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.
MLAB 1415: Hematology
Cell Type
Relative
Reference
Range
Neutrophil
40-80%
Lymphocyte
25-35%
Monocyte
Counting to 100
Term for
Increased
Increase
Few Potential
causes
Neutrophilia
Term for
Decreased
Decrease
Few
potential
causes
Bacterial
infection,
arthritis,
trauma,
vasculitis,
surgery, AML,
CML
Lymphocytosis Viral infection,
ALL, CLL,
Multiple
Myeloma, TB,
Crohn’s Disease
Neutropenia
Often
associated
with
chemotherapy
Lymphopenia or
Lymphocytopenia
Following a
recent
infection,
Immune
suppression
drugs, HIV
infection,
acute stress
2-10%
Monocytosis
Monocytopenia
Acute
infections,
stress,
glucocorticoid
s, aplastic
anemia
Eosinophil
0-5%
Eosinophilia
Basophil
0-1%
Basophilia
Bacterial
infection,
malaria,
sarcoidosis,
ulcerative
colitis,
neoplasms
Allergic
reaction,
helminth
infection, fungal
infections,
Eczema,
Eosinophilic
leukemia
Allergic
reactions,
Helminth
infections,IDA,
AML, CML,
Myelodysplasia
Not applicable
Not applicable
MLAB 1415: Hematology
Counting to 100
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.
2.
3.
4.
5.
Prepared slides
Manual cell counter designed for differential counts
Microscope
Immersion oil
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.
MLAB 1415: Hematology
Counting to 100
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:
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 %
Pro:
Lymphocyte %
Atyp:
Myelo:
Meta:
Lymph
Band:
Seg:
Total:
Total:
Pro:
Atyp:
Myelo:
Meta:
Lymph
Band:
Seg:
Total:
Total:
Monocyte %
Eosinophil %
Basophil %
Others %
# of
nRBCs/100
WBC
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.