LABORATORYNOTES-
Morphological Classification of the Anemias
Wintrobe introduced the hematocrit method for
estimation of the volume percent of erythrocytes (PCV)
in peripheral blood. Using a combination of PCV,
hemoglobin (Hb) concentration, and erythrocyte count
(RBC), Wintrobe developed three erythrocytic indexes.
Mean compuscular hemoglobin (MCH), which is the
weight of Hb in the average red cell; and mean
corpuscular hemoglobin concentration which expresses
the ratio of weight of Hb t o volume of the erythrocyte.
Originally, MCV was stated in cubic microns, MCH in
micromicrograms, and MCHC in percent. More recently
a change has been made t o an international system based
on the meter and liter, i.e., femtoliters (fl) for MCV,
picograms (pg) for MCH, and grams/deciliter (g/dl) for
MCHC. The cubic millimeter (cmm) has been changed to
microliter (p1)of blood.
Given a blood with a PCV of 42%. Hb of 14 g/dl, and
RBC of 6.0 million /pl, the erythrocytic indexes are
derived as follows:
MCH=
HbX10
RBC
-
- 140
6.0 - 23.3pg
Hb 14
MCHC= X 1 0 0 ~42 X 100=33.3 g/dl
Validty of the indexes depends upon accuracy with
which the erythrocytic parameters are obtained. PCV
from the microhematocrit test is accurate. Spectrophoto-
metric methods of HB determination are generally
accurate when the blood plasma is clear. Counting
erythrocytes by electronic methods is considerably more
accurate than the hemocytometer method particularly
when the latter method is conducted by persons not
skilled in its use.
The Wintrobe erythrocytic indexes serve a useful
purpose in classifying an anemia on the basis of MCV and
MCHC. The normal range for MCHC is 30 to 36 g/dl for
all mammals with the exception of some strains of sheep
and all members of the family Camellidae. These latter
animals have MCHC valles of 40 t o 45%. Normal ranges
for MCV and MCH vary with species as follows:
MCV (fl] MCH [pgl
Dog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60-77 19.5-24.5
Pig . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50-68 17.0-21.0
Horse . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34-58
Cat . . . . . . . . . . . . . . . . . . . . . . . . . . . . :.39-55
COW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40-60
Sheep . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23-48
Goat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15-30
Erythrocytes larger than the maximum normal for
MCV are macrocytic, those normal in size are
0. W. Schalm
normocytic, and cells less than the minimum normal
MCV are microcytic. Erythrocytes having MCHC less
than 30% are hypochromic. When the MCHC fall within
the normal range of 30 to 36% the cells are called
normochromic. A hyperchromic state is not possible
according t o Wintrobe. An error in technic is indicated
when MCHC exceeds 36%. Hemolysis, lipemic plasma,
and the presence of many Heinz bodies in erythrocytes
are conditions leading to erroneously increased MCHC
values.
Morphologic Classification of the Anemias
Classification of an anemia on a morphologic basis is
useful for giving a clue to possible cause, e.g., blood loss,
hemolytic, iron deficiency, or marrow deficiency. Table 1
presents examples of anemias representative of the
several different morphologic classifications. Table 2
presents selected cases of anemia in canine patients
which will form a basis for the discussion to follow.
Macrocytic normochromic anemia. The anemias in
which the erythrocytes are macrocytic normochromic are
the result of a Dartial arrest of maturation at the
prorubricyte-basophilic stages. The problem is one of a
deficiency in replication of nucleoprotein (DNA) for
normal mitotic division. The erythrocytic precursor cells
remain large due t o decreased - mitotic division.
Hemoglobin synthesis proceeds normally leading to
production of macrocytic normochromic erythrocytes.
In man, vitamin B12 and folate deficiencies lead to
macrocytic normochromic anemias. In the few instances
in the dog and cat where we have suspected a vitamin
B,, folate deficiency, the anemia was not macrocytic
normochromnic although macrocytic erythrocytes could
be found within a population of normocytic erythrocytes
and a careful search for hypersegmentation of neutrophil
leukocytes would reveal the presence of a few such cells.
In erythremic myelosis and erthroleukemia in the cat,
t h e e r y t h r o c y t e population may be macrocytic
normochromic. Among miniature poodles, some may
have maturation defects demonstrable by bone marrow
aspiration that lead to macrocytic normochromic
erythrocytes. The PCV remains well within the normal
range although erythrocyte number is near or below the
minimum normal. Neutrophil numbers also are reduced
and some hypersegmentation may be seen. Administra-
tion of vitamin B by intramuscular injection and folic
acid orally has not corrected the maturation defect.
Neither has it been shown that the defect has clinical
12.3-19.7
12.5-17.5
11.0-17.0 Professor Enittrit us.
8.0-12.0 D e p a r t m e n t of ('linical l'iit hology
5.2- 8.0 School of Veterinary Mtdicintl
Dnv is, California .
.
Rcprod u w d hy kind pvr mi s5ion of t hv b:d i t 4 )r.
from Thv ('aliforniii Yvtvrinarian V2X. 1974.
significance. Macrocytic normochromic anemia may
develop in cattle on cobalt deficient pasture since cobalt
is required for synthesis of vitamin B12 by the rumen
flora.
Macrocytic hypochromic anemia. A rapid decrease
in RBC number, Hb concentration, and PCV due t o
hemorrhage or hemolytic destruction (dogs 1 and 2,
Table 2) results in hypoxemia. Synthesis of renal
erythropoietic factor is increased in response to the
reduced oxygen tension. Erythropoietin is produced by
fusion of the renal factor with an alpha-2 globulin
produced by the liver.' Erythropoietin stimulates
erythropoiesis and causes premature extrusion of the
nucleus from the large, polychromatophilic rubricytes of
t h e bone marrow. By t h i s means, macrocytic
polychromatophilic erythrocytes (reticulocytes) are
released to the circulation in 3 to 4 days after the first
massive erythrocyte loss or destruction. MCHC is below
30% and t h u s t h e newly released macrocytic
erythrocytes are hypochromic. The weight of the
hemoglobin (MCH) in these large cells, however, may
exceed the maximum normal (dogs 1and 2, Table 2).
Macrocytic hypochromic anemias are characteristic
of blood loss or hemolytic anemia in remission in all
domestic animals with exception of the horse. The horse
retains erythrocytes in the bone marrow until they are
fully hemoglobinized even when responding to the most
severe blood loss. MCV of the equine erythrocyte
increases only to a limited extent (frbm 50 fl to 6 5 f l )
under these circumstances.
The total plasma protein concentration is useful to
distinguish between blood loss and hemolytic anemia.
Icterus index is not always elevated in the latter and,
therefore, cannot be depended upon as a distinguishing
feature. The escape of erythrocytes from the circulation
in hemorrhage is accompanied by the loss of protein. The
finding of anemia, hypoproteinemia and reticulocytosis
(dog 1, Table 2) is a triad characteristic of blood loss. In II.
hemolytic anemia the concentration of plasma protein is
normal or slightly increased (dogs 2 and 3, Table 2) since
there is no loss of plasma from the circulation.
The macrocytic hypochromic state rapidly returns to
normal as the crisis for oxygen transport is met. Thus,
macrocytic hypochromic anemia of blood loss or hemo-
lytic disease in remission is pseudomacrocytic for the
macrocytic state is temporary. On the other hand, a true
macrocytic hypochromic anemia is encountered in a
congenital anemia in the Basenji breed? Erythrocyte
life span is short due t o a deficiency of the enzyme
pyruvate kinase. There is a continuous release of
macrocytic hypochromic reticulocytes (20-50%) to the
circulation throughout life of the dog (dog 3, Table 2).
Normocytic normochromic anemia. Anemias char-
acterized by erythrocytes of normal size and Hb content 111.
are secondary to a variety of chronic disorders (Table 1).
Dogs 4 , 5 , 6 and 7 (Table 2) are representative examples.
The normocytic normochromic anemias are generally
unresponsive to therapy as long as the primary disease is
active. Administration of hematinics, vitamins and/or
anabolic steroids is of little avail. Blood transfusions are
indicated when the PCV falls to below 40% of minimum
normal value. Plasma protein concentration may be
reduced but more commonly is normal or somewhat
7
elevated. Reticulocyte respoonse generally is nil.
Chronic secondary anemias may also be normocytic
hypochromic when appetite is poor leading to
malnutritiion.
Microcytic hypochromic anemia. Iron deficiency,
either as a result of faulty intake (dog 8, Table 2), chronic
blood loss (dogs 9 and 10) or faulty utilization of iron as in
vitamin Bg or copper deficiency (Table 1) leads to the
production of small erythrocytes deficient in hemoglobin.
Extrusion of the nucleus from metarubricytes appears to
be controlled by attainment of a critical concentration of
hemoglobin within the cell. In iron deficiency, Hb
synthesis is reduced. The rubricyte nucleus remains
viable beyond the normal time and some late rubricytes
may undergo an additional mitosis giving rise to small
erythrocytes. Erythrocytes in iron deficiency are thin
and pack more completely during centrifugation
resulting in a small PCV value in relation to number of
cells. This fact contributes additionally t o the small MCV
value (40 to 40 fl) in iron deficiency.
The demonstration of a microcytic hypochromic
anemia indicates that iron deficiency exists. Blood
sucking parasites, a bleeding ulcer, or a hemorrhaging
leiomyoma of the duodenum (dog 10, Table 2) should be
searched for. Correction of the primary cause,
transfusions of whole blood and iron therapy are
indicated.
Table 1. Morphollogic Classification of the Anemias
I. Macrocytic Normochromic
A. Penicious anemia in man and primates.
B. Vitamin B12 and folate deficiencies.
C. Cobalt deficiency in ruminants.
D. Erythremic myelosis a myeloproliferative dis-
order in cats.
E. Macrocytosis of Poodles.
Macrocytic Hypochromic
A. During recovery from massive erythrocyte
loss.
1. Hemorrhage.
a. Injury.
b. Neoplasms.
c. Thrombocytopenia.
d. Blood clotting disorders.
2. Hemolytic destruction of erythrocytes.
a. Hemoparasites (anaplasmosis, haemo-
bartonellosis) .
b. Autoimmune hemolytic anemia.
c. Heinz body anemia from drug toxicity.
d. Erythrocyte pyruvate kinase deficiency
in Basenji dogs.
Normocytic Normochromic
A. Chronic diseases leading t o depression of
erythrogenesis as in hypoplastic and aplastic
bone marrow.
1. Chronic infections, particularly suppurative
and granulomatous.
2. Nephritis with uremia.
3. Malignancies.
4. Hormone deficiency (hypothyroidism,
hypoadrenocorticism).
IV. Microcytic Hypochromic
A. Iron deficiency (also copper deficiency).
B. Vitamin B6 (pyridoxine) deficiency.
C. Chronic blood loss (ulcer, leiomyoma of duo-
denum, blood sucking parasites).
SUMMARY
The Wintrobe erythrocytic indexes MCV and MCHC
are used in the morphologic classification of an anemia.
MCV is a measure of mean cell sue and provides a basis
for classifying an erythrocyte population as macrocytic,
normocytic or microcytic. MCHC is a measure of mean
hemoglobin concentration within the erythrocyte and
permits describing a population or red blood cells as
either normochromic or hypochromic.
The morphologic classification of an anemia is an aid
to speculation of possible cause. Macrocytic normo-
chromic indicates an eryhthrocytic maturation defect as

Table 2. Representative Types of Anemias in the Dog and Their Morphologic

Classifications.

Range 37-55 5.5-8.5 12-18 60-77 32-36 19.5-24.5 0-1.5

Normal dog Mean 45 6.8 15 70 34 22.8 0.8 6.0-8.0*

1 4 yrs. Warfarin poisoning 19 2.00 5.4 95.0 28.4 27.00 25.0 5.3
2 3yrs. Autoimmune 17 1.49 4.3 114.0 25.7 28.8 36.0 8.5 Macrocytic
3 10mo. Erythrocytepyruvate 19 2.11 4.9 90.0 25.8 23.2 21.8 7.8 hypochromic
kinase deficiency
4 8 yrs. Chronic interstitial 17 2.61 5.9 65.1 34.7 22.6 0.0 6.7
nephritis with uremia Normocytic
5 5 yrs. Chronic infection 29 4.10 10.6 70.7 36.6 25.8 Oe2 7-5 normochromic
6 5 yrs. Lymphosarcoma 28 4.29 8.7 65.3 31.1 20.3 4.6 6.4
7 8yrs. Pyometra 25 3.48 8.0 71.8 32.0 23.0 1.8 7.3
8 2 mo. Iron deficiency 15 3.37 3.7 43.2 24.7 10.7 6.8 4.0
9 8 m o . Coiccidiosis 26 6.04 7.3 43.0 28.1 12.1 5.1 2.8 Microcytic
10 8 yrs. Bleeding neoplasm, 8 1.57 2.3 50.9 28.7 14.6 12.4 6.4 hypochromic
leiomyoma of nut
*Age dependent, 6.0 to 7.0 for young dogs, 7.0 to 7.5 for young adults, and 7.5to 8.0 for old dogs.

seen in vitamin B12 and folic acid deficiencies, in
erythremic myelosis and erythroleukemia of cats, or
macrocytosis in miniature poddles. Macrocytic hypo-
chromic is characteristic of erythrocytes in response t o
blood loss or hemolytic anemia. Normocytic normo-
chromic indicates a depression of erythrogenesis
secondary t o a variety of chromic diseases, e.g.,
infections, malignancies, hormone deficiences, and
nephritis with uremia. A classification of microcytic
hypochromic is the hallmark of iron deficiency.

References
1. Gordon, A. S.. and Zanjani, E. D.: Some Aspects of
Erythropoietin Physiology. In Regulation of Hematopoiesis. A. S.
Gordon (ed), Vol. I, p. 413. Appleton-Century-Crofts. New York,
1970.
2. Searcy, G. P., Miller, D. R., and Tasker, J. R.: Congenital
Hemolytic Anemia in the Basenji Dog Due to Erythrocyte (RBC)
Pyruvate Kinase (PK)Deficiency. Canad. J. Comp. Med.. 35:67,1971.
Abst. Blood. 34:860,1969.