An unusual variant of hereditary spherocytosis

An Unusual Variant of Hereditary Spherocytosis*

JAMES

S. WILEY,

BARRY G. FIRKIN,

M.B., M.B.,

B.Sc. M.R.A.C.P.t

Sydney, Australia

* From the Department of Medicine, University of Sydney, Sydney, New South Wales, 2006, and Clinical Research Unit, Royal Prince Alfred Hospital, Australia. Requests for reprints should be addressed to Dr. J. S. Wiley. Royal North Shore Hospital, St. Leonard% Sydney, New South Wales 2065. Australia. Manuscript received February 18, 1969. t PraSant address: Department of Medicine, Alfred Hospital, Prahran, Victoria 3181, Australia. Volume

48, January

1970

A variant of hereditary spherocytosis is described in which red cell survival studies indicated severe splenic conditioning of nearly all the circulating red cells. Clinical features also suggested a severe form of the disease with anemia, splenomegaly, a history of leg ulcers and retardation of growth in childhood. Osmotic fragility testing showed a high median corpuscular fragility which decreased appreciably after removal of the spleen. Autohemolysis was atypical for hereditary spherocytosis in that the increased hemolysis of incubated blood was not corrected by glucose supplementation, although after splenectomy the autohemolysis reverted to the typical pattern. Transfusion of the patient’s red cells to a normal compatible recipient resulted in their removal by the spleen with a half time (T1/2) of twelve and a half minutes, equivalent to complete removal of these cells during a single passage through the spleen. After the patient’s spleen had been removed, however, his red cells survived in a normal recipient with a Tl/a of ninety minutes. This difference in survival times clearly demonstrates that splenic conditioning had a deleterious effect on the in vivo survival of the patient’s cells; moreover splenic conditioning also seemed responsible for the atypical autohemolysis of these cells. Since the patient’s cells survived with a T1/2 of ten days in his own circulation but with a Tl/a of only twelve and a half minutes in a normal recipient, the efficiency of the patient’s spleen in removing his own circulating red cells was severely limited. This inefficiency in the sequestering function of the patient’s spleen was attributed to reticuloendothelial blockade. The observation of Howell-Jolly bodies in the patient’s red cells before splenectomy was also consistent with reticuloendothelial blockade and reduction in the “pitting” function of the spleen.

Hereditary spherocytosis varies in its manifestations and clinical severity, even though it is unusual for the anemia in this condition to be severe. Although it is likely that much of the variability results from genetic heterogeneity, this can be established with certainty only when the underlying biochemical lesion is defined. Nevertheless there appear to be subgroups within this disease, since atypical cases of hereditary spherocytosis have been reported with a variety of anomalous features. In two cases in a South African family with hereditary spherocytosis and in a sporadic case found in Wisconsin osmotic fragility was shown to be normal, and although the survival of radioactive chromium labeled red cells was shortened, the spleen did not sequester these cells [l,Z]. Another report describes three cases of an unusual spherocytic anemia in which the spherocytes disappeared within one year of splenectomy [3]. Still another distinct subgroup are the two cases labeled as “atypical (group B) spherocytosis” by Young and colleagues together with three similar cases mentioned by Dacie and a recent report of two more such cases [4-61. Autohemolysis of blood in these cases was increased but not corrected by the addition of glucose and thus differed from the usual pattern seen in hereditary spherocytosis. Finally, mild forms of the disease characterized by the absence of anemia or splenomegaly and the presence of only minor changes in osmotic fragility have been recognized since their description by Gansslen, Zipperlen and Schijz but they are not common [5,7]. The patient described here represents a variant of hereditary spherocytosis with unusual features resulting from a major degree of red cell 63

VARIANT

OF HEREDITARY

SPHEROCYTOSIS

-

WILEY, FIRKIN

conditioning by the spleen. Such was the severity of this red cell damage that transfusion of the patient’s cells to tiormal compatible recipients resulted in their complete removal during a single passage through the spleen, a finding in marked contrast to other patients with hereditary spherocytosis whose cells survive for several days in the circulation [8,9]. Despite this extreme degree of trapping of the patient’s red cells in a normal spleen, the life span of these cells within the patient’s own circulation was similar to that of other patients with hereditary spherocytosis, thus indicating gross inefficiency of the patient’s spleen in sequestering his own red cells. Reticuloendothelial blockade is suggested as a possible explanation for the splenic inefficiency and this interpretation is supported by the finding of inclusion bodies in the circulating red cells. Moreover, the autohemolysis test was of the “atypical (group B) type” [4] since the considerable hemolysis occurring in vitro was actually increased by glucose supplementation. The autohemolysis test reverted to the typical pattern after splenectomy, indicating splenic conditioning as the cause of the atypical autohemolysis results. Investigation of hereditary spherocytosis in the mother and brother disclosed a similarity to the propositus in some of the hematologic features. This variant of hereditary spherocytosis emphasizes the wide range that can occur in the magnitude of the cellular defect and is further evidence for the variability observed in this disease. CASE

REPORTS

Propositus

(11-l).

The

patient,

a fifty-eight

year

old white

man (H.C.H.), was asymptomatic and referred for investigation only because splenomegaly was found on a routine insurance examination. He gave a past history of retarded growth in childhood. Although he weighed about 50 pounds at six years of age, his weight was only 77 pounds at fifteen and a half years, well below the 3 percentile for this age. Within one year, however, his weight increased to within the normal range. Growth in height was always normal. Throughout childhood he frequently felt weak and unwell, and at the age of twelve he had numerous leg ulcers from the midcalf level down to and including the dorsum of both feet. This leg ulceration persisted for nine months but did not recur subsequently. Adult life was uneventful until the age of fifty when he was awakened one night with the abrupt onset of pain and tinnitus in the right ear. Audiometric testing at the time, which was confirmed during this investigation, showed a severe nerve deafness in the right ear and a milder nerve deafness in the left ear. There was a strong family history of hereditary spherocy tosis (Fig. 1). The mother (l-2), two brothers (11-3, 11-4) one nephew (Ill-l) and a niece (111-3) of the propositus were

Fig. 1. Pedigree of propositus (11-1) showing family hereditary spherocytosis. l = hereditary spherocytosis.

64

history

of

investigated Brisbane

in

was confirmed osmotic

the

Children’s from

fragility

the blood screening.

(Christiansen-Wolf) South

Jutland

Royal Hospital:

region

had

Brisbane the

smear, Both

Hospital

presence

and

of the

reticulocyte maternal

emigrated

to

of Denmark,

but

Royal disease

count

and

grandparents

Australia it was

from not

the

possible

to establish if either suffered from anemia. Examination of the patient revealed 6 cm of nontender splenomegaly and numerous scars left by the leg ulcers in childhood. Hemoglobin was 12.5 gm per cent with 6 per cent reticulocytes. A peripheral film showed numerous spherocytes and microspherocytes and was typical of hereditary spherocytosis apart from the presence of red cell inclusion bodies. White blood cell and platelet counts and the white cell differential count were normal; the white cell lobe count was normal although the direct Coombs test was negative. The serum bilirubin level was elevated to 1.4 mg per cent (0.6 mg per cent conjugated). Results of the following tests were within normal limits: serum alkaline phosphatase, thymol and zinc sulphate flocculation tests, serum protein concentration, serum glutamic-oxaloacetic transaminase, serum cholesterol, blood urea, serum uric acid and blood glucose. The result of Schumm’s test was also negative and neither urinary porphyrins nor porphobilinogen were detected. A serum electrophoretogram showed slight decrease in the alphan-globulin fraction. A chest x-ray film was normal. An oral cholecystogram revealed calculi in a functioning gallbladder. A screening test for pyruvate kinase deficiency [lo] and a methemoglobin reduction test [ll]

were both normal. Hemoglobin electrophoresis revealed no abnormal hemoglobins and hemoglobin A, was not increased. In April 1968 splenectomy was performed, and an enlarged spleen weighing 1,027 gm was removed. Microscopically the architecture of the spleen was normal, and the red pulp was congested with spherocytic cells. There were no unexpected findings on either light or electron microscopy of splenic tissue, and there was little evidence of erythrophagocytosis. One unit of blood was transfused during the operation, and it could be calculated that the transfused cells contributed less than 15 per cent of the red cell volume for the immediate postoperative tests. The platelet count rose temporarily following the operation to a maximum of 850,000 per cu mm after nine days. The patient’s ability for sustained work and sense of well-being improved greatly after the operation and after nine weeks his hemoglobin was 15.0 gm per 100 ml, reticulocytes 1.5 per cent and platelet count normal. In this eighty-nine year old widow (M.H.) the Mother (l-2). diagnosis of hereditary spherocytosis was made seventeen years breviously, but she did not elect to undergo splenectomy. During childhood she once noticed slight jaundice, and this had recurred at infrequent intervals thereafter although she never had symptoms of anemia. Leg ulceration had begun in her mid-forties, was bilateral and had recurred over the ensuing forty years. Examination in 1968 showed 6 cm nontender splenomegaly, 2 cm hepatomegaly and induration and pigmentation on both shins. Hemoglobin was 9.7 gm per cent with 12 per cent reticulocytes, white blood cell and platelet counts were normal and a peripheral film showed spherocytes, microspherocytes and red cell inclusion bodies similar to those observed in her son’s red cells. A direct Coombs test was negative, the serum bilirubin level was raised to 3.9 mg per cent (1.5 mg per cent conjugated) whereas serum alkaline phosphatase, thymol and zinc flocculation tests, serum protein concentration, blood urea and blood glucose were normal. Serum folate concentration (2.2 ng per ml) was below the normal range (3.2 to 24.0 ng per ml) whereas serum iron, total iron-binding capacity and serum vitamin B13 concentrations were all normal. Brother (11-4). This forty-two year old man (R.T.H.) had The AmLrican

Journal

of Medicine

VARIANT

OF

HEREDITARY

SPHEROCYTOSIS

-

WILEY.

FIRKIN

suffered episodic attacks of left hypochondria1 pain, jaundice with vomiting and anemia since childhood. At the age of twenty hereditary spherocytosis was diagnosed, and splenomegaly was noticed on examination. Leg ulceration had never occurred. Splenectomy was performed of thirty following which he had complete

at the age relief from

symptoms.

METHODS Red cell survivals were carried out using the chromium-51 technic [12,13]. Ten milliliters of blood was withdrawn into a sterile syringe containing 3 ml of acid-citrate-dextrose solution. The blood was then centrifuged for three minutes at 1,000 g in a sterile McCartney bottle, the plasma and huffy coat were removed and 50 PC Na8CrOl added to the packed cells which were allowed to stand at room temperature for twenty minutes. Eighty-eight per cent of the radioactivity was shown to attach to red cells under these conditions. The packed cells were then washed twice in sterile saline solution to remove extracellular “*Cr, made up to 12 ml volume with saline solution and an injection of 10 ml was given to the patient. An aliquot of the remaining red cells was diluted for counting to allow calculation of the body red cell volume. Fifteen to twenty minutes after injection of the labeled cells the initial sample was taken. Samples consisted of blood withdrawn into a heparinized syringe, 10 ml of which was hemolyzed with saponin for counting in a Packard automatic scintillation counter. Survivals were followed until the Tl/a was reached. All samples were counted together and the count rates corrected for background and the hematocrit of each sample. No correction was made for elution of %r. When sampling at intervals of several minutes was necessary, an indwelling Cournand needle was inserted into the antecubital vein to avoid repeated venepunctures. Normal red cell survival Tl/z fell in the range of twenty-six to thirty days. Measurements

of

osmotic

fragility

were performed

by standard

was determined

using 22NaCI [15].

tions were measured the

cells

having

methods

by flame

been

ment

into erythrocytes

cell sodium

during

spension

with

Glycolytic

rate was measured

cell

suspension

incubations

on

imidazole.

for at

37°C

by hypotonic

hemolysis

were then

for twenty

MgC13 (3 (100

mM),

sodium

chloride

pH 7.5,

both in the absence

PM) and the ATPase of the increase concentration The both

mM) activity

in inorganic of the mixture

morphology by light

and

of the electron

All

Erythrocyte

stromas

of red cells and re-

minutes

at 37°C

potassium

chloride

calculated

with ATP (10

and imidazole-chloride and presence

pH 7.5. Stromas mM),

(5 mM),

of ouabain

from

(100

z

measurement

(Pi) and the protein

[17]. patient’s

spleen

microscopy.

was

A small

examined block

1

0.5

of

(

.I

The

The

standard methods and the sections Phillips 200 electron microscope. 1970

was

prepared

were examined

by

under a

3.

(. (--.-.-.-.-.-_)

Osmotic

fragility

curve

of

blood

) and fragility and

curve the

0.9

0.7

NaCl

%

and fixed for Fig.

January

propositus

of

splenectomy

46,

the

20 mM

sample

Volume

of

by a

(6.5 per cent v/v) buffered

cacodylate.

blood

peripheral

Red Cell Inclusion Bodies. Examination of peripheral blood films both of the propositus (11-l) prior to splenectomy and his mother (l-2), who had an intact spleen, showed the presence of small red cell inclusion bodies (Fig. 2) in addition to spherocytes, microspherocytes and punctate basophilia. These inclusion bodies did not stain for iron but were positively identified as Howell-Jolly bodies by a modification of the Feulgen stain [18]. Similar red cell inclusion bodies in a case of hereditary spherocytosis have been noted by Dacie [5, Fig. 381. Osmotic Fragility and Autohemolysis. Osmotic fragility testing of fresh blood from the patient drawn into heparin showed an unusually high median corpuscular fragility of 0.68 per cent sodium chloride (Fig. 3). Two weeks after splenectomy the median corpuscular fragility was unaltered, and although the curve differed slightly from that obtained before splenectomy, this difference could be attributed to the cells that were transfused during operation. Nine weeks after splenectomy the osmotic

three hours in cold glutaraldehyde with

the

RESULTS

to

7.4

in

at 20 to

chloride,

spleen was cut within seconds after its removal pH

body

37°C.

composed

was removed.

phosphate

in

Howell-Jolly splenectomy.

in vitro

in 6 mM Tris chloride,

of hemoglobin

incubated

at

production

[16].

5 mM potassium

peated washing of the stroma

mM),

hours

medium

pH 7.5 and 10 mM glucose.

prepared

0.12 move-

2.

before

of the cell su-

red cells suspended

until all visible trace (1.5

four

in a buffered

chloride,

in cold

from the changes

as the lactate

washed

hematocrit

145 mM sodium

times

incubation

ouabain

incubation

employed

40 per cent

were

pM

cation concentraof an hemolyzate.

The net passive sodium

was calculated

concentration 100

Cellular three

autohemolysis

and sodium influx

photometry

washed

M MgCI, prior to hemolysis.

and

[14]

Fig.

mother

from

nine

the

weeks

of

blood

(-

-------_)

propositus after

from

before

splenectomy the

brother is

also

shown.

65

VARIANT

OF HEREDITARY

20-1 0

SPHEROCYTOSIS

8

-

WILEY,

FIRKIN

I 36

32

24

16 DAYS

Fig.

Autologous

4.

splenectomy tomy

(0)

was

ten

survivals and

days

after and

of

red

cells

splenectomy after

in

(0).

splenectomy

the

The

propositus T%

before

before

twenty-nine

splenec-

days.

7001

0

0

4

12

20

16

MINUTES Fig.

5.

Rapid

following were

disappearance

injection

sequestered

a half

into by

a

the

of labeled normal

red

cells

compatible

recipient’s

spleen

from

the

recipient. with

a

propositus

Labeled

cells

TX/2 of twelve

and

minutes.

100

80

0”

sm

60

z-

fragility had decreased appreciably to a median corpuscular fragility of 0.65 per cent sodium chloride. Since the shape of the fragility curve was the same before and after splenectomy (Fig. 3), it is evident that most if not all of the patient’s cells now had a lower fragility. No further change in osmotic fragility was detected six months later. The autohemolysis test was performed twice before splenectomy and gave atypical results since, on both occasions, the increased hemolysis in the absence of glucose (30 and 16 per cent) was not reduced by glucose supplementation (50 and 21 per cent, respectively). One week after splenectomy the autohemolysis results were still atypical (18 per cent hemolysis without glucose, 28 per cent with glucose supplement) but nine weeks after operation the autohemolysis had reverted to the pattern typical of hereditary spherocytosis (10 per cent hemolysis without glucose, 3 per cent with glucose supplement). Recovery of the typical pattern of autohemolysis following splenectomy thus followed a time-course similar to the decrease in osmotic fragility. Red Cell Survivals. Survival of the patient’s red cells in his own circulation was shortened, with a Tl,s of about ten days (Fig. 4). Hemolysis of this degree is typical of hereditary spherocytosis, moreover surface counting of organ activities showed the expected degree of sequestration and destruction of labeled red cells in the spleen since the ratio of splenic to precordial count rates rose from 2.3 one hour following injection to reach 4.4 after ten days [8]. The rate of disappearance of the patient’s red cells was then measured in a normal volunteer subject of the same blood group (A positive) in whom compatibility had been established by routine crossmatching technics. The labeled cells were rapidly removed at an exponential rate with a Tl/s of twelve and a half minutes (Fig. 5). This unexpected result was confirmed in two more compatible normal subjects in whom Tl/s values of eleven and twelve minutes were obtained. Surface counting over the liver, spleen and precordium at the conclusion of each survival showed an accumulation of radioactivity only in the recipient’s spleen. Since the patient’s hereditary spherocytes were very fragile, it seemed possible that centrifuging the cells during the labeling procedure in vitro could have damaged them and shortened their subsequent survival. This possibility was eliminated by modifying the procedure so as to avoid centrifugation. Blood from the patient was withdrawn into acid-citrate-dextrose solution containing 100 pc NazS1Cr04 and the syringe was left at room

20 0 Fig.

6.

normal T$$

Survival recipients.

of

six

of eighteen

66

lb

days days

of

red

cells

Before (0). (0).

from

the

splenectomy

After

splenectomy,

24

propositus the

in

patient’s his

red

28

splenectomized red

cells

cells

had

had a

T%

a

temperature for twenty minutes. The contents of the syringe were then given as an injection to a normal compatible recipient immediately after the intravenous administration of 500 mg ascorbic acid to prevent further labeling of red cells by the chromate anion. The decrease in radioactivity in the blood showed a Tl/a of thirteen and a half minutes and again the activity accumulated in the recipient’s spleen. Although not all the 51Cr was present inside red cells, the close correspondence between this Tl/a value and those obtained earlier indicates that the multiple centrifugations used in the routine labeling technic did not spuriously shorten the subsequent survival of these hereditary spherocytes. The

American

Journal

of

Medicine

VARIANT

OF

HERE:DITARY

SPHEROCYTOSIS

-

WILEY,

FIRKIN

60

HOURS Fig. only

7.

Survival

eleven

or

of twelve

red

cells

from

minutes

(0).

the After

propositus splenectomy

in

compatible his

red

normal cells

The specificity of the spleen in destroying these hereditary spherocytes was further demonstrated by measuring their survival in a compatible patient who had undergone splenectomy for idiopathic thrombocytopenic purpura one year previously. The survival in this case was prolonged to a Tl/s of six days (Fig. 6) and the disappearance of the labeled red cells occurred at a rate comparable to the disappearance rate of the patient’s cells in his own circulation. Obviously a normal intact spleen was required for the rapid removal of the patient’s cells. Nevertheless the survival of the patient’s red cells in this splenectomized recipient (Tl,s six days) was still far short of the normal survival (Tl/a twenty-six to thirty days). The reason for this became evident from subsequent survival studies which demonstrated a major degree of irreversible damage to the patient’s red cells as a result of their exposure to his own spleen. Following removal of the patient’s spleen the survival ot his red cells was again measured in himself, in normal compatible recipients and in a splenectomized compatible recipient. Nine days after splenectomy the Tl/a of the patient’s cells given as an injection to a normal subject had increased to ninety minutes (Fig. 7) with solely Volume 48,

January

1970

had

recipients. a Tl/,

of from

Before sixty

splenectomy to

180

the

patient’s

red

cells

had

a

T%

of

(0).

splenic destruction of the injected red cells. Nine and twelve weeks after operation this survival was repeated in different compatible recipients with similar results (T1/2 180 and sixty minutes, respectively). Not only had the viability of the patient’s cells in normal recipients improved, but also the shape of the survival curve had changed from a purely exponential to a biphasic type of survival curve indicating the presence of two or more populations of red cells with different survival times. Graphic analysis of the survival curve obtained twelve weeks after splenectomy showed that 55 per cent of the patient’s cells were removed exponentially with a T*/s of sixty minutes whereas another 30 per cent disappeared with a Tl/’ of eight hours. These results reveal a remarkable increase in the transfusion viability of the patient’s red cells after his spleen had been removed. Compared to a Tl/s of twelve and a half minutes on transfusion of the patient’s cells before splenectomy, the T1/2 of the patient’s cells was lengthened to ninety minutes after splenectomy. Survival of the patient’s red cells in himself and in a splenectomized compatible recipient was measured ten to eleven weeks after the patient’s splenectomy. AS 67

VARIANT

OF HEREDITARY

TABLE

I

SPHEROCYTOSIS

-

WILEY, FIRKIN

Sodium Movements and Glycolytic Rate of Red Cells from the Propositus (11-l) Measured During in Vitro Incubation for Four Hours at 37oc Mean Rate Over 4 Hours (p Eqlml cellslhr)

zzNa-influx Net change in cell sodium content Lactate production

No Ouabain

+ Ouabain

3.5

3.6

-0.1 2.80

+1.3 2.30

expected, the patient’s autosurvival Tl/z was lengtened from ten days before splenectomy to twenty-nine days after splenectomy (Fig. 4). In the splenectomized recipient a survival Tl,z of eighteen days was obtained (Fig. 6) and if the initial destruction of some 20 per cent of the injected cells in the first hour is ignored, it can be seen that the remaining 80 per cent survived completely normally with a Tl/z of twenty-seven days. Comparison of this figure with the T1/2 of six days obtained with the patient’s cells before splenectomy leads to the conclusion that the patient’s cells were capable of surviving normally in a splenectomized recipient only when they had not suffered recent exposure to the environment of the patient’s spleen. Sodium Movements. Since “sodium leak” into the cell has been proposed as the basic lesion in hereditary spherocytosis [19,20] it was of interest to study sodium movements in a patient with hereditary spherocytes who had such a severe hemolytic tendency. Blood was taken from the patient before splenectomy, washed, and the red cells were incubated at 37°C both with and without the addition of ouabain. Sodium influx and cell sodium concentration were measured at zero, two and four hour incubation. As anticipated, sodium influx was increased to 3.6 PEq per ml cells per hour (Table I) compared to a range for normal cells of 2.0 to 2.7 PEq per ml cells per hour. During incubation the sodium influx was constant with ouabain (3.6 PEq per ml cells per hour), although without ouabain the influx declined slightly from 3.5 to 3.1 PEq per ml cells per hour after four hours at 37°C. The presence of a “sodium leak” was also confirmed by measuring the increase in sodium content of these cells incubated with ouabain to inhibit extrusion of sodium by the cation pump. The hereditary spherocytes showed a net gain of sodium averaging 1.3 PEq per ml cells per hour over four hours whereas the corresponding figure for normal cells was 0.9 PEq per ml cells per hour. Despite this increased passive entry of sodium the intracellular sodium concentration of freshly drawn red cells from the patient (6.7 FEq per ml cells) was not greater than normal (mean 8.0 PEq per ml cells). Since autohemolysis, osmotic fragility and survival characteristics of the patient’s cells were altered following splenectomy measurement of the 22Na influx into his red cells was also repeated 9 weeks postoperatively. At each of the four different external sodium concentrations used the 22Na influx into the cells was less after splenectomy 68

than it was before splenectomy (Fig. 8). Sodium influx into the patient’s cells after splenectomy was measured a second time a week later, and the difference was confirmed. The extent of this reduction in the 22Na influx was significant (P < 0.001) and the fall was from 3.6 ,uEq per ml cells per hour before splenectomy (on two occasions) to a mean of 3.1 PEq per ml cells per hour after splenectomy (3.2 and 2.95 on separate occasions) when the external medium contained 145 mM sodium chloride. If splenic conditioning increases the sodium permeability of red cells, this fall in sodium influx after splenectomy would reflect the removal of this damaging influence. Another possibility, however, which is equally likely is that reticulocytes have a higher sodium influx in which case the fall in reticulocyte count after splenectomy would account for the decreased sodium influx. Further experiments are necessary to distinguish between these two possibilities. Glycolysis and Active Cation Transport. The metabolism of the patient’s red cells was also studied when these cells were incubated in vitro with glucose (Table I). Both the rate of lactate production of these cells (2.80 pmole (PM) per ml per hour) and the decrement in this rate produced by addition of 100 PM ouabain (0.50 PM per ml per hour) were typical of hereditary spherocytes [16,19]. The ATP-ase activity of isolated erythrocyte stroma from the patient was also measured both in the absence and presence of 100 ,uM ouabain, the difference between these two values being a measure of the ATP-ase activity associated with the active cation pump. The value for this ouabainsensitive ATP-ase from the patient (0.47 PM Pi per mg protein per hour) lay close to the mean value for a group of ten patients with hereditary spherocytes (0.45 PM Pi per mg protein per hour) and exceeded the range of values for normal red cells (0.24 to 0.40 PM Pi per mg protein per hour: mean 0.30) [17]. It was concluded that the glycolytic activity of these cells was not impaired and that the activity of the active cation pump was increased to the same extent as in other hereditary spherocytes so as to maintain normal levels of intracellular sodium ions. Since the hereditary spherocytosis in Family Studies. many of the subjects found to have atypical autohemolysis was apparently sporadic [5], the proved pedigree of spherocytosis in this case made investigation of other family members of special interest. Cell survival in vivo, osmotic fragility, autohemolysis and 22Na influx were therefore measured in red cells from both the brother (11-4) and the mother (l-2) of the propositus. The brother, who had been splenectomized twelve years previously, had red cells with a high osmotic fragility and a median corpuscular fragility (0.575 per cent sodium chloride) which was well above the normal range (Fig. 3). Autohemolysis of his blood was increased, but the hemolysis was partially corrected by glucose supplementation, as is typical for hereditary spherocytosis. 22Na influx measured at four different external sodium concentrations gave a curve for influx against concentration which closely resembled that for the patient’s red cells (Fig. 8). The survival of Wr-labeled red cells from the brother was then tested in a normal compatible recipient. The labeled cells disappeared rapidly at first and the Tl/z was reached in ninety minutes, but the rate of removal of labeled cells slowed considerably thereafter (Table II). Red cells from The American

Journal

of Medicine

VARIANT

the brother thus survived in a normal recipient with a time.course remarkably similar to cells from the patient (11.1). It was concluded that close similarities existed in certain properties of red cells from the patient and his brother since both had short and identical in vivo survivals in a compatible recipient, the sodium influxes were similar and cells from both had a high osmotic fragility. Investigation of the mother, who had not had splenectomy, showed fewer similarities to the patient although some features did suggest splenic conditioning of her circulating red cells. The autohemolysis of blood tiNas increased and was atypical since glucose did not correct this hemolysis (10 per cent without glucose and 10 per cent with glucose supplement). The median corpuscular fragility was only moderately increased (0.525 per cent sodium chloride), but there was a “tail” of osmotically fragile cells which constituted more than 40 per cent of the total (Fig. 3). Iron deficiency could not have accounted for the shape of the fragility curve since both serum iron and total iron-binding capacity were normal. Such a large “tail” of fragile cells indicates considerable splenic conditioning [21,22] and the presence of so many damaged cells probably accounts for the atypical autohemolysis noted, a conclusion which has recently been reached by other investigators 163. The curve of 2”Na influx against sodium concentration was also similar to that for the patient’s red cells (Fig. 8). The autologous survival of the mother’s hereditary spherocytes was considerably shortened (Tl,z of six days), although the survival of the same cells in compatible recipients was even shorter (Tl/’ values of 3.0 and 3.2 days in two different subjects (Table II). These values represent about a twofold difference in the rate of destruction of spherocytes in the donor compared with a compatible recipient. This contrasts with the 1,OOOfold difference in the corresponding survival times for hereditary spherocytes obtained from the patient before splenectomy. Thus the patient and his mother both had a history of leg ulceration and evidence of splenic conditioning of red cells although their red cells differed widely in their survival times in compatible recipients.

OF HEREDITARY

0

48,

January 1970

-

WILEY, FIRKIN

110

150

NaCl IN MEDIUM

Fig. 8. Sodium influx into red cells from the propositus, his brother and mother measured at four different external sodium concentra. tions. Influx into the patient’s cells before splenectomy (01 was significantly higher than after splenectomy (0). Influx into red cells from both the brother (0) and mother (A) was similar to that of the patient before splenectomy.

cases (including the patient and his mother) seen at this hospital in the past two years was there a history of leg ulcers. Although both the patient and his mother showed an association of leg ulcers and splenic conditioning ot red cells, a definitive statement as to whether leg ulcera. tion reflects the severity of splenic conditioning must await description of variants similar to this. The most unexpected finding in this study was that the red cell survival T1/2 was only twelve and a half minutes for clearance of the patient’s red cells by a compatible recipient. This figure corresponds to 4 per cent of the blood volume per minute which is roughly equal to the splenic blood flow [25]. Since surface counting showed accumulation of labeled cells only over the spleen it can be concluded that hereditary spherocytes from this patient were cleared in a single passage through a normal spleen. The rapidity of clearance of these cells is com-

Summary of in Vivo Survivals of Red Cells from the Propositus, His Brother and Mother as Measured by the 51Cr Technic

II

COMMENTS

Volume

70

50

mM

TABLE

The diagnosis of hereditary spherocytosis in the propositus is unequivocal. Spherocytosis, clinical and laboratory evidence of hemolysis, splenomegaly, a positive family history and increased osmotic fragility are typical although the extreme susceptibility of the patient’s red cells to trapping and destruction in a normal spleen seems unique. Several features in the clinical history are unusual and may suggest a severe form of the disease. Retardation of growth in childhood was well documented in this patient and contrasts with his siblings who had shown normal physical maturation. Slowing of growth is not generally recognized as a manifestation of hereditary spherocytosis but it has been described [23], and it seems quite possible that severe chronic anemia did retard growth in this patient. Numerous bilateral leg ulcers occurred when the patient was aged twelve and persisted for nine months. Leg ulceration is an uncommon manifestation of hereditary spherocytosis. Young [24] found no leg ulcers in fifty cases whereas in only three of thirty-one

SPHEROCYTOSIS

Survival T% of 51Cr-Labeled Red Cells

Propositus

10 days

29 days

12.5 min

90 min

6 days

18 days

(I l-4)

Survival ir, compatible recipient Mother

After SpleneCtomy

(I l-l)

Autologous survival Survival in compatible recipient Survival in splenectomized compatible recipient Brother

Before Splenectomy

90 min

...

(l-2)

Autologous survival Survival in compatible recipient

...

6 days

...

3 days 69

VARIANT

OF HEREDITARY

SPHEROCYTOSIS

-

WILEY, FIRKIN

parable to the clearance rates of mildly heat-damaged red cells by the spleen, with a Tl/z as low as ten to sixteen minutes [26,27]. However, the specificity of the spleen in removing these hereditary spherocytes was even greater than for heat-damaged cells, which are also cleared by the liver to a minor extent. Such a severe hemolytic tendency might at first suggest the presence of two coexisting disorders of the red cell. Hereditary spherocytosis has been described in association with a Coombs’-positive hemolytic process [28], beta thalassemia (291 and sickle cell trait [30,31]. However the presence of a second defect superimposed on hereditary spherocytosis seemed unlikely as the Coombs test was negative, no abnormal hemoglobins were detected, the hemoglobin A? level was not raised, glucose-6-phosphate dehydrogenase was not decreased and energy metabolism and active cation transport in these cells was typical of hereditary spherocytes. A vital compensatory mechanism in this patient was the relative failure of his spleen to sequester his red cells, so that they survived with a Tl/z of ten days compared with a T1/2 of twelve and a half minutes in a normal recipient, a 1000 fold difference in survival times (see Table II for summary of survivals). This relative inefficiency of the patient’s spleen was further illustrated by the finding that his red cells survived longer in his own blood (T1/2 ten days) than in a splenectomized recipient (T1/2 six days). Although a different partition of splenic blood flow between the “slow and rapid transit” compartments may explain minor differences in the survival

Another feature of this case is the lengthened survival of the hereditary spherocytes in a normal subject after the patient’s spleen had been removed. This enhanced viability was considerable, from a Tl/z of twelve and a half minutes before splenectomy to ninety minutes after splenectomy so that even though splenic conditioning was not the major cause of hemolysis in these cells, it made an important contribution. Thus the spleen, although relatively incompetent in respect to removing the patient’s cells from his own circulation somehow altered nearly all of them irreversibly, so that on subsequent circulation through a normal spleen they were removed immediately. The action of this patient’s spleen in predisposing hereditary spherocytes for rapid removal by a normal spleen is analogous to the mechanism of hemolysis in pyruvate-kinase deficiency. In this hemolytic anemia, the enzyme-deficient red cells are irreversibly conditioned by the spleen for their subsequent removal by the liver [37]. The deleterious effect of the patient’s spleen on his own red cells was also evident from the change in osmotic fragility after splenectomy since the high median corpuscular fragility of the patient’s red cells decreased from 0.68 per cent sodium chloride to 0.65 per cent sodium chloride after splenectomy (Fig. 3). It is known that the spleen may render hereditary spherocytic blood more osmotically fragile since after splenectomy a more fragile “tail” of cells on the osmotic fragility curve is often lost [21,22]. No fragile “tail” of cells was evident however, in any osmotic fragility curve of the patient’s blood, which again indicates that splenic

of spherocytes between donor and normal recipient, it could not explain the thousandfold difference in survival times. A selective splenic inefficiency in removing circublockade, lating spherocytes, i.e., a reticuloendothelial

conditioning affected most if not all of the patient’s red cells. In contrast the osmotic fragility curve obtained in

must be postulated of the patient’s

to explain

the relatively

cells in his own circulation.

long survival Hence it is

of interest that both light and electron microscopy of splenic tissue from the patient showed almost complete absence of erythrophagocytosis. Howell-Jolly bodies in the patient’s nectomy sequent

The observation of red cells before sple-

may be related to this gross blockade and subreduction of the usual “pitting” function of the

spleen [32]. Similar inclusion bodies were also observed in red cells from the patient’s mother (l-2). Although in the latter case the serum folate level was below normal, red cell inclusion bodies in both cases were more numerous than could be explained entirely by folate deficiency, and reticuloendothelial blockade was a more likely reason for the persistence of Howell-Jolly bodies, Evidently a major degree of blockade must have operated in this patient since birth and slowed the rate of hemolysis sufficiently for the marrow to compensate. Reticula. endothelial blockade may also occur in infants with hemolytic disease of the newborn since anti-D-sensitized fetal red cells can survive longer than predicted from the degree of antibody coating [33]. A failure of splenic function .also occurs in sickle cell disease [34] and in thrombocythemia [35], but the underlying mechanism in these diseases is infarction of splenic tissue rather than reticuloendothelial blockade. Circulating Howell-Jolly bodies have also been described in idiopathic steatorrhoea due to associated splenic atrophy [36]. 70

the mother (l-2) showed an osmotically fragile “tail” which suggested that only about 40 per cent of her cells had been changed

by splenic conditioning

[6]. The mech-

anism of splenic conditioning of red cells is not precisely known but may be related to the loss of membrane lipid. Not only is the lipid content of hereditary spherocytes before splenectomy less than after splenectomy [38] but also,

as a recent

study

of two

patients

with

“atypical

(group B) hereditary spherocytosis” showed, their red cell lipid content is considerably less than normal [6]. Moreover transfusion of hereditary spherocytes to patients with obstructive jaundice produces a decrease in the osmotic fragility of these cells and improves their in vivo survival, presumably due to the addition of lipid to the cell membrane [39]. The failure of added glucose

to correct

the increased

autohemolysis of the patient’s red cells is unusual, and only seven cases of hereditary spherocytosis have been documented with this atypical autohemolysis result [4-61. Following splenectomy autohemolysis in the patient reverted to the typical pattern described in previous cases. However this change in the behaviour of autohemolysis was not immediate, since atypical autohemolysis was observed one week after splenectomy and the more typical pattern was evident only after nine weeks. Such alteration in the autohemolysis pattern was similar in time course to the decrease in the median corpuscular fragility and presumably reflected the gradual replacement of circulating red cells by fresh cells which had not been subject to splenic conditioning. In contrast to the slow changes in The

American

Journal

of

Medicine

VARIANT

autohemolysis and osmotic fragility, the red cell survival had already lengthened in a normal recipient when tested only nine days after the patient’s splenectomy. It is likely, therefore, that the deleterious effect of the spleen on the cross survival characteristics of these hereditary spherocytes was readily reversible and may be a distinct phenomenon from that underlying the changes in autohemolysis and osmotic fragility. An important implication can be drawn from this case by comparing the relative severity of the “sodium leak” with the rate of hemolysis, i.e., the rate of disappearance of circulating red cells. Sodium influx into these hereditary spherocytes certainly exceeds the normal, although leak” is no greater the magnitude of the extra “sodium than that observed in hereditary spherocytes from many other patients with far milder susceptibility to hemolysis in a normal recipient [9]. It follows that the “sodium leak” cannot be the major cause for the rapid sequestration of these cells in a recipient’s spleen. Once these hereditary spherocytes have been trapped in the recipient’s spleen, however, the inward “sodium leak” and osmotic swelling of the cell may contribute to its destruction although its importance relative to loss of whole membrane fragments as shown by symmetrical reduction

OF

HEREDITARY

SPHEROCYTOSIS

-

WILEY,

FIRKIN

in cell lipid is uncertain [40]. What: property of the cell then is responsible for the rapid trapping of these hereditary spherocytes in the spleen? It may be significant that in a series of patients with hereditary spherocytosis studied in our laboratory the propositus (11-l) and his brother (11-4) had both the shortest cell survivals in a normal recipient and the highest osmotic fragilities [9]. Cell shape or some other property of the cell reflected in its osmotic fragility may thus be of importance in determining the rate of hemolysis of hereditary spherocytes in a normal recipient. Nevertheless the development of anemia in patients with hereditary spherocytosis must depend on several factors in addition to cell shape, and this patient well illustrates the role of reticuloendothelial blockade in mitigating the severity of anemia.

ACKNOWLEDGMENT We thank Dr. Ross Winton, Castle Hill, for referring the patient to this hospital and Dr. H. Kronenberg and Miss Helen Robin for performing the autohemolysis and screening tests for pyruvate kinase and G6P dehydrogenase deficiency. Without the full and most willing cooperation of Mr. H. C. Huish this investigation would not have been possible. This work was supported by a grant from the National Health and Medical Research Council of Australia.

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