Hematology Differentials: Not Just a Smear

Performing hematology differentials (sometimes referred to as a "peripheral smear" or a "diff") is an essential component of laboratory diagnostics, requiring a comprehensive examination of a stained blood smear under the microscope. This process provides valuable insights into the health and functioning of a patient’s hematological system. Generally, it will need to be performed when a hematology analyzer flags for certain parameters (to be set up by the laboratory).

Smear

The first step is to smear it on a slide:

1. Place a small drop of freshly mixed whole blood on a slide (it should be small, such as using a capillary tube or else it will be too thick).

   
   

2. Use another fresh slide to "smear" it into a thin layer. Holding it at a 45 degree or lower angle, pull back until you pull it into the blood drop, then at a steady, but quick rate, push it forward along the surface of the slide. This requires lots of practice, but you'll get the hang of it.

Stain

The next step is a stain. Generally, this means a Wright's stain that will stain the red cells red and other elements such as nuclei a purple/blue. It entails soaking the slide in a stain for minute or two and then soaking it in clean water to clear the excess stain. Allow it to completely dry before placing it on the microscope.

Differential (the "diff")

The analysis encompasses several aspects, including the differential count, white blood cell morphology, red blood cell morphology, platelet estimation, and the identification of cases that necessitate referral to a pathologist for further investigation.

This should be done with oil immersion under 100x, 50x, or 40x objective, but no less.

The differential process involves the careful counting and classification of white blood cells into their various subtypes: neutrophils, lymphocytes, monocytes, eosinophils, and basophils. This usually means a count to 100 total cells and reporting it as a percentage. For example, a pretty normal differential would be as follows:

"Normal" diff

Neutrophils: 66

Lymphocytes: 22

Monocytes: 9

Eosinophils: 4

Basophils: 1

Total: 100 cells

This step provides a detailed snapshot of the immune system’s status, revealing patterns indicative of infections, inflammation, or hematological disorders. There are many other cells that can be seen in a differential. These could be immature cells such as myelocytes or blasts, but also "reactive" cells due to some process such as infection. Often reactive lymphocytes will be seen in viral infections.

The microscopic examination begins at a lower magnification to identify an appropriate region of the smear, often referred to as the “feathered edge,” where cells are evenly distributed. Once located, the evaluation transitions to higher magnification to ensure accurate identification and categorization of each cell type. The relative proportions of the different white blood cell types are then calculated, offering clinicians valuable diagnostic clues.

CML (chronic myeloid leukemia) in a blast crisis. A very abnormal smear.

White cell morphology

White blood cell morphology is another critical element of the hematology differential. Observing the size, shape, and granularity of these cells can uncover abnormalities that suggest specific conditions. For instance, hypersegmented neutrophils may indicate a megaloblastic process, while atypical lymphocytes are commonly associated with viral infections. Toxic granulation or vacuolation within neutrophils might point to sepsis or severe inflammation. Accurate morphological assessment requires a well-prepared smear and the use of high-quality staining techniques to distinguish subtle features.

Red cell morphology

The examination of red blood cell morphology focuses on identifying variations in size, shape, and color, which can indicate underlying anemia or other hematological conditions. The presence of anisocytosis, poikilocytosis, or hypochromasia can provide important diagnostic information. Schistocytes, for example, may signal microangiopathic hemolytic anemia, while target cells are often associated with liver disease or hemoglobinopathies. A detailed review of red cell morphology can also reveal inclusions such as Howell-Jolly bodies, basophilic stippling, or malaria parasites, which carry significant clinical implications.

Platelet estimate

Platelet estimation involves evaluating both the number and appearance of platelets on the blood smear. This step complements automated platelet counts by providing visual confirmation and additional information about platelet morphology. The identification of giant platelets or platelet clumps can help explain discrepancies in automated counts. Platelet abnormalities, such as the presence of agranular forms, may also suggest specific platelet disorders or bone marrow pathology.

When to Refer to a Pathologist

In some cases, the findings from the differential or morphological assessment warrant referral to a pathologist. Situations that necessitate expert review include the presence of blasts, unexplained cytopenias, or significant morphological abnormalities in any cell line. Pathologists bring specialized expertise to interpret complex findings and correlate them with clinical information, enabling a more definitive diagnosis. This collaborative approach ensures that rare or critical conditions are not overlooked. I've referred hundreds of slides that turned out to be nothing serious, but I've also submitted hundreds of slides that turned out to be leukemia. Submitting slides to further scrutiny is part of the process and it's the due diligence that you owe the patients.

Performing hematology differentials requires meticulous attention to detail and a systematic approach. Each component—from differential counts to morphology assessments and platelet estimates—contributes to a comprehensive understanding of the patient’s hematological health. When performed correctly, this procedure not only aids in diagnosis but also guides treatment decisions and supports patient management.

How do I get better as performing differentials?

This is simple. Do more of them. But also, get feedback from an experienced tech or pathologist. And when in doubt, refer smears to a pathologist until you are comfortable knowing what is "normal" vs. "abnormal". This is part of the process of learning, and you should not be embarrassed to be too careful.