The Natural History of Iron Deficiency

Induced by Phlebotomy

By MARCEL E. CONRAD AND WILLIAM H. CROSBY

With the technical assistance of N. R. Benjamin

S YDENHAM’S
patients was
report
the basis
of
for
the
the
therapeutic
rational use
effectiveness
of this agent
of
and
iron in
its recognition
chiorotic

as an essential body constituent and requirement. Subsequently, the demonstra-

tion of microcytic and hypochromic red cells led to a method of differentiating

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iron deficiencies from other anemias.’ Newer technics such as serum iron
determination, radioactive iron studies and quantitation of iron in tissue stores
have provided more precise means of distinguishing iron deficiency from other
disorders.28 We have attempted to study the temporal relation of the char-
acteristic changes of iron deficiency and to learn how they relate to severity.
Phlebotomy was selected as the method of studying the natural history of iron
deficiency in humans.

METHODS

Subjects of the experiment were nine healthy volunteers and three patients with un-
treated polycythemia vera.
Phlebotomy was performed in 500 ml. increments, one to three times weekly, until
each person had been bled 2.0 to 7.5 L. Twenty ml. of blood was taken at each phlebotomy
and subsequently at weekly intervals for chemical analyses and blood counts. The loss
of hemoglobin was computed by multiplying volume of the shed blood by hemoglobin
concentration. The loss of iron was calculated on the basis of 3.38 mg. of iron per Gm. Hb.
Red cell indices were computed from counts of at least 2000 cells,9 duplicate high speed
microhematocnts1#{176} and calibrated hemoglobinometry.11 Reticulocyte counts were per-
formed by the method of Brecher.’2 Serum iron was measured by the method of Ramsay
(normal: 70-170 g./100 ml. )2 and the unsaturated iron binding capacity also by the
method of Ramsay3 or, in some of the earlier studies, that of Ventura (normal: 250-400
j.g./100 ml. ).13 Bone marrow iron was demonstrated by the Prussian blue reaction.9
Gastrointestinal absorption of iron was determined by measuring body retention of orally
administered radioactive iron59 (1 jc in a 1 mg. carrier dose) using a whole-body liquid
scintillation counter. Normal volunteers absorbed less than 10 per cent of the administered
dose.7

RESULTS

Iron deficiency was gradually induced in nine normal and three poly-
cythemic adult males by repeated phlebotomy. Bloodletting was performed
once weekly in seven of the normal volunteers (table 1: 2-8). The polycythemic
patients and one normal volunteer (case 1) were bled 1-1.5 L. weekly. There

From the Department of Hematology, Walter Reed Army Institute of Research, Wash-
ington, D. C.
This material was presented in part at the Seventh International Congress of the Inter-
national Society of Hematology, Rome, September 1958.29
Submitted Feb. 16, 1962; accepted for publication Apr. 11, 1962.

173

BLOOD, VOL. 20, No. 2 (AUGUST), 1982

174 CONRAD AND CROSBY

Table 1.-Phlebotomy in Normal Volunteers

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la 55 10 589 1.96 0 5.0 14.7 45 90 29 32 0.5 140 330

50 3.3 9.7 31 94 29 31 1.0 65 400
116 5.3 12.4 41 77 23 30 0.5 60 450
lb 5 :322 1.09 290 4.8 14.6 45 93 30 32 0.4 125 350 64.0
340 4.0 11.2 35 87 28 32 0.8 49 435

436 5.4 12.6 43 79 23 29 0.8 48 385

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480 5.2 14.5 45 87 27 32 0.6 75 321

2 70 5 340 1.15 0 4.95 15.5 47 95 31 32 0.5 165 400

41 4.3 12.9 40 93 30 32 0.8 70 525
92 5.2 13.3 43 82 26 31 0.3 85 495
:i so 5 337 1.14 0 4.6 14.6 46 99 31 32 0.7 122 235 2.4
35 3.2 11.0 35 110 34 32 1.3 41 420
126 4.9 11.5 40 81 23 28 0.5 30 445
190 5.1 13.9 45 88 27 31 0.7 109 365 71.0

4 60 5 277 .94 0 4.3 12.8 40 93 30 32 0.3 93 242 0.40

42 3.4 9.4 30 87 30 31 0.9 24 423
119 5.2 12.7 40 77 24 32 0.9 46 355
190 4.2 i:.i 40 95 31 32 0.7 117 335 13.0
5 85 5 293 .99 0 5.2 13.9 44 86 26 31 0.3 117 347 I .20
:15 3.5 9.3 31 58 26 30 0.9 26 486
119 4.8 10.8 35 73 22 30 0.8 24 504 40.0
6 64 5 270 .92 0 4.2 12.1 41 97 28 30 0.8 132 294 1.60
35 2.9 8.1 26 89 28 31 0.8 19 476
87 5.3 11.2 42 75 22 27 0.5 37 405 13.2
7 70 5 322 1.09 0 5.2 15.3 48 92 29 31 0.4 141 316 5.4
28 3.1 10.3 33 105 33 31 1.1 35 422
$4 5.2 12.2 42 80 24 29 0.7 26 422 65.4
S 81 5 295 1.00 0 4.6 13.1 42.5 92 28 31 0.6 88 280 1.7
28 3.9 10.5 33 84 27 32 1.3 49 341
to 4. 12.5 40 83 26 31 0.5 50 423
9 76 4 242 .85 0 4.6 13.5 42 91 29 32 1.0 94 294 30.5
42 3.4 9.3 30 88 30 31 1.8 32 472
105 4.7 11.0 37.5 80 23 29 0.5 38 444
1#{149}0 4.7 13.3 42.5 93 28 31 0.5 79 397

were no untoward reactions to phlebotomy. Each volunteer continued per-

forming normal daily activities without difficulty.
After three to five phlebotomies, the rate of decline in hemoglobin concentra-
tion slowed despite continued bleeding. Increased production of hemoglobin
persisted until the subject had lost approximately 1.5 Gm. of iron. Then the
hemogichin concentration again fell at an accelerated rate if phlebotomy was
continued ( fig. 1 ) . Reticulocytosis was maximal during the period of ac-
celerated hemoglobin production but never exceeded 4 per cent. Volunteers
with partially depleted stores due to previous phlebotomy had less pronounced
reticulocytosis ( cases ib, 2 and 9).
The plasma iron concentration gradually fell but did not fall below the
normal range until at least 500 mg. of iron had been removed. Decreased
serum iron was the earliest evidence of iron depletion. Subsequent to a de-
crease in the serum iron, there was a pronounced increase in the total iron
binding capacity of the serum in each person. These changes persisted until
IRON DEFICIENCY INDUCED BY PHLEBOTOMY 175

EFFECT OF REPEATED PHLEBOTOMY

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Fig. 1.-Following the initial three phlebotomies, a rapid fall in hemoglobin
concentration was observed. The rate of decline iii hemoglobin slowed with con-
tinued bleeding. When hemoglobin production kept pace with loss, an average of
20.8 Gm. of hemoglobin was produced daily (3-4 times normal) . Maximum reticu-
locytosis occurred during this period. As available iron stores were depleted, hemo-
globin again declined with each blood-letting. ( All illustrations in this article are
U. S. Army photographs.)

he had recovered from the induced anemia and the hemoglobin concentration
had returned to prephlebotomy values.
Ten to twenty days after the initiation of phlebotomy and coinciding with
the reticulocyte response, there was an increase in the mean corpuscular vol-
ume of the red cells. Then the cells gradually decreased in size with the most
marked microcytosis occurring 90 to 120 days following the initial bleeding in
all subjects. Recognizable hypochromia was seen in five of the volunteers. De-
creases in the mean corpuscular hemoglobin concentration were less pro-
nounced than changes in cell volume, and occurred subsequent to them.
Two subjects had no change in mean corpuscular hemoglobin concentration
despite significant decrease in the mean corpuscular volume (fig. 4). Despite
the loss of 850 to 1960 mg. of iron by these normal volunteers, only moderate
changes in cell size and shape were demonstrable.
Gastrointestinal absorption of iron was studied by means of orally admin-
istered radioactive iron59 and a whole body liquid scintillation counter. Prior
to phlebotomy, the iron-replete volunteers absorbed less than 10 per cent of
the radionuclide. Increased absorption (over 10 per cent) was observed
during recovery from iron loss in all six of the volunteers tested. Three of
these subjects (cases 1, 3 and 4) absorbed increased iron after their hemo-
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CROSBY

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IRON DEFICIENCY INDUCED BY PHLEBOTOM’i 177

100 35 INDICES

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80 25

60 20

Phlebotomy

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DAYS FOLLOWING FIRST PHLEBOTOMY

Fig. 3.-Subject lb. Subject la (fig. 1) bled an additional 2.5 L. after initial
recovery from abnormalities in the peripheral blood suggestive of iron deficiency.

globin, cellular indices and serum iron had returned to approximate pre-
phlebotomy levels.
One normal subject (fig. 7) absorbed 64 per cent of the test dose of iron 300
days following the first phlebotomy. At that time there was no other demon-
strable evidence of iron deficiency except for the absence of stainable iron in
bone marrow sections. This subject was subsequently phlebotomized an addi-
tional 2.5 L. with an additional loss of 1.09 Gm. of iron. The hemoglobin re-
turned to normal prephiebotomy levels in 190 days (fig. 3). The hemoglobin
iron in this case was replenished from an ordinary diet at a rate of 5.7 mg
daily.14
Three patients with polycythemia vera were bled for therapeutic reasons.
This permitted scrutiny of the rate of red cell regeneration when hemoglobin
synthesis was limited by iron deficiency. Though these bled patients showed
the same temporal relation of changes in cellular indices and serum iron as
did the normal subjects, the changes in cell size were more profound. The
rapid return of the red cell count to supranormal levels while hemoglobin
concentration remained low was associated with marked depression of the
 178 CONRAD AND CROSBY

00 35 N

Phlebotomy

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Fig. 4.-Subject 2. Chronic blood donor bled 2.5 L. Minimal reticulocytosis and
absence of an increase in MCV were attributed to a decrease in available stored
iron. Red cells decreased in size without change in mean corpuscular hemoglobin
concentration.

mean corpuscular volume and mean corpuscular hemoglobin and the appear-
ance of extremely small erythrocytes. The microcytosis in these polycythemic
patients was greater than that observed in our normal volunteers and in
nonpolycythemic patients with more profound iron deficiency (figs. 5 and 6) 15

DISCUSSION

The total iron present in the adult human is not great, amounting to 3-5
Gm. Approximately 55 per cent is present in circulating hemoglobin, a small
fraction is incorporated in bone marrow hemoglobin, myoglobin and respira-
tory enzymes (10-20 per cent), and the remainder is found in iron stores.16
The normal human preserves a constant level of total body iron throughout
adult life by efficient conservation of this element, maintaining rigid control
over absorption to balance losses.17’ The adult male loses approximately 1
mg. of iron daily, mostly in desquamated epithelium.’5 The balance of absorp-
tion and loss may be referred to as iron equilibrium. In case of chronic con-

tinued bleeding, the increased loss may be compensated by increased absorp-

IRON DEFICIENCY INDUCED BY PHLEBOTOMY 179

Table 2.-Phlebotomy in Polycythemia Vera

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A 65 15 1130 3.81 0 8.8 20.9 73 83 25 29 0.8 108 415
84 4.5 8.5 32 73 19 26 1.2 64 450
154 6.5 9.7 40 62 15 24 0.8 56 435
400 7.6 17.8 60 78 24 30 0.6 115 398
B 63 9 564 1.90 0 6.9 18.4 64 93 27 29 0.8 350 420
:34 3.5 8.8 30 85 25 29 2.0 55 525

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114 6.1 11.3 43.5 71 19 26 1.2 60 535
300 6.5 14.1 50 77 22 28 1.0 81 340
C 82 12 884 2.98 0 7.0 20.2 64 91 29 32 0.5 175 280
70 5.0 10.8 38 76 22 28 0.7 72 410

tion, and the patient thereby maintains iron equilibrium. Normal menstruating
women are examples of this.
Iron deficiency may be defined as a reduction of total body iron below levels
which are normal for the subject in question. To restore iron repletion, the
absorption must temporarily exceed loss until the total body iron becomes
normal once again. The demonstration of increased absorption implies iron
deficiency ( or hemochromatosis ) . Iron deficiency may exist even in the
presence of normal hemoglobin and red cell mass and may be confirmed by
finding depleted iron stores.5 Thus demonstration of increased absorption and
reduced iron stores permits the diagnosis of iron deficiency in the absence of
anemia, hypoferricemia or changes in erythrocyte morphology.7’9 Iron de-
ficiency develops when iron equilibrium is upset, either by insufficient intake
or by excessive loss.1hi The most common causes are, on the one hand, pro-
longed dietary deficiency or poor absorption secondary to gastrointestinal
pathology,2#{176} and, on the other hand, loss by bleeding and childbirth.
The term iron-deficiency anemia is properly used when the total body iron
is insufficient to provide a normal mass of hemoglobin. It is not accurately used
to describe blood-loss anemia when iron stores are still sufficient, as in the
early phase of the present studies. It should not be used to describe depletion
of iron stores where there is no anemia, as in the final phase of these studies.
This is iron deficiency without anemia, characterized only by depletion of the
iron stores.
The concentration of iron in circulating hemoglobin (3.38 mg. iron/Gm. of
Hb.) makes hemorrhage an ideal method of rapidly depleting body iron. In-
duction of iron deficiency in normal subjects by bloodletting produces a con-
trolled disorder which permits planned scrutiny of the characteristic abnor-
malities of this state. The initially normocytic, normochromic anemia is due
to loss of hemoglobin. A mild reticulocytosis, maximal 10-20 days after the
initial phlebotomy, is probably responsible for the increase in mean corpuscular
volume seen at that time.2’ The earliest evidence of iron depletion is diminu-
tion of the serum iron. As the serum iron concentration falls, the mean corpuscu-
180 CONRAD AND CROSBY

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IRON DEFICIENCY INDUCED BY PHLEBOTOMY 181

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p
31

Fig. 6.-Microcytosis was more pronounced 100 days postphlebotomy in polycy-

themic patients (above) than in normal volunteers (below) (X784).

lar volume and mean corpuscular hemoglobin begin to diminish, and the un-
saturated iron binding capacity of the plasma increases. Only subsequently is
there a decrease in the mean corpuscular hemoglobin concentration. This
seems to indicate that cellular hemoglobin concentration is preserved at the
expense of erythrocyte size.
182 CONRAD AND CROSBY

IRON ABSORPTION

U) IRON DEFICIENT
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DAYS

Fig. 7.-A normal volunteer who absorbed 5 per cent of an oral dose of iron
when iron replete, and absorbed 64 per cent 274 days following phlebotomy.
Increased absorption was demonstrated after the indications of iron deficiency in
the peripheral blood had returned to approximate prephlebotomy levels, but with
bone marrow stores still iron depleted. (Subject 1, figs. 1 and 2.)

The cellular indices and morphology of the peripheral smear are useful
tools in establishing the diagnosis of iron deficiency. However, the diagnosis
will be missed frequently if departure from “normal values” is used as the
principal criterion.22 Others have emphasized the relation of the severity of
iron depletion to the degree of hypochromia, microcytosis and the intensity of
the anemia.2324 The time of examination of the blood with relation to the
loss of iron is equally important. Despite significant loss of iron, our patients
had “normal indices” for at least 60 days following phlebotomy. Maximum
changes in circulating erythrocytes occurred when the normal cells produced
prior to iron loss were completely replaced by newly formed microcytes (90-
120 days). As iron deficiency becomes even more severe, the quality of the
red cells deteriorates further. At first they are only smaller. When the stores
have been completely exhausted, the mean corpuscular hemoglobin concentra-
tion begins to diminish. When the iron deficiency becomes extremely severe,
the life span of the red cells may be shortened, complicating the situation with
a true hemolytic disease.’5
Estimation of iron in marrow stores and radioiron technics provided methods
IRON DEFICIENCY INDUCED BY PHLEBOTOMY 183

of detecting iron deficiency when there were no diagnostic abnormalities in

the peripheral blood. Increased absorption of iron from the gut can be demon-
strated early in the course of iron depletion and these changes persist until
iron stores are repleted. Depletion of marrow hemosiderin cannot be used as
an early criterion of iron deficiency, but this change persists after peripheral
blood abnormalities return to prephiebotomy levels.57 The mechanism by
which the depleted depots transmit their requirement to the epithelium of the
gastrointestinal tract remains unknown.2528

SUMMARY

1. The sequence of characteristic changes of progressive iron deficiency was

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demonstrated by serial bleeding of normal volunteers and polycythemic pa-
tients.
2. After bloodletting, changes occurred in peripheral blood in the following
order: a ) fall in hemoglobin concentration; b ) decreased plasma iron; c)
reticulocytosis, increased MCV and MCH; d ) diminution of MCV and MCH,
increased total iron-binding protein; and e ) decreased MCHC.
3. Characteristic changes of iron deficiency returned to prephlebotomy
levels in the following sequence: a ) hemoglobin concentration; b ) cellular
indices; c) serum iron; d) serum iron binding protein; and e) bone marrow
hemosiderin, and finally the increased gastrointestinal absorption of iron
reverted to normal.
4. Accelerated production of red cells continued in polycythemic patients
despite the induction of moderate iron deficiency. Quality was sacrificed for
quantity, and thereby a more profound microcytosis occurred than in normal
subjects with a similar degree of iron deficiency.

SUMMARIO IN INTERLINGUA

1. Le sequentia del alterationes characteristic de progressive deficientia de

ferro esseva demonstrate per le sanguination serial de voluntarios normal e
de patientes con polycythemia.
2. Post le sanguination, alterationes in le sanguine peripheric occurreva in
le sequente ordine: a) declino del concentration de hemoglobina; b) declino
del nivello de ferro in le plasma; c) reticulocytose, augmento del volumine
corpuscular medie e del hemoglohina corpuscular medie; d) declino del
volumine corpuscular medie e del hemoglobina corpuscular medie, augmento
del total proteina ferro-ligatori; e) declino del concentration medie de hemo-
globina corpuscular.
3. Le alterationes characteristic de deficientia de ferro retornava al nivellos
de ante le phlebotomia in le sequente ordine: a) concentration de hemoglobina;
h) indices cellular; c) ferro seral; d) proteina ferro-ligatori del sero; e) hemo-
siderina del medulla ossee; e, finalmente, f) renormalisation del augmentate
absorption gastrointestinal de ferro.
4. Production accelerate de erythrocytos continuava in patientes polycythemic
in despecto del induction de moderate grados de deficientia de ferro. In illos
qualitate esseva sacrificate pro quantitate, e assi ii occurreva un plus profunde
microcytose que in sub jectos normal con un simile grado de deficientia de ferro.
184 CONRAD AND CROSBY

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IRON DEFICIENCY INDUCED BY PHLEBOTOMY 185

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XIV. The relationship of humoral 29. Conrad, M. E., Jr., and Benjamin, N. R.:
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Ma/or Marcel E. Conrad, MC, Chief, Department of Gastro-

enterology, Walter Reed Army Institute of Research,
Washington, D.C.

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Colonel William H. Crosby, MC, Chief, Department of Hem-
atology, Walter Reed Army Institute of Research, Washingtcm,
D.C.