The Red Cell and Anemia

The Red Cell and Anemia
Part One: Introduction to Hematopoiesis and Its
Routine Clinical Evaluation
I. Introduction
Hematopathology is not only the study of disease of the blood and bone marrow, but
also of the organs and tissues which employ blood cells as principal effectors of their
physiologic functions. Such would include the lymph nodes, spleen, thymus, and the
many foci of lymphoid tissue found along the aerodigestive tract. Generally two
types of medical subspecialists intensively practice in this area, the hematologist
and the hematopathologist. The hematologist usually is a Board-certi?ed internist
who has completed additional years of training in hematology, usually as part of a
combined fellowship in hematology and oncology. The thrust of this individual’s
work is toward the diagnosis and medical management of patients with hematologic
disease, especially neoplasms, and medical management of other nonhematologic
cancer. The hematopathologist, on the other hand, is usually Board-certi?ed in
anatomic and clinical pathology and has taken additional years of training in
hematopathology. His or her principal activity is the morphologic diagnosis of condi-
tions of the hematopoietic and lymphocyte-rich tissues and in the performance of
laboratory testing that assists such diagnosis.1
Hematopathology is somewhat unique in its approach to the patient and the dis-
ease, in that 1) many diseases are understood at the molecular level, 2) the patient’s
tissue is easily obtainable in large quantities (in the case of peripheral blood, at
least) and easily kept viable for special studies, and 3) the function of the blood (or
at least the erythroid component) is relatively simple when compared to that of oth-
er organ systems. Because it is a scienti?cally integrated discipline hematolo-
gy/hematopathology is an area which is intellectually gratifying to the eclectic
individual who is well-rounded in various biomedical endeavors, including
biochemistry, physiology, pharmacology, microanatomy, morphologic diagnosis, and
patient care.
II. The Blood
A few nights working in a trauma center would tend to convince one that the body is
just a huge bag of blood. In fact, an “average” 70 liter human body contains only
about 5 liters of blood, or 7% by volume. In the normal state, blood has no business
anywhere except in the con?nes of the heart and blood vessels and in the sinusoids
of the marrow, liver, and spleen. Of the average 5 L of blood, only 2.25 L, or 45%,
1If you are destined to become a dean or departmental chairman, you are probably sensing a turf war
here somewhere. Actually, if this is war, it is one of great chivalry compared to some of the other ju-
risdictional altercations in medicine. For instance, the war between plastic surgeons and cosmetic
surgeons (yes, there is a difference) makes any tiff between internal medicine and pathology look like
the War of Jenkins’ Ear (no pun intended).
version 2.0, copyright © 1995-96, Edward O. Uthman (uthman@domi.net)

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The Red Cell and Anemia
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consists of cells. The rest is plasma, which itself consists of 93% water (by weight)
and 7% solids (mostly proteins, the greatest proportion of which is albumin). Of the
2.25 L of cells, only 0.037 L (1.6%) are leukocytes. The entire circulating leukocyte
population, if puri?ed, would ?t in a bartender’s jigger. The total circulating platelet
volume is even less — about 0.0065 L — or a little over one teaspoonful.
III. Erythrocytes
Structurally the simplest cell in the body, volumes have been written about the low-
ly red blood cell. The basic function of the rbc is the creation and maintenance of an
environment salutary to the physical integrity and functionality of hemoglobin. In
the normal state, erythrocytes are produced only in the skeleton (in adults only in
the axial skeleton), but in pathologic states (especially myelo?brosis, which will be
covered subsequently) almost any organ can become the site of erythropoiesis. Nu-
merous substances are necessary for creation of erythrocytes, including metals
(iron, cobalt, manganese), vitamins (B12, B6, C, E, folate, ribo?avin, pantothenic
acid, thiamin), and amino acids. Regulatory substances necessary for normal eryth-
ropoiesis include erythropoietin, thyroid hormones, and androgens. Erythrocytes
progress from blast precursors in the marrow over a period of ?ve days. Then they
are released into the blood as reticulocytes, distinguishable from regular
erythrocytes only with special supravital stains. The reticulocyte changes to an
erythrocyte in one day and circulates for 120 days before being destroyed in the
reticuloendothelial system.
Clinical laboratories measure several important parameters that re?ect rbc struc-
ture and function. These measurements are used to 1) evaluate the adequacy of oxy-
gen delivery to the tissues, at least as is related to hematologic (as opposed to car-
diopulmonary) factors, and 2) detect abnormalities in rbc size and shape that may
provide clues to the diagnosis of a variety of hematologic conditions. Most of these
tests are performed using automated equipment to analyze a simple venipuncture
sample collected in a universal lavender- (or purple-) top tube containing EDTA as
an anticoagulant. Let us consider each of these tests.
A. Hemoglobin concentration in whole blood
Referred to simply as “hemoglobin,” this test involves lysing the erythrocytes,
thus producing an evenly distributed solution of hemoglobin in the sample. The
hemoglobin is chemically converted mole-for-mole to the more stable and easily
measured cyanmethemoglobin, which is a colored compound that can be
measured colorimetrically, its concentration being calculated from its amount
of light absorption using Beer’s Law 2. The normal range for hemoglobin is
highly age and sex dependent, with men having higher values than women,
2Beer's law expresses the mathematical relationship between the molar concentration of a colored
substance in solution and the amount of monochromatic light it absorbs. This relationship is the ba-
sis of the great majority of automated chemical tests run in the clinical laboratory.

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The Red Cell and Anemia
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and adults having higher values than children (except neonates, which have
the highest values of all). At Hermann Hospital the young adult female normal
range is 12 – 16 g/dL; for adult males it is 14 – 18 g/dL.
This is an easy test to perform, as hemoglobin is present in the blood in higher
concentration than that of any other measured substance in laboratory
medicine3. The result is traditionally expressed as unit mass per volume,
speci?cally grams per deciliter (g/dL). Ideologues in lab medicine have been
maintaining for years that this unit will be replaced by Système Internationale
(SI) units of moles per liter, but this has not gained any signi?cant acceptance
in clinical medicine except in the most nerdly circles.
B. Erythrocyte count
Also referred to as just “rbc,” this simply involves counting the number of rbcs
per unit volume of whole blood. Manual methods using the hated
hemocytometer4 have been universally replaced by automated counting. The
major source of error in the rbc count is an arti?cially reduced result that oc-
curs in some conditions where rbcs stick together in the sample tube, with two
or more cells being counted as one. The result of the test is expressed as num-
ber of cells per unit volume, speci?cally cells/µL. At Hermann Hospital, the
normal range is 4.2 – 5.4 × 106/µL for females; for adult males it is 4.7 – 6.1 ×
106 /µL.
C. Hematocrit
This is also called the packed cell volume or PCV 5. It is a measure of the total
volume of the erythrocytes relative to the total volume of whole blood in a
sample. The result is expressed as a proportion, either unitless (e.g., 0.42) or
with volume units (e.g., 0.42 L/L, or 42 cL/L [centiliters/liter]). An archaic way
of expressing hematocrit is “volumes per cent” or just “percent” (42%, in the
above illustration). Small of?ce labs and stat labs measure hematocrit simply
by spinning down a whole blood sample in a capillary tube and measuring the
length of the column of rbcs relative to the length of the column of the whole
specimen. Larger labs use automated methods that actually measure the vol-
ume individually of each of thousands of red cells in a measured volume of
whole blood and add them up. The volume of individual erythrocytes can be
3For instance, the concentration of hemoglobin in blood is approximately 300,000,000,000 times that
of estradiol, the principal female sex hormone, which is also routinely measured in clinical laborato-
ries.
4Doctors who demand that the lab perform a manual count for anything except platelets are usually
found at the bottom of an elevator shaft the next morning.
5Not to be permutatively confused with “PVC” (premature ventricular contraction), or “CVP” (central
venous pressure)

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The Red Cell and Anemia
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electronically determined by measurement of their electrical impedance or
their light-scattering properties. The normal range is 0.37 – 0.47 L/L for
females, and 0.42 – 0.52 L/L for males.
D. Erythrocyte indices
The three cardinal rbc measurements described above (hemoglobin, hematocrit,
and rbc count) are used to arithmetically derive the erythrocyte indices – mean
corpuscular volume, mean corpuscular hemoglobin, and mean corpus-
cular hemoglobin concentration. As much as we all hate memorization, it
is important to know how to calculate these indices and have some idea of the
normal ranges. We will consider these individually.
1. Mean corpuscular volume (MCV)
This is the mean volume of all the erythrocytes counted in the sample. The
value is expressed in volume units, in this case very small ones – femtoliters
(fL, 10-15 liter). The normal range is 80 – 94 fL. The formula for the calcula-
tion in general terms is
hematocrit
MCV = rbc count
When using speci?c units, decimal fudge factors are required; for example,
hematocrit (in L/L) × 1000
MCV (in fL) = rbc count (in millions/µL)
I think that it is easier to forget the fudge factors, use the first formula,
multiply out the values while ignoring the bothersome decimal, and
reposition the decimal in the ?nal result so as to approximate the order of
magnitude of the normal range. This is safe, since you will not see an MCV
of 8 fL, or one of 800 fL.6
When the MCV is low, the blood is said to be microcytic7, when high,
macrocytic. Normocytic refers to blood with a normal MCV. Keep in mind
that the MCV measures only average cell volume. The MCV can be normal
while the individual red cells of the population vary wildly in volume from
6Except maybe in Lilliput or Brobdingnag, respectively.
7For some reason the “mic” in “microcytic” is pronounced like the “mic” in “Mickey Mouse,” not like
the “mic” in “Michael Jackson.” The problem with “micro-” and “macro-” is similar to that with such
medical antonymic pairs as “adduct” and “abduct,” and “hypo-” and “hyper-.” These roots sound
similar (especially when pronounced by a fatigued Texan) but have exactly opposite meanings. I
would imagine that this produced analogous problems in the Classical languages from which these
terms sprang. For instance: “Yes, Leonidas. The messenger was a Macedonian with a brutish accent,
but I distinctly understood him to say that the Persians are approaching Thermopylae with a micro
army which is hypo equipped. I say take 300 hoplites up there to polish them off, and the rest of us
can stay here in Sparta and, uh, initiate the new recruits.”

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The Red Cell and Anemia
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one to the next. Such an abnormal variation in cell volume is called
anisocytosis. Some machines can measure the degree of anisocytosis by use
of a parameter called the red cell distribution width (RDW). This is sim-
ply a standardized parameter (similar to the standard deviation) for mathe-
matically expressing magnitude of dispersion of a population about a mean.
The normal range for RDW is 11.5 – 14.5 %.
2. Mean corpuscular hemoglobin (MCH)
The MCH represents the mean mass of hemoglobin in the RBC and is ex-
pressed in the mass unit, picograms (pg, 10-12 gram). The value is deter-
mined by the formula,
hemoglobin (in g/dL) × 10
MCH (in pg) = rbc count (in millions/µL)
Again, a fudge factor is required in this equation, so it helps to get some feel
for the normal range (27 – 31 pg) and gestalt the decimal point, as described
for MCV, above. Since small cells have less hemoglobin than large cells,
variation in the MCH tends to track along with that of the MCV. The MCH
is something of a minor leaguer among the indices in that it adds little infor-
mation independent of the MCV.
3. Mean corpuscular hemoglobin concentration (MCHC)
This is the mean concentration of hemoglobin in the red cell. Since whole
blood is about one-half cells by volume, and all of the hemoglobin is con?ned
to the cells, you would correctly expect the MCHC to be roughly twice the
value for hemoglobin in whole blood and to be expressed in the same units;
the normal range is 32 – 36 g/dL. The value is calculated using the formula,
hemoglobin (in g/dL)
MCHC (in g/dL) = hematocrit (in L/L)
Cells with normal, high, and low MCHC are referred to as normochromic,
hyperchromic, and hypochromic, respectively. Again, these terms will
have importance in anemia classi?cation.
IV. Leukocytes and the leukocyte differential count
To consider the leukocytes together as a group is something of a granfalloon8, be-
cause 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 any-
8Granfalloon, noun, a false or misleading grouping together of objects or concepts based on
irrelevant commonalities; from Kurt Vonnegut's Cat's Cradle (1963), in which it is offered:
“If you wish to study a granfalloon
Just remove the skin of a toy balloon.”
An essential goal of medical nosology is the elimination of granfalloons from our body of knowledge.

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The Red Cell and Anemia
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thing to the doctor is a travesty, yet the “wbc” count has traditionally been consid-
ered 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
?rst 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
/µL
segmented neutrophils
60
%
band neutrophils
2
%
lymphocytes
25
%
monocytes
8
%
eosinophils
3
%
basophils
2
%
Your ?rst 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
/µL
segmented neutrophils
3600
/µL
band neutrophils
120
/µL
lymphocytes
1500
/µL
monocytes
480
/µL
eosinophils
180
/µL
basophils
120
/µL
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 ?ne 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.

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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 de-
velop deep blue cytoplasmic granules, vacuoles, and vague blue cytoplasmic in-
clusions called Döhle bodies, which consist of aggregates of ribosomes and en-
doplasmic reticulum. All of these features are easily seen (except possibly the
Döhle bodies), even by neophytes.
The normal range for neutrophil (band + seg) count is 1160 – 8300 /µL for
blacks, and 1700 – 8100 /µL for other groups. Keeping in mind the lower ex-
pected 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 /µL.
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 ?ve
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”9 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 /µL.
9This sounds very high-tech and diplomatic to describe activities that must be very gloopy and
unsympathetic. I think more descriptive phraseology would be “…capable of ‘busting’ foreign anti-
gens and ‘taking them downtown.’”

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The Red Cell and Anemia
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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 /µL.
Eosinophils are capable of ameboid motion (in response to chemotactic sub-
stances 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 sub-
stance 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 esthetically pleasing of all the leukocytes, the basophils are also the
least numerous, the normal range of their count in peripheral blood being 0 –
200/µL. They are easily recognized by their very large, deep purple cytoplasmic
granules which overlie, as well as ?ank, the nucleus (eosinophil granules, by
contrast, only ?ank 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. The following
table presents some of the contrasts between mast cells and basophils:

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The Red Cell and Anemia
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Basophils
Mast cells
Nuclear morphology
segmented
round or ovoid
Mitotic potential
no
yes
Peroxidase content10
+
-
Acid phosphatase11
-
+
Alkaline phosphatase
-
+
PAS reaction12
++++
+
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 differ-
entiating 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 occa-
sionally death for the hapless host.
E. Lymphocytes
In the immune/in?ammatory 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 de?ciency. 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
10Peroxidase catalyzes the following reaction:
H
O
2 R C OH
H O
2 2
2 R C
2 H O
2
H
H
The “purpose” of the enzyme is to generate bactericidal aldehydes from peroxide generated naturally
by phagocytes. Whether peroxidase is important to basophil function is not known.
11Acid and alkaline phosphatases are among the most commonly measured enzymes in the
laboratory. Nevertheless, their functions in physiology are unknown.
12Periodic acid-Schiff reaction (PAS) is an oft-used stain to detect the presence of large quantities of
complex polysaccharides in anatomic structures. The reaction is positive for any molecular species
that has a large number of diglycol (…-CHOH-CHOH-…) linkages.

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The Red Cell and Anemia
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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 fol-
lows are a few general remarks concerning examination of lymphocytes in pe-
ripheral blood.
After neutrophils, lymphocytes are the most numerous of the circulating
leukocytes. The normal range of the lymphocyte count is 1000 – 4800/µL. 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 capa-
ble 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? 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 identi?cation.
When activated by whatever means, lymphocytes can become very large (ap-
proaching or exceeding the diameter of monocytes) and basophilic (re?ecting
the increased amount of synthesized cytoplasmic RNA and protein). The cyto-
plasm becomes ?nely granular (re?ecting 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. Al-
though such cells are classically associated with viral infection (particularly in-
fectious mononucleosis), they may also be seen in bacterial and other infections
and in allergic conditions. A morphologic pitfall is mistaking them for mono-
cytes (a harmless mistake) or leukemic blasts (not so harmless).
V. 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 hemato-
logical abnormality, never to realize there is nary a platelet in sight. It is therefore
necessary to discipline yourself to ?rst check for a normal number of platelets when
sitting down with a slide, before being seduced by the midnight beauty of the baso-
phil’s alluring granules or the monocyte’s monolithic sovereignty. The normal plate-
let count is 133 – 333 × 103/µL.
Platelets are counted by machine in most hospital labs and by direct phase micros-
copy in smaller facilities. Since platelets are easily mistaken for garbage (and vice

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