ERYTHROPOISES.docx - Ning by wuyyok






                                  RELATED INFORMATION


The burst-forming unit - erythrocyte (BFU-E) represents the next stage in
differentiation from the myeloid stem cell for the RBC line. At this stage, this cell is
committed to eventually become an erythrocyte. The cell has the potential to rapidly
proliferate, forming up to 1,000 replicates. This is the most primitive dedicated
erythrocyte precursor cell. This cell, if observed on a slide, would be designated as an
undifferentiated blast cell.


The colony-forming unit - erythrocyte (CFU-E) is the next maturation step toward an
erythrocyte. It is an immature cell capable of proliferating up to 64 cells. This cell if
observed on a slide would be designated as an undifferentiated blast cell.

              Erythrocytes: Morphology and Physiology

This teaching syllabus discusses the morphology and physiology of the erythrocytes
appropriate for basic hematology curriculum. All objectives listed are


All blood cells originate from the undifferentiated or pluripotential stem cell. There
are three general maturation sequences that are common to all cells.
[1] All immature cells become progressively smaller as they mature.
      A. The cytoplasm undergoes changes that affects it staining properties. The
           cytoplasm in the mature cell demonstrates strong basophilia or blue
           coloration. This is due to the amount of large amounts of RNA present
           As the cell matures, the cytoplasmic RNA content decreases and the
          cytoplasmic hemoglobin content increases.

B. There are changes in the nucleus. In essence, the nucleus undergoes a decrease in
mass and becomes smaller. The nuclear material in the immature cell has an affinity
for eosinophilic dye to give it a predominately red color. As the nuclear chromatin
condenses, the color changes to a predominately dark blue color. The nucleus, as it
condenses, becomes more and more coarse and clumped. This causes much variation
in the leukocytes. In the erythrocyte, the nuclear mass is ejected, leaving the
erythrocyte as a anucleate cell.

The nucleus becomes small and there is variation between the erythroid, myeloid,
and lymphoid lines.

C. The nuclear chromatin material in the immature cell changes from its loose,
delicate, and spreading strands to form wider strands that are more coarse. The
nucleoli that are present in the immature cell as islands of metabolic activity do not
have a definite membrane. The nuclear chromatin compresses around the nucleoli to
give the appearance of membrane-like structures. The nucleolus consists of RNA that
decreases as the cell matures. The function of the nucleolus is to synthesize
cytoplasmic RNA. This function ceases in all cell lines, except the lymphocyte,.as the
cell matures and the nucleus condenses.

D. Once the maturing erythrocyte can be identified by cytochemical staining, there
are eight days in the maturation sequence of the rubriblast(ERYTHROBLAST) to the
mature erythrocyte. There is a cell volume decrease in the rubriblast of 500 fL to
about 90 fL in the erythrocyte.

Synonym: Pronormoblast. This is the earliest immature cell that can be recognized as
an erythrocyte precursor. Its size is 14 to 20 μM. The cytoplasm is deeply basophilic
and exists as a small band about the nucleus. The cytoplasmic area adjacent to the
nucleus stains lightly and is designated as a perinuclear halo. The cytoplasm is
nongranular. The nucleus is large, occupying a large part of the cell. The nuclear to
cytoplasm (N/C) ratio is 8/1. The nucleus is round or slightly oval with a fine
chromatin pattern characterized by fine clumping. Wright’s stain gives a reddish-
purple color. Usually 1-2 nucleoli may be observed. The nucleoli tend to larger (when
compared to the nucleoli of the myeloblast) and have a bluish tint. The nucleus may
be eccentric. This cell is capable of mitosis, producing two rubriblasts.


Synonym: basophilic erythroblast) The prorubricyte has an average diameter of 12 to
17 μM. It cytoplasm is very basophilic. Hemoglobin synthesis is occurring but is
masked out the large amount of RNA synthesis. The cytoplasm is basophilic and
continues to be nongranular. The nucleus is large with a round to slightly oval shape
and eccentric. The nuclear to cytoplasm (N/C) ratio is 6/1. The chromatin is
condensing, becoming more coarse. Parachromatin areas are appearing. The nucleus
takes on a deep purple-red color. Nucleoli (as a rule) are not visible. This cell can
undergo mitosis yielding two prorubricytes.

Parachromatin is a chromatophilic substance that stains lightly in the nucleus. It is
non-gene bearing. Euchromatin is the chromatophilic gene-bearing material (DNA)
and globulins. It stains darkly.

06.    DISCUSS THE POLYCHROMATIC ERYTHROBLAST                         .

Synonym: Polychromatic erythroblast/intermediate normoblast. The rubricyte
averages 10 to 15 μM in diameter. Its cytoplasm is taking on a pink hue as synthesis in
RNA and hemoglobin shifts. The cytoplasm will be variable with colors between blue
gray to pink-gray. The cytoplasm continue to be non granular and a perinuclear halo
may be present. The nucleus is undergoing changes, becoming more pycnotic, round,
and smaller as the chromatin condenses and increases the intensity of its clumping.
There are distinct areas of parachromatin. The nucleus will stain darker, a deep blue-
purple.. Dependent upon the degree of clumping, the nucleus of some rubricyte’s may
take on a clumped appearance. The N/C ratio is around 4/1. This cell represents the
last stage in the maturation sequence of erythrocytes in which mitosis can occur. It
can divide forming two rubricytes.


Synonym: Orthochromic normoblast. The metarubricyte measures from 7 to 12 μM in
diameter. Its cytoplasm is pink to pink-orange. Gray or bluish tones, if observed are
due to the presence of residual organelles scattered in the cytoplasm. Hemoglobin
production is increased and the cytoplasm has increased in relation to the nucleus.
N/C ratio = 1:2. The nucleus is condensed and the chromatin in pycnotic with a
homogenous appearance. The nucleus stain blue-black and either centric or eccentric.
It is not unusual to find an occasional metarubricyte on the slide with the nucleus
protruding from the cell. This cell can squeeze through the capillary walls into
systemic circulation by diapedesis.


Synonym: immature erythrocyte, “retic”, juvenile RBC, or neocyte. Its size ranges
from 7 to 10 μM, usually slightly larger than a mature RBC. The cytoplasm stains from
pink to pink-gray and contains aggregates of RNA reticulum. This reticulum can be
seen when the cell is stained with a vital stain. The nucleus is absent. The
reticulocyte will quickly eliminate the reticulum, becoming a mature RBC and living
an average of 120 days.

The reticulocyte is an index to RBC turnover. Once the nucleus is extruded from the
metarubricyte, it takes 4 - 5 days for the reticulocyte to loose it reticulum and
become a mature RBC. The “retic” cell spends about three days in the bone marrow.
It is slightly larger than the mature RBC and when stained with Wright’s stain, it
appears polychromatic (the grayish or bluish tones). The blue tones are referred to as
polychromasia or diffuse basophilia. Wright’s stain does not stain or demonstrate the
reticulum present in this cell. It is referred to as a polychromatic erythrocyte and
represents the “retic” cell. About the third day, the “retic” cell moves into general
blood circulation. It will take approximately another 24 hours for the RNA reticulum
to disappear from the cell. If there is a demand by the body for more RBC’s in general
circulation, then more “retic” cells will be shifted into the blood stream earlier than
normal. These larger and more immature forms will be seen on the blood smear as
polychromatic “retics”. Because they are the more immature forms, they will
circulate in the general circulation longer. The more severe the anemia or blood loss,
the greater the number of shifted retics. The normal retic count for the adult is 0.5%
to 1.5%. For the newborn, retic counts of 2.5% to 6.5% are considered normal. The
newborn’s retic count will fall to the adult level in about two weeks.

The Retic-Production Index (RPI) is a mathematical manipulation to measure
erythropoietic activity when “stress” or “prematurely released” reticulocytes
are present. It eliminates the error of using the simple reticulocyte
calculations that gives it answers in percentages. If the bone marrow
production is increased due to erythrocyte stimulation, reticulocytes are
being prematurely released into blood circulation (before their usual 2 to 3
day maturation period. These immature retic cells appear as large
polychromatophilic erythrocytes. The more reticulum in the cell, the more
immature the cell.


Synonyms: RBC, discocyte, normocyte, akaryocyte, erythroplastid. The mature
erythrocyte ranges from 6 to 8 μM. Its cytoplasm stains pink to pink-orange. (NOTE:
The color will vary dependent upon the quality of stain and type of stain used. This
will be true for all cells seen in a stained blood smear.) It is a non-nucleated,
nongranular cell, and contains no inclusions. If inclusions are observed, then
something is wrong. It as round and biconcave shape. It is a membrane sack filled
with hemoglobin (90%) and water (10%). Enzymes are present so that glycolysis can
occur. The membrane is semi-permeable and deformable. RBC’s are subjected to a
variety of osmotic forces, undergoing mechanical stressing when passing through the
liver, and capillaries. Some textbooks reports that the RBC will travel about 300
miles before being “recycled.


In normal conditions, the reticulocyte will take about 3.5 days to mature in the bone
marrow then they are released into peripheral circulation where they will eliminate
the reticulum in about 24 hours to become a mature RBC. If anemia is present in an
individual, there is a corresponding increase in erythropoietin production. This
hormone decreased the maturation time spent in the bone marrow by the reticulocyte
and it precursors. The early released reticulocytes are larger than the normal RBC and
take on a bluish hue in Wright’s stain. These cells are called shift cells.

The hematocrit is an index to the degree of anemia. Hematocrit values have been
interpolated into maturation time factors for calculating the reticulocyte production
index (RPI). A hematocrit range of 40% to 49% has the designation of 1.0. For a
hematocrit of 30% to 39%, the designation is 1.5. A hematocrit of 20% to 29% become
2.0 and a hematocrit of 10 to 19 become 2.5. Consider the following table to show the
correlation of the hematocrit with bone marrow and peripheral blood.

The RPI become an indicator of the degree of bone marrow response to anemia. This
calculation is a shift correction for the corrected reticulocyte count. The formula is as

         % retic count (X) hematocrit (L/L)/0.45 (LL)
RPI = ------------------------------------------------------
            maturation time in peripheral blood

Sample problem: A patient has a reticulocyte count of 6.5% with a hematocrit of
26%. Calculate the Reticulocyte Production Index (RPI)

              6.5 (X) 0.26 (L/L)/0.45 (L/L)
        RPI = -------------------------------------
                 6.5 (X) 0.58         3.76
        RPI = ----------------  = --------- = 1.88
                     2.0              2.0
This means that the RBC production rate has increased by 1.88 times. This would be
deemed to be an inadequate response. By convention, it is agreed that a RPI value less
than 2.0 is an inadequate response. A RPI value of 3.0 is considered to be an
appropriate bone marrow response to anemia.


The number of reticulocytes must be determined by counting the retic cells in 1,000
RBC’s. The next step is to divide the number of retic cells by 1,000 and multiply by
100. This will give an answer in percent. Example problem. If you counted 65 retic
cells per 1000 RBC’s then divide 65 by 1000. Your answer is 0.065. Multiply this by 100
and obtain 6.5. Your answer is in percent and is the relative count.


The corrected reticulocyte count (CRC) is also called the hematocrit correction and
reticulocyte index. A lab report of a relative reticulocyte count may give the
appearance of an elevated retic count when it isn’t. the CRC adjusts the actual
number of reticulocytes to the hematocrit, giving a more reliable estimate. The
formula is as follows:
                                           Hematocrit (L/L)
              CRC = % retic cells (X) ------------------------------------
                                        normal hematocrit (0.45 L/L)

If a patient’s retic count is 6.5% and the hematocrit is 0.28 (L/L), then the formula
sets up as

CRC = 6.5% (X) 0.28 / 0.45 = 6.5% (X) 0.62 = 4.04%

A normal CRC is 1% if the hematocrit is between 40 to 48%. If the hematocrit is from
25 to 35% and the CRC falls between 2 and 3%, then “retic” cell production is normal.
For a hematocrit that is less than 25%, the CRC should be between 3 and 5 %. The
reticulocyte production index (RPI) is the preferred method for determining if there is
normal production of reticulocytes.


The round, biconcave nature of the erythrocyte membrane gives it maximum surface
area that is advantageous for gaseous exchange and increased deformability. It
composition is approximately 50% protein, 40% lipids, and 10% carbohydrates.
Morphologically it is composed of two layers of phospholipids, arranged so that the
polar surfaces face the inside and outside of the cell. The non-polar groups are
directed to the center of the membrane layer.

The proteins in the RBC membrane account for its shape, structure, and ability to
change shape. These proteins are also the channels and pumps to move ions and other
molecules in, out, and across the membrane. Some of the proteins function as
receptors, many of the proteins function as the RBC antigens (ABO, and Rh), other
proteins have enzymatic capability, and all in some degree or another help to
stabilize the membrane.

If the molecular composition of the RBC membrane changes, the membrane is
affected inducing changes in its shape or ability to transport ions and molecules. If
the cholesterol content of the membrane increases, the membrane takes on the
appearance of a target cell or spicules develop to form the acanthocyte. If abnormal
proteins are incorporated into the membrane, the cell may become an elliptocyte or
spherocyte. If proteins are lost, for whatever reason, the integrity of the membrane is
compromised and hemolysis will result. It has been found that some of the RBC
membrane antigens are essential for membrane integrity.


Erythrocytes must be able to metabolize in order to remain viable. The cell has the
ability to metabolize glucose through the glycolysis cycle (Embden-Meyerhof
anaerobic pathway) for ATP production. ATP is needed to run the membrane pumps
(example: Na+ and K+ exchange) which helps to control membrane integrity and cell
osmolarity. Energy is required to maintain cell function, membrane shape, and to
protect the lipid composition of the cell.

The glycolysis of glucose to ATP provides energy through the Rapoport-Leubering
pathway. This is a metabolic strategy to produce 2,3-Diphosphoglycerate (2,3-DPG).
2,3-DPG has an affinity for oxyhemoglobin, which causes the hemoglobin molecule to
release it oxygen to the tissues. 2,3-DPG inserts itself between the β-chains of
hemoglobin, causing electrostatic interactions, which facilitates displacement of
oxygen molecules into the tissues. With increased oxygen pressure in the lungs, the
increased number of oxygen molecules displace the 2,3-DPG molecule. Note: 2,3-DPG
is also known as 2,3-Bisphosphoglycerate (2,3-BPG).

The Pentose-Phosphate pathway converts oxidized glutathione to it reduced form.
Reduced glutathione stabilizes the reduced state of hemoglobin. If reduced
hemoglobin changes to the oxidized form, then it will denature and precipitate out as
Heinz bodies. Glutathione maintains hemoglobin in its reduced state (Fe++),
preventing oxidation of the sulfhydryl groups in the hemoglobin molecules and further
reduction to Fe+++ (methemoglobin). Accumulation of methemoglobin will change the
structure of the cell membrane, weakening it, and rendering it susceptible to
rupture/hemolysis. Increased methemoglobin will eventually precipitate to form
Heinz bodies. As a rule, no more than 1% of the cell’s hemoglobin is in the
methemoglobin form (some textbooks suggests the 3% may be the upper normal limits
for methemoglobin concentration).


Such evaluations enable a conformation of a diagnosis, provides visual criteria for
classifying anemias, and describes RBC anomalies in terms of size, shape, and degree
of hemoglobinization.

Erythrocyte size varies from microcytic to normocytic to macrocytic. Microcytic is
characterized by [1] a MCV = <80 fL and [2] size = <6 μM. Microcytes are observed in
iron-deficiency anemias. Normocytic is characterized by [1] MCV = 80 to 100 fL and [2]
size = 6 to 9 μM. Macrocytosis, observed in hepatic diseases and vitamin B12 and folic
acid deficiency anemias are distinguished by an MCV = >100 fL and size = >9 μM. In
macrocytosis, cells tend to maintain a round shape. (Refer to objective 69 for an
explanation of the indices.)

Erythrocyte size is described by the term anisocytosis. Anisocytosis is one of the most
common forms of abnormal RBC’s and can be associated with a variety of disorders
(leukemia, pernicious anemia and other forms of anemia). See Objective #18 to grade
the degree of anisocytosis present.

Poikilocytosis indicates a variation in the shape of erythrocytes. A deviation for the
normal discoid shape of the erythrocyte is the result of a chemical or physical
alteration in the red blood cell membrane or the actual contents of the cell. Because
of the variety of shapes seen in erythrocytes, specific names have been assigned to
the red blood cell to describe it shape. Examples are: acanthocytes, blister cells,
echinocytes, elliptocytes, and target cells. Poikilocytosis may be associated with a
variety of anemias.

Erythrocytes hemoglobinization is describes as either normochromic, hypochromic, or
hyperchromic. A normochromic RBC describes the presence of a normal amount of
hemoglobin in the cell and that it stains uniformly, evenly. Its MCH = 27 to 32 pg
(μμgm) and the MCHC = 31 to 37%. (Refer to Objective #69 for an explanation of the
indices.) Hypochromasia (also known as hypochromia) indicates that the RBC contains
a decreased amount of hemoglobin. Visually, a larger than normal central area of
pallor or paleness will be present, with an thin rim of hemoglobin . This is one of the
most common forms of abnormal erythrocytes, seen in iron-deficiency anemia and
thalassemia. It may also be seen in any hemoglobinopathy. The MCH = <27 pg (μμg)
and the MCHC = <31%. The following illustration will assist you in grading the degree
of hypochromia.

Hyperchromasia (also called hyperchromia) implies a heavy staining of the red blood
cell. Usually it is difficult to correlate over saturation of hemoglobin in the
erythrocyte. This type of appearance is seen in extra thick RBC’s or spherocytosis.
Indices values may be of little value in this condition. MCH values usually do not
differentiate macrocytosis, however MCV values will be increased. MCHC values do
not exceed 36 or 37% as a rule. Values of 38%, when obtained, are a maximum value
and for all practical purposes are of no value. Polychromasia (also called
polychromatophilia) describes the erythrocyte that has taken on a bluish hue. The
presence of polychromasia can be correlated to the number of reticulocytes and
indicates that the younger and larger RBC’s are being shifted into general
circulation.. If polychromasia is observed, a “retic” stain may be needful. Also note
the MCV values, these should be slightly increased (slightly greater than 100 fL’s.
Refer to the grading scale in Objective #18 for hypochromasia, hyperchromasia, and


Cell Type Diameter Thickness Volume    Example
Spherocyte 6.18 μ   3.02 μ    90 fL  Chronic hemolytic jaundice
Microcyte I 7.07 μ   1.63 μ   63 fL  Simple microcytic anemia
Macrocyte I 8.89 μ   2.20 μ 135 fL    Pernicious anemia
Macrocyte II 8.58 μ  1.60 μ    92 fL  Obstructive jaundice
Normocyte 7.7 μ      2.00 μ    90 fL  Normal
Microcyte II 6.5 μ   1.50 μ   <60 fL  Thalassemia

One method that is used in some laboratories is the following description. Red blood
cell anomalies may be graded on a scale of normal, slight, and 1+ to 4+ as follows:
Normal = less than 5% of RBC’s differ in shape, size, or hemoglobin intensity from the
surrounding pattern of normal round, discoid RBC’s. Slight = approximately 5% to 10%
of the RBC’s differ from the normal cells. 1+ = 10% to 25% differ from the normal
cells, 2+ = 25% to 50% difference from the normal cells, 3+ = 50% to 75% differ from
the normal cells, and 4+ = >75% differences from the normal cells.

The following scale is used by many laboratories in the delta region of the U. S.
morphology                  normal      1+           2+       3+         4+
characteristics              limits
Macrocytes (>9 μ dia.)         0-5     5 - 10    10 - 20    20 - 50      >50
Microcytes (<6 μ dia.)         0-5     5 - 10     10 - 20    0 - 50      >50
Hypochromia                   0-2      3 - 10    10 - 50    50 - 75      >75
Poikilocytosis                0 -2     3 - 10    10 - 20     20 - 50     >50
Anisocytosis                   0-2     3 - 10     10 - 20   20 - 50       >50
Acanthocyte                   none     1-5         5 - 10   10 - 20       >20
Burr Cell                      0-2      3 - 10     10 - 20    20 - 50     >50
Target cell (codocyte)         0-2      3 - 10     10 - 20    20 - 50     >50
Tear drop cell (dacryocyte)    0-2       2-5        5 - 10    10 - 50     >50
Sickle Cell (depranocyte)      none (If present in any number, report as positive.)
Elliptocyte/Ovalocyte          0-2       2 - 10     10 - 20   20 - 50     >50
Helmet cell / Bite cell         none      1-5        5 - 10    10 - 20     >20
Schistocytes                   none      1-5        5 - 10     10 - 20    >20
Spherocytes                 0-2          2 - 10   10 - 20    20 - 50    >50
Stomatocytes                0-2          2 - 10   10 - 20     20 - 50   >50
Basophilic stippling         0-1         1-5       5 - 10     10 - 20   >20
Polychromatophilia, adult    0-1         2-5       5 - 10     10 - 20   >20
Polychromatophilia, infant    1-6        7 - 15   15 - 20     20 - 50    >50
Howell-Jolly (HoJo) body      none       1- 2      3-5         5 - 10   >10
Pappenheimer body (siderocyte) none       1-2      3-5         5 - 10    >10

These enumerating values are expresses as the number of occurrences per OIF
assuming an average of 200 to 250 RBC’s per the 100X objective.

For grading rouleaux formation, use the following criteria:
   [1] 1+ = aggregates of 3 to 4 cells
   [2] 2+ = aggregates of 5 to 10 cells
   [3] 3+ = numerous aggregates of RBC’s. Only a few free RBC’s are observed.


Poikilocytosis (poikilocytes) describes the variety of nonspecific shapes that may be
observed in RBC’s. Poikilocytosis is an irreversible alteration of the cell membrane
and is an indicator of abnormal erythropoiesis due to bone marrow effects and/or
abnormal RBC destruction. This is one of the most common forms of abnormal RBC
morphology. There is a poikilocytosis expression that occurs as the RBC ages
(senescence). The RBC will become pinched, pitted, or notched as the membrane
breaks down and sloughs off.


The spherocyte is an erythrocyte in which the biconcave disc profile is lost. It appears
as a smaller and more dense RBC. It is also called a hyperchromic microspherocyte.
The spherocyte is formed when there is a defect in the membrane function. The
sodium pump causes Na+ retention which increases water retention, increasing the
intravascular volume. This cell is observed in immune induced hemolysis, post blood
transfusions, and congenital anemia. Comment: Fine needle like projections have
been reported as being observed in the membrane surface.

The echinocyte is a crenated erythrocyte. Laboratory vernacular will refer to this cell
as a crenated RBC. Crenation is usually not an indicator of a pathological problem. It
is usually an artifact due to [1] loss of intracellular fluid, [2] increased anti-coagulant
blood ratios (due to a technique during a phlebotomy or manufacturing error in
measuring anticoagulant in the vacutainer tube), [3] slow drying of the blood film, or
[4] the patient being dehydrated. Look for rounded, regular, smooth,-tipped
projections all around the periphery of the cell. Report as crenated cells present.
Sometimes these are graded. Follow lab policy.


The acanthocyte is an abnormally crenated RBC. It is the consequences of a defect in
the cell membrane. Projections from the cell membrane are irregular and distorted
with the apex of the projections being pointed. Synonyms include: thorn cell, spur
cell, and spicule. These cell types are observed in abetalipoproteinemia, liver
disorders, and lipid metabolism disorders. Their presence has been reported in
patients on heparin therapy. Use the grading scale described in Objective #18.
Remember that acanthocytes should not be observed in a normal stained blood film.
When they are seen, be sure to indicate their presence. Follow lab policy for

The burr cells is characterized by abnormal cytoplasmic projections, but not to the
same extent as that of the acanthocyte. It is characterized by regular pointed
projections with regular shaped curves. There is an overall uniform spacing. These
cells are observed in uremia, acute blood loss, stomach cancer, and pyruvate kinase
deficiency. Note: Some labs consider the burr cell and the crenated cell as the same
cell, therefore do not distinguish between the two.


Schistocytes (fragmented cells) are fragments of erythrocytes with wide variation in
sizes and shapes, ususally microcytic in size. Schistocytes are seen in vascular
lesions, uremia, microangiopathic hemolytic anemias, hemolytic anemias cause by
physical agents, and disseminated intravascular coagulation (DIC), whenever there is
blood vessel pathology present. Schistocytosis is the result of mechanical trauma in
the spleen and interaction with intravascular fibrin strands.


Synonyms: ovalocyte, pencil cell, or cigar cells. Normally about 5% to 10% of the
circulating RBC’s are oval. These cells are formed after the erythrocytes matures and
leaves the bone marrow. Patients diagnosed with elliptocytosis tend to have normal
shaped reticulocytes. The mechanism that causes elliptocytes is not known. There is
known to be a hereditary defect present in the RBC cytoskeletal proteins (the spectrin
chain). These cells observed in varying percentages in iron deficiency anemia,
leukemia associated anemias, thalassemia, and dyserythropoiesis. In most anemias,
elliptocytes may make up 10% of the RBC population. Patients with congenital
elliptocytosis may demonstrate up to 90% distinctly oval shaped cells.

Synonym: Mexican hat cell and codocyte. This cell is characterized by an abnormally
thin membrane with an increase incorporation of cholesterol into the cell membrane.
It appears because of maldistribution of abnormal hemoglobin or certain materials
being deposited into the cell membrane. These cells are more resistant to hypotonic
lysis. It is observed in hemoglobinopathy, hepatic diseases, iron deficiency anemia,
hemolytic anemia, and splenectomy.

Leptocytes are thin cell that is large in diameter and generally displays a thin rim of
hemoglobin at the periphery and a large area of central pallor. This cell is cup-
shaped as is the stomatocyte but it has very little depth. Target cells are thought to
from from this cell type if the cup depth increases. This cell is seen in liver disease
and hypochromic anemias.


Synonym: sickle cell and meniscocyte. Lab vernacular refers to these cells as sickle
cells. They are associated with the disorder, sickle cell anemia. Like the target cell,
sickle cells are resistant to hypotonic lysis. There are two basic types of sickle cells;
[1] the oat cell, slightly sickled variation, and/or holly leaf. These RBC collapses into
these shapes when there is a reduced oxygen atmosphere. In the presence of a normal
oxygen atmosphere, the cells revert to the normal discoid shape. [2] The second type
form very distorted filamentous forms. In the presence of a reduced oxygen
atmosphere these cells form, but when the oxygen pressure is normalized, they do
NOT revert back to the normal discoid shape. Sickle cells are also observed in
hemoglobin Sβ-thalassemia anemia and hemoglobin SC anemia. Doe not grade these
cells. They are to be reported out as positive, if present.


The stomatocyte is characterized by a slit-like or narrow rectangular area of pallor in
the cell. This cell will be concave on one side and convex on the other. These cells
are characterized by a alteration in the permeability of the cell membrane to sodium.
It is observed in liver disease, alcoholism, electrolyte imbalance, hereditary
stomatocytosis, infectious mononucleosis, lead poisoning, malignancies, and
thalassemia minor. Note that these cells may appear as artifacts on a stained blood
smear. One textbook states stomatocytes when seen are more apt to be an artifact
than a pathological process.


The spheroidocyte is a thicker than normal erythrocyte. It does have an increased
amount of hemoglobin. There is usually a smaller area of pallor in the center of the
cell and the area of pallor may be located eccentric.


Synonym: Tear-drop cell or tennis racket cell. The cell has a definite tear drop shape
and the length of the “tail” may vary from cell to cell. Small areas of pallor may be
present on the cell. The tear-drop cell is observed in pernicious anemia, thalassemia,
myeloid dysplasia, severe anemia, and hemolytic anemia.


The siderocyte is an erythrocyte that contains deposits of iron in the cytoplasm that
stain dark blue with Prussian blue stain. The number of granules vary, often there are
more than one granule present in a cell. These granules (which are aggregates of
mitochondria, ribosomes and iron particles) may be called Pappenheimer bodies. If
more than 10% of the RBC’s contain these granules, then abnormal hemoglobin
synthesis is present as seen in hyposplenism and hemolytic anemia. Pappenheimer
bodies may aggregate so that they resemble a stack of cannonballs. It is not necessary
to enumerate the Pappenheimer bodies, it is sufficient to indicate the presence as
positive or negative.


Basophilic stippling (also called punctate basophilia), is characterized by the presence
of numerous granules in the erythrocyte. These blue granules may be fine or coarse
and may be intense in color. The granules are aggregates of ribosomes and are evenly
distributed in the cell. They are observed in lead poisoning, hemoglobinopathy,
alcoholism, and megaloblastic anemias. If the “stippling” is coarse, it may be referred
to as punctate stippling. Do not grade basophilic stippling. Report is as “positive” if it
is present.

The Howell-Jolly body (or HoJo bodies) are round, purple staining nuclear DNA
fragments. They may be 1.0 μM in diameter. They are usually observed in the mature
erythrocyte, but may also be seen in the nucleated and immature red blood cell. As a
rule, only one Howell-Jolly body is seen per cell and some times two. More than
two/cell are not the rule. They are formed during the process of karyorrhexis, usually
in the megaloblast. If more than two “HoJo bodies” are present in the red cells, then
the patient may have megaloblastic anemia. They are also observed in hemolytic
anemias, pernicious anemia, post-operative conditions, splenectomy, or splenic
atrophy. If Howell-Jolly bodies are present, report out as positive.


The Cabot ring is a purple staining ring-like filament or figure-8 and is thought to be
formed from the microtubules of the mitotic spindle. This inclusion may be present as
a double or triple ring. If observed, it is most likely to be seen in severe anemias
(example: pernicious anemia) and lead poisoning. It is generally thought to be due to
abnormal erythropoiesis. If present, report it out as positive for Cabot rings.


Hemoglobin crystals are seen as tetragonal shaped crystals, found in Hemoglobin C
and Hemoglobin SC disease. If the condition is severe, then up to 10% of the RBC’s
may contain these crystals. In the case of Hemoglobin SC disease, the crystals may
show greater variation. Hemoglobin C crystals may be demonstrated by washing the
red blood cells and suspending them in sodium citrate. Hemoglobin C crystals are
precipitated polymers of the beta chains of hemoglobin A. If hemoglobin C crystals
are observed, then so are target cells (as a rule). If Hemoglobin C crystals are
present, do not enumerate, just report that they are present.


Rouleaux formation are RBC’s arranged in rows or stacks. They are sometimes present
as a slide artifact due to a delay in the spreading of blood or the settling out
phenomenon in the thick portion of the blood smear. Rouleaux appears in chronic
inflammatory disorders, multiple myeloma, hyperproteinemia, and Waldenström’s
macroglobulinemia. Increased amount of fibrinogen in the blood can cause rouleaux
formation. If rouleaux is noted in the thick portion of the stained blood film but not in
the thin portion, it is probably an artifact. If rouleaux is noted to extend into the thin
monolayer portion of the smear, then it is pathological. If rouleaux is NOT an artifact,
but represents some pathologic problem, report as follows: If the cells are arranged in
aggregates of 3 to 4 RBC’s, report as 1+; if aggregates of 5 to 10 RBC’s, then report as
2+, and if the aggregates are so numerous that only a few free RBC’s, report as 3+.


Agglutination occurs when cold agglutinins or autoimmune hemolytic anemia are
present. The RBC’s do not stack as in rouleaux, they will clump randomly. If
agglutination is present then the automated RBC counts and cell sizing will not be

Synonyms: Half-moon cell, semilunar body, crescent cell. These are faintly staining
RBC’s that have a quarter-moon shape. They are thought to be ruptured RBC’s. Their
size approaches that of a WBC. They are observed in malaria and hemolytic anemias.
If an occasional crescent shape is seen, do not report. If a significant number of
crescent bodies are noted on the blood spear, include the observation in your report.

Do not grade unless required by the laboratory. View the “neighborhood” for


Microspherocytes appear in the blood as small round cells and are the result of
intravascular hemolysis. This cell type is seen in patient who receive burns over a
minimum of 15% of their bodies. It is thought that the heat, that the burned part of
the body experiences exerts a direct effect upon the RBC’s to produce fragmentation,
budding, and microspherocyte formation. Experiments conducted by heating RBC’s to
49 oC demonstrated this fragmentation phenomenon of erythrocytes. These cells are
mechanically and osmotically fragile and are rapidly removed from circulation. This
phenomenon is characterized by tiny cell diameters of 2 to 4 μM and a MCV that is <60

There is a hemolytic anemia disorder in which the cell membrane
protein (spectrin) is abnormal. The RBC will fragment, producing
similar fragments as seen in burn patients. These RBC fragments
are called “pyropoikilocytes.


Helmet cells are fragmented cells or schistocytes. They are also called “bite” cells.
They are cells that are defective and when traveling through the spleen, the
macrophages failed to remove the total cell. They are observed in pulmonary emboli,
myeloid metaplasia, and disseminated intravascular coagulation (DIC).


Knizocytes are RBC’s with more than two concavities. They tend to appear on the
blood smear as having a dark staining bar in the center of the cell with two areas of
pallor on either side. They have been describes as resembling a pinched bottle How
these are formed is not understood. They are observed in hemolytic anemia and
hereditary spherocytosis.


The blister cell is formed when the cell is injured and a portion of the hemoglobin
leaks out. If the blister breaks, a keratocyte is formed. Once the keratocyte is
formed, they are a fragile cell and will disappear from circulation in a few hours. It is
seen in end-stage renal disease, as an indicator of pulmonary emboli, also seen in
sickle cell anemia, and microangiopathic hemolytic anemia. Also, the vacuole in
blister cells is known to rupture and form keratocytes and/or schistocytes.

Synonym: horn cell. These cells form when an erythrocyte is “snagged” by a fibrin
strand and the cell is partially cut into. Part of the cell fuses back leaving two or
three horn-like projections. The keratocyte is a fragile cell and remains in circulation
for only a few hours. These are observed in diffuse intravascular coagulation (DIC).
There is a recommendation that keratocytes be reported as schistocytes. Follow lab
protocol in reporting these cell types.


Hemoglobin (hgb) is a red colored, conjugated, large molecular weight protein (mw =
64,458) that makes up about 28% of the RBC mass. Most of the RBC mass is water.
Each adult hemoglobin molecule (designated as hgb A) consists of a quaternary
protein molecule that consists of four globulin (polypeptide) sub-units. The four
globulin chains constitute a tetramer. Two of the sub-units are designated as α-chains
and the other two subunits are the β-chains. Each subunit contains one heme
structure which binds the oxygen molecule to form oxyhemoglobin. See the following
The hemoglobin chain is manufactured in the cytoplasm of the cell by the ribosomes.
Hemoglobin synthesis begin in the prorubricyte (basophilic normoblast). By the time
the developing erythrocyte has matured to the metarubricyte stage, about 66% of the
hemoglobin formation has been completed. The completion of hemoglobin synthesis
occurs in the reticulocyte. The heme structure is manufactured in the mitochondria
and cytoplasm in five basic steps. [Step 1] The Kreb’s cycle provide a porphyrin
precursor in the mitochondrion, [Step 2] The formation of the porphyrin ring occurs in
the cytoplasm, [Step 3] The porphyrin rings are assembled into the
coproporphyrinogen III (CPG). [Step 4] The CPG molecule is transferred into the
mitochondrion for transforming to protoporphyrinogen IX (PPG). [Step 5] The final
step is inserting a single ferrous (Fe++) molecule to form heme. Heme is expelled
from the mitochondrion to the cytoplasm where it combines with an α- or β-globulin
subunit to form a hemoglobin monomer. Two α-hgb monomers and two β-hgb
monomers combine to form the hemoglobin tetramer.

The function of hemoglobin is to transport oxygen to the tissues and return carbon
dioxide to the lungs. Each erythrocyte contains about 300 million hemoglobin
molecules and there are about 30 trillion RBC in the average adult body. One gram of
hemoglobin can combine with 1.34 mL of oxygen. In one liter of blood, about 195 mLs
of oxygen is bound to hemoglobin and 3 mLs. of oxygen is carried in the free form.

The globulin chain determines the classification of the type of hemoglobin.

Hemoglobin Structure Stage of Life     % in Adult        % in Newborn
Gower I      ζ2ε2   0-5 weeks Embryo    None             up to 40
Gower II     α2ε2   4-13 weeks Embryo    None             up to 35
Portland      ζ2γ2  4-13 weeks Embryo    None             up to 35
Fetal (F)    α2γ2    Newborn and Adult    <1.0                 80
A1           α2β2   Newborn and Adult     97                  20
A2           α2δ2   Newborn and Adult      2.5               <0.5

Iron is necessary for the production of hemoglobin. There is about 4.0 grams of iron in
the body. An estimated 65% of the iron is bound up as hemoglobin and up to 30% is
stored in the liver, spleen, and bone marrow. The remainder of the iron is bound to
myoglobin, transferrin, and ferritin. Iron is not synthesized in the body and must be
incorporated through the diet on a regular basis. Dietary iron is in both the ferrous
(Fe++) and ferric (Fe+++) forms in the GI tract. Only the ferrous form is absorbed and
this occurs primarily in the jejunum and duodenum. The body does an excellent job of
recycling iron so that only about 10% of dietary iron is being absorbed in the healthy
individual.. Some of the ferric iron is reduced to its ferrous form by the acidity of the
stomach. Once the ferrous iron is absorbed by the intestinal mucosal cells, it is
reduced to the ferric state as it combines with the protein apoferritin (a β1-globulin)
to form the storage molecule ferritin. It has been estimated that one apoferritin
molecule can bind up to 4,000 iron molecules. Once the mucosal cells are saturated
with iron, absorption ceases and any unabsorbed iron is excreted in the feces. The
mucosal cells release ferric (Fe+++) iron on body demand and release from ferritin
allows Fe+++ to be reduced to its ferrous (Fe++) form as it is taken up by the plasma
transport molecule, transferrin
(TRF) to form a ferric-transferrin complex. This complex is taken to the bone marrow
or other cells needing iron. The complex attaches to the cell’s receptor, then the
membrane invaginates, and the iron molecules are encased within a vacuole within
the cytoplasm. The iron dissociates from the complex and goes to the mitochondria
where it is incorporated into heme or bound with apoferritin to form ferritin. The
transferrin is ejected from the cell to repeat its transport function. The iron
incorporated as ferritin forms aggregates as it is stored in nucleated RBC’s. Note: Fe
and Fe+++ does not exist in the free form. With special stains, these iron stores can be
seen in the erythrocytes designated as siderocytes.

Transferrin is a glycoprotein (a β1-globulin) and is synthesized in the liver. Transferrin
(TRF) is known to exist in more than 30 variant forms, yet each variant can bind and
transport iron. TRF transports iron from the intestinal mucosal cells and the
mononuclear phagocyte system (originally called the reticuloendothelial system [RES])
to other cellular sites for metabolic activity. Most of the iron is delivered to the
developing rubriblast and the remainder for the synthesis of myoglobin, cytochromes,
catalases, peroxidases, and flavoproteins. TRF (type C) is the predominant from in
American Caucasians and Afro-Americans.


The total iron-binding capacity test (TIBC) is a test procedure that totally saturates
the protein transferrin (TRF) [but it will include other proteins with iron binding
capability] with iron. The TIBC test measures the resulting iron concentration to
arrive at the available amount of transferrin (hence it is functional measurement of
transferrin concentration). The normal value for the adult male and female is 250 to
460 μg/dL. Actually this test tends to over-measure the actual amount of transferrin
because iron can also be bound by albumin and certain other plasma proteins. In
normal conditions, when iron values are about 70 to 180 μg/dL, an estimated 30% to
35% of the transferrin molecules are bound with iron. In the TIBC test, from 15% to
50% transferrin saturation can be demonstrated in the normal individual with values
for female being somewhat lower than for the male. This test can be useful to help
diagnose disorders involving iron metabolism or anemias. The TIBC (ug/dL) can be
calculated indirectly by multiplying the serum transferrin value times 1.25.

The adult body contains about 3 to 5 grams of iron, with 2 to 2.5 grams being located
in the hemoglobin. A small amount (less than 150 mg) is found in myoglobin (an
oxygen binding protein of muscle). Iron is also a ligand or cofactor in enzymes,
enabling the enzyme (such as peroxidases, catalases, and cytochromes) to be
functionally active. Iron can be stored as ferritin and hemosiderin, which serves as an
important storage pool of iron. If an iron deficiency condition arises, these two pools
will become diminished. Iron deficiency is one of the most prevalent disorders,
affecting 15% of the world population. Iron is decreased in iron deficiency anemia,
malnutrition, malignancy, chronic infection, and anemia of chronic disease. Normal
values are shows as follows:
Male (adult):              50 to 160 μL/dL
Female (16 to 40 y/o): 45 to 150 μL/dL
Child:                     50 to 120 μL/dL
Newborn:                  100 to 250 μL/dL
Iron is increased in iron poisoning, hematochromatosis, viral hepatitis, and
sideroblastic anemia.


This test is also known as the percent saturation test and is simply a mathematical
comparision of the serum iron to the TIBC. Calculate it as follows: Divide the Total
Iron (μg/dL) by TIBC (μg/dL) and multiply by 100% to obtain the % saturation.
Example problem: if the total iron is 100 μg/dL and divided by TIBC of 350 μg/dL, a
result of 0.2857 is obtained. Multiple 0.2857 by 100% and the % saturation (or
transferrin saturation) becomes 28.57 %. The μg/dL for both total iron and TIBC cancel
out and vanish to leave a percent value. Normal values are as follows:
Male (adult):                20 to 55%
Female (16 to 40 y/o)        15 to 50%
Child, newborn, and infant: 12 to 50%


Ferritin is a multi-unit (24-subunits) protein that takes on the form of a shell and
contains approximately 4,500 iron atoms. The iron is deposited in the core of this
large molecle as a ferric hydroxyphosphate complex. In this way, the toxic action of
extra iron is prevented. If the ferritin shell is void of iron, it is then known as
apoferritin. It is found in most cells and can be quickly mobilized to store iron. Little
ferritin is found in human plasma, but will be elevated if there is an excess of iron.
The amount of ferritin in plasma is used as an index of body iron stores. The synthesis
of ferritin is connected to the level of iron in the cells. When iron levels are high,
mRNA (ferritin type) is activated (or stabilized) to produce more ferritin. When iron
stores are decrease, the ferritin type mRNA is destablilized (deactivated) and
production of ferritin ceases. Normal levels of ferritin are as follows:
Adult (male):             20 to 250 μg/L
Female (16 to 40 y/o): 10 to 120 μg/L
Child:                     7 to 140 μg/L
Newborn:                   25 to 200 μg/L
Ferritin is elevated in sideroblastic anemia and hemochromatosis, but it is decreased in
iron deficiency anemia.


Transferrin is a beta-1-globulin, a glycoprotein that is synthesized in the liver. It
transports iron through the circulatory system to cells that require iron. There are
transferrin receptors on the cell allowing the binding the molecule to attach. The
transferrin enters the cell via endocytosis and the iron detaches from the transferrin
molecule and is passed into the cytoplasm where it is bound to ferritin to be held
until ready for use. The transferrin molecule does not detach from the cytoplasmic
membrane receptor and it will be returned back to outside the cell where it
dissasociates from the receptor and reenters the plasma to pick up more
iron. Normal values for plasma transferrin are:
Adult (male):               200 to 380 mg/dL
Female (16 to 40 y/o)        200 to 380 mg/dL
Child:                     200 to 360 mg/dL
Newborn:                    130 to 275 mg/dL
Transferrin is increased in iron deficiency anemia and decreased in iron overload and
hemochromatosis. If transferrin is tested for, it may be to access the nutritional
health of the patient. If there is an inflammatory process going on in the body, its
concentration will decrease.


Hemochromatosis can express itself an autosomal recessive disorder with a
progressive increase in iron stores. This creates a toxic condition and can lead to
organ impairment and damage. Males are affected more than females and the
disorder usually expresses itself around the age of 40 (or older). When the disorder
expresses itself, up to 4 mg of iron (possibly more) can be absorbed daily and the iron
is deposited directly to parenchymal cells (liver, pancreas, heart, and other organs).
Consequently, patients are at high risk for cardiac damage, liver cancer, and diabetes
(type I).

Hemochromatosis may be of the acquired type and in this case the iron is deposited
into the reticuoendothelial cells of the liver, spleen, and bone marrow. If the
condition persists, then iron is deposited in the parenchymal cells as in the hereditary
type. The acquired form of hemochromatosis may be a secondary response to
anemias or multiple blood transfusions. Chronic liver disease with alcoholism may
present with increased iron levels in the tissues.

In hemochromatosis the serum iron test, transferrin saturation, and serum ferritin,
are increased. The TIBC is decreased.

Free erythrocyte protoporphyrin is s product of the heme synthesis pathway. This
molecule escapes from the synthesis pathway and exists as a nonheme protoporphyrin
in the erythrocyte. It can be measured and the information it provides is clinically
useful. Normal reference levels range from 170 to 770 μg/L. If a condition arises in
which there is a iron deficiency or an impairment of the utilization of iron, FEP will
increase. Also lead poisoning will signficantly increase this product. This testing
procedure is a helpful screening test for iron deficiency. It was used to screen for
lead posioning, but more sensitive tests have replace it as a screening test. It is also
increased in anemia of chronic disease and may be in siderblastic anemia (but not
always). It is usually normal in hemochromatosis and thalassemia trait.


Hemosiderin is a poorly defined molecule that is formed by the degradation of
ferritin, yet still contains iron. It can be detected by Prussian Blue stain and is seen
histologically with there is excessive storage of iron. The iron stored in the cell is an
insoluble cellular inslusion of Fe3+ complexed with ferritin. These complexes form
granules (from 1 to 2 μm in diameter) that serve as as storage form of iron when there
is insufficient levels of apoferritin. The iron to ferritin ratio is much greater as
hemosiderin than in ferritin. Hemosiderin can release it iron, but does so at a very
slow rate. Hemosiderin, when seen on unstained smears, appears as yellow granules.
When present in Wright's or Wright's-Giemsa stain, the granules are bownish-blue
coloration. The presence or absence of hemosiderin is determined by cytochemical
staining of bone marrow using Prussian blue stain which is the preferred and precise
technique, which yields bluish granules. It is used to assess body iron stores. If
hemosiderin granules is absent, then the body iron stores are depleted. The presence
of granules may be reported as a numerical value from 0 to 4+, where 2+ represents a
normal or adequate iron store. Normal marrow will have 30 to 50% of the
erythroblasts containing specks of hemosiderin. These cells are called sideroblasts.

In the normal metabolism of hemoglobin, iron can be captured by the renal tubules
and complexed with storage proteins and ferritin to form tubular hemosiderin. When
these cells are sloughed off, hemosiderin can be demonstrated in the urine
(hemosiderinuria). Normally hemosiderin is not found in the urine. If present, it
indicates an unexplained anemia or chronic intravascular hemolysis.


The life span of an erythrocytes averages about 120 days for an adult male but only
109 days for an adult female. As the RBC circulates in vessels with a larger diameter,
the cell is a typical biconcave cell. When it circulates through capillaries, the
diameter reduces to 2 to 3 μM and the cell must “squeeze” through. In this process,
the cell experiences physical and osmotic stresses that causes the loss of much of it
internal plasma. This ability to migrate through a channel smaller than the cell is
called deformability. Repeated passages through the capillaries is traumatic and
results in the significant changes in the cell membrane which leads to its removal
from circulation. These changes includes membrane damage and decreased efficiency
of the metabolic pathways. RBC destruction occurs primarily in the spleen, liver, and
bone marrow. The rupture of the membrane allow the escape of hemoglobin.
Hemoglobin is degraded to heme and globulin. The “globin” polypeptide chain is
degraded to smaller peptide units and joins the amino acid pool.

The amino acid pool is not an actual storage place for amino acids. Amino acids
cannot be stored in special sites as is fat in adipocytes. This is a conceptual concept
in which it is understood that there are small amounts of amino acids present in cells
and in circulating blood. Remember that proteins are present in all cells and tissues.
These proteins can be mobilized during times of stress (starvation or times of fasting).
Also, amino acids can be metabolized to glycogen to be used as a source of energy or
to triacylglycerol to be stored as fat.

The heme of hemoglobin is degraded to iron and the porphyrin ring in the
macrophage. The ring is opened by the enzyme heme oxidase to produce carbon
monoxide and biliverdin. Carbon monoxide is formed when the α-carbon in the
methane bridge of the ring structure is broken to form biliverdin. CO escapes and
some will bind with hemoglobin, but most is eventually exhaled in the lungs.
Biliverdin is quickly reduced to bilirubin. Most of the released bilirubin is immediately
bound to albumin to form an unconjugated bilirubin. When the unconjugated bilirubin
enters the liver, the bilirubin-albumin complex is broken and bilirubin recombined
with glucuronic acid to form conjugated bilirubin. In the conjugated state, bilirubin is
now polar and lipid insoluble. It is in this form that bilirubin is excreted into the bile
and passes into the small intestine. In the small intestine, the conjugated bilirubin is
called mesobilinogen and it is acted upon by bacteria to form stercobilinogen, then
stercobilin. These are pigmented compounds. Up to 80% of the conjugated bilirubin
will be excreted as stercobilinogen and stercobilin. The remainder is reabsorbed into
general circulation and recycles through the liver or is excreted through the kidneys.
In the urinary system, stercobilinogen and stercobilin are secreted as urobilinogen and
urobilin, even though they are the same compounds.

Some bilirubin will circulate in the plasma as a free form (unbound). The albumin-
bilirubin complex and the free bilirubin are referred to as unconjugated bilirubin.
These are water insoluble and alcohol soluble. These give the indirect van den Bergh
reaction. Bilirubin complexed with glucuronic acid is designated as conjugated
bilirubin and is water soluble and alcohol insoluble. The conjugated form gives the
direct van den Bergh reaction.

[1]   increased membrane bound IgG        [8] cell becomes more spherical
[2]   increased cell density             [9] increased intracellular viscosity
[3]   increased intracellular sodium      10] increased methemoglobin
[4]   decrease enzyme activity         [11] decrease intracellular potassium
[5]   decrease hgb affinity for oxygen [12] decrease phospholipids
[6]   decreased cell cholesterol [      [13] decrease in sialic acid
[7]    changes in MCHC and MCV


Birth . . . . . . . . 17 to 23 g/dL     6 to 10 years . . . . . 10.5 to 14.6 g/dL
2 months . . . . . . 9 to 14 g/dL       11 to 15 years . . . . 11.4 to 15.4 g/dL
12 months . . . . . . . 11.8 g/dL       >16 to 50 (male) . . .. . . 14. to 18 g/dL
five years .. . . . . . . 12.6 g/dL    >16 to 50 (female) . . . . 12 to 16 g/dL
                 > 50 years . . . . . values may increase slightly


[1]    Physiological. Taller and heavier individuals tend to have increased RBC
[2]     Psychic. Fear and excitement tend to increase the RBC count.
[3]     Sexual differences. Males tend to have higher RBC counts. Testosterone
        promotes erythropoiesis. Estrogen tend to decrease erythropoiesis.
[4]     Barometric pressure. Increasing altitudes has an augmenting effect that
        increases the RBC count.
[5]     Muscular activity. Physical conditioning and training leads to elevated RBC
        counts. RBC values drop to normal ranges as the person settles back into a
        routine that maintains his physical conditioning.

NOTE: Anything that will increase the RBC count will also have the same effect the
hemoglobin and hematocrit determinations.


The globin chains that are part of the hemoglobin molecule are sequences of amino
acids which may be designated as peptide chains. The different globulin chains are
designated by an Greek letter. Examine the following listing for the types of globin
chains found in hemoglobin

Greek letter        # of amino acids                comments
Alpha (α)                141
Beta (β)                  46
Delta (δ)                146             differs from β-form by 10 amino acids
Gamma (γ)                146             differs from β-form by 39 amino acids
Epsilon (ε)              146             embryonic stage only
Zeta (ζ)               146           embryonic stage only

The embryonic forms of globin (ζ and ε) appear only in the first three months of
embryonic development and then disappears. The globin portion of the hemoglobin
molecule will consist of two sets of globin pairs. For example, two α-globin and two β-
globin molecules is characteristic of hemoglobin A. Two ζ-globin and two γ-globin
molecules is characteristic of the Portland type of hemoglobin.


[1] See Objective #43 for the description of Hemoglobin A1, the normal adult
hemoglobin. This hemoglobin is the major oxygen carrier in the human from about
three months to death. It appears in the fifth month of gestation.
[2] Hemoglobin F. This is a hemoglobin normal to the fetus. Appearing about the fifth
week of gestation, it will increase and peak about the seventh month, and can make
up to 95% of the total hemoglobin. About the time of birth the amount of Hgb F will
be reduced to about 80% of the total hemoglobin, as the developing body increased
the rate of Hgb A1 production. About six months after birth, Hgb A1 has almost totally
replaced Hgb F. By the child’s third birthday, <1.0% of the total hemoglobin is the F-
type. Hemoglobin F has a higher affinity for oxygen and can “pick-up” the low levels
of placental or uterine oxygen. It consists of two α-globins and two γ-globins. Hgb F is
easier to oxidize to methemoglobin and it is also more resistant to alkaline
denaturation than other hemoglobins.
[3] Hemoglobin Gower I. This is an embryonic hemoglobin, found in trace amounts. It
consists of two ζ-globins and two ε-globins. It can be detected on in the first three
months of embryonic life.
[4] Hemoglobin Gower II. This is the most important of the embryonic hemoglobins
and will make up as much as 60% of the total embryonic hemoglobin. It persist only
during the first three months of life. It consists of two ε-globins and two α-globins.
[5] Hemoglobin Portland. This is an embryonic hemoglobin, found in trace amounts. It
consists of two γ-globins and two ζ-globins.
[6] Hemoglobin A2. This hemoglobin type makes up to 3% of normal adult hemoglobin
and consists of two α-globins and two δ-globins. It appears late in the fetal life and is
not produced in any significant quantities. It is designated as a minor hemoglobin.


Synonyms: hemiglobin (Hi). Methemoglobin (non-functional hemoglobin) is the result
of the oxidation of Fe++ (ferrous iron) in the hemoglobin to the ferric state (Fe +++).
This oxidized form of hemoglobin cannot bind oxygen. Up to 3% of the total
hemoglobin in the body may be converted to methemoglobin daily. The body has a
means of counteracting this oxidation process through the enzyme, NADH dependent
methemoglobin reductase (also called diaphorase). This is an efficient reduction
system, reducing methemoglobin 250 times faster than a hemoglobin molecule can be
oxidized. As a rule, the methemoglobin levels never exceed 1.0%. If methemoglobin
levels reach 10%, cyanosis can develop. Blood plasma will begin to take on a brownish
appearance. If levels approach 60%, then hypoxia occurs. Heinz bodies may be seen in
this anomaly.

In Hemoglobin M disease, the affected individual will demonstrate greater than 2%
methemoglobin levels. This disorder can be acquired (caused by toxic substances),
presence of a Hemoglobin M variant, the presence of a defective enzyme (NADH-
diaphorase), or a NADH-diaphorase deficiency. The normal mechanism for converting
methemoglobin to normal functional hemoglobin is as shown.

Oxidation is the loss of electrons. A molecule can give up an electron or hydrogen ion
to another molecule. Reduction is to gain an electron. It can mean for something to
undergo a reaction.

In the laboratory, a popular testing method for determine the amount of hemoglobin
is the cyanmethemoglobin method. This method requires reducing hemoglobin to the
methemoglobin form before it can be reduced to cyanmethemoglobin. Hemoglobin
electrophoresis is diagnostic for this disorder. Nitrates and chlorates can cause
oxidation of hemoglobin to methemoglobin.


In the oxidation of hemoglobin to methemoglobin, if an electron is lost, the ferrous
iron ( Fe++) is oxidized to ferric iron (Fe+++). Conversely, if an electron is added to
ferric iron, ferrous iron is formed. The Embden-Meyerhof pathway (Glycolysis cycle)
produces NADH, a nucleotide molecule that can function as an electron donor. RBC’s
contain the enzyme, NADH-methemoglobin reductase (also knows as diaphorase).
NADH is produced as glyceraldehyde (which gives up an electron and NAD captures it)
is converted to 1,3-diphosphoglycerate. The resulting NADH molecule can then donate
its electron to methemoglobin reducing it to functional hemoglobin.


Sulfhemoglobin is formed with sulfur levels build up in the body. This is a stable
hemoglobin that cannot combine with or carry oxygen. Sulfhemoglobin is usually seen
when methemoglobin is present in higher than normal amounts. It is formed with
hemoglobin is exposed to drugs, such as trinitrotoluene, acetanilid, aniline dyes,
sulfur containing cathartics, inorganic sulfides, phenacetin, or sulfonamides.
Sulfhemoglobin is found in normal blood, but in concentrations <1.0%. This type of
hemoglobin once formed, remains with the cell until the cell is removed from
circulation, at which time the sulfhemoglobin is removed. Sulfhemoglobin cannot be
reduced by ascorbic acid or methylene blue as can methemoglobin, but can combine
with carbon monoxide to form carboxysulfhemoglobin. Sulfhemoglobin cannot be
converted to cyanmethemoglobin and measured by this standard procedure.
Sulfhemoglobin can be measured examining a prepared hemolysate at 610 nm


Carboxyhemoglobin (Hgb∙CO)is a carbon monoxide derivative of hemoglobin and is
usually found in normal blood at concentrations <1.0% of the total hemoglobin.
Carbon monoxide has an affinity for hemoglobin that is 200 time greater than that of
oxygen. People who smoke have concentrations of carboxyhemoglobin of
approximately 5.0% of the total hemoglobin. People who live in cities have a higher
level of Hgb∙CO than those who live in rural areas. When Hgb∙CO concentrations reach
10%, judgment is impaired and at 50% concentration, unconsciousness is a real risk.
Also other symptoms include respiratory failure, followed by the risk of death. A
simple procedure for testing for carbon monoxide poisoning is to hemolyze 0.5 mLs of
blood with 20 mLs of distilled water and adding 1.0 mL of 1N NaOH. If a light cherry
red color appears, then the blood contains more than 20% carboxyhemoglobin.
Carboxyhemoglobin can be measured spectrometrically by comparing the δ-absorption
bands with that of oxyhemoglobin.


Dacie’s reagent is made with distilled water, potassium ferricyanide, and potassium
cyanide. Drabkin’s reagent is similar except it contains sodium bicarbonate. Drabkin’s
reagent is preferred over the Dacie reagent. The sodium bicarbonate facilitates the
lysis of the RBC’s.


When whole blood is added to the reagent, potassium ferricyanide converts
hemoglobin to methemoglobin. In the next reaction step, methemoglobin reacts with
potassium cyanide to form cyanmethemoglobin (a stable colored compound). The
amount of cyanmethemoglobin formed is measured spectrophotometrically at 540 nm
wavelength. Cyanmethemoglobin is the standard for hemoglobin determinations
because it measures all hemoglobins except sulfhemoglobin. It is very easy to
standardize, forms a stable solution, and is a rapid and easily reproducible procedure.
There are some sources of concern, these being, [1] cyanide is toxic, but it takes from
4 to 6 liters of Drabkin’s solution to form a lethal dose. [2] Commercial solutions tend
to be unstable and have a short shelf life. To avoid this problem, refrigerate and/or
keep in the dark. [3] Testing blood from heavy smokers tend to introduce significant
error into the procedure. As much as 10% of their blood can be in the form of
carboxyhemoglobin. It takes about one hour for cyanmethemoglobin reagent to
convert Hgb∙CO to cyanmethemoglobin. [4] If lipemic blood is used is used to
determine hemoglobin concentrations (may that never happen), there will be
increased turbidity in the reagent-blood mixture and the reading will be increased. [5]
Globulin diseases (examples: multiple myeloma and Waldenstrom’s macroglobinemia)
cause high readings. In Drabkin’s reagent the abnormal proteins will precipitate,
causing increased turbidity. (Note: K2CO3 , if added to the Drabkin’s solution, it will
prevent these proteins from precipitating out.)

NOTE: Objectives 65-68 are abbreviated descriptions of obsolete technologies that are included
for their historical interest. These four objectives will not be included in any tests.


This is a method in which dilute HCl is added to blood to form acid hematin. This is a
slightly inaccurate method and requires a Sahli-Hellige hemoglobinometer that
contains a colored glass standard for comparison.


This is a method in which dilute NaOH is added to blood and heated. It’s major
disadvantage was that it did not measure hemoglobin F. The color of the alkali
hematin solution is compared to known standards or measured in a colorimeter at a
wavelength of 610.


This is a method where whole blood is mixed with 0.007 NH4OH (N/150) and
hemoglobin is converted to the oxyhemoglobin form. It is an accurate and stable
method. It’s disadvantage lies in its sensitivity to copper. Any copper present will
convert oxyhemoglobin to methemoglobin. Sodium carbonate (0.1%) may be
substitute for ammonium hydroxide. The oxyhemoglobin solution is measured
photometrically at 550 wavelength.


This is an interesting, but outmoded method of evaluating the amount of hemoglobin
in the cell. It is a mathematical method that requires the RBC count and hemoglobin
value. It was calculated by dividing the Hgb reading by the first two numbers in the
RBC count. It is based upon the principle that a 100% reading is equal to a hemoglobin
concentration of 14.5 g/dL..

Synonyms: packed cell volume, “crit”. The hematocrit (HCT) is the percentage of the
total volume of blood that is occupied by packed red blood cells. It is the simplest and
most accurate of the laboratory procedures. The hematocrit results are preferred (as
a rule) over that of the RBC count and allows for calculation of the indices. Regarding
the manual hematocrit, the following is applicable:
[1] The buffy coat can provide a “rough” estimate of the WBC count. A rule-of -thumb
estimate is a buffy coat of ≤1.0% is generally represents a normal WBC count.
[2] The buffy coat is not to be included as part of hematocrit reading.
[3] A distinctly colored plasma layer can indicate the presence an icteric condition.
     A. Normal values are as follows:
           • Adult male = 42% to 53%
           • Adult female = 36% to 46%
           • Newborn = 50% to 62%
           • One year = 31% to 39%


The buffy coat is a distinct white layer or “button” between the plasma and RBC’s.
Platelets (a very thin yellow-white layer) will layer over WBC cells (the thicker
grayish-white layer). The buffy coat is a convenient way to collect a large number of
leukocytes for evaluating a blood film for leukemia, leukopenia, megaloblasts, LE
cells, etc. The buffy coat from a normal healthy individual will be 1% or less. If the
buffy coat is greater than 1%, then this indicates an increased WBC count and a
possible pathology.


[1] Improper sealing of tube.
[2] Increased anticoagulant ratio causing increased cell shrinkage which
    causes a decreased hematocrit. [3] Time and speed of the centrifuge.
[4]   Improper mix of blood.
[5]   Including the buffy coat into the hematocrit determination.,
[6]   Incorrectly using the hematocrit reader.
[7]   If the patient has an anemic condition (See comment below).
[8]   Hemorrhage (decreases hct).
[9]   Not removing first drop of blood of a fingerstick (Tissue fluid is in the
      first drop and will dilute the specimen).

Comment: Anemias can affect the hematocrit by increasing or decreasing the reading. For
example, if the patient has microcytosis with anemia, the HCT will be decreased. Other
examples include: [1] macrocytosis will cause an increased HCT and [2] poikilocytosis may
facilitate trapping of plasma in the spaces about the cells, causing an increase in the hematocrit.


Being by preparing several hematocrits for centrifugation using the same blood
specimen. On the first centrifugation, spin the specimens for two minutes. Repeat
centrifugation steps by increasing centrifuge time at 30 second intervals. Look for
consistent readings and a translucent appearance in the packed RBC’s. Normal
centrifugation time should fall between three and five minutes.


[1] Pathological polycythemia, [2] physiological polycythemia, [3] shock associated
with surgery, burns, or traumas, and [4] dehydration.


[1] anemias, [2] hydration, [3] pregnancy, [4] receiving IV fluids, and [5] cardiac
decompensation (a failure to maintain a good blood circulation).


The indices are a set of mathematical calculations that define the size and
hemoglobin content of erythrocytes. The requires measurement of hemoglobin (g/dL),
hematocrit (%), and the RBC count (μL). Synonyms for the indices are {1] red cell
indices, [2] mean cell values, [3] average cells values, [4] mean corpuscular values, [5]
blood indices, and [6] erythrocyte indices. All indices should be confirmed by visual
examination of a stained peripheral blood smear. The indices calculations are a
valuable means of helping to differentiate anemias. The three indices are as follows:

Mean Corpuscular Volume (MCV). This calculates the average volume of the
RBC in a given sample of blood. Calculate as follows: HCT / RBC/μL (X) 10 = MCV. The
MCV is expresses in fL. fL = femtoliter (10-15/L). (Note: fL = μM3). Sample problem:
HCT = 45% and RBC count = 4.85 X 106/μL (use only the first three digits, 4.85)?
Answer = 92.78 fL
          MCV = 45 divided by 4.85 times 10 = 92.7835 OR 92.78 fL.

MCV values are significant in determining if the RBC is microcytic, normocytic, or
macrocytic. If <80 fL, then microcytic; if between 80 and 100 fL, then normocytic;
and if >100 fL, then macrocytic. Examples: [1] pernicious anemia = 99.7 to 146.7 fL,
[2] iron deficiency anemia = 56.7 to 88.8 fL, and β-thalassemia minor = 53.7 to 73.5 fL
There may be a rare occasion in which femtoliters must be calculated in femtomoles.
If this is the case, simply multiply fL times 0.0155.

Mean Corpuscular Hemoglobin (MCH) represents the amount of hemoglobin
(by weight) in the average erythrocyte in a sample of blood. Calculate as follows:
MCH (pg) = Hgb (g/dL) / RBC (μL). pg = picogram (10-12). (NOTE: pg = μμg) Sample
problem: Hgb = 15.0 g/dL, RBC = 4.85 (x) 106. Answer = 30.93 pG.
          MCH = 15 divided by 4.85 times 10 = 30.9278 OR 30.93 pG

Both the MCV and MCH have the same denominators (RBC count/μL). A relationship
between the Hgb and Hct is maintained. Because the Hct is usually 3X that of Hgb,
the MCV will usually be 3X the MCH in normal individuals. The MCH is considered to be
of lesser importance in describing anemias then the MCV and MCHC. If the MCH is <27
pg, then it tends to be characteristic of microcytic anemias and normocytic,
hypochromic anemias. If the MCH falls between 27 to 32 pg, then this indices is
normal. A MCH >32 pg has been perceived as being indicative of macrocytic anemias.
Because the MCH does NOT take into account the size of the cell, it must be
interpreted by considering the MCV. The reason why the MCH has been deemed to
less clinically significant in evaluating anemias is that in some anemias, there is a
somewhat proportional change in the hemoglobin content (based on the MCH) with a
change in the size of the cell (based on the MCV), BUT the MCHC remains normal,
therefore the anemia is designated as normochromic. In other anemias, with a
decrease in cell size (MCV) and a corresponding decrease in the MCH, If the MCHC is
also decreased the anemia is hypochromic. It is found that a decrease in the MCH
(<26 pG) or increase in MCH (>32 pG) does not mean that the anemia should be
respectively classified as hypochromic or hyperchromic. Remember that under
certain conditions, the MCH can be falsely elevated. This is also true for the MCV and

Mean Corpuscular Hemoglobin Concentration (MCHC) is considered to
be an absolute value and expresses the concentration of hemoglobin in terms of
average weight in the average RBC in a sample of blood and expressed as a percent
value. The MCHC is now being expressed as g/dL. It is the ratio of the hemoglobin
weight to RBC volume. Calculate the MCHC (%) as follows: Hgb (g/dL) / Hct (%) (X)
100. Sample problem: Hgb = 15 g/dL and Hct = 45%. Answer = 33.3 g/dL (%).
        MCHC = 15 divided by 45 times 100 = 33.3333 or 33.3 g/dL

If the MCHC values <31%, then it is suggestive of iron deficiency anemia. A value of 31
to 37% is normal. Values >37 are suggestive of hyperchromia. (NOTE: MCHC values >38
should not occur. Normal RBC’s contain maximum amounts of hemoglobin.) If a MCHC value
>38% occurs, then revisit the indices, an error in calculation is very likely. If a blood
sample contains rouleaux or RBC agglutination, then an electronically counted and
determined Hct may be falsely low, which will affect the MCH. A blood specimen with
lipemic plasma may result in abnormally low MCHC values. Hypochromia
characteristically occurs in iron deficiency anemias, thalassemia, and defective iron
metabolism. Hyperchromic values usually indicates shape changes. It is not the usual
rule to find the term hyperchromia included in many anemia classification schemes.
Note: In the past the MCHC has normally been reported as a percent value. Lab’s
continue to report the MCHC value this way. There is a trend to report this indices as
g/dL because it represents the average concentration of hemoglobin per cell size and
hemoglobin is classically reported as g/dL.

Accurate calculations of hemoglobin, hematocrit, and RBC count
determinations are required for accurate indices calculations. If an error
of 5% or greater occurs in any of the three parameters, the indices results
are invalid. It is important for the laboratory to use electronic counters,
quality reagents, reliable controls, and precision volumes in measurement
and glassware.


[1]   Normochromic, normocytic erythrocytes.
Hgb = 14.0 g/dL      MCV = 91.9 fL (80 - 100 fL)
Hct = 41%            MCH = 31.3 pg (27 - 32 pg)
RBC = 4.5 X 106/μL   MCHC = 34.1% (31 - 36 %)
Erythrocytes are normal in size and contain a normal concentration of hemoglobin.

[2] Hypochromic, normocytic erythrocytes.
Hgb = 9.6 g/dL                MCV = 85.4 fL (80 - 100 fL)
Hct = 38%                     MCH = 21.6 pg (27 - 32 pg)
RBC = 4.45 X 106/μL           MCHC = 25.3% (31 - 36 %)
Erythrocytes are normal in size but contain less hemoglobin than normal

[3] Hypochromic, microcytic erythrocytes.
Hgb = 8.9 g/dL        MCV = 65.9 fL (80 - 100 fL)
Hct = 29%             MCH = 20.2 pg (27 - 32 pg)
RBC = 4.40 X 106/μL       MCHC = 30.7% (31 - 36 %)
Erythrocytes are smaller than normal and contain less hemoglobin than normal.

[4] Normochromic, macrocytic erythrocytes.
Hgb = 8.0 g/dL         MCV = 112.6 fL (80 - 100 fL)
Hct = 26%              MCH = 34.6 pg (27 - 32 pg)
RBC = 2.31 X 106/μL    MCHC = 30.8% (31 - 36 %)
Erythrocytes are larger than normal (MCV) and contain a larger
amount of hemoglobin (MCH). Notice that the MCHC is normal,
therefore the RBC’s are normochromic.

[5] Hypochromic, macrocytic erythrocytes.
Hgb = 5.6 g/dL         MCV = 113.7 fL (80 - 100 fL)
Hct = 24%             MCH = 26.5 pg (27 - 32 pg)
RBC = 2.11 X 106/μL    MCHC = 23.3% (31 - 36 %)
Erythrocytes are large (MCV). Both MCHC and MCV are low,
therefore hypochromia.


A general application of mathematics can be used to verify the validity of the RBC
count, along with the hemoglobin and hematocrit values. This concept is valid as long
as the visual inspection of the stained blood film demonstrates normochromic,
normocytic red blood cells. Look at the following examples for how the “rule of
three” is suppose to work. Assume that the lab reported the following normal values
for a pre-surgery patient:
RBC count = 4.85 X 106/μL
Hgb = 14.5 g/dL
Hct = 44%

[1] If the RBC count is 4.85 X 106/μL, then 3 X 4.85 = 14.55 (g/dL) for hemoglobin.
This is a confirming value because it falls within ± 3 units.
[2] If the hemoglobin is 14.5%, then 3 X 14.5 = 43.5. This is a confirming value
because it falls within ± 3 units
[3] Divide the hematocrit (44%) by 3 to obtain an answer of 14.7 (g/dL). This is a
confirming value for hemoglobin because it falls within ± 3 units.
[4] Divide the hemoglobin (14.5) by 3 to obtain an answer of 4.83. This is a
confirming value of the RBC count because it falls within ± 3 units.

The rule of three will not function for a patient with a normocytic, hypochromic
anemia. If the lab reported the following values:
RBC count = 4.20 X 106/μL
Hct = 39%
Hgb = 8.9 g/dL

[1] 4.20 X 3 = 12.6 (g/dL for hemoglobin) Does not follow the rule of three
[2] 8.9 X 3 = 26.7 (% for the hematocrit). Does not follow the rule of three
[3] 39 ÷ 3 = 13 (g/dL for hemoglobin). Does not follow the rule of three.
[4] 8.9 ÷ 3 = 2.96 (X 106/μL for the RBC count). Does not follow the rule of three.

If the rule of three indicates a discrepancy, then an investigation should be
undertaken to determine this represents the results of a pathology, in which case no
action is required. If not a pathology, then corrective action must be implemented,
whether the problem is due to the instrument or the specimen.


The red cell distribution width (RDW) is a mathematical calculation (built into the
automated instrumentation) that uses the MCV and RBC count to measure the
variation in the RBC volume distribution. The following formula is used:
              SD [RBC volume distribution] (x) 100/MCV = RDW (%)

The normal range is 11.5% to 14.5%. If the RDW value is low, the RBC population is
even, all cells are in the same size range. Low readings suggest that there is little
clinical significance because there is little or no deviation in the size of the
erythrocytes. If the RDW is increased, then it is clinically significant, indicating
increased sizes with variation. Examination of a blood smear with an increased RDW
should reveal a picture of anisocytosis. RDW values should be interpreted with
caution and only after evaluating the blood smear and histogram. If there is a true
increase in the variation of cell sizes, the base of the RBC histogram should be
broader. Remember, when you are examining a blood smear, to see up to 6% variation
in RBC sizes is not abnormal.


The erythrocyte sedimentation rate (ESR) is a simple and commonly performed
laboratory test that is useful in monitoring inflammatory disorders or differentiating
between diseases. This procedure is nonspecific. German physician (Christian
Friedrich Nasse) in 1836, discovered that changes in the plasma proteins caused the
variable rates at which RBC’s wold fall in whole blood. It would be a century before
the significance of this finding would be recognized. The ESR procedure was
introduced to the medical profession about 1915 by a Swedish physician. Alf
Westergren (Swedish physician) developed the basic sedimentation rate procedure
that is still in current use and bears his name. A popular synonym for ESR is “sed

In inflammatory changes or other disorders, the plasma will under go changes in
fibrinogen, α-globulins, and/or β-globulins. These plasma changes will enhance
rouleaux formation and speed the fall of the erythrocytes. Plasma changes causes
changes in the RBC surface permitting aggregation of the RBC (rouleaux formation)
which in turn increases RBC mass. The rate at which RBC’s fall is directly proportional
to the erythrocyte mass and also inversely proportional to the plasma viscosity. In the
normal healthy individual, the red blood cell does not form rouleaux. Absence of
rouleaux leaves the RBC as a small mass and settling phenomenon of whole blood will
proceed slowly.

There are two procedures employed by the laboratory:
[1] Westergren method. This is the preferred procedure and is in more common use
than the other method. A blood specimen drawn in EDTA or Sodium Citrate is
required. The original method required diluting four parts of whole blood with one
part of sodium citrate. The method has been modified to combining 2.0 mLs of whole
blood (drawn in EDTA) with 0.5 mLs of 0.85% NaCl. Air bubbles invalidate the test.
The diluted blood is placed in a special Westergren pipet and allowed to “settle out”
during a one hour interval. Normal ESR Westergren values are:
Males:     <50 years = 0 to 15 mm/hr      >50 years = 0 to 20 mm/hr
Females: <50 years = 0 to 20 mm/hr         >50 years = 0 to 30 mm/hr
Children: 0 to 10 mm/hr

NCCLS specifications for the Westergren method are strict. The tube must
be a thick wall plastic or glass tube at least 300 mm in length. The tube
must support a column of blood 200 mm in length with a bore of 2.65 mm (±
0.15 mm). Markings must be from 0 to 180. The supporting rack must hold
the tube exactly vertical with no more than a 2o variation. The rack must
also be able to support the tube vibration free.

[2] The Wintrobe method uses a heavy walled tube that is 115 mm in length and a
bore of 3.0 mm. This tube was originally used to perform a macrohematocrit which
has become an obsolete test. The tube was also designed to perform an ESR. The
tube is marked with two series of graduations from 0 to 100 graduations that run in
opposite directions. The rack used to hold the Wintrobe tube must be vertical and
vibration free. This test procedure uses EDTA anticoagulant whole blood without
dilution and no air bubbles.

Normal reference ranges are:
   men:      0 to 9 mm/hr
   women:     0 to 20 mm/hr
   children: 0 to 13 mm/hr

Cautions to be applied in performing the ESR test are:
[1] If clots, any size, are in the tube, it invalidate the test.
[2] The concentration of anticoagulant must be correct or the results will
     be skewed. Too much anticoagulant will increase the ESR. To low a
     concentration will results in clots.
[3] Invert the tube of blood after collection several times to assure a good
    mixing of anticoagulant.
[4] Patients diagnosed with severe anemia should not be tested by this
       technique. Results are invalid.
[5]    If sodium or potassium oxalate and heparin are used as anticoagulants,
       the RBC’s will undergo some degree of shrinkage, causing a falsely
       elevated ESR.
[6]    Tilting of the ESR tube will increase the ESR.
[7]    The blood (at the beginning of the test) must be set at the zero mark
       for accuracy.
[8]    Bubbles in the tub invalidate the test.
[9]    If the blood sets at room temperature for more than two hours, the
       cells will become more spherical, skewing the test. Such cells cannot
       under rouleaux.
[10]    Do not test patients with such diagnosis as sickle cell anemia,
        thalassemia, or spherocytosis. These conditions causes decreased ESR

The ESR is increased in the following   conditions:
  acute infections                       pregnancy
  acute appendicitis (after 24 hours)      pyogenic arthritis
  chronic infections                     rheumatic fever
  hepatitis                              rheumatoidal arthritis
  inflammation                            ruptured ectopic pregnancy
  menses                                  subacute bacterial endocarditis
  myocardial infarction                    tuberculosis
  multiple myeloma                         nephrosis
  Waldenstrom’s macroglobulinemia


Also referred to as the ZSR test, this is a procedure that requires the uses of special
75 mm long capillary tubes (with an inner diameter of 2.0 mm) and a “Zetafuge”. This
is a technique of using series of controlled centrifuge speeds to compact and disperse
the erythrocytes. The first spin is at low speed for 45 seconds which forces the cells
against the vertical wall of the capillary tube to increase the rate of rouleaux
formation. The “zetafuge” will stop and the tubes are rotated 180O and spun a second
time for 45 seconds to move the rouleaux formed cells back across the inner diameter
of the tube. This step is repeated two more times. The result is that the inter-face of
the red blood cell column and plasma forms a unique curve dependent upon the
negative charge on the RBC membrane that normally keeps the RBC’s apart. The
degree of the net charge on the membrane affects the degree of packing and spacing
between the RBC’s that results in the formation of a unique curve that contains a
meniscus design
called the “knee of the curve”. It is at this curve that the measurement is taken. An
ordinary hematocrit reader may be used. See the above illustration. A
standard hematocrit is also performed and the results used in the calculation of the
ZSR. The formula is ZSR% = Hct% ÷ Zetacrit X 100. This procedure is dependent upon
the behavior of RBC’s as they approach each other under a specific, standardized
gravitational force. The reference range for males and females are as follows:
      Normal =               40% to 51%
      Borderline =           51% to 54%
      Slightly elevated =     55% to 59%
      Moderately elevated = 60% to 64%
      Markedly elevated =     > 64%

The procedure takes about four minutes to run and is not affected by anemias. It is
affected by fibrinogen and gamma globulin concentrations which will reduce the
normal negative charges on the surface of the RBC. It is an alternate technique for
obtaining the erythrocyte sedimentation rate.


Electrophoresis is a testing principle that take advantage of charged particles
migrating in an electric field. The hemoglobin molecule carries an electrical charge
because it contains both carboxyl (COO−) and protonated nitrogen (NH3+) groups. The
number of these two groups determines the strength of the charge on the molecule. If
the hemoglobin molecule contains more carboxyl groups, the molecule will carry a
stronger negative charge that cancels out the weaker positive charge from the
protonated groups. In an electric field this molecule will migrate toward the positive
electrode (the anode). If the protonated groups predominate, then the molecule will
migrate toward the cathode (negative charged electrode). There are several factors
that affect the rate at which the molecule will migrate toward an electrode. These
are the net charge on the molecule, the size (mass) of the molecule, the shape of the
molecule, the strength of the electrical field, the chemical and physical properties of
the supporting medium, and the temperature of the electrophoresis system.

The hemoglobin molecule is made up of repeating units of amino acids and the
number and type of amino acids determine its charge. There are more than 450
known variants of hemoglobin that result form amino acid substitutions made in the
hemoglobin molecule. For example, sickle cell hemoglobin differs from normal (A 1)
hemoglobin by a single amino acid. Sickle cell hemoglobin contains valine instead of
glutamic acid at the sixth position on the β-globulin chain. Substitutions can alter the
solubility, stability, and function of the hemoglobin molecule. As a result of this one
amino acid molecule difference, sickle cell hemoglobin will migrate slower than
normal adult hemoglobin.

If a hemoglobin migrates in an electric field faster than normal adult hemoglobin, it is
designated as a fast hemoglobin. If it migrates slower, then it is a slow hemoglobin.
The rate at which a hemoglobin molecule migrates in an electric field is known as its
Rf value.

Routine hemoglobin electrophoresis tests are routinely performed first on cellulose
acetate strips at a pH of 8.6. When two or more hemoglobin species (such as Hgb D,
Hgb S, and Hgb G) are found to migrate at the same speed, then the electrophoresis
may be repeated on a different or secondary system such as citrate agar at a pH of
6.0. Note that hemoglobins A2, C, E, and O Arab will migrate at the same Rf value on
cellulose acetate strips at a pH of 8.6 and have to be differentiated using citrate agar
at a pH of 6.0.

To set up a hemoglobin electrophoresis procedure, a blood sample (venous or
capillary blood) is obtained from the patient and a hemolysate prepared using red
blood cells. Using EDTA or heparin as anticoagulants does not interfere with the
electrophoresis procedure. The hemolysate can be stored frozen for future use. The
major source of error in performing an electrophoresis procedure is poor technique.
Other sources of error include deteriorated reagents, incorrect pH, equipment
malfunction, contamination of agar or acetate strips, improperly stored or prepared
hemolysates, and bacterial contamination.

The interpretation of results is based upon known migration patterns of the different
types of hemoglobins and comparing these patterns with known controls. Refer to the
following illustration of hemoglobin electrophoresis patterns. Remember that normal
blood contains A1, A2, and F hemoglobins.
           Table 1. Hemoglobin Types and Percentages


Anemia is a disorder in which there is a decrease in the ability of blood to carry and
deliver oxygen to the tissues/cells. In the clinical laboratory, when anemia is
present, there will be a decrease in the number of erythrocytes/μL (RBC count) or in
grams of hemoglobin or a decrease in the red blood cells mass (hematocrit). Usually
anemia is an expression of a primary disorder or disease. The physician's role (with
clinical laboratory testing) then become that of determining the primary cause and
treating the anemia if necessary.

As a "rule of thumb", the body will lose about 0.85% of its red blood cells mass daily.
This represents about 20 mls of blood that is lost. The body in turn must replenish that
loss. If this loss cannot be replenished, then anemia will develop. Anemia can develop
with the loss of RBC's exceeds the ability of the body to maintain an appropriate level of
production or there is some event occurring in the body that prevents RBC production.

The physician will evaluate the patient's clinical symptoms and medical history to help
him/her to diagnose the presence and the type of anemia. The physician's review of the
patient's medical history will include any or all of the following:
A. family history, if any members experienced anemic conditions
B. the patient's diet and consumption of snack foods
C. any medications that are currently prescribed
D. if their home/work environment exposes them to chemicals

The physician will consider the symptoms that are being described:
A. fatigue and weakness
B. appetite loss
C. discomfort in breathing
D. changes in the rate of the heart beat or unusual ryhthms
E. headaches or other body discomfort
F. fainting and/or dizziness

The evaluation will include physical examination to include:
A. enlargement of the spleen and/or liver
B.   pallor in the skin, cheeks, and/or fingernails
C.   blood pressure
D.   changes in the nails of the fingers and toes
E.   neural changes and irritability

The physician will turn to the laboratory to collect blood and perform all or any
combination of the following tests:
A. CBC (hemoglobin, RBC count, WBC count, hemoglobin, hematocrit, indices. |
    platelet count, RDW, PMV, and a stained peripherial blood smear
B. Reticulocyte count and a retic stained blood smear evaluation.
C. Serum iron, TIBC, transferrin test, percent saturation, ferritin test, and
    free erythrocyte protoprophyrin (FEP) test.
D. Hemosiderin in the urine.

The information obtained in the medical history and physical evaluation along with that
provided by the laboratory will help the physician classify the type of anemia. Anemia's
may be classified as either functional (pathophysiological), morphological, maturation
defect, proliferation defect, or a possible combination of two or more of these


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