thrombocytosis


● Chronic myeloproliferative diseases, e.g. essential thrombocythemia, polycythaemia vera, chronic myeloid leukaemia, myelofibrosis.
● Carcinoma (disseminated)
● Chronic inflammatory disease, e.g. tuberculosis
● Haemorrhage
● Sickle cell disease associated with a nonfunctioning spleen or after splenectomy.
● Iron deficiency anaemia, associated with active bleeding.

URIC ACID MEASUREMENT IN BLOOD

It is tested to evaluate renal failure, gout and leukemia

What are the normal ranges for Uric acid?

  • Male: 3.9 – 9.0 mg/dl
  • Female:. 2.2 – 7.7 mg/dl

What causes increase in Serum Uric acid

  • Renal failure
  • Gout
  • Leukemia
  • Severe eclampsia
  • Lymphomas

What causes decrease in Serum Uric acid

  • Patients undergoing treatment with uricosuric drugs

MEASUREMENT OF SERUM TRIGLYCERIDES

Tested to evaluate patients with suspected atherosclerosis

What are normal ranges for Serum Triglycerides

  • Below 150mg/dl. Desirable
  • 150 – 199mg/dl. Borderline high
  • >500mg/dl. Very high

What causes increase in Serum Triglycerides

  • Liver disease
  • Nephrotic syndrome
  • Hypothyroidism
  • Poorly controlled diabetes
  • Pancreatitis

What causes decrease in Serum Triglycerides

  • Malnutrition
  • Congenital lipoproteinemia

SERUM PHOSPHORUS TEST

Assists in proper evaluation and interpretation of calcium levels. It is used to detect disorders of endocrine system, bone diseases and kidney dysfunction.

What are the normal ranges of Phosphorus

2.5mg/dl – 4.5mg/dl

What causes high levels of Serum Phosphorus?

  • Renal insufficiency
  • Severe nephritis
  • Hypoparathyroidism
  • Addison’s disease

What causes low levels of serum Phosphorus?

  • Hyperparathyroidism
  • Rickets
  • Osteomalacia
  • Diabetic coma
  • Hyperinsulinism

SOURCES OF ERROR WHEN MEASURING HAEMOGLOBIN PHOTOMETRICALLY

● Not measuring the correct volume of blood due to poor technique or using a wet or chipped pipette.

● When using anticoagulated venous blood, not mixing the sample sufficiently.

● Not ensuring that the optical surfaces of a
cuvette are clean and dry and there are no air bubbles in the solution.

● Not protecting a colorimeter or haemoglobin meter from direct sunlight and not checking the performance of an instrument or maintaining it
as instructed by the manufacturer. A common error when using a filter colorimeter is using a glass filter which is not clean.

● Not checking a diluting fluid such as Drabkin’s for signs of deterioration as explained in the HiCN techniques.

Technique to prevent cuvette-related errors

1. Hold a clean cuvette only by its frosted (matt) or ridged sides. When transferring a solution to a cuvette, allow the fluid to run down the inside wall of the cuvette. This will help to avoid air bubbles in the solution. Do not fill a cuvette more than three quarters full.

2. Using a tissue or soft clean cloth, wipe clean the clear optical surfaces of the cuvette.
Carefully insert the cuvette in the colorimeter or haemoglobin meter (optical surfaces facing the light source).
Note: Ensure a solution is at room temperature before reading its absorbance otherwise condensation will form on the outside of the cuvette which will give an incorrect reading.

COLLECTION OF 24-HOUR URINE SPECIMEN

1. Sanitize your hands and assemble the equipments

2. Greet and identify the patient. Introduce yourself to patient and explain the procedure

Instruct the patient as follows:

3. When you get up the in the morning empty your bladder into the commode just as you normally do In other words, this urine is not to be saved. Make note of the time.

4. The next time you need to urinate, void the urine directly into the plastic container.

5. Tightly screw the lid onto the container, and put into refrigerator or into an ice chest.

6. Repeat steps 4 & 5 each time you urinate.

7. On the next morning, get up exactly the same time ( exactly 24hrs after beginning the test). Void into the container for the last time.

8. Put the lid on the container tightly. Return the urine collection container to the office the same morning you complete the urine collection.

Provide the patient with the collection container and written instructions.

HAEMOGLOBIN MEASUREMENT USING HEMOCUE METER


1. Pull out the microcuvette holder to its loading position.

2. Press and hold down the left button of the meter until the display is activated. the meter automatically carries out a performance check. after 10 seconds the meter will show three flashing dashes, indicating it is ready for use.

3. Fill a microcuvette in one continuous process with capillary blood or well mixed venous blood.

4. Wipe off excess blood from the outside of the microcuvette tip, making sure no blood is withdrawn.

5. Within 10 minutes of filling the microcuvette, measure the haemoglobin. Place the microcuvette in the microcuvette holder and push in the holder to its measuring position.
After 15–60 seconds, the haemoglobin value
will be displayed (it will remain displayed for as long as the holder is left in its measuring position.)

THE EDTA ANTICOAGULATED BLOOD


When EDTA anticoagulated blood cannot be tested within 1–2 hours, it must be refrigerated at 4–8 C to prevent cellular changes affecting test results.
Manual or automated blood cell counts, reticulocyte counts, and PCV change little in EDTA blood at
4–8 C when stored for up to 24 hours. Haemoglobin concentration is stable for 2–3 days at 4–8 C providing there is no haemolysis

Blood films:

In EDTA anticoagulated blood, morphological blood cell changes occur soon after blood is collected when it is stored at room temperature (18–25 C) and within 3 hours when stored at
4–8 C.

It is therefore recommended that blood films be made and methanol-fixed as soon as possible after blood is collected and never made after overnight storage. Some of the blood cell changes which occur in EDTA blood include:

● Neutrophil degeneration with neutrophils
becoming more irregular in shape, nuclear lobes separating, and vacuoles appearing in the cytoplasm. There is also loss of granules.

● Segmentation (budding) of the nucleus of lymphocytes and monocytes and vacuoles appearing in the cytoplasm.

● Erythrocytes becoming crenated and spherocytic.

● Platelets disintegrating.

HANDLING AND STORAGE OF BIOLOGICAL SPECIMEN

BLOOD

Handling

All blood specimen

  • Prevent haemolysis
  • Collect specimen in a tube at room temperature

Serum

  • Separate serum from blood within 30-45 min after collection

Plasma

  • Mix anticoagulant gently but thoroughly with blood specimen immediately after collection

Storage

For blood specimen:

  • Refrigerate at 4°C to retard alterations in physical and chemical composition of specimen

Plasma and serum

  • These can be frozen but whole blood should not be frozen as it may cause Haemolysis

URINE

Handling

Avoid contamination of inside of specimen contamination

Storage

If urine cannot be tested within one hour after collection, refrigerate it or add an appropriate preservative

MICROBIOLOGICAL SPECIMEN

Handling

  • Avoid contamination of swab used to collect specimen
  • Avoid contamination of inside of the specimen container.
  • Protect yourself from contamination from biological specimen
  • Protect anaerobic specimen from exposure to air.

Storage

  • Transport the specimen as soon as possible.
  • If not possible, place the specimen in a transport medium or inoculate in suitable culture media.
  • For most specimen: Place in refrigerator at 4°C to prevent drying, death or overgrowth of specimen with extraneous microorganisms.

STOOL

Handling

  • Collect the specimen in a clean container.
  • For detection of ova and parasites, keep the specimen warm

Storage

  • For most accurate results, deliver specimen to the laboratory immediately
  • For a delayed transportation, mix with appropriate preservative or place in a transport medium.