Radionuclide Techniques in Pediatric Hematology

Philip A. Bardfeld

A variety of hematologic studies using radionuclides are suitable for use in pediatrics. These include the major tests currently in use in adults and consist of red cell survival and splenic sequestration studies, the Schilling test, iron absorption, and ferrokinetic studies and imaging procedures using radiocolloid to demonstrate the spleen and splenic function. Although certain specific indications are unique to pediatric practice, in general the procedures are approached in a manner identical to that in adults. That is, the indications and methods for

carrying out the test require only min- imum modification. The major require- ment in pediatrics is that the admin- istered dose must be proportionately scaled down to the size of the patient in the study in order to avoid unneces- sary radiation. The new use of nuclear medicine techniques in pediatric hematology permits the identification of causes of various types of obscure anemias, evaluation of platelet func- tion, the detection of juvenile perni- cious anemia and the detection of abnormalities of red blood cell forma- tion and of splenic function.

T HE HEMATOLOGIST, in choosing nuclear medicine procedures, is faced with a decision regarding the risk of radiation close to the patient and

the probability of obtaining an answer to a clinical question. This dilemma is especially evident in pediatric hematology because greater weight must be given to questions of dose in children as discussed elsewhere in this series on pediatric nuclear medicine.

TESTS FOR RED CELL SURVIVAL AND SPLENIC SEQUESTRATION

Tests for red cell survival and splenic sequestration are useful in a variety of pediatric blood disorders including red cell enzymopathies, autoimmune hemolytic anemias, and hypersplenism. Although there is no completely standardized method for the performance of red cell survival studies, the most commonly used technique involves labeling with 51Cr and measurement of the time taken for one-half the label to leave the circulation.

The procedure is essentially the same as that followed in the adult. 1"9" Twenty milliliters of whole blood is withdrawn and mixed with 10 ml of acid citrate dextrose. The labeling is performed with 5ZCr in the form of sodium chromate (hexavalent state) which is allowed to incubate with the blood sample for 30-45 min at room temperature. The addition of 50 mg of ascorbate reduces the chromate (-[-6) to chromic ion (+3) and terminates the labeling of the hemoglobin molecule. Twenty milliliters of the labeled mixture is then reinjected into the patient. At 24 hr after injection, a sample of heparinized blood is withdrawn and sampling is continued daily or every other day, depending on the anticipated severity of hemolysis until a level corresponding to one half of the original activity specimen (at 24 hr) has been

From Montefiore Hospital, Brbnx, N. Y. 10467. Philip A. Bardfeld, M.D.: Head, Nuclear Medicine Section, Department of Oncology,

Montefiore Hospital, Bronx, N. Y. �9 1973 by Grune & Stratton, Inc.

Seminars in Nuclear Medicine, Vol. 3, No. 1 (January), 1973 81

82 PHILLIP A. BARDFELD

100_

I0_ ! ! | !

T~t DAYS Fig. 1. Markedly short- ened red cell survival.

reached. Each of the samples is counted in a NaI (Tl) Well detector together with the specimen obtained at 24 hr to obviate the necessity for decay correction. If these data are plotted on semilogarithmic paper a normal SZCr red cell survival would be 25-35 days (see Fig. 1).

The adult close of 100 ~Ci of radioactive chromium should be reduced in proportion to surface area in children using a nomogram or body weight. The absorbed close to the blood from this procedure is approximately 240 mrads. It is possible that this radiation close may be significantly reduced in the future by measurement of the urinary excretion of SlCr after the injection of 5 #Ci in 1 ml of blood. 3"4 The only departure from the con- ventional SlCr survival test is that four consecutive 24-hr urine specimens are collected and the time required for one-half the 5ZCr label to be excreted in the urine is determined. In addition, variability in SZCr urine excretion in the first 24--48 hr is minimized by two washings of the cells in isotonic saline prior to reinjection. A longer counting time is needed to insure statistical significance. The urine excretion test is promising not only because it may reduce the radiation dosage, but it would also provide a means of distinguish- ing acute blood loss from hemolysis since only a hemolytic process will affect the urine activity. This technique has only been used in adults so far, but it would seem to be worthy of application in pediatric nuclear medicine.

Another potential approach towards reducing the radiation close in red cell survival studies is the labeling of red cells with stable chromium (S~ followed by neutron activation of the blood specimens, s The resultant SlCr is detected by high resolution Ge (Li) spectrometry. At the present time, this technique requires the availability of a reactor and it is generally too time consuming for ordinary practice. However, further work is war- ranted to bring it to the level of clinical feasibility.

The determination of sequestration of red blood cells in the spleen is possible by external counting over the liver, spleen and precordium on several occa- sions till the half-time of the routine red cell survival test is reached. The criteria of significant splenic sequestration are a spleen to liver ratio

HEMATOLOGY 83

2.0

I.r

0.5

SPLEEN: PRECORDIUM RATIO

LIVER: PRECORDIUM RATIO

�9 . . . . . , 4 P . . 6 . ,.@ . . . . . " ~ " . . . . . . . "11" - ' ~

F i g . 2 . T y p i c a l p a t t e r n o f s i g n i f i c a n t s p l e n i c s e - ' ~ ' ~, ' ~ ' ~ ' t'o ' t'2 ' I~ q u e s t r a t i o n . DAYS

of 2.5:1 or greater, a spleen to precordial ratio of 2:1 or higher and a spleen to liver curve which tends to rise from the horizontal (Fig. 2).

BLOOD LOSS STUDIES

The 51Cr labeling of erythrocytes may also be used in the pediatric patient to quantitate chronic blood loss. The 200 /~Ci close recommended in adults is reduced in proportion to surface area or weight, and a 72-hr fecal collection is initiated on the clay after injection of the nZCr-labeled cells. Blood specimens are withdrawn at the beginning and at the end of the 72-hr period. The blood loss is derived from

Total cpm in 72-hr stool collection Average cpm/ml of blood from the first and last collection day.

This technique has been sensitive enough to permit the measurement of minimal blood loss from drugs, helminthic infection, milk allergy or gastro- intestinal lesions. It is described in detail in Vol. II, No. 3 of this Journal.

PLATELET STUDIES

Chromium-51 labeling of plate]ets and Ieukocytes has also been per- formed. In the case of leukocytes, labeling has been used in kinetic studies but has not been of value in clinical practice. I n contrast, platelet survival and sequestration studies ~ have achieved a more widespread recognition although chromium has a lesser affinity for the platelet than it has for erythrocytes or leukocytes. 8

Basically, the labeling is performed by incubating a suspension of platelet rich plasma with 250/~Ci of high specific activity 5]Cr in the form of sodium chromate (hexavalent) for 90 rain at 37~ This suspension is then washed with plasm~i together with ascorbic acid which will reduce the excess chromate. The labeled platelets are then centrifuged, the supernatant plasma is removed and the remaining platelets are resuspended in plasma and injected. Blood samples are obtained at 5 min, 1-hr, 2-hr, and sequential

84 PHILLIP A. BARDFELD

12-hr intervals after injection. The platelet half-life is plotted on semi- logarithmic graph paper. Normal values with this procedure are 7-10 days.

External counting may be performed simultaneously as with the red blood cell survival studies. If a spleen-to-liver ratio of 2.5 or greater is found, then splenic sequestration is definitely established. A pattern of splenic sequestration has been shown to correlate with a successful response to splenectomy in idiopathic thrombocytopenic purpura and secondary thrombo- cytopenias of varying etiology.

Recent evidence suggests that even the trace amounts of chromate used in labeling may effect platelet function and thus alter the normal platelet survival or half-time. 9 However, even though the ~lCr labeling of platelets may not provide as exact an estimate of platelet life-span as might be desired, the technique does provide some valuable information particularly in the determination of splenic sequestration.

Both red cells and platelets may be labeled with DFa2P. DF a2p is not commonly used because of the necessity for liquid scintillation counting which is not commonly available in most nuclear medicine laboratories.

THE SCHILLING TEST

The Schilling Test may be of value in several interesting but uncommon clinical entities: 1~ (1) Juvenile Pernicious Anemia clue to a deficiency of intrinsic factor; (2) Vitamin B12 deficiency caused by abnormal intrinsic factor; (3) Vitamin B12 deficiency resulting from selective malabsorption. This abnormality is presumed to occur at a point somewhere between binding of the B~2 to ileal receptor sites and attachment to transcobalamin II. (4) Vitamin B~2 deficiency clue to lack of transcobalamin II.

The procedure for the Schilling test is identical to the procedure used in the adult. 0.5-2.0 ~g containing 0.5 to 1.0 ~Ci of STCo--vitamin B12 is given by mouth to a fasting patient. A flushing dose containing 1 mg of non- radioactive Vitamin B12 is injected intramuscularly 3 hr after the oral dose of labeled vitamin. A 48-hr urine collection is started after the latter dose. 57Co is preferable to 6~ as a label for the B~2 because it provides less of a radiation close (80 mrads/0.5 /~Ci) to the liver in comparison to 8~ (2100 mrads/0.5 ~Ci).

If a 2.0/~g dose of 57Co-vitamin B~ has been administered, then a 48-hr urine collection should contain at least 5% of the administered dose. Any values less than 5% are an indication for a repetition of the test procedure using intrinsic factor (second phase). If the second phase of the Schilling test results in a correction of the urine excretion values to normal levels then the classical requirements for the diagnosis of pernicious anemia have been met. Malabsorption must be considered in those situations in which intrinsic factor administration does not lead to an augmentation of urinary excretion.

IRON STUDIES

Iron absorption and ferrokinetic studies may also be of value in certain circumstances. In idiopathic hemochromatosis, iron absorption is increased,

HEMATOLOGY

~.~ 50.

r

8O Fig. 3. Normal iron T -tl/Z

clearance half,time. IRON CLEARANCE

..-ZERO TIME

TIME COUNTS

o do t~o 160 MINUTES

but there is no significant anemia. In anemias clue to iron deficiency or hemolysis, iron absorption may also be increased.

There is no one technique for quantifying iron absorption which meets the requirements of both accuracy and technical simplicity. One commonly used method involves the oral administration of 1.0 /tCi of 59Fe as ferrous dtrate and 700 /~g of carrier iron (ferrous ammonium sulfate) and the meastirement of activity in a 7-day stool collection. The normal range with this procedure is 50% absorption. Variability has been associated with inaccurate stool collections and changes in the amount of carrier iron.

A more reliable dual isotope technique has been described in which one radionuclide (5~ is given orally and compared with the levels of intra- venously injected 55Fe. The per cent absorption is calculated as

59Fe activity in blood SSFe activity given i.v. x x lOO.

S~ activity given orally SSFe activity in blood

The whole-body counter is also a quite reliable tool for quantifying iron absorption but its availability is limited.

T h e r e are four components in the standard ferrokinetic evaluation: (1) Iron clearance half-time (T 1/2); (2) Plasma iron ttirnover rate (P.I.T.); (3) Iron red cell use or incorporation; (4) Surface counting.

The iron clearance half-time is initiated by the intravenous administration of 5 /~Ci of 59Fe in the form of ferrous citrate. Serial blood specimens are drawn at 15, 30, 60, 90, and 120 min after injectiori. The time required to reach the 50% level of activity is read on a semilogarithmic graph (Fig. 3). The normal range is 1-2 hr (Fig. 3). A slow iron clearance half, time is char- acteristic of aplastic anemia, hemochromatosis, and acute viral hepatitis. Rapid clearance is seen in iron deficiency anemia, anemias of infection and cancer, and in some hemolytic anemias.

86 PHILLIP A. BARDFELD

The daily plasma iron turnover rate in mg/kg is calculated from

0.693 X plasma iron (mg/ml) X plasma volume (ml) X 24

iron clearance half-time (hr)

The normal range is 27-42 mg with an average of 37 rag. The plasma iron turnover rate is an index of total erythropoiesis.

The iron red cell incorporation is determined by counting blood specimens obtained at 3, 7, and 10 days after the original injection of 59Fe. The per cent incorporation is derived from

counts/ml X blood volume (ml) X 100 zero-time counts (see vertical axis of Fig. 3)

The iron red cell incorporation is a measure of effective erythropoiesis and normal values are 80%-90% at 10 days.

Surface counting over the sacrum (bone marrow), liver and spleen is also performed during the 10-day period of the ferrokinetic tests. Normally the bone marrow uptake is increased within the first 2-3 days but decreases as radioactive red ceils leave the marrow. Liver and spleen uptake are minimal. Characteristic patterns are noted in iron deficiency anemia, ineffec- tive erythropoiesis, and hemochromatosis (Fig. 4).

Activation analysis has been applied to ferrokinetic studies in an effort to reduce radiation dose. Approximately 0.05 /~g of 58Fe is injected and appro- priate samples are withdrawn and irradiated to produce S~ via an (n, y) reaction. 11 Although this technique is promising, long delays prior to clinical availability of results, necessity of proximity to a reactor, and difficulties with radiochemical separation have hampered its current use in ferrokinetic studies. Here again further improvements would be worthwhile.

21 ~ f -,,v~R ,~- ~ ~ - ~

-~3 . . . . r 21" ~ IRON-DEFICIENCY ANEMIA

_~ I ",LIVER

3 . . . . .~ - : ~ [NEFFECTIVE ERYTHROPOIES~S

~ UVER , r~ , ,

3 HEMOCHROMATOSIS

g ib I'5 ~'o TIME, DAYS

Fig. 4. Characteristic surface count- ing patterns over liver, spleen, and bone marrow in normals, iron defi- ciency anemias, ineffective erythro- poiesis and hemochromatosis.

HEMATOLOGY 87

IMAGING PROCEDURES

Imaging procedures, part icularly radiocolloid scans of the Spleen, have also had a Significant impact upon pediatric hematology. A state of func- tional asplenia has been recently described in young children with homo- zygous Hgb S disease. 12

This defect is characterized by (1) splenomegaiy (evident by palpat ion or radiographic examination); (2) hematological evidence of asplenia in the RBC smear, i.e., Howell-Jol ly bodies (This was present in most of the cases.); (3) absence of uptake of ~ colloid (~ in the area of the spleen.

This defect was shown to be reversible after the t ransfusion of normal red cells. In contrast, the phenomenon of asplenia has been observed only rarely in patients with the sickle cell variants (Hgb SC, Hgb S thalaSsemia). The pathogenetic mechanism of functional asplenia is believed to be a sickling within the splenic circulation leading to occlusion, subsequent opening of ar ter io-venous Shunts and bypass ing of the phagocytic cells within the spleen.

The survey has been directed toward methods and clinical utility of various nuc l ea r hematological techniques in children. Even if one, were to allow for a multiplication factor of ten in the newborn related to differences in organ mass and distribution and differences in metabolic activity, 13 the dose f rom each of the tests described would be well within the suggested limit of 5 reins in 1 yr for an infant o r child. Refinement of activation analysis for ferrokinetic and red blood cell survival studies as well as use of the ur inary excretion test for red blood cell survival may substantially reduce this dose in the future and permit more widespread utilization of pediatric nuclear hematologic techniques.

REFERENCES

1. Wagner, H. N., Jr., and Bardfeld, P. A.: Evaluation of structure and function of spleen with radioactive tracers. JAMA 199: 202, 1967.

2. International Committee for Standard- ization in Hematology: Recommended meth- ods for radioisotope red cell survival studies. Blood 38:378 , 1971.

3. Shih, S. C., Tauxe; W. N., and Fair- banks, V. F.: The kinetics of urinary excre- tion of 51Cr from labeled erythrocytes in hemolytic anemia and the anemia of blood loss. Am. J. CIin. Pathol. 55:431, 1971.

4 . . . . and Taswell, H. F.: Urinary excretion of 51Cr from labeled erythrocytes. JAMA 220:814, 1972.

5. Glomski, C. A., Pillay, S. K. K ' and Hagle, R. E.: Survival of 50Cr-labeled erythrocytes as studied by instrumental activation analysis. J. Nuel. Med. 12:31, 1971.

6. Aster, P. H., and Jandi, j. H.: Ptatelet sequestration in man. I. Ciin. Invest. 43: 843, 1964.

7. Cooper, M. R., Hansen, K. S., Maynard, C. D., Elrod J. W., and Spurr, C. L. : Platelet survival and sequestration patterns in thrombocytopenic disorders. Radiology 102: 89, 1972.

8. Eyre, H. J., Rosen, P. J., and Perry S. : Relative labeling of leukocytes, erythro- cytes, and platelets in human blood by 51- chromium. Blood 36:250, 1970.

9. Kattlove, H. E., and Spaet, T. H.: The effect of chromium on platelet function in vitro. Blood 35:659, 1970.

10. MacKenzie, I. L., Donaldson, R. M., Jr., Trier, J. S., and Mathan, M. I.: Ileal mucosa in familial selective Vitamin B-12 malabsorption. New Eng. J. Med. 286:1021, 1972.

11. Lowman, J. T:, and Krivit, W.: New

88 PHILLIP A. BARDFELD

in vivo tracer method with the use of non- radioactive isotopes and activation analysis. J. Lab. Clin. Med. 61:1042, 1963.

12. Pearson, H. A., Spencer, R. P., and Cornelius, E. A.: Functional AsFlenia in Sickle Cell Anemia. New Eng. J. ivied. 281:

923, 1969. 13. Saenger, E. L., and Kereiakes, J. G.:

In Lauwrence, J. H. (Ed.): Recent Advances in Nuclear Medicine, Vol. 3. New York, Grune & Stratton, 1971, p. 158.