FREE ESSAY ON LEUKOCYTES

LEUKOCYTES

Leukocytes and the leukocyte differential count
To consider the leukocytes together as a group is something of a granfalloon, because
each type of leukocyte has its own function and ontogeny semi-independent of the others.
To measure the total leukocyte count and allow this term to mean anything to the doctor
is a travesty, yet the wbc count has traditionally been considered a cardinal measurement
in a routine laboratory workup for just about any condition. I cannot emphasize too much
that to evaluate critically the hematologic status of a patient, one must consider the
individual absolute counts of each of the leukocyte types rather than the total wbc
count. For such a critical evaluation, the first step is to order a wbc count with
differential. In many labs, the result will be reported as a relative differential,
something like this: 
WBC 6000/?L 
segmented neutrophils 60% 
band neutrophils 2% 
lymphocytes 25% 
monocytes 8% 
eosinophils 3% 
basophils 2% 
Your first task is to multiply the wbc count by each of the percentages given for the
cell types; this gives you an absolute differential. Now you're in business to get some
idea as to the pathophysiologic status of the patient's blood and marrow. Thus, the
illustration above becomes: 
WBC 6000/?L 
segmented neutrophils 3600/?L 
band neutrophils 120/?L 
lymphocytes 1500/?L 
monocytes 480/?L 
eosinophils 180/?L 
basophils 120/?L 
The total wbc count is invariably done using an automated method. Routinely, the
differential count is done by hand (i.e., through the microscope) in smaller labs, and by
automated methods in larger facilities. The automated methods are amazingly accurate,
considering the fine distinctions that must often be made in discerning one type of
leukocyte from the other. One manufacturer's machine can quite reliably pick out one
leukemic blast cell in eight hundred or more leukocytes. Now we shall consider each of
the leukocyte types individually. 
A. Neutrophils 
The most populous of the circulating white cells, they are also the most short lived in
circulation. After production and release by the marrow, they only circulate for about
eight hours before proceeding to the tissues (via diapedesis), where they live for about
a week, if all goes well. They are produced as a response to acute body stress, whether
from infection, infarction, trauma, emotional distress, or other noxious stimuli. When
called to a site of injury, they phagocytose invaders and other undesirable substances
and usually kill themselves in the act of doing in the bad guys. 
Normally, the circulating neutrophil series consists only of band neutrophils and
segmented neutrophils, the latter being the most mature type. In stress situations (i.e.,
the acute phase reaction), earlier forms (usually no earlier than myelocytes) can be seen
in the blood. This picture is called a left shift. The band count has been used as an
indicator of acute stress. In practice, band counts tend to be less than reliable due to
tremendous interobserver variability, even among seasoned medical technologists, in
discriminating bands from segs by microscopy. Other morphologic clues to acute stress may
be more helpful: in the acute phase reaction, any of the neutrophil forms may develop
deep blue cytoplasmic granules, vacuoles, and vague blue cytoplasmic inclusions called
Dohle bodies, which consist of aggregates of ribosomes and endoplasmic reticulum. All of
these features are easily seen (except possibly the Dohle bodies), even by neophytes. 
The normal range for neutrophil (band + seg) count is 1160 - 8300 /?L for blacks, and
1700 - 8100 /?L for other groups. Keeping in mind the lower expected low-end value for
blacks will save you much time (and patients much expense and pain) over the course of
your career. Obesity and cigarette smoking are associated an increased neutrophil count.
It is said that for each pack per day of cigarettes smoked, the granulocyte count may be
expected to rise by 1000 /?L. 
B. Monocytes 
These large cells are actually more closely related to neutrophils than are the other
granulocytes, the basophil and eosinophil. Monocytes and neutrophils share the same stem
cell. Monocytes are to histiocytes (or macrophages) what Bruce Wayne is to Batman. They
are produced by the marrow, circulate for five to eight days, and then enter the tissues
where they are mysteriously transformed into histiocytes. Here they serve as the welcome
wagon for any outside invaders and are capable of processing foreign antigens and
presenting them to the immunocompetent lymphocytes. They are also capable of the more
brutal activity of phagocytosis. Unlike neutrophils, histiocytes can usually survive the
phagocytosis of microbes. What they trade off is killing power. For instance,
mycobacteria can live in histiocytes (following phagocytosis) for years. 
The normal range for the monocyte count is 200 - 950 /?L. 
C. Eosinophils 
These comely cells are traditionally grouped with the neutrophils and basophils as
granulocytes, another granfalloon. Current thinking is that eosinophils and neutrophils
are derived from different stem cells, which are not distinguishable from each other by
currently available techniques of examination. Although the hallmark of the eosinophil is
the presence of bright orange, large, refractile granules, another feature helpful in
identifying them (especially on H&E-stained routine histologic sections) is that they
rarely have more than two nuclear lobes (unlike the neutrophil, which usually has three
or four). The normal range of the absolute eosinophil count is 0 - 450 /?L. 
Eosinophils are capable of ameboid motion (in response to chemotactic substances released
by bacteria and components of the complement system) and phagocytosis. They are often
seen at the site of invasive parasitic infestations and allergic (immediate
hypersensitivity) responses. Individuals with chronic allergic conditions (such as atopic
rhinitis or extrinsic asthma) typically have elevated circulating eosinophil counts. The
eos may serve a critical function in mitigating allergic responses, since they can 1)
inactivate slow reacting substance of anaphylaxis (SRS-A), 2) neutralize histamine, and
3) inhibit mast cell degranulation. The life span of eos in the peripheral blood is about
the same as that of neutrophils. Following a classic acute phase reaction, as the
granulocyte count in the peripheral blood drops, the eosinophil count temporarily rises.

D. Basophils 
The most aesthetically pleasing of all the leukocytes, the basophils are also the least
numerous, the normal range of their count in peripheral blood being 0 - 200/?L. They are
easily recognized by their very large, deep purple cytoplasmic granules which overlie, as
well as flank, the nucleus (eosinophil granules, by contrast, only flank the nucleus but
do not overlie it). It is tempting to assume that the basophil and the mast cell are the
blood and tissue versions, respectively, of the same cell type. Actually it is
controversial as to whether this concept is true or whether these are two different cell
types. 
Feature Basophils Mast cells 
Nuclear morphology segmented round or ovoid 
Mitotic potential no yes 
Peroxidase content + - 
Acid phosphatase - + 
Alkaline phosphatase - + 
PAS reaction ++++ + 
The table at right presents some of the contrasts between mast cells and basophils. 
In active allergic reactions, blood basophils decrease in number, while tissue mast cells
increase. This reciprocal relationship suggests that they represent the same cell type
(i.e., an allergen stimulates the passage of the cells from the blood to the site of the
allergen in the tissues). Some experiments with animals have also shown that mast cells
are marrow-derived and are capable of differentiating into cells that resemble basophils.
Conversely, some recent evidence suggests that basophils (as well as eosinophils) can
differentiate from metachromatic precursor cells that reside among epithelial cells in
the nasal mucosa 
Without invoking religion or Alexander Pope (Whatever is, is right, An Essay on Man,
1732-34) it is hard to see any useful role of the basophil/mast cell in human physiology.
The mast cell is the essential effector of immediate (Type 1) hypersensitivity reactions,
which produce only misery, dysfunction, and occasionally death for the hapless host. 
E. Lymphocytes 
In the immune/inflammatory response, if the neutrophils and monocytes are the brutes, the
lymphocytes are the brains. It is possible to observe the horror of life without
lymphocyte function by studying the unfortunate few with hereditary, X-linked, severe
combined immune deficiency. Such individuals uniformly die of systemic infections at an
early age (except for the bubble boys of yesteryear, who lived out their short lives in
antiseptic prisons). The functions of lymphocytes are so diverse and complex that they
are beyond the scope of this text (and the scope of the author, it must be admitted).
What follows are a few general remarks concerning examination of lymphocytes in
peripheral blood. 
After neutrophils, lymphocytes are the most numerous of the circulating leukocytes. The
normal range of the lymphocyte count is 1000 - 4800/?L. Their life span may vary from
several days to a lifetime (as for memory lymphocytes). Unlike neutrophils, monocytes,
and eosinophils, the lymphocytes 1) can move back and forth between the vessels and the
extravascular tissues, 2) are capable of reverting to blast-like cells, and 3) when so
transformed, can multiply as the immunologic need arises. 
In normal people, most of lymphocytes are small, innocent-looking round cells with
heavily painted-on nuclear chromatin, scant watery cytoplasm, and no granules. A small
proportion of normal lymphs are larger and have more opaque, busy-looking cytoplasm and
slightly irregular nuclei. Some of these have a few large, dark blue granules, the so
called azurophilic granules. It has been maintained that these granulated cells are
T-gamma cells (i.e., T-cells that have a surface receptor for the IgG Fc region) or
natural killer (NK) null-cells. Other phenotypes of lymphocytes are not recognizable as
such on the routine, Wright-stained smear and require special techniques for
identification. 
When activated by whatever means, lymphocytes can become very large (approaching or
exceeding the diameter of monocytes) and basophilic (reflecting the increased amount of
synthesized cytoplasmic RNA and protein). The cytoplasm becomes finely granular
(reflecting increased numbers of organelles), and the nuclear chromatin becomes less
clumped (the better to transcribe you with, my dear!). Such cells are called transformed
lymphocytes, atypical lymphocytes, or viral lymphocytes by various votaries of blood
smears. Although such cells are classically associated with viral infection (particularly
infectious mononucleosis), they may also be seen in bacterial and other infections and in
allergic conditions. A morphologic pitfall is mistaking them for monocytes (a harmless
mistake) or leukemic blasts (not so harmless). 
Platelets
The main thing to remember about platelets is to look for them first! A typical tyro
maneuver is to study a blood smear for an hour looking for some profound hematological
abnormality, never to realize there is nary a platelet in sight. It is therefore
necessary to discipline yourself to first check for a normal number of platelets when
sitting down with a slide, before being seduced by the midnight beauty of the basophil's
alluring granules or the monocyte's monolithic sovereignty. The normal platelet count is
133 - 333 x 103/?L. 
Platelets are counted by machine in most hospital labs and by direct phase microscopy in
smaller facilities. Since platelets are easily mistaken for garbage (and vice versa) by
both techniques, the platelet count is probably the most inaccurate of all the routinely
measured hematologic parameters. Actually, you can estimate the platelet count fairly
accurately (up to an absolute value of about 500 x 103/?L) by multiplying the average
number of platelets per oil immersion field by a factor of 20,000. For instance, an
average of ten platelets per oil immersion field (derived from the counting of ten
fields) would translate to 200,000/?L (10 x 20,000). Abnormal bleeding generally does not
occur unless the platelet count is less than 30,000/?L, if the platelets are functioning
properly. Screening for proper platelet function is accomplished by use of the bleeding
time test. 
Other cells in peripheral blood
Plasma cells sometimes appear in the peripheral blood in states characterized by
reactivity of lymphocytes. Old time hematologists often maintain that the cells that look
exactly like plasma cells on the smear are really plasmacytoid lymphs, and it is usually
nonproductive to argue this point with them. Endothelial cells occasionally get scooped
up into the phlebotomy needle during blood collection and show up on the slide. They are
huge and tend to be present in groups. Histiocytes, complete with pseudopodia and
phagocytic vacuoles, may appear in states of extreme reactivity, especially in septic
neonates. Nucleated red cells may also be seen in small numbers in the peripheral blood
of newborns; however, in adults, even a single nucleated rbc on the slide is abnormal,
indicating some sort of serious marrow stress, from hemolytic anemia to metastatic
cancer. Myeloblasts are always abnormal and usually indicate leukemia or an allied
neoplastic disease. Rarely they may be seen in non-neoplastic conditions, such as
recovery from marrow shutdown (aplasia). Later stages of myeloid development
(promyelocyte, myelocyte, metamyelocyte) may be represented in the peripheral blood in
both reactive states and leukemias. 
Bone marrow examination 
This is one of the most common biopsy procedures performed on both outpatients and the
hospitalized. Two types of specimens are generally obtained, the aspirate and the core
biopsy. The site of biopsy is usually the posterior iliac crest (via the posterior
superior iliac spine) in adults and the anterior tibia in children, although other sites
are available. After local anesthesia is applied to the periosteum and overlying skin, a
small needle (usually the University of Illinois needle) is introduced (or crunched
actually) into the medullary space through a small skin incision. About 0.5 mL of marrow
material is aspirated and smeared onto several glass slides and stained with a stain
identical or similar to the Wright stain used on peripheral blood. Some material usually
remains in the syringe where it is allowed to clot. It is then fished out of the syringe,
processed like all other biopsy tissue, embedded in paraffin, sectioned, and stained with
hematoxylin/eosin and other selected stains. The core biopsy, generally performed after
the aspirate is done, is taken with a larger, tapered needle, typically the Jamshidi
needle. This yields a core of bone (similar to a geologic core sample) which is fixed,
decalcified, processed, and sectioned. The H&E-stained core biopsy and aspirate clot
sections are best for assessment of marrow cellularity and the presence of metastatic
neoplasms or granulomas. The Wright-stained aspirate smears are best for studying the
detailed cytology of hematopoietic cells. 
The bone marrow biopsy procedure produces some pain for the patient, since it is
impossible to anesthetize the inside of bone. The level of pain ranges from mild
discomfort to agony, depending on the individual's pain threshold and level of
apprehension. Some physicians elect to precede the biopsy with a benzodiazepine or other
minor tranquilizer. Generally the aspiration action produces much more pain than the core
biopsy. 
For a procedure that involves invasion of bone, the marrow biopsy is remarkably free of
complications. Bleeding and infection may occur but are rare, even in severely
thrombocytopenic and immunosuppressed patients. It is highly recommended that med
students learn how to perform this useful procedure during the clinical years of their
training.