MANUAL DIFFERENTIAL, SMEAR REVIEW

Effective Date: 
Sun, 07/01/2012
Reviewed: 
Mon, 04/28/2014
Revised: 
Fri, 09/06/2013
Policy: 

 

Principle:

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]) .

Materials:

  • CAMCO QUIK STAIN  Buffered Differential Wright Stain (Cambridge Diagnostic Products, lnc)

Methanol 89%  by weight

Wright Stain, certified 0.4% by weigh (polychrome stain) 

  • Microscope slides
  • Coplin Jar
  • Immersion  Oil
  • Microscope w/high dry and high power oil lens

 

Procedure: 
  1. Prepare two thin films of blood at conclusion of venipuncture or from  EDTA tube.  Label slide  in pencil with patient's full name and date.  Allow slides to adequately air dry.
  2. Dip slide  in  stain  for 10 seconds.
  3. Dip slide  in distilled water for 20 seconds  or more for darker staining.

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: 

  • Erythrocytes:   pink to  red-orange biconcave discoid  forms (usually)
  • Lymphocytes:  dark violet nucleus with medium  blue cytoplasm
  • Monocytes:  lobated nucleus,  medium purple with  light blue cytoplasm
  • Neutrophils;  dark blue to purple  nucleus  (3 or more lobes),  pale pink to almost colorless cytoplasm,  red to  lavender small granules
  • Eosinophils  bright red or reddish  orange granules in pale pink cytoplasm,  blue to blue-purple nucleus (multilobed)
  • Basophils:  deep purple and violet black granules in  pale  blue or neutral cytoplasm,  dark blue to purple  nucleus (often bilobed)
  • Platelets:  clearly demarcated blue violet -  purple granules in  light blue cytoplasm

 

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                                    

Rouleaux            

Slight = aggregates of 3 to 4 RBC

Moderate = aggregates of 5 to 10 RBC

Marked = numerous aggregates  

                   

                                                          

Key Points: 

Limitations:  

  • lt is of utmost importance that the  blood film be well prepared.  Smears that are poorly made and / or are too thick will show unequal distribution of white  blood cell types.
  • If clumps of platelets are observed, the EDTA tube should be vortexed for 15 seconds and the slide remade. (Note: CBC should also be rerun on vortexed tube to recheck platelet count.)
  • Slides should  be stained within four hours of preparation. 
  • Precipitate formation may be due to  inadequate or incorrect washing, dust,  or a dirty slide.   Excessive blue stain may be due to overstaining,  excessive  alkalinity of the distilled water, or inadequate washing.  Excessive red stain may be due to  understaining or distilled water being too acidic.

References:

  • Brown,  B. A.,  Hematology Principles and Procedures,  sixth edition,  1993,  Lea and Febiger, Philadelphia, pp.  102-105.
  • Camco Quik Stain Wright Stain package  instructions  (Cambridge Diagnostic Products, Inc.)
  • Carr, J.H. and Rodak, B.F. Clinical Hematology Atlas, 3rd edition Saunders Elsevier 2009