Skip to 0 minutes and 7 secondsVANESSA YARDLEY: This video will describe the procedures used when looking for Leishmania amastigotes in tissue samples with a patient with suspected visceral leishmaniasis or post kala-azar dermal leishmaniasis.

Skip to 0 minutes and 23 secondsThe labelled slides are brought to the lab and placed on the staining rack. Gloves and a lab coat should be worn by the operator. The slide should first be flooded with a 100% methanol for a minimum of 30 seconds. The excess methanol can then be tipped off and the Giemsa stain applied, ideally before the fixed slide is completely dry. The Giemsa stain is used as a concentration of 10%. One part of stock Giemsa solution should be diluted with nine parts of distilled water. For example, 1 ml of Giemsa plus 9 mls of water. You may wish to use buffered filtered water for the dilution. The preparation should be mixed by inversion and used promptly.

Skip to 1 minute and 9 secondsThe slides should be flooded with Giemsa solution and left for approximately 20 to 25 minutes. As conditions vary from one laboratory to another, it is good practice to establish an optimal time for staining in your setting.

Skip to 1 minute and 27 secondsTo stop the staining, the Giemsa solution is washed off with clean running water, and the slides are placed on a drying rack, left to air dry at room temperature before examination under the microscope. Ensure the slides to be used have been labelled with appropriate patient identifiers, and then examine under a light microscope.

Skip to 1 minute and 49 secondsFirst, examine the slide under a 10 times magnification. Then repeat the process with a 40 times. This will allow you to systematically examine the tissue and observe how it is spread onto the slide. To begin reading the slide, you will have to move to a greater magnification of 100 times. Add a few drops of oil to the slide and shift to the 100 times objective. Photos here are from a splenic aspiration sample from a patient positive for visceral leishmaniasis. Notice that the tissue may often have dense aggregates of cells, which can be difficult to count. Leishmania amastigotes reside within monocytes and macrophages of the tissue.

Skip to 2 minutes and 34 secondsUsually, the host cells are ruptured during the smear process, so the amastigotes will appear free of the cells. Leishmania amastigotes are oval or round organisms around 2 by 4 micrometres with delicate cell margins. They contain two visible structures, a nucleus and a kinetoplast. The nuclei and kinetoplasts of amastigotes stain dark red or purple, while the cytoplasm of the amastigotes stains paler pink. To maintain consistency of reading and in order to avoid reading the same area twice, it is best to start reading from a top corner of the slide and move slowly across towards the end of the sample in a zig-zag fashion. Keep proceeding this way until the reading is complete.

Skip to 3 minutes and 24 secondsTo report the results, use a grading system that indicates the number of Leishmania amastigotes in a specific set of microscopic view fields. For example, in a splenic aspiration sample, if you counted 5 amastigotes in 100 fields, you would report this is grade 2 plus, whereas if you count 5 amastigotes in 10 fields, the result would be reported as grade 3 plus. Make sure to note down the results in your record book carefully. If abnormal or potentially malignant cells are seen, these should also be reported. After finishing the examination, remove the slide and carefully blot the oil off before storing the slide appropriately. Clean the microscope lenses with proper cleaning material and do not forget to switch the microscope off.

Skip to 4 minutes and 15 secondsDo remember that in a diagnostic lab, it is important that internal quality control is done routinely. Slides should be stored. And regardless of their positive or negative results, 10% of slides should be randomly selected for a second reading by a second person. If the readings of the same slide by two people are not in agreement, an investigation should be started to understand why.

Parasitological diagnosis demonstration: Giemsa staining and microscopic detection of LD bodies

Once the criteria of VL case definition have been fulfilled and VL is suspected, confirmation is ideally by parasitological diagnosis. This involves microscope examination of biopsy material in order to identify Leishmania amastigotes (LDUs) in the sample. Material may be sampled from the spleen, bone marrow or lymph node and requires a high level of skill to conduct as well as hospital facilities to deal with any adverse events following an invasive procedure.

For the parasitological diagnosis of PKDL a thin skin biopsy, skin snip or slit skin sample is collected, smeared onto a slide and similarly examined under the microscope.

Panel of photos showing method of collection of biopsy samples from the spleen, bone marrow, lymph node or skin and the respective microscopic images of the samples stained with Giemsa for the identification of Leishmania amastigotes (LDUs).

Examining Giemsa-stained aspiration samples

The sample is fixed with 100% methanol and stained with Giemsa dye before being examined with a light microscope, under the x100 objective with oil immersion. Watch the following video for the procedure.

Microscopic view-field of infected macrophages with Leishmania amastigotes under the 100x objective, after Giemsa staining.

Notes

  1. In a busy diagnostic lab, when the turnover of samples is high, it may not be possible to review 10% of slides as this could lead to excessive workload. In such cases, it is best to agree upon a capped number of slides for systematic review.
  2. DISCLAIMER: LSHTM and partners collaborating in this video can take no legal responsibility for the use of any information contained. Users implementing the techniques shown do so at their own risk and must take responsibility for local verification/ validation of these techniques before they are used for the diagnosis of patients.

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This video is from the free online course:

Control and Elimination of Visceral Leishmaniasis

London School of Hygiene & Tropical Medicine