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Encephalopathy and fatty metamorphosis of the liver associated with cold-agglutinin autoimmune hemolytic anemia
April 1979 The Journal o f P E D I A T R I C S
569
Encephalopathy and fatty metamorphosis of the liver associated with cold-agglutinin autoimmune hemolytic anemia Three patients with encephalopathy clinically indistinguishable from Reye syndrome but associated with elevated cold-agglutinin titers and antiglobulin-I autoimmune hemolytic anemia are reported The patients were treated with exchange transfusions, dexamethasone, controlled hyperventilation, and intracranial pressure monitoring. Liver biopsy specimens in two of the three patients showed fatty infiltration of the hepatocytes, but the light microscopic and electron microscopic appearance of the liver was not typical for Reye syndrome. These patients are reported to alert physicians to a metabolic encephalopathy in children which mimics Reye syndrome, but is distinctly different in clinical course and complicated by cold agglutinins and an autoimmune hemolytic anemia.
James P. Orlowski, M.D.,* Johann H. Johannsson, M.D., and Nancy G. Ellis, M . D . , Cleveland, Ohio
R E V E S VN D R O ME is a distinct clinicopathologic entity in children characterized by a rapidly progressive encephalopathy and fatty degeneration of the viscera? Recent reports have emphasized encephalopathies which clinically mimic Reye syndrome but have different histopathologic findings3 -4 This report describes three patients seen over a sixmonth interval with an encephalopathy mimicking Reye syndrome but distinctly different because of the associated abnormalities of elevated cold agglutinins, an antiglobulin-I associated a u t o i m m u n e hemolytic anemia, and a prolonged clinical course. The appearance of the liver biopsy specimen in two of the children was not typical for Reye syndrome. Therapeutic interventions for encephalopathies associated with cold agglutinins and hemolytic anemia are discussed. We believe that these patients had a unique and previously unrecognized encephalopathic condition. From The Pediatric Intensive Care Unit and The Department of Pathology, Rainbow Babies and Childrens Hospital, Case Western Reserve University. *Reprint address: The Cleveland Clinic Foundation, Departments of Pediatrics and Anesthesia, 9500 Euclid Ave,, Cleveland, OH 44106.
0022-3476/79/400569+07500.70/0 9 1979 The C. V. Mosby Co.
Abbreviations used CT: computed tomography EEG: electroencephalogram ICP: intracranial pressure AIHA: autoimmune hemolytic anemia URI: upper respiratory infection
CASE R E P O R T S
Patient 1. An I 1-month-old boy developed URI symptoms with cough but no fever five days prior to admission. He was treated with ampicitlin 125 mg by mouth four times a day after his private physician was contacted by phone. One day before admission he began to vomit repeatedly, and within the next 12 hours he became tachypneic with periods of lethargy alternating with periods of screaming. His mental status deteriorated rapidly, and when he came to the emergency room he was comatose, flaccid, unresponsive to noxious stimuli, but had an intact gag reflex and coughed with oral and nasotracheal suctioning. He was very pale and edematous. Temperature was 37.5~ heart rate 180, respiration rate 76, blood pressure 60/40 tort. Pupils were dilated but reactive to light, corneal reflexes were absent, but the oculocephalic reflexes were present. Optic fundi were normal, deep tendon reflexes were symmetric but hyperactive in the lower extremities, and the Babinski sign was present bilaterally. The liver was enlarged to 3 cm below the right costal margin.
Vol. 94, No. 4, pp. 569-575
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Orlowski,Johannsson, and Ellis
The Journal of Pediatrics April 1979
Table. A comparison o f the laboratory features of Reye syndrome with the three cases of cold-agglutinin anti-I autoimmune hemolytic anemia
Reye syndrome Increased Increased Both increased Normal or slight increase Increased Normal Normal Normal Variable Negative Negative Normal or increased/ normal Negative Negative
Microdroplet lipid infiltration Abnormal swollen mitochondria with loss of dense bodies Needs to be less than 48 hr Normal
Study (normal values) SGOT (0-32 U/l) NH 3 (20-80 mg/dl) PT/PTT (12-13/27-30 sec) Bilirubin, total/direct (0.1-1.2/0.0-0.3 mg/dl) LDH (90-180 u/l) Hgb/Hct (12-15/36-45%) Reticulocytes (0.5-1.5%) Platelets/mm ~ (150,000.350,000) WBC/mm ~ (4800-10,800) Cold agglutinins ( < 1:32) Mycoplasma titer (negative) BUN/creatinine (6-23/0.4-1.4 mg/dl) Antiglobulin-I (negative) Blood smear showing erythrocyte fragmentation (negative) Light microscopy with fat stain of liver biopsy specimen Electron microscopy of liver biopsy specimen
Time after admission that liver biopsy was performed Bone marrow aspirate
Patient 1
I
Patient 3
Patient2 146
399 1,200 22/35 1.6/0.5
270 24/27 6.1/1.0
120 344 21/56 3.1/1.6
1,050 10.5/26 16.3 350,000 13,700 1:112 1:8 7/0.6
2,220 3.1/8.9 8.4 232,000 36,000 1:16 1:16 45/0.8
600 10.1/29.5 ND 369,000 35,400 1:128 1:16 70/2.5
+ +
+ +
Large lipid lakes
ND
Large cytoplasmic fat
Swollen mitochondria but no disruption of mitochondrial matrix 20 hr
ND
Swollen mitochondria but no flocculation of mitochondrial matrix 40 hr
Erythroid hyperplasia
Erythroid hyperplasia
ND
ND
Abbreviations used: SGOT ffi Serum glutamic oxalacetic transammase; PT = prothrombin time; FIT ffi partial thromboplastin time; LDH = lactic dehydrogenase; Hgb ffi hemoglobin; Hct ffi hematocrit; WBC ffi white blood count; BUN = blood urea nitrogen; ND ffi not determined. A C T scan of the brain showed slitlike ventricles. Lumbar puncture revealed an opening pressure of 23 torr with no cells, normal protein, and a CSF glucose concentration of 35 mg/dl. He was given ampicillin, chloramphenicol, manuitol, and dexamethasone intravenously. Blood NH~ was 1,200 mg/dl, SGOT 399 U/l, and the prothrombin time and partial thromboplastin time were both prolonged. He became apneic and was promptly intubated and taken to the operating room, where an external ventriculostomy catheter was placed for monitoring intracranial pressure. A peripheral blood smear on admission showed RBC fragments, and a direct Coombs test in preparation for exchange transfusion was positive with an IgM, antiglobulin-I antibody. Cold agglutinins were positive in 1:112 dilution. Eighteen hours after admission the liver was palpable 9 cm below the right costal margin, and after the dotting abnormalities were corrected with exchange transfusion and fresh frozen plasma, a liver biopsy was performed percutaneously approximately 20 hours after hospitalization. The patient received five two-volume exchange transfusions over the first 36 hours of hospitalization. An EEG was grossly abnormal with decreased physiologic rhythm, increased delta
activity (R > L), and 15 Hz spikes on the right. Phenobarbital therapy was commenced and dexamethasone continued. The protracted course was characterized by episodes of intracranial hypertension requiring mannitol therapy, and the intraventricular ICP monitor was employed for 24 days. Despite apparently adequate control of intracranial hypertension, the patient remained stuporous and required traeheostomy because of inadequate cough reflex, an~d gastrostomy because of inab~llty to feed orally. He remains in a vegetative state in a chronic care facility. Viral titers showed no change between acute and convalescent sera, but the mycoplasma titer increased from 1:8 acutely to 1:64 on convalescent serum. Patient 2. A 16-month-old girl seemed to be recovering from an upper respiratory infection when she began to vomit every one to two hours and became lethargic. She remained in bed for the 24 hours prior to admission and was given 2-grain aspirin suppositories three times over 24 hours for a "slight fever." The mother found the child suddenly pale, tachypneic, and unresponsive, and rushed her to a community hospital where she was noted to be afebrile, hyperpneic, and unresponsive to stimuli. Temperature was 36.6~ heart rate 160, respiratory rate 70, and
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Encephalopathy and fatty metamorphosis of liver in A IHA
57 1
Fig. 1. Hematoxylin-eosin-stained section from the liver biopsy in Patient 1. Diffuse swelling and vacuolization of hepatocyte cytoplasm is evident. (Original magnification 200x .) blood pressure 110/60 torr. Hemoglobin was 3.1 gm/dl, the. hematocrit was 8.9%, and her blood glucose concentration was 8.0 mg/dl. She was treated with glucose intravenously and transferred to our hospital where she was noted to be extremely pale but without cyanosis or edema. Pupils were asymmetric (right eye 3 mm; left eye 2 mm) and reacted sluggishly to light; fundi were normal. The liver was palpable 2 cm below the right costal margin. She was hyperreflexic with sustained ankle clonus, had no spontaneous motor activity, and was unresponsive to painful stimuli. She had an episode of decorticate posturing followed by unobtainable blood pressure. She was treated with 0-negative blood transfusions and responded quickly. A blood sample prior to transfusion had a positive direct Coombs test with a complement-fixing dntibody subsequently determined to be an IgM, anti-I, and cold agglutinins were positive at a 1:64 titer. The salicylate level was 2.5 mg/dl, blood ammonia was 270 mg/dl, SGOT 146 U/l, and a peripheral blood smear showed RBC fragments. CSF obtained by lumbar puncture after intravenous use of mannitol and dexamethasone contained no cells; CSF protein was 70 mg/dl, and CSF glucose concentration was 150 mg/dl with a simultaneous blood sugar of 180 mg/dl. A CT scan showed cerebral edema. To remove circulating antibodies, she was treated with 1.5 volume exchange transfusions on each of the subsequent two days, with improvement of clotting studies and an increase in hemoglobin and hematocrit. The hematocrit twice fell to less than 20% between exchange transfusions. An EEG was abnormal, with rhythmic slowing and 1 to 2 Hz delta activity. An intraventricular catheter for ICP monitoring was inserted with an opening CSF pressure of 18 torr. She did very well after two days of therapy with mannitol, glycerol, and dexamethasone. The ICP monitor was discontinued after 48 hours, and the external ventriculostomy catheter was removed four days later. She
recovered slowly and was discharged home one month later. On examination four months after discharge there was no neurologic or developmental abnormality. There was no change between acute and convalescent viral or mycoplasma titers. Patient 3. A 7-year-old boy was well until six days before admission when URI symptoms developed. He was treated with 4 grains of aspirin every six hours until 36 hours prior to admission when he began to vomit repeatedly every 15 to 30 minutes. He became gradually unresponsive and tachypneic, and when examined in the emergency room, he was alternately stuporous and combative. Pupils were 3 mm and reactive, temperature 38.9~ heart rate 180, respiratory rate 32, and blood pressure 150/90 torr. Over the next 90 minutes he became intermittently opisthotonic with 7 mm equal pupils, spontaneous hippus, and intact oculocephalic reflexes. The deep tendon reflexes were hyperactive, with sustained ankle clonus and bilateral extensor plantar responses. Two hours after admission (30 minutes later) decerebrate posturing and central neurogenic hyperventilation developed. He was immediately taken to the operating room for intraventricular catheter placement for ICP monitoring. The serum salicylate level was 1.0 mg/dl, blood ammonia 344 mg/dl, SGOT 120 U/l, hemoglobin 10.1 gm/dl, and hematocrit 29.5% with evidence of hemolysis on peripheral blood smear. CSF examination showed no WBC, protein 17 mg/dl, and glucose 91 mg/dl. After ICP monitoring was begun, he was prepared for exchange transfusion through a Scribner shunt placed in the operating room. Cross-matching for exchange transfusion revealed a cold antibody of anti-I specificity. Exchange transfusions were given every eight hours for two days, and a liver biopsy was performed on the second hospital day when the clotting studies had corrected. He died on the fourth , hospital day of intracranial hypertension refractory to mannitol,
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Orlowski, Johannsson, and Ellis
The Journal
of Pediatrics April 1979
Fig. 2. Oil-red-O stained cryostat section of' liver in Patient 1, showing fat infiltration of hepatocytes ranging from microdroplets to fat "lakes." (Original magnification 200 x .)
glycerol, and furosemide therapy. Postmortem examination showed mild cerebral edema, fatty metamorphosis of the liver, and focal interstitial pneumonitis. Postmortem viral and mycoplasma titers were unchanged from admission values. The Table details the clinical and laboratory aspects of the three patients and compares them with the classic description of Reye syndrome. HISTOLOGIC
METHODS
The liver biopsy specimens (Patients 1 and 3) were immediately brought to ,the laboratory for processing. A portion was frozen in isopentane cooled in liquid nitrogen. Cryostat sections were prepared and stained with oil-red-O for lipids. A second portion was fixed ifi cold 2.5% glutaraldehyde in 4% sucrose buffered to pH 7.2 with 0.05M phosphate buffer. It was postfixed in phosphatebuffered, 1% osmium tetroxide, dehydrated with acetone, embedded in Spurr-mixture (ERL-4206), ~ and stained with 1% uranyl acetate. The remainder of each specimen was fixed in 10% formalin, embedded in paraffin, and sectioned at 6 microns. The sections were stained with routine histologic stains including hematoxylin-eosin, periodic-acid-Schiff procedure with and without diastase treatment, Wilder reticulin stain, and Masson trichrome. The liver specimen from the autopsy (Patient 3) was treated similarly. RESULTS Light microscopy. Both liver biopsy specimens (Patients I and 3) showed uniform swelling of hepatocytes and coarse vacuolization of the cytoplasm in many cells
pushing the nucleus to the periphery. Oil-red-O stain revealed a diffuse fatty infiltration of hepatocytes progressing from small vesicles around central veins to fat lakes in periportal areas. The fat droplets were somewhat smaller in Patient 3 but mostly larger than hepatocyte nuclei. Glycogen was reduced, and no cytoplasmic or nuclear inclusions were seen. There was no inflammation, necrosisl or fibrosis. Bile ducts were'preserved. Patient 1 had no bile stasis; Patient 3 had mild centrilobular canalicular bile plugging. Liver sections from the autopsy of Patient 3 were similar except for slight periportal mononuclear cell infiltrates and occasional small intralobular infiltrates. No acute inflammation or necrosis was seen except in the biopsy site (Figs. 1 and 2). Electron microscopy. Electron microscopy was done on the biopsy specimen from Patient 1 and on an autopsy specimen from Patient 3. Glycogen was greatly reduced, and multivesicular cytoplasmic fat was seen, similar to that in the light microscopy specimens. Mitochondria were uniformly swollen, with reduction of eristae and matrical dense bodies. There was no flocculation of mitochondrial matrix, and no ameboid mitochondria or vesicular structures were seen. Peroxisomes did not appear increased. Viruslike inclusions were not seen. Some hepatocytes had irregular areas of clear, watery cytoplasm, especially in Patient 1 (Fig. 3). DISCUSSION Autoimmune hemolytic anemia is uncommon in children, 6 and disease due to cold-reactive autoantibodies of anti-I specificity accounts for only I0 to 30% of pediatric
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Encephalopathy and fatty metamorphosis of liver in AIHA
573
Fig. 3. Electron photomicrograph from postmortem liver specimen in Patient 3. Nucleus at left and fat droplet below to right. Mitochondrial are swollen. Cristea and occasional dense bodies are seen. (Magnification 9,100• .)
patients with AIHA. 6. 7 The occurrence of cold-agglutinin AIHA in association with an encephalopathy mimicking Reye syndrome in three patients over a six-month period of time seems more than coincidental. The clinical and laboratory findings of our three patients were consistent with Reye syndrome, and therapy proceeded upon that presumptive diagnosis. Each of the patients had a prodromal upper respiratory tract infection followed by apparent recovery. Pernicious vomiting and deranged mental state marked the onset of encephalopathy, which rapidly progressed from agitation and delirium to unresponsiveness and coma. The biochemical aberrations were characteristic of Reye syndrome? .9 Indirect hyperbilirubinemia, hemolysis, elevated cold agglutinins, and the presence of an antiglobulin-I cold antibody are not typical of Reye syndrome, however, and prompted us to perform liver biopsies and bone marrow aspirations on two of the patients. The histopathologic findings of the liver in Reye syndrome include diffuse fat infiltration of hepatocytes of variable severity, which may be "masked ''~ in hematoxylin-eosin-stained sections but is easily demonstrated with lipid stains. L.~-" The fat droplets are small, multiple in each hepatocyte, and displace the nucleus very little. They tend to be larger toward the portal areas than around central veins. Inflammation and cell necrosis may be present H but is minimal, ~ and there is also glycogen depletion and often mild canalicular bile stasis? Ultrastructural studies 1~ have revealed some addi-
tional and fairly characteristic changes consisting of severely swotlen mitochondria, often ameboid in shape, with flocculated matrix, preserved cristae, reduced or absent dense bodies, and sometimes containing vesicular structures? ' These mitochondrial changes appear to parallel the clinical severity of the disease? *. ~ Other electron microscropic findings are reduction of glycogen; proliferation, swelling, and flocculation of peroxisome~; proliferation of endoplasmic reticulum; and viral inclusions? 2 These findings have varied from one study to another, as have conclusions about their specificity. 9"1' It appears that the findings change rapidly with time and severity of the disease and require immediate and careful processing of the tissues for their demonstration. When the liver biopsy is done within 48 hours after the onset of neurologic deterioration, the microvesicular lipid pattern and the mitochondrial changes appear to be reliable criteria for the diagnosis?- x" Both have still to be interpreted in the light of the clinical findings, since microvesicular fatty infiltration is a common pattern of fatty liver in childhood, and mitochondrial changes can be produced with a variety of agents. 2, ,4 The liver biopsies in both of our patients were obtained within 48 hours. In both there was unmasking of hepatocyte lipid which in periportal areas had already formed lipid lakes. Except for the size of the lipid droplets the fight microscopy was very similar to the severe lesion of Reye syndrome, 9 with a lack of necrosis, inflammation, or fibrosis. Electron microscopy of the liver biopsy from
574
Orlowski,Johannsson, and Ellis
Patient 1 did not show the characteristic mitochondrial changes listed above; the liver specimen from Patient 3 also showed nonspecific mitochondrial changes and glycogen depletion but none of the specific features. Although the histologic and ultrastructural changes in these two children are not typical for Reye syndrome, that diagnosis can probably not be ruled out on morphologic grounds alone. Other disease states producing encephalopathy and liver dysfunction resembling Reye syndrome, but with distinctly different liver histopathology, have been describedY -~Although several authors have suggested that Reye syndrome can be diagnosed with confidence by history and routine laboratory data without the necessity for confirmation of fatty degeneration of the liver, 16. 17our data confirm the impression of others that liver biopsy is necessary for accurate diagnosis. 3, is Percutaneous needle biopsy of the liver can be performed safely after correction of the coagulation defects with fresh frozen plasma or exchange transfusion. Our patients' clinical and laboratory findings were virtually identical to those of classic Reye syndrome, unlike other reported encephalopathies mimicking Reye syndrome. 3 To our knowledge, the association of a cold-antibody AIHA with Reye syndrome has not been reported previously; we believe that these patients represent a unique encephalopathic condition. Wintrobe et al TM state that in severe acute forms of hemolytic anemia, semicoma and shocklike prostration may develop, but a syndrome masquerading as Reye syndrome has never been previously reported. We were unable to determine the etiology of the cold agglutinins and hemolytic anemia in our patients. Mycoplasma titers remained constant in two patients and rose only slightly in one patient. Titers for influenza A and B, cytomegalovirus, Coxsackie, measles, and mumps viruses remained constant between acute and convalescent sera. Results of the monospot test were negative in all three patients, and none of them had posterior cervical adenopathy 6r an absolute lymphocytosis. Occult viral infections have been implicated in the pathogenesis of AIHA both experimentally and clinically. 2~ ~ Cold antibodies frequently develop in high concentrations in Mycoplasma pneumoniae infections but an associated hemolytic anemia is rare and usually occurs toward the end of the second week of illness. 2~AIHA has been described during the course of infectious mononucleosis, ~3 acute viral hepatitis, and herpes simplex infections?" Anemia usually developed during the convalescent phase of these illnesses and was attributed to cold agglutinins or to cold hemolysins. Associated severe granulocytopenia and thrombocytopenia were observed
The Journal of Pediatrics April 1979
in a few instances. Acquired hemolytic anemia has been associated with cytomegalovirus infections in children, but these are usually warm antibody-type AIHA, and most patients are immunologically compromised, s~ 2~ Hemolytic anemia occurring early in the course of an infection is rare and AIHA due to cold autoantibodies is uncommon in children. Cold-antibody disease is generally mild and occurs most frequently in elderly subjects. 1~ Zupanska et al 6 reported 44 children with AIHA and only two patients with acute AIHA had anti-I specific autoantibodies. Buchanan et al 2. reviewed the records of 22 children with AIHA and were unable to find elevated titers Of cold agglutinins in any of their patients. A simple screening test for the presence of high-titer cold agglutinins consists of rubbing a test tube of citrated blood on the freezer wall of a refrigerator. If coarse, floccular agglutination occurs and can be reversed by warming the test tube, the cold agglutinin titer is usually in excess of 1:64.... s and the antibody probably contributes to the hemolysisY9 This simple test was positive in our three patients. Most clinically important antibodies agglutinate erythrocytes up to 32~ 3~ 31 Barcroft and Edholm 32 showed that the subcutaneous temperature of the bared forearm readily fails to below 30~ if the forearm is exposed to air at cool room temperature (18.5~ Transient antibodyantigen interaction can therefore be expected to occur as blood circulates through areas of the body exposed to a cool environment. For this reason the use of controlled hypothermia as therapy for cerebral edema is contraindicated in patients with cold-agglutinin AIHA; it is also essential to use warmed donor blood for transfusion. 33 Exchange transfusion, plasma exchange, 3' and plasmapheresis 33 have been reported to be effective in reducing the level of autolog0us serum cold agglutinins and improving the clinical status of these patients. We thank Drs. Elizabeth M. Kurczynski and Morris W. Levinsohn for their advice and assistance in the management of these patients, and Ms. Rita Feran for editorial assistance. REFERENCES
1. Reye RDK, Morgan G, and Baral J: Encephalopathy and fatty degeneration of the viscera: a disease entity in childhood, Lancet 2:749, 1963. 2. Partin JC, Partin JS, and Schubert WK: Fatty liver in Reye's syndrome: is it a distinct morphologic entity? Gastroenterology 65:563, 1973. 3. Gall DG, Cutz E, McClung HJ, and Greenberg ML: Acute liver disease and encephalopathy mimicking Reye's syndrome, J PEDIATR87:869, 1975. 4. Truscott R, Pullin C, Halperin B, et al: Suspected defect in fatty acid oxidation, N Engl J Med 299:152, 1978. 5. Hayat MA: Principles and techniques of electron micros-
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Encephalopathy and fatty metamorphosis of liver in A I H A
copy, vol. I, New York, 1970, Van Nostrand Reinhold Company, p 161. 6. Zupanska B, Lawkowicz W, Gorska B, et al: Autoimmune hemolytic anemia in children, Br J Haematol 34:511, 1976. 7. Habibi B, Homberg JC, Schaison G, and Salmon C: Autoimmune hemolytic anemia in children. A review of 80 cases, Am J Med 56:61, 1974. 8. LovejoyFH, Smith AL, Bresnan MJ, et al: Clinical staging in Reye's syndrome, Am J DIS Chad 128:36, 1974. 9. BoveKE, McAdams AJ, Partin JC~ et al: The hepatic lesion in Reye's syndrome, Gastroenterology 69:685, 1975. 10. Partin JC, Schubert WK, and Partin JS: Mitochondrial ultrastructure in Reye's syndrome (encephalopathy and fatty degeneration of the viscera), N Engl J Med 285:1339, 1971. 11. Svoboda DJ, and Reddy JK: Pathology of the liver in 9 Reye's syndrome, Lab Invest 32:571, 1975. 12. Tang TT, Siegesmund KA, Sedmak GV; et al: Reye's syndrome. A correlated electronmicroscopic, viral and biochemical observation, JAMA 232:1339, 1975. 13. BradelEJ, and Reiner CB: The fine structure ofhepatocytes in Reye's syndrome, in Pollack JD editor: Reye's syndrome, New York, 1975, Grune & St~atton, Inc., p 147. 14. Iancu TC, Mason WH, and Neustein HB: Ultrastructural abnormalities of liver cells in Reye's syndrome, Hum Pathol 8:421, 1977. 15. Partin JC: Liver ultrastructure in Reye's syndrome, in Pollack JD, editor: Reye's syndrome, New York, 1975, Grune & Stratton, Inc., p 117. 16. Glasgow AM, Cotton RB, and Dhiensire K: Reye's syndrome. I. Blood ammonia and consideration of the nonhistologic diagnostic, Am J Dis Child 124:827, 1972. 17. Samaha FJ, Glau E, and Berardinelli JL: Reye's syndrome: Clinical diagnosis and treatment with peritoneal dialysis, Pediatrics 53:336, 1974. 18. Schubert WK: Commentary: The diagnosis of Reye syndrome, J PEDIATg87:867, 1975. 19. Wintrobe MM, Lee GR, Boggs DR, et ah Immunohemolytic anemias, in Clinical hemaotology, ed 7, Philadelphia, 1974, Lea & Febiger, Chapter 27.
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20. Zuelzer WW, Mastrangelo R, Stulberg CS,.et al: Autoimmune hemolytic anemia: natural history and viral-immunologic interactions in children, Am J Med 49:80, i970. 21. Murdock JMcC, and Smith CC: Haemolytic anemias, Clin Haematol 1:619, 1972. 22. Neely FL, Baria WH, Smith C, and Stone CF Jr: Primary atypical pneumonia with high titer cold hemaglutinins, hemolytic anemia, and false positive Donath-Landsteiner test, J Lab Clin Med 37:382, 1951. 23. Mengel CE, Wallace AG, and McDaniel HG:. Infectious mononucleosis, hemolysis and megaloblastic arrest, Arch Intern Med 114:333, 1964. 24. Finland M, Peterson OL, Allen HE, et al: Cold agglutinins: occurrence of cold isohemagglutinins in various conditions, J Clin Invest 24:451, 1945. 25. Zuelzer WW, Stulberg CS, Page RH, et al: Etiology and pathogenesis of acquired hemolytic anemia, Transfusion 6:438, 1966. 26. Buchanan GR, Boxer LA, and Nathan DG: The acute and transient nature of idiopathic immune hemolytic anemia in children, J P~DL~TR88:780, 1976. 27. CosteaN: The differential diagnosis of hemolytic anemias, Med Clin North Am 57:289, 1972. 28. C-arrowDH: A rapid test for the presence of increased cold agglutinins, Br Med J 2:206, 1958. 29. Jacobson LB, Longstreth GF, and Edginton TS: Clinical and immunologic features of transient cold agglutinin hemolytic anemia, Am J Med 54:514, 1973. 30. Pirofsky B: Clinical aspects of autoimmune hemolytic anemia, Semin Hematol 13:251, 1976. 31. Dacie JV: Autoimmune hemolytic anaemias, Br Med J 2:381, 1970. 32. BarcroflH, and Edholm O~.. Temperature and blood flow in the human forearm, J Physiol 104:366, 1946. 33. Roseniield RE, and Jagathambal J: Transfusion therapy for autoimmune hemoltyic anemia, Semin Hematol 13:311, 1976. 34. Taft EG, Propp RP, and Sullivan SA: Plasma exchange for cold agglutinin hemolytic anemia, Transfusion 17:173, 1977.
569
Encephalopathy and fatty metamorphosis of the liver associated with cold-agglutinin autoimmune hemolytic anemia Three patients with encephalopathy clinically indistinguishable from Reye syndrome but associated with elevated cold-agglutinin titers and antiglobulin-I autoimmune hemolytic anemia are reported The patients were treated with exchange transfusions, dexamethasone, controlled hyperventilation, and intracranial pressure monitoring. Liver biopsy specimens in two of the three patients showed fatty infiltration of the hepatocytes, but the light microscopic and electron microscopic appearance of the liver was not typical for Reye syndrome. These patients are reported to alert physicians to a metabolic encephalopathy in children which mimics Reye syndrome, but is distinctly different in clinical course and complicated by cold agglutinins and an autoimmune hemolytic anemia.
James P. Orlowski, M.D.,* Johann H. Johannsson, M.D., and Nancy G. Ellis, M . D . , Cleveland, Ohio
R E V E S VN D R O ME is a distinct clinicopathologic entity in children characterized by a rapidly progressive encephalopathy and fatty degeneration of the viscera? Recent reports have emphasized encephalopathies which clinically mimic Reye syndrome but have different histopathologic findings3 -4 This report describes three patients seen over a sixmonth interval with an encephalopathy mimicking Reye syndrome but distinctly different because of the associated abnormalities of elevated cold agglutinins, an antiglobulin-I associated a u t o i m m u n e hemolytic anemia, and a prolonged clinical course. The appearance of the liver biopsy specimen in two of the children was not typical for Reye syndrome. Therapeutic interventions for encephalopathies associated with cold agglutinins and hemolytic anemia are discussed. We believe that these patients had a unique and previously unrecognized encephalopathic condition. From The Pediatric Intensive Care Unit and The Department of Pathology, Rainbow Babies and Childrens Hospital, Case Western Reserve University. *Reprint address: The Cleveland Clinic Foundation, Departments of Pediatrics and Anesthesia, 9500 Euclid Ave,, Cleveland, OH 44106.
0022-3476/79/400569+07500.70/0 9 1979 The C. V. Mosby Co.
Abbreviations used CT: computed tomography EEG: electroencephalogram ICP: intracranial pressure AIHA: autoimmune hemolytic anemia URI: upper respiratory infection
CASE R E P O R T S
Patient 1. An I 1-month-old boy developed URI symptoms with cough but no fever five days prior to admission. He was treated with ampicitlin 125 mg by mouth four times a day after his private physician was contacted by phone. One day before admission he began to vomit repeatedly, and within the next 12 hours he became tachypneic with periods of lethargy alternating with periods of screaming. His mental status deteriorated rapidly, and when he came to the emergency room he was comatose, flaccid, unresponsive to noxious stimuli, but had an intact gag reflex and coughed with oral and nasotracheal suctioning. He was very pale and edematous. Temperature was 37.5~ heart rate 180, respiration rate 76, blood pressure 60/40 tort. Pupils were dilated but reactive to light, corneal reflexes were absent, but the oculocephalic reflexes were present. Optic fundi were normal, deep tendon reflexes were symmetric but hyperactive in the lower extremities, and the Babinski sign was present bilaterally. The liver was enlarged to 3 cm below the right costal margin.
Vol. 94, No. 4, pp. 569-575
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Orlowski,Johannsson, and Ellis
The Journal of Pediatrics April 1979
Table. A comparison o f the laboratory features of Reye syndrome with the three cases of cold-agglutinin anti-I autoimmune hemolytic anemia
Reye syndrome Increased Increased Both increased Normal or slight increase Increased Normal Normal Normal Variable Negative Negative Normal or increased/ normal Negative Negative
Microdroplet lipid infiltration Abnormal swollen mitochondria with loss of dense bodies Needs to be less than 48 hr Normal
Study (normal values) SGOT (0-32 U/l) NH 3 (20-80 mg/dl) PT/PTT (12-13/27-30 sec) Bilirubin, total/direct (0.1-1.2/0.0-0.3 mg/dl) LDH (90-180 u/l) Hgb/Hct (12-15/36-45%) Reticulocytes (0.5-1.5%) Platelets/mm ~ (150,000.350,000) WBC/mm ~ (4800-10,800) Cold agglutinins ( < 1:32) Mycoplasma titer (negative) BUN/creatinine (6-23/0.4-1.4 mg/dl) Antiglobulin-I (negative) Blood smear showing erythrocyte fragmentation (negative) Light microscopy with fat stain of liver biopsy specimen Electron microscopy of liver biopsy specimen
Time after admission that liver biopsy was performed Bone marrow aspirate
Patient 1
I
Patient 3
Patient2 146
399 1,200 22/35 1.6/0.5
270 24/27 6.1/1.0
120 344 21/56 3.1/1.6
1,050 10.5/26 16.3 350,000 13,700 1:112 1:8 7/0.6
2,220 3.1/8.9 8.4 232,000 36,000 1:16 1:16 45/0.8
600 10.1/29.5 ND 369,000 35,400 1:128 1:16 70/2.5
+ +
+ +
Large lipid lakes
ND
Large cytoplasmic fat
Swollen mitochondria but no disruption of mitochondrial matrix 20 hr
ND
Swollen mitochondria but no flocculation of mitochondrial matrix 40 hr
Erythroid hyperplasia
Erythroid hyperplasia
ND
ND
Abbreviations used: SGOT ffi Serum glutamic oxalacetic transammase; PT = prothrombin time; FIT ffi partial thromboplastin time; LDH = lactic dehydrogenase; Hgb ffi hemoglobin; Hct ffi hematocrit; WBC ffi white blood count; BUN = blood urea nitrogen; ND ffi not determined. A C T scan of the brain showed slitlike ventricles. Lumbar puncture revealed an opening pressure of 23 torr with no cells, normal protein, and a CSF glucose concentration of 35 mg/dl. He was given ampicillin, chloramphenicol, manuitol, and dexamethasone intravenously. Blood NH~ was 1,200 mg/dl, SGOT 399 U/l, and the prothrombin time and partial thromboplastin time were both prolonged. He became apneic and was promptly intubated and taken to the operating room, where an external ventriculostomy catheter was placed for monitoring intracranial pressure. A peripheral blood smear on admission showed RBC fragments, and a direct Coombs test in preparation for exchange transfusion was positive with an IgM, antiglobulin-I antibody. Cold agglutinins were positive in 1:112 dilution. Eighteen hours after admission the liver was palpable 9 cm below the right costal margin, and after the dotting abnormalities were corrected with exchange transfusion and fresh frozen plasma, a liver biopsy was performed percutaneously approximately 20 hours after hospitalization. The patient received five two-volume exchange transfusions over the first 36 hours of hospitalization. An EEG was grossly abnormal with decreased physiologic rhythm, increased delta
activity (R > L), and 15 Hz spikes on the right. Phenobarbital therapy was commenced and dexamethasone continued. The protracted course was characterized by episodes of intracranial hypertension requiring mannitol therapy, and the intraventricular ICP monitor was employed for 24 days. Despite apparently adequate control of intracranial hypertension, the patient remained stuporous and required traeheostomy because of inadequate cough reflex, an~d gastrostomy because of inab~llty to feed orally. He remains in a vegetative state in a chronic care facility. Viral titers showed no change between acute and convalescent sera, but the mycoplasma titer increased from 1:8 acutely to 1:64 on convalescent serum. Patient 2. A 16-month-old girl seemed to be recovering from an upper respiratory infection when she began to vomit every one to two hours and became lethargic. She remained in bed for the 24 hours prior to admission and was given 2-grain aspirin suppositories three times over 24 hours for a "slight fever." The mother found the child suddenly pale, tachypneic, and unresponsive, and rushed her to a community hospital where she was noted to be afebrile, hyperpneic, and unresponsive to stimuli. Temperature was 36.6~ heart rate 160, respiratory rate 70, and
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Fig. 1. Hematoxylin-eosin-stained section from the liver biopsy in Patient 1. Diffuse swelling and vacuolization of hepatocyte cytoplasm is evident. (Original magnification 200x .) blood pressure 110/60 torr. Hemoglobin was 3.1 gm/dl, the. hematocrit was 8.9%, and her blood glucose concentration was 8.0 mg/dl. She was treated with glucose intravenously and transferred to our hospital where she was noted to be extremely pale but without cyanosis or edema. Pupils were asymmetric (right eye 3 mm; left eye 2 mm) and reacted sluggishly to light; fundi were normal. The liver was palpable 2 cm below the right costal margin. She was hyperreflexic with sustained ankle clonus, had no spontaneous motor activity, and was unresponsive to painful stimuli. She had an episode of decorticate posturing followed by unobtainable blood pressure. She was treated with 0-negative blood transfusions and responded quickly. A blood sample prior to transfusion had a positive direct Coombs test with a complement-fixing dntibody subsequently determined to be an IgM, anti-I, and cold agglutinins were positive at a 1:64 titer. The salicylate level was 2.5 mg/dl, blood ammonia was 270 mg/dl, SGOT 146 U/l, and a peripheral blood smear showed RBC fragments. CSF obtained by lumbar puncture after intravenous use of mannitol and dexamethasone contained no cells; CSF protein was 70 mg/dl, and CSF glucose concentration was 150 mg/dl with a simultaneous blood sugar of 180 mg/dl. A CT scan showed cerebral edema. To remove circulating antibodies, she was treated with 1.5 volume exchange transfusions on each of the subsequent two days, with improvement of clotting studies and an increase in hemoglobin and hematocrit. The hematocrit twice fell to less than 20% between exchange transfusions. An EEG was abnormal, with rhythmic slowing and 1 to 2 Hz delta activity. An intraventricular catheter for ICP monitoring was inserted with an opening CSF pressure of 18 torr. She did very well after two days of therapy with mannitol, glycerol, and dexamethasone. The ICP monitor was discontinued after 48 hours, and the external ventriculostomy catheter was removed four days later. She
recovered slowly and was discharged home one month later. On examination four months after discharge there was no neurologic or developmental abnormality. There was no change between acute and convalescent viral or mycoplasma titers. Patient 3. A 7-year-old boy was well until six days before admission when URI symptoms developed. He was treated with 4 grains of aspirin every six hours until 36 hours prior to admission when he began to vomit repeatedly every 15 to 30 minutes. He became gradually unresponsive and tachypneic, and when examined in the emergency room, he was alternately stuporous and combative. Pupils were 3 mm and reactive, temperature 38.9~ heart rate 180, respiratory rate 32, and blood pressure 150/90 torr. Over the next 90 minutes he became intermittently opisthotonic with 7 mm equal pupils, spontaneous hippus, and intact oculocephalic reflexes. The deep tendon reflexes were hyperactive, with sustained ankle clonus and bilateral extensor plantar responses. Two hours after admission (30 minutes later) decerebrate posturing and central neurogenic hyperventilation developed. He was immediately taken to the operating room for intraventricular catheter placement for ICP monitoring. The serum salicylate level was 1.0 mg/dl, blood ammonia 344 mg/dl, SGOT 120 U/l, hemoglobin 10.1 gm/dl, and hematocrit 29.5% with evidence of hemolysis on peripheral blood smear. CSF examination showed no WBC, protein 17 mg/dl, and glucose 91 mg/dl. After ICP monitoring was begun, he was prepared for exchange transfusion through a Scribner shunt placed in the operating room. Cross-matching for exchange transfusion revealed a cold antibody of anti-I specificity. Exchange transfusions were given every eight hours for two days, and a liver biopsy was performed on the second hospital day when the clotting studies had corrected. He died on the fourth , hospital day of intracranial hypertension refractory to mannitol,
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Fig. 2. Oil-red-O stained cryostat section of' liver in Patient 1, showing fat infiltration of hepatocytes ranging from microdroplets to fat "lakes." (Original magnification 200 x .)
glycerol, and furosemide therapy. Postmortem examination showed mild cerebral edema, fatty metamorphosis of the liver, and focal interstitial pneumonitis. Postmortem viral and mycoplasma titers were unchanged from admission values. The Table details the clinical and laboratory aspects of the three patients and compares them with the classic description of Reye syndrome. HISTOLOGIC
METHODS
The liver biopsy specimens (Patients 1 and 3) were immediately brought to ,the laboratory for processing. A portion was frozen in isopentane cooled in liquid nitrogen. Cryostat sections were prepared and stained with oil-red-O for lipids. A second portion was fixed ifi cold 2.5% glutaraldehyde in 4% sucrose buffered to pH 7.2 with 0.05M phosphate buffer. It was postfixed in phosphatebuffered, 1% osmium tetroxide, dehydrated with acetone, embedded in Spurr-mixture (ERL-4206), ~ and stained with 1% uranyl acetate. The remainder of each specimen was fixed in 10% formalin, embedded in paraffin, and sectioned at 6 microns. The sections were stained with routine histologic stains including hematoxylin-eosin, periodic-acid-Schiff procedure with and without diastase treatment, Wilder reticulin stain, and Masson trichrome. The liver specimen from the autopsy (Patient 3) was treated similarly. RESULTS Light microscopy. Both liver biopsy specimens (Patients I and 3) showed uniform swelling of hepatocytes and coarse vacuolization of the cytoplasm in many cells
pushing the nucleus to the periphery. Oil-red-O stain revealed a diffuse fatty infiltration of hepatocytes progressing from small vesicles around central veins to fat lakes in periportal areas. The fat droplets were somewhat smaller in Patient 3 but mostly larger than hepatocyte nuclei. Glycogen was reduced, and no cytoplasmic or nuclear inclusions were seen. There was no inflammation, necrosisl or fibrosis. Bile ducts were'preserved. Patient 1 had no bile stasis; Patient 3 had mild centrilobular canalicular bile plugging. Liver sections from the autopsy of Patient 3 were similar except for slight periportal mononuclear cell infiltrates and occasional small intralobular infiltrates. No acute inflammation or necrosis was seen except in the biopsy site (Figs. 1 and 2). Electron microscopy. Electron microscopy was done on the biopsy specimen from Patient 1 and on an autopsy specimen from Patient 3. Glycogen was greatly reduced, and multivesicular cytoplasmic fat was seen, similar to that in the light microscopy specimens. Mitochondria were uniformly swollen, with reduction of eristae and matrical dense bodies. There was no flocculation of mitochondrial matrix, and no ameboid mitochondria or vesicular structures were seen. Peroxisomes did not appear increased. Viruslike inclusions were not seen. Some hepatocytes had irregular areas of clear, watery cytoplasm, especially in Patient 1 (Fig. 3). DISCUSSION Autoimmune hemolytic anemia is uncommon in children, 6 and disease due to cold-reactive autoantibodies of anti-I specificity accounts for only I0 to 30% of pediatric
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Fig. 3. Electron photomicrograph from postmortem liver specimen in Patient 3. Nucleus at left and fat droplet below to right. Mitochondrial are swollen. Cristea and occasional dense bodies are seen. (Magnification 9,100• .)
patients with AIHA. 6. 7 The occurrence of cold-agglutinin AIHA in association with an encephalopathy mimicking Reye syndrome in three patients over a six-month period of time seems more than coincidental. The clinical and laboratory findings of our three patients were consistent with Reye syndrome, and therapy proceeded upon that presumptive diagnosis. Each of the patients had a prodromal upper respiratory tract infection followed by apparent recovery. Pernicious vomiting and deranged mental state marked the onset of encephalopathy, which rapidly progressed from agitation and delirium to unresponsiveness and coma. The biochemical aberrations were characteristic of Reye syndrome? .9 Indirect hyperbilirubinemia, hemolysis, elevated cold agglutinins, and the presence of an antiglobulin-I cold antibody are not typical of Reye syndrome, however, and prompted us to perform liver biopsies and bone marrow aspirations on two of the patients. The histopathologic findings of the liver in Reye syndrome include diffuse fat infiltration of hepatocytes of variable severity, which may be "masked ''~ in hematoxylin-eosin-stained sections but is easily demonstrated with lipid stains. L.~-" The fat droplets are small, multiple in each hepatocyte, and displace the nucleus very little. They tend to be larger toward the portal areas than around central veins. Inflammation and cell necrosis may be present H but is minimal, ~ and there is also glycogen depletion and often mild canalicular bile stasis? Ultrastructural studies 1~ have revealed some addi-
tional and fairly characteristic changes consisting of severely swotlen mitochondria, often ameboid in shape, with flocculated matrix, preserved cristae, reduced or absent dense bodies, and sometimes containing vesicular structures? ' These mitochondrial changes appear to parallel the clinical severity of the disease? *. ~ Other electron microscropic findings are reduction of glycogen; proliferation, swelling, and flocculation of peroxisome~; proliferation of endoplasmic reticulum; and viral inclusions? 2 These findings have varied from one study to another, as have conclusions about their specificity. 9"1' It appears that the findings change rapidly with time and severity of the disease and require immediate and careful processing of the tissues for their demonstration. When the liver biopsy is done within 48 hours after the onset of neurologic deterioration, the microvesicular lipid pattern and the mitochondrial changes appear to be reliable criteria for the diagnosis?- x" Both have still to be interpreted in the light of the clinical findings, since microvesicular fatty infiltration is a common pattern of fatty liver in childhood, and mitochondrial changes can be produced with a variety of agents. 2, ,4 The liver biopsies in both of our patients were obtained within 48 hours. In both there was unmasking of hepatocyte lipid which in periportal areas had already formed lipid lakes. Except for the size of the lipid droplets the fight microscopy was very similar to the severe lesion of Reye syndrome, 9 with a lack of necrosis, inflammation, or fibrosis. Electron microscopy of the liver biopsy from
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Patient 1 did not show the characteristic mitochondrial changes listed above; the liver specimen from Patient 3 also showed nonspecific mitochondrial changes and glycogen depletion but none of the specific features. Although the histologic and ultrastructural changes in these two children are not typical for Reye syndrome, that diagnosis can probably not be ruled out on morphologic grounds alone. Other disease states producing encephalopathy and liver dysfunction resembling Reye syndrome, but with distinctly different liver histopathology, have been describedY -~Although several authors have suggested that Reye syndrome can be diagnosed with confidence by history and routine laboratory data without the necessity for confirmation of fatty degeneration of the liver, 16. 17our data confirm the impression of others that liver biopsy is necessary for accurate diagnosis. 3, is Percutaneous needle biopsy of the liver can be performed safely after correction of the coagulation defects with fresh frozen plasma or exchange transfusion. Our patients' clinical and laboratory findings were virtually identical to those of classic Reye syndrome, unlike other reported encephalopathies mimicking Reye syndrome. 3 To our knowledge, the association of a cold-antibody AIHA with Reye syndrome has not been reported previously; we believe that these patients represent a unique encephalopathic condition. Wintrobe et al TM state that in severe acute forms of hemolytic anemia, semicoma and shocklike prostration may develop, but a syndrome masquerading as Reye syndrome has never been previously reported. We were unable to determine the etiology of the cold agglutinins and hemolytic anemia in our patients. Mycoplasma titers remained constant in two patients and rose only slightly in one patient. Titers for influenza A and B, cytomegalovirus, Coxsackie, measles, and mumps viruses remained constant between acute and convalescent sera. Results of the monospot test were negative in all three patients, and none of them had posterior cervical adenopathy 6r an absolute lymphocytosis. Occult viral infections have been implicated in the pathogenesis of AIHA both experimentally and clinically. 2~ ~ Cold antibodies frequently develop in high concentrations in Mycoplasma pneumoniae infections but an associated hemolytic anemia is rare and usually occurs toward the end of the second week of illness. 2~AIHA has been described during the course of infectious mononucleosis, ~3 acute viral hepatitis, and herpes simplex infections?" Anemia usually developed during the convalescent phase of these illnesses and was attributed to cold agglutinins or to cold hemolysins. Associated severe granulocytopenia and thrombocytopenia were observed
The Journal of Pediatrics April 1979
in a few instances. Acquired hemolytic anemia has been associated with cytomegalovirus infections in children, but these are usually warm antibody-type AIHA, and most patients are immunologically compromised, s~ 2~ Hemolytic anemia occurring early in the course of an infection is rare and AIHA due to cold autoantibodies is uncommon in children. Cold-antibody disease is generally mild and occurs most frequently in elderly subjects. 1~ Zupanska et al 6 reported 44 children with AIHA and only two patients with acute AIHA had anti-I specific autoantibodies. Buchanan et al 2. reviewed the records of 22 children with AIHA and were unable to find elevated titers Of cold agglutinins in any of their patients. A simple screening test for the presence of high-titer cold agglutinins consists of rubbing a test tube of citrated blood on the freezer wall of a refrigerator. If coarse, floccular agglutination occurs and can be reversed by warming the test tube, the cold agglutinin titer is usually in excess of 1:64.... s and the antibody probably contributes to the hemolysisY9 This simple test was positive in our three patients. Most clinically important antibodies agglutinate erythrocytes up to 32~ 3~ 31 Barcroft and Edholm 32 showed that the subcutaneous temperature of the bared forearm readily fails to below 30~ if the forearm is exposed to air at cool room temperature (18.5~ Transient antibodyantigen interaction can therefore be expected to occur as blood circulates through areas of the body exposed to a cool environment. For this reason the use of controlled hypothermia as therapy for cerebral edema is contraindicated in patients with cold-agglutinin AIHA; it is also essential to use warmed donor blood for transfusion. 33 Exchange transfusion, plasma exchange, 3' and plasmapheresis 33 have been reported to be effective in reducing the level of autolog0us serum cold agglutinins and improving the clinical status of these patients. We thank Drs. Elizabeth M. Kurczynski and Morris W. Levinsohn for their advice and assistance in the management of these patients, and Ms. Rita Feran for editorial assistance. REFERENCES
1. Reye RDK, Morgan G, and Baral J: Encephalopathy and fatty degeneration of the viscera: a disease entity in childhood, Lancet 2:749, 1963. 2. Partin JC, Partin JS, and Schubert WK: Fatty liver in Reye's syndrome: is it a distinct morphologic entity? Gastroenterology 65:563, 1973. 3. Gall DG, Cutz E, McClung HJ, and Greenberg ML: Acute liver disease and encephalopathy mimicking Reye's syndrome, J PEDIATR87:869, 1975. 4. Truscott R, Pullin C, Halperin B, et al: Suspected defect in fatty acid oxidation, N Engl J Med 299:152, 1978. 5. Hayat MA: Principles and techniques of electron micros-
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Encephalopathy and fatty metamorphosis of liver in A I H A
copy, vol. I, New York, 1970, Van Nostrand Reinhold Company, p 161. 6. Zupanska B, Lawkowicz W, Gorska B, et al: Autoimmune hemolytic anemia in children, Br J Haematol 34:511, 1976. 7. Habibi B, Homberg JC, Schaison G, and Salmon C: Autoimmune hemolytic anemia in children. A review of 80 cases, Am J Med 56:61, 1974. 8. LovejoyFH, Smith AL, Bresnan MJ, et al: Clinical staging in Reye's syndrome, Am J DIS Chad 128:36, 1974. 9. BoveKE, McAdams AJ, Partin JC~ et al: The hepatic lesion in Reye's syndrome, Gastroenterology 69:685, 1975. 10. Partin JC, Schubert WK, and Partin JS: Mitochondrial ultrastructure in Reye's syndrome (encephalopathy and fatty degeneration of the viscera), N Engl J Med 285:1339, 1971. 11. Svoboda DJ, and Reddy JK: Pathology of the liver in 9 Reye's syndrome, Lab Invest 32:571, 1975. 12. Tang TT, Siegesmund KA, Sedmak GV; et al: Reye's syndrome. A correlated electronmicroscopic, viral and biochemical observation, JAMA 232:1339, 1975. 13. BradelEJ, and Reiner CB: The fine structure ofhepatocytes in Reye's syndrome, in Pollack JD editor: Reye's syndrome, New York, 1975, Grune & St~atton, Inc., p 147. 14. Iancu TC, Mason WH, and Neustein HB: Ultrastructural abnormalities of liver cells in Reye's syndrome, Hum Pathol 8:421, 1977. 15. Partin JC: Liver ultrastructure in Reye's syndrome, in Pollack JD, editor: Reye's syndrome, New York, 1975, Grune & Stratton, Inc., p 117. 16. Glasgow AM, Cotton RB, and Dhiensire K: Reye's syndrome. I. Blood ammonia and consideration of the nonhistologic diagnostic, Am J Dis Child 124:827, 1972. 17. Samaha FJ, Glau E, and Berardinelli JL: Reye's syndrome: Clinical diagnosis and treatment with peritoneal dialysis, Pediatrics 53:336, 1974. 18. Schubert WK: Commentary: The diagnosis of Reye syndrome, J PEDIATg87:867, 1975. 19. Wintrobe MM, Lee GR, Boggs DR, et ah Immunohemolytic anemias, in Clinical hemaotology, ed 7, Philadelphia, 1974, Lea & Febiger, Chapter 27.
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20. Zuelzer WW, Mastrangelo R, Stulberg CS,.et al: Autoimmune hemolytic anemia: natural history and viral-immunologic interactions in children, Am J Med 49:80, i970. 21. Murdock JMcC, and Smith CC: Haemolytic anemias, Clin Haematol 1:619, 1972. 22. Neely FL, Baria WH, Smith C, and Stone CF Jr: Primary atypical pneumonia with high titer cold hemaglutinins, hemolytic anemia, and false positive Donath-Landsteiner test, J Lab Clin Med 37:382, 1951. 23. Mengel CE, Wallace AG, and McDaniel HG:. Infectious mononucleosis, hemolysis and megaloblastic arrest, Arch Intern Med 114:333, 1964. 24. Finland M, Peterson OL, Allen HE, et al: Cold agglutinins: occurrence of cold isohemagglutinins in various conditions, J Clin Invest 24:451, 1945. 25. Zuelzer WW, Stulberg CS, Page RH, et al: Etiology and pathogenesis of acquired hemolytic anemia, Transfusion 6:438, 1966. 26. Buchanan GR, Boxer LA, and Nathan DG: The acute and transient nature of idiopathic immune hemolytic anemia in children, J P~DL~TR88:780, 1976. 27. CosteaN: The differential diagnosis of hemolytic anemias, Med Clin North Am 57:289, 1972. 28. C-arrowDH: A rapid test for the presence of increased cold agglutinins, Br Med J 2:206, 1958. 29. Jacobson LB, Longstreth GF, and Edginton TS: Clinical and immunologic features of transient cold agglutinin hemolytic anemia, Am J Med 54:514, 1973. 30. Pirofsky B: Clinical aspects of autoimmune hemolytic anemia, Semin Hematol 13:251, 1976. 31. Dacie JV: Autoimmune hemolytic anaemias, Br Med J 2:381, 1970. 32. BarcroflH, and Edholm O~.. Temperature and blood flow in the human forearm, J Physiol 104:366, 1946. 33. Roseniield RE, and Jagathambal J: Transfusion therapy for autoimmune hemoltyic anemia, Semin Hematol 13:311, 1976. 34. Taft EG, Propp RP, and Sullivan SA: Plasma exchange for cold agglutinin hemolytic anemia, Transfusion 17:173, 1977.