The manual differential white blood cell count is performed to determine the relative number of each type of white blood cell present in the blood. A study of red blood cell, white blood cell, and platelet morphology is also performed. The differential count may be performed after the wbc blood count has been determined by the automated 3 part differential , and may be used as a double check on the white blood cell count. The combination of polychrome methylene blue and eosin stains has selective staining properties. The differential staining allows one to identify the types of white blood cells on the smear.
The smear review is performed same as manual differential but rather than quantitating the different types of cells the technologist will look for abnormal cells and semiquantitate (few,moderate,many) if present.
A smear review will revert to a manual differential if it is estimated that there is greater than 5% immature WBCs (bands,metas,myelos,blasts etc.) or greater than 5% nucleated RBCs. (Note: If greater than 5 % nRBCs the WBC count from the instrument will have to be corrected as follows: Corrected WBC = obtained nucleated cell count x (100 ÷ [nRBC + 100]) .
Methanol 89% by weight
Wright Stain, certified 0.4% by weigh (polychrome stain)
Perform smear review or differential of white blood cells:
Observe slide under low dry for overall impression and general appearance of blood cells. Check for even distribution of white blood cells and correct staining of cells.
Observe slide under high dry lens after smearing oil droplet over length of slide. Estimate wbc count by noting number of white cells per high power field X 1000. This number should agree with automated results. lf there is a large discrepancy between the two numbers, the white count should be repeated.
For smear review, scan at least 10 fields on high dry looking for the following abnormal cells:
Immature granulocytes, dysplastic granulocytes, abnormal granulocytes (toxic granulation, dohle bodies, hypersegmentation, pelger-huet anomaly), atypical lymphs, immature lymphs, immature monocytes, abnormal platelets, blood parasites.
Semiquantitate abnormal cells accordingly:
0-2 per high dry field = Few
3-6 per high dry field = Moderate
>6 per high dry field = Many
If it is estimated that there is > 5% immature granulocytes then a manual differential should be performed.
If it is estimated that there is >10% eosinophils or >5% basophils then a manual differential should be performed.
For manual differential count at least 100 white blood cells under high dry lens using the LIS keyboard. Alternatively, differential may be done under oil immersion lens.
(Note: If greater than 5 % nRBCs the WBC count from the instrument will have to be corrected as follows: Corrected WBC = obtained nucleated cell count x (100 ÷ [nRBC + 100]) .
Refer to "Clinical Hematology Atlas" (see references) for guidance and illustrations of abnormal WBC morphology
Stain quality: For slide review and manual diff answer the question in the line "Stain Quality Acceptable?" using the pull down choice list. Answer "Yes" if stained cells appear according to the following:
Perform a platelet estimate:
The estimate is made by counting the average number of platelets seen per 100x oil immersion field in the monolayer of a well-spread smear. This number multiplied by 15,000 equals the approximate platelet count/µL. This value would then be compared to the reference interval for the sample in question. For the estimate, an actual count is not provided but platelets are designated into specific categories:
Increased - the platelet count is estimated to be above the reference interval.
Adequate - the platelet count is estimated to be within the reference interval.
Decreased - the platelet count is estimated to be below the reference interval.
Perform RBC morphology evaluation:
Whenever possible, a red blood cell abnormality should be described in as much detail as possible.
For example, when poikilocytosis is present, the type(s) of irregularly shaped cells should be noted; also anisocytosis, the amount of variation in the size of the red blood cells should be noted. The generally accepted methods of reporting red blood cell irregularities include commenting on the degree of variability present (Slight,moderate,marked). Regardless of the method chosen, it should be used consistently. The reporting of red blood cell morphology varies widely between technologists. It is, therefore, helpful for each laboratory to have a uniform grading system.
Also, the significance of different types of abnormal morphology will vary, and therefore, the degree of grading may depend on the abnormality present. In addition it is important to select the proper area of the smear when determining morphology. The recommended areas on wedge smears are those fields in which some red blood cells begin to overlap.
Refer to "Clinical Hematology Atlas" (see references) for guidance and illustrations of abnormal RBC morphology.
Grading will be performed according to the following:
Polychromatophilia, Helmet Cells, Tear drop RBC, Acanthocytes, Schistocytes, Spherocytes, Poikilocytosis, Ovalocytes, Elliptocytes, Burr Cells, Target Cells, Stomatocytes, Basophilic Stippling, Pappenheimer bodies, Howell Jolly bodies:
0-2 cells per high dry field = Slight
3-6 cells per high dry field = Moderate
>6 cells per high dry field = Marked
Slight = aggregates of 3 to 4 RBC
Moderate = aggregates of 5 to 10 RBC
Marked = numerous aggregates