MLAB 1415: Hematology Counting to 100
Laboratory: Counting to 100
Skills= 20pts
Objectives:
1. Correctly identify white blood cells in a peripheral blood smear.
2. Determine the correct percentage of each type of WBC out of 100 WBCs within 15% of the automated result.
Principle:
Peripheral blood smears are evaluated to determine cell morphology, verify automated cell counts, and determine the percentage of each type of WBC. Today’s lab focuses on counting the percentage of each type of WBC.
Fewer WBC differentials are performed today than in previous years because of the improved accuracy of 5-part differentials by automated analyzers; however, if the instrument “flags” a parameter as abnormal, most institutions will reflex a peripheral smear scan or, depending on the abnormality, a full manual differential which includes a WBC differential, evaluation of RBC, WBC, and PLT morphology, and the WBC and PLT estimates (covered in previous labs).
In the examination area of the slide, the “battlement” track pattern is used to ensure that each cell is counted once. One hundred WBCs are counted and classified using a differential counter or computer keyboard.
In the event that the automated WBC count is >40 X 109 /L, the technician should increase the count to a 200 WBC differential to increase accuracy. It is important to verify that the sum of the percentages always equals 100.
When a patient has an extremely low WBC count a 100 cell differential can be difficult. Some institutions will make exceptions to the 100 cell differential when the patient’s WBC count is <1.0 X 109 /L and allow the technician to do a 50 cell WBC differential. The results are then multiplied by two (2); however, the accuracy of the 50 cell differential is questionable and not recommended. In these cases of <1.0 X 109 /L, other laboratories will centrifuge the sample, harvest the buffy coat, and evaluate the buffy coat for blast cells, but the differential is not performed on a buffy coat due to distribution errors from centrifugation.
The proportion of each cell type is calculated as a percentage of the total WBC count and represents the relative differential count. If one cell line is increased or decreased beyond the relative differential count reference range, the terms in the following table should be used to describe the patient’s peripheral blood status.
Cell Type / Relative Reference Range / Term for Increased / IncreaseFew Potential causes / Term for Decreased / Decrease Few potential causes
Neutrophil / 40-80% / Neutrophilia / Bacterial infection, arthritis, trauma, vasculitis, surgery, AML, CML / Neutropenia / Often associated with chemotherapy
Lymphocyte / 25-35% / Lymphocytosis / Viral infection,
ALL, CLL, Multiple Myeloma, TB, Crohn’s Disease / Lymphopenia or Lymphocytopenia / Following a recent infection, Immune suppression drugs, HIV infection, acute stress
Monocyte / 2-10% / Monocytosis / Bacterial infection, malaria, sarcoidosis, ulcerative colitis, neoplasms / Monocytopenia / Acute infections, stress, glucocorticoids, aplastic anemia
Eosinophil / 0-5% / Eosinophilia / Allergic reaction, helminth infection, fungal infections, Eczema, Eosinophilic leukemia / Not applicable
Basophil / 0-1% / Basophilia / Allergic reactions, Helminth infections,IDA, AML, CML, Myelodysplasia / Not applicable
Each cell line should be examined for immature cells. Immature WBCs are not a normal finding in the peripheral blood. Presence of immature WBCs can indicate infections or malignancies such as leukemia. The neutrophilic line is the only cell line that immature cells are differentiated and identified in most laboratories. The term “left shift” refers to the presence of bands and younger neutrophilic cells.
Specimen:
Peripheral blood smear made from EDTA-anticoagulated blood. Smears should be made within 4 hours of blood collection from EDTA specimens stored at room temperature to avoid distortion of cell morphology. Unstained smears can be stored for indefinite periods in a dry environment, but stained smears gradually fade unless coverslipped.
Reagents, supplies, and equipment:
1. Prepared slides
2. Manual cell counter designed for differential counts
3. Microscope
4. Immersion oil
5. Lens paper
100 Cell Differential for Cell counts <40 X 109 /L:
1. Take the first of two of the instructor-supplied prepared slides.
2. Set a timer for 20 minutes.
3. Focus the microscope on the 10X objective (low power). Scan the smear to check for cell distribution, clumping, and abnormal cells. In scanning the smear it is important to note anything unusual or irregular, such as rouleaux or RBC clumping.
4. Examine the peripheral edge of the smear. The number of WBCs should NOT exceed 3X the number of cells in the proper examination area. Large cells such as neutrophils and monocytes can be pushed to the edges. If this occurs, the distribution of the cells is poor and the smear is unacceptable.
5. If the smear is acceptable as determined by observation on 10x, change to the 100x oil objective. Find an area of the smear where ½ the RBCs are overlapping and ½ are not overlapping.
6. Using a cell counter, begin the count in the thin area of the slide and utilize the battlement track to read the slide. Carefully push the button that corresponds to the cell you are counting until you reach 100 total WBCs.
7. Record results on the report form. Also record the actual or known values for each category on the report sheet.
Procedural Notes:
1. If nucleated red blood cells (nRBCs) are observed, they are enumerated; however, they are NOT included in the 100 WBC total. If the cell counter has a nRBC button, the counter will not be include the nRBCs in the 100 WBCs total. If the cell counter does NOT have a nRBC button, you must keep a separate count of nRBCs you observe until you reach 100 WBCs. If you observe >5 nRBCs/100 WBC diff, the automated WBC count must be corrected by using a calculation you will learn in the Manual Differential Lab.
2. If atypical lymphocytes are observed, count them using the specified atypical/reactive lymphocyte button. If there is not a specified button, use a blank button on the cell counter. These are included in the total lymphocyte percentage, but need to be differentiated from normal lymphocytes.
3. If immature neutrophils (promyelocytes-bands) are observed, they must be identified and counted separately. These count toward the neutrophil percentage, but must be differentiated from mature neutrophils. In all other granulocytic or monocytic cell lines, immature cells (other than blasts) are included with the mature cell count.
4. Blasts of all cell lines will be classified as “others” in this lab. They will be counted by pressing a blank or “other” button. They will count toward the 100 WBC total and recorded in the “Other %” column on the report form. In most clinical laboratories, these would be referred to a pathologist for identification.
Examples of Differential Cell Counters:
(Rev 10/7/2016) Page 5
MLAB 1415: Hematology Counting to 100
Name:______Date:______
Laboratory: Counting to 100
Skills: 20 pts.
Report Form
Patient Name and ID or Slide Number / Neutrophil % / Lymphocyte % / Monocyte % / Eosinophil % / Basophil % / Others % / # of nRBCs/100 WBCActual Count
Pro: / Atyp:
Myelo:
Meta: / Lymph
Band:
Seg: / Total:
Total:
Actual Count
Pro: / Atyp:
Myelo:
Meta: / Lymph
Band:
Seg: / Total:
Total:
(Rev 10/7/2016) Page 5
MLAB 1415: Hematology Counting to 100
Laboratory: Counting to 100
Study Questions
10 pts. (1 pt each answer)
1. If you observed a promyelocyte on your smear, what button would you press during your WBC differential?
2. According to ACC procedure, how would you classify the following cell at the arrow?
(what column on report form)
3. Under what conditions would a 200 WBC differential be performed AND WHY?
4. List four (4) causes of Neutrophilia.
a.
b.
c.
d.
5. List three (3) causes of Lymphocytopenia.
a.
b.
c.
(Rev 10/7/2016) Page 5