Manual microscopy procedure

Automated hematology analyzers constitute quick and effective screening tools to monitor the overall blood status of a patient. However, such instruments do not identify all morphologically abnormal cell forms. Instead, the analyzers indicate cell abnormalities through system messages, so called parameter flags. In such cases, a manual examination of the sample by a microscopic morphological count is recommended.

For an accurate manual cell count, the quality of the blood smear is important. This entails the practice of good slide preparation and staining procedure.

Top tips from the laboratory technician

Here we list a few things to consider when preparing a microscopy slide.

Blood sampling procedure

Manual microscopy can be done using both venous and capillary samples. For skin puncture and capillary blood sampling, see capillary blood sample collection

Slide preparation

 Tips for things to do to get the optimal thin blood film:

  • Clean the base slide (frosted glass microscope slide) with a napkin before starting to make the blood to later slide more easily.
  • Place approximately 8–10 μl of blood on the base slide.
  • Touch the drop of blood with the spreader slide (this slide should have chipped edges) until blood is evenly spread across the spreader slide.
  • Quickly but gently, without exerting too much pressure on the actual slide, slide the blood across to make a thin film of blood that is evenly spread.
  • The smear should cover two-thirds of the base slide and should have a feathered end.

It is essential to get a good monolayer to accurately count the WBCs (Fig 1). One tip to get a good monolayer is to not use too much blood for the slide preparation.


Figure 1. Preparation of a blood smear.
Staining procedure

There are different staining methods where one of the more common ones is the Romanowsky staining. There are variations such as Leishman and Giemsa. The staining instructions for use should be followed but may need to be adjusted slightly depending on the buffer and pH of the water used.

Sample age

Samples should be prepared and stained preferably within four hours since the blood cells grow old and EDTA changes the morphology of the cells.

Learn more

To read more in-depth about the staining and slide preparation procedures, different staining types, equipment and helpful counting tools, see WBC differential manual microscopy slide preparation.