Performing A Manual Differential And Assessing Red Blood Cell Morphology


When blood samples are evaluated by the use of automated hematology analyzers, this analysis includes automated differentials. Specific criteria pertaining to normal, abnormal, and critical values have been programmed into the analyzers by the institution, and if the differentials do not meet these criteria, verification is necessary. This is done by performing manual differentials and further evaluating the peripheral smear. First, a differential white blood cell (WBC) count is performed to determine the relative number of each type of white cell present. Technologists/technicians must recognize and properly record the type(s) of white cell observed. Simultaneously, red cell, white cell, and platelet morphology is noted and recorded. Also, a rough estimate of platelets and WBC counts is made to determine if these numbers generally correlate with the automated hematology analyzer. Technologists/technicians must be proficient at recognizing red and white cell abnormalities, identifying them correctly, and quantifying them.

Reagents and Equipment

1. Microscope

2. Immersion oil

3. Differential cell counter

Specimen Collection and Storage

Well-made stained blood smear obtained from a capillary puncture or an EDTA tube at least three-fourths full.

Quality Control

The slide should have three zones: head, body, and tail (Fig. 20.7). In the tail area, neutrophils and monocytes predominate, while red cells lie singly. In the body area, lymphocytes predominate, and red cells overlap each other to some extent.

306 PartV • Laboratory Procedures



Tail (includes feathered edge)



Tail (includes feathered edge)

306 PartV • Laboratory Procedures

Figure 20.7 Three zones of wedge preparation.

Table 20.2 O Platelet Estimate From Peripheral Smear

Average No. of

Platelets per X100 Field Platelet Count Estimate

Figure 20.7 Three zones of wedge preparation.

1. WBCs should contain a blue nucleus along with a lighter staining cytoplasm.

2. RBCs should have good quality of color ranging from buff pink to orange.

3. Platelets should be blue with granules and no nucleus.

5 to 8 100,000 to 160,000

10 to 15 200,000 to 300,000

16 to 20 320,000 to 400,000


Observations Under X10

1. Place a well-stained slide on the stage of the microscope, smear side up, and focus using the low-power objective (X10).

2. Check to see if there are good counting areas available free of ragged edges and cell clumps.

3. Check the WBC distribution over the smear.

4. Check that the slide is properly stained.

5. Check for the presence of large platelets, platelet clumps, and fibrin strands.

Observations Under X40 x-: WBC Estimates

1. Place a drop of immersion oil on the slide and change the objective to X50 oil. (In cases where no X50 is available, use the X40 high dry with no oil.)

2. Choose a portion of the peripheral smear where there is only slight overlapping of the RBCs. Count 10 fields, take the total number of white cells and divide by 10, and refer to Table 20.1 to determine the WBC estimate.

Table 20.1 O Estimated WBC Count From Peripheral Smear

WBC/High-Power Field Estimated WBC Count

3. An alternative technique is to do a WBC estimate by taking the average number of white cells and multiplying by 2000.

Observations Under X100: Platelet Estimates

1. Platelet estimates are done under X 100 with the RBCs barely touching, approximately 200 RBCs. This takes place under the X 100 objective (oil). On average there are 8 to 20 platelets per field. See Table 20.2.

2. Ten fields are counted using the zigzag method. This method of counting is done by going back and forth lengthwise or sidewise (Fig. 20.8).

Platelets per oil immersion field (OIF) <8 platelets/OIF = decreased 8 to 20 platelets/OIF = adequate

>20 platelets/OIF = increased

3. After the 10 fields are counted, the number of platelets is divided by 10 to get the average. The average number is now multiplied by a factor of 20,000 for wedge preparations. For monolayer preparations, use a factor of 15,000.

Figure 20.8 Zigzag method of performing differential.

Example: 120 platelets/10 fields = 12 platelets per field

12 X 20,000 = 240,000 platelets

Manual Differential Counts

1. These counts are done in the same area as WBC and platelet estimates with the red cells barely touching.

2. This takes place under X100 (oil) using the zigzag method previously described in the platelet estimate (see Fig. 20.8).

3. Count 100 WBCs including all cell lines from immature to mature. Normal values for WBCs can be found in Table 20.3.

Observing and Recording Nucleated Red Blood Cells (nRBCs)

1. If nRBCs are observed while performing the differential, they need to be reported. These elements in a peripheral smear are indicative of increased erythropoietic activity and usually a pathologic condition. Additionally, the presence of nRBCs per 100 white cells will falsely elevate the white count and is clinically significant.

2. Correct the WBC count if the nRBC count is greater than 5 nRBCs/100. The following formula is applied for correcting NRBCs:

WBC X 100/NRBC + 100

Example : If WBC = 5000 and 10 NRBCs have been counted

Table 20.3

Normal Differential

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