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The Widespread Application of Red Cell Survival Sive Red CLINICAL DETERMINATION OF THE SITES OF RED CELL SEQUESTRATION IN HEMOLYTIC ANEMIAS1 By JAMES H. JANDL, MORTIMER S. GREENBERG, ROBERT H. YONEMOTO, AND WILLIAM B. CASTLE (From the Thorndike Memorial Laboratory and Second and Fourth (Harvard), Medical Services Boston City Hospital, and the Department of Medicine, Harvard Medical School, Boston, Mass.) (Submitted for publication January 30, 1956; accepted April 3, 1956) The widespread application of red cell survival greater than that of other tissues even when cor- techniques has revealed the importance of exces- rection was made for the Cr5l activity of the re- sive red cell destruction in the pathologic physi- sidual red cells. Moreover, the radioactivity of ology of many of the anemias. An increasing ar- the packed red cells removed from the spleen ex- ray of in vitro methods for detecting red cell or ceeded that of a comparable sample of packed red serum abnormalities has provided insight into the cells from the peripheral blood. In order to in- in vivo mechanisms underlying some of these proc- vestigate the possibility that Cr51-labelled red cell esses. In certain disease states the presence of deposition could be determined by measuring body visible or physically measurable alterations of the surface radioactivity, several questions required red cells has permitted detection of the sites and exploration: 1) Are the emanations of Cr5 suit- to some extent of the mechanisms of sequestration able for external body scanning at safe dosage of these cells. Such valuable observations have levels? 2) Does the site of tissue deposition of been made upon pathologic material from patients Cr65 following the intravenous injection of Cr51- with congenital hemolytic anemia (1-5) and labelled red cells necessarily indicate the site of sickle cell anemia (1, 5-7). The need has con- deposition of whole red cells? 3) Is the turnover tinued to exist, however, for a clinical method by of Cr5I deposited in tissues sufficiently slow to which to determine the sites of red cell destruction, provide a detectable accumulation? 4) Finally, both for an understanding of the relationship be- do the results obtained confirm the information tween red cell abnormalities and the body's red already established in certain specific instances, cell clearing mechanisms, and for pragmatic aid for example, in congenital hemolytic anemia and in the often difficult decisions concerning splenec- in sickle cell anemia? In order to investigate tomy. these questions, observations were first made on The potential usefulness of Na2Cr5104-labelled the distribution of Cr5' in normal subjects. The red cells in the clinical determination of the sites fate of Cr51-labelled autogenous red cells was then of red cell destruction was suggested to us by ob- observed in patients with known intracorpuscular servations on postmortem tissues taken from a defects and, lastly, in patients with certain diseases patient with the hemolytic anemia of liver disease, generally characterized by excessive red cell de- who succumbed during observation of the sur- struction and a preliminary report (10) was made. vival of Cr5'-labelled autogenous red cells.2 The Crp5 activity of the patient's splenic tissue was METHODS Cr' labelling technique. All red cell labelling was per- 1 This investigation was supported in part by a grant formed under sterile conditions with Na,CrMO43 diluted from the Helen Hay Whitney Foundation and by Re- with normal saline to a concentration of about 30 micro- search Grant RG3507 (C3) from the National Institutes of grams of chromium per cubic centimeter. Between 100 Health, Public Health Service. and 150 microcuries of Cr5' in this form were added with 2 Cr' either as Cr"OJ- (8) or as Cr5' "' (9) is immediate mixing to 50 cc. of whole blood in a siliconized capable under appropriate conditions of firmly labelling flask containing 12 cc. of acid-citrate-dextrose solution. red cells by chemical attachment to the hemoglobin and The flask was then gently agitated continuously for 45 to the red cell membrane, respectively. For brevity, how- ever, red cells labelled with Na,Cr"04 will be described 3 "Rachromate," Abbott Laboratories, North Chicago, here as "Cr"-labelled red cells." Illinois. 842 DETERMINATION OF THE SITES OF RED CELL SEQUESTRATION 843 Cr5I-LABELLED AUTOGENOUS RED CELLS SUBJECT NORMAL 90 z w 70 I0-60 - BLOOD X. so 0 0 z 30 o _ _ _ ~~~~~~~~~~~~~TISSUE z __ - ~~~~~~~~~ _ o ~~~URINE o 4 31.-03 0 o , O * _ SPLEEN P LIVER 14 e 00 i'o l's 2 o £O DAYS FIGURE 1 The gradual, almost linear, disappearance of Cr'-labelled normal autogenous red cells from the blood stream shown in the upper portion of the figure is reflected by ,a gradual accumulation of Cr' in the tissues. Little difference exists in the levels of relative radioactivity over liver and spleen, as depicted in the lower portion of the figure. minutes. Unbound or reduced chromium was removed A solution of a reduction product of Na2CrkO,, Cr'G13,5 by washing the red cells once in sterile normal saline, was diluted with normal saline to a suitable volume; this after which the red cells were suspended in 2 volumes of was injected directly intravenously in one instance and normal saline preparatory to injection. Over 99 per after preliminary incubation with serum in another. cent of the Cr' injected was in the red cells. Specimen collection and Crt determination. Following Solutions of Cr'-labelled hemoglobin were prepared injection of Crc-containing preparations, venous blood by similarly labelling red cells which were thereafter specimens were collected at appropriate intervals in washed 5 times in normal saline and then hemolyzed with bottles containing dry "balanced" oxalate. Their radio- 4 volumes of distilled water.4 After adjusting the sodium activity was determined after hemolysis by freezing and chloride concentration of the suspending fluid to 0.85 gm. thawing. Specimens for the measurement of plasma ra- per cent, the red cell "ghosts" were centrifuged down and dioactivity were drawn into saline-wetted syringes and the supernatant solution was Seitz-filtered prior to the citrated plasma removed after centrifugation of the injection. specimens within one hour of their procurement. The plasma samples were then frozen and thawed along with 4 It is equally feasible to label the hemnoglobin in solu- tion after hemolysis; this may be done either with 5 Radioactive chromic chloride, Abbott Laboratories, Na2CrMO4 or Cr'Cl.. North Chicago, Illinois. 844 J. H. JANDL, M. S. GREENBERG, R. H. YONEMOTO, AND W. B. CASTLE the whole blood samples. Plasma Cru activity was ex- of whole blood Cr' was plotted against time. From this pressed as a percentage of the whole blood activity by value the initial blood volume was calculated by the dilu- correction for the plasma volume of a sample of whole tion principle and the CrM concentration of the circulating blood. When possible, all urine was collected as indi- blood was thereafter expressed as a percentage of the vidual specimens for about 18 hours after the Cr' in- zero time value. The Cr'1 content of the circulating blood jection, and thereafter 24-hour urine collections were was calculated as the product of the blood Cr' concen- made. In several subjects serial 2- or 4-day total stool tration and the blood volume, by making the assumption collections were made. The stool specimens were di- that the latter was unchanged during the period of study. gested with 10 per cent sodium hydroxide and mixed in Red cell survival at any time was then taken as the per- a blender before sampling. centage of the injected Cr's which remained in the cir- The radioactivity of all types of materials was deter- culating blood. For graphic presentation data so ob- mined with a well-type scintillation counter. The "zero- tained were plotted against time on rectilinear graph time" peripheral blood radioactivity was estimated by ex- paper without correction for Cr' "elution" from the red trapolation of a line drawn visually through points on cells. The blood Cr'1 activity was followed in most sub- semilogarithmic graph paper on which the concentration jects until its disappearance. However, for easier com- Cr5l- LABELLED HEMOGLOBIN SUBJECT NORMAL I.. too I- 'a. SKS 0SW -V o < 0 3w .. DAYS FIGUE 2 Following intravenous injection of Crh-labelled hemoglobin solution, radioac- tivity disappeared from the blood at a continuously slowing rate with a half-disap- pearance time of 3 hours, whereas by chemical determination plasma hemoglobin was removed from the circulation "exponentially" with a half-time of from 1% to 2 hours. With the removal of Cr' from the peripheral blood, there was a rapid, prominent rise in radioactivit-y over the liver and to a lesser extent over the spleen. -if DETERMINATION OF THE SITES OF RED CELL SEQUESTRATION 845 Cr51 C13 SUBJECT: NORMAL loo 900 so o 0 r SO .1-O40s .510 - - - - - - - - - - z 30 - - - - - - - - - go0 AL o -i I - FL LIVTISSE hi 0~ ~ ~ ~ ~ ~ DY FIGURE 3 The upper portion of this figure indicates the great rapidity with which injected cationic trivalent Crm in saline is cleared from the circulation. Most of this chrom- ium appeared in the urine, with only a slight accumulation by the liver and spleen. Preliminary incubation of Cr'Cl, and serum before injection enhanced the rela- tive Cr's uptake by the liver. parison of data, a time axis of only 30 days was em- fore, surface projections of these organs were deter- ployed in the presentation of the studies of individual pa- mined by physical examination and their approximate tients shown below in Figures 1 to 13. The urinary centers were marked with a skin pencil. In some patients excretion of Cr' was expressed as the cumulative per- radioactivity levels were also followed over the thoracic, centage of the injected dose of Cr'.
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