COAGULATION AND TRANSFUSION MEDICINE Single Case Report
Cold Agglutinin Disease After Chicken Pox An Uncommon Complication of a Common Disease HENRY D. FRIEDMAN, M.D.,1 AND ROBERT A. DRACKER, M.D.1'2
fifth reported case of autoimmune hemolytic anemia and the second reported case of cold agglutinin disease with anti-Pr specificity to be associated with chicken pox. (Key words: Chicken pox; Varicella; Autoimmune hemolytic anemia; Cold agglutinin disease; Anti-Pr antibody; Erythrophagocytosis) Am J Clin Pathol 1992;97:92-96
Common infections may be associated with autoimmune hemolytic anemia (AIHA), especially in children. Although the hemolytic antibody may be either warm or cold reactive, the cold-reactive type is more common. Two types of cold-reactive hemolytic antibodies occur: those that only lyse erythrocytes and those that lyse and agglutinate erythrocytes. Most pure cold-reactive hemolysins are Donath-Landsteiner antibodies. These are of immunoglobulin (Ig) G class, specific for the P antigen, and exhibit biphasic hemolysis. They sometimes cause an explosive hemolysis that is referred to as paroxysmal cold hemoglobinuria. Historically, most cases of paroxysmal cold hemoglobinuria have been associated with chronic syphilitic infection.' Today it is a relatively common form of childhood AIHA and accompanies a variety of nonsyphilitic infections and vaccinations.' Cold agglutinins (CA) are typically of the IgM class. Those associated with infection are usually polyclonal. Immunoglobulin G and A class CAs are uncommon but also occur.2 Immunoglobulin M is a powerful complement-fixing and -activating class of immunoglobulin and causes intravascular hemolysis by classical complement activation. Most CAs react with the polysaccharide antigen "I" that is present on all adult erythrocytes. The next
most common CA specificity is directed against the i antigen that is strongly expressed on fetal red blood cells and belongs to the same antigenic family as I. Less common target antigens are Pr and Gd. Most infection-associated cold agglutinin disease (CAD) is transient. The association with certain infectious agents is particularly strong and these tend to elicit CAs with a particular specificity. Mycoplasma pneumoniae is the most commonly associated microbe and elicits anti-I CAs. Epstein-Barr viral disease is associated with anti-i CAs. Although less common, CAD has been observed in a variety of other viral, bacterial, and protozoal disease.3 We present a case of CAD that was associated with chicken pox. As far as we are aware, this is the fifth reported case of AIHA associated with chicken pox. CASE REPORT Preadmission History
An 8-year-old boy was referred to our institution for suspected hemolytic anemia. He had been in his usual state of good health until 10 days before admission when he developed chicken pox. This illness was worst 7 or 8 days before admission, with disseminated pox, fever, catarrh, and general discomfort. His only medication was aspirin. The patient had been feeling well for approximately 3 days and his pox were healing when he became pale, jaundiced, lethargic, and thirsty within a period of 24 hours. His pediatrician measured a hematocrit value of 0.19 and referred the patient to our facility for further evaluation. 1 2 From the Departments of Pathology and Pediatrics, State University of New York-Health Science Center at Syracuse. Syracuse, New York.
Physical Examination
Received February 25, 1991; received revised manuscript and accepted for publication May 21, 1991. Address reprint requests to Dr. Friedman: Department of Pathology, SUNY Health Science Center; 750 East Adams Street, Syracuse, New York 13210.
When he came to our institution, the patient was pale and jaundiced. His oral temperature was 38.1 °C, with a pulse of 150 bpm. Blood pressure, respiration frequency, and auscultation of lungs and heart were unremarkable. There was no hepatosplenomegaly or peripheral lymph-
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Chicken pox, although common, is rarely associated with autoimmune hemolytic anemia. Reported here is the case of an 8year-old boy who was found to have cold agglutinin disease and severe anemia several days after he contracted chicken pox. The cold agglutinin appeared to be a polyclonal immunoglobulin M antibody with anti-Pr specificity. To our knowledge, this is the
FRIEDMAN ET AL. Cold Agglutinin Disease After Chicken Pox adenopathy. He had no complaints of nausea, vomiting, or diarrhea and was not of Mediterranean descent. His initial hemoglobin level was 42 g/L, with a hematocrit value of 0.12 and a relative reticulocyte count of 30 X 10"3 (calculated reticulocyte production index was about 0.3; the normal value in a nonanemic state is 1.0). The white blood cell count was 26 X 109/L, with 85% neutrophils, 11% lymphocytes, 2% monocytes, and 2% bands. The peripheral blood film was made from an anticoagulated blood sample (ethylenediaminetetraacetic acid) that was incubated for approximately 60 minutes at room temperature. It was remarkable for erythrocyte agglutination and stunning erythrophagocytosis by 9% of the segmented neutrophils (Fig. 1). The erythrocytes were normocytic; anisocytosis and spherocytosis were minimal. The serum lactate dehydrogenase value was 1,430 U/L and the bilirubin level was 120 Mmol/L (7.1 mg/dL), of which 80% was unconjugated. Urine was brown tinged and contained 4+ protein and hemoglobin. Liver enzymes were normal and neither heterophil nor antinuclear antibodies were detected.
Blood group testing was performed by accepted tube test techniques.4 The direct and indirect antiglobulin tests were performed using polyspecific anti-IgG and anti-C3b/ C3d complement anti-human globulin reagent (OrthoDiagnostic Systems, Raritan, NJ), according to accepted blood banking procedures.4 The antibody detection test was performed using albumin (two drops of 22% bovine albumin [OrthoDiagnostic Systems]) with incubation at 37 °C followed by the indirect antiglobulin test.4 The CA specificity was determined using a commercial reagent red blood cell panel (OrthoDiagnostic Systems) and cord
FIG. 1. Photomicrograph of the peripheral blood smear at admission. This film was made within 60 minutes of the blood draw; ethylene diaminetetracetic acid as the anticoagulant and the specimen was left at room temperature. There is striking erythrophagocytosis by neutrophilic granulocytes; one neutrophil contains three engulfed erythrocytes (arrow). Note the erythrocyte agglutination and the rosette-like pattern with which red blood cells surround neutrophils. (Wright Giemsa; 870x; reproduced with permission from the American Medical Association. Friedman HD, Dracker RA. Intravascular erythrophagocytosis. JAMA 1991 ;265:1082).
erythrocytes. The serum/test cell mixture was incubated for 30 minutes at room temperature, centrifuged, and observed for agglutination and hemolysis. Positive reactions were graded using a standard protocol.4 After the addition of two drops of 22% bovine albumin, the reaction mixture was incubated for 30 minutes at 37 °C. After centrifugation and grading for agglutination and hemolysis, the specimen was tested in the "Coombs' phase" (by washing the serum/test cell mixture three times with normal saline and adding two drops of polyspecific antihuman-globulin reagent, centrifuging, and grading reactions). The CA titer was determined using fresh adult 0/Rh 0 (D) positive red cells. Two drops of a 3% red blood cell saline suspension were washed with normal saline. After centrifugation, the saline was decanted and the tubes were blotted to yield a dry cell button. The cells were resuspended in two drops of serum or serum/saline dilution, incubated for 18 hours at 4 °C, centrifuged, and read at 4 °C. Two-step protease (ficin [Organon Teknika Corp., Durham, NC] and papain [Sigma Chemical Co., St. Louis, MO]) and sialidase (neuraminidase from Vibrio cholera source, Calibiochem, San Diego, CA) enzyme treatments were performed on 0.3-mL aliquots of fresh packed O/ Rh 0 (D) positive adult red blood cells by the method of Judd. 5 A method for the detection of low-titer anti-Pr agglutinins6 was used in an adapted form to evaluate target antigen sensitivity to sialidase. Briefly, equal volumes of packed sialidase-treated adult 0/Rh 0 (D) positive erythrocytes and patient serum were mixed and incubated for 1 hour at 0 °C to absorb out anti-T antibodies and CA (if its antigenic target is sialidase resistant). The mixture was centrifuged in the cold and the supernatant serum was tested for the presence of residual agglutinating antibody, as previously described. Biphasic hemolysis was evaluated with and without addition of complement (in the form of fresh serum from a healthy volunteer) according to the method of Judd. 5 Serum was treated with the reducing agent dithiothreitol according to the method of Judd 5 and subsequently tested for (retained) agglutinating ability at 4 °C, as previously described. Serum immunonxation electrophoresis was performed on both untreated serum and eluted agglutinin. The elution procedure was as follows: 0.5 mL of packed washed fresh adult 0/Rh 0 (D) positive erythrocytes were mixed with 0.5 mL of untreated patient serum. The mixture was incubated for 5 hours at 4 °C; the red blood cells were washed twice with saline cooled to 4 °C. The washed cells were resuspended in 0.5 mL saline and incubated for 4 hours at 37 °C, with periodic agitation. The mixture was centrifuged and the supernatant was immediately recovered for analysis. The serum immunonxation electrophoresis procedure used was a kit that employed antisera directed against IgG, IgA, IgM, kappa, and lambda (Paragon IFE, Beckman, Brea, CA).
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Immunohematologic
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TABLE 1. COLD AGGLUTININ TITER AND TARGET ANTIGEN ENZYME SUSCEPTIBILITY Cold Agglutinin Test RBC
Preabsorption
Untreated Sialidase treated Ficin treated Papain treated
32 128 0 1
Titer Postabsorption*
* Absorption was performed using an equal volume of sialidase-treated adult erythrocytes. Table adapted from NorthofT et al.9 RBC = red blood cell.
absolute Lymphocytes
20
30
40
50
60
immun* globulin
Day of Illness FIG. 2. Clinical summary and medical treatment. The first day of hospitalization is counted as the first day of illness. Leukocyte and reticulocyte counts are in 109/L.
untreated serum and a predominance of polyclonal IgM in the eluted antibody. Hospital Treatment The clinical presentation and laboratory findings strongly suggested cold agglutinin disease with recent acute hemolysis. To prevent further hemolysis, the patient was kept warm. Because of tachycardia and diaphoresis, oxygen was administered and one unit of red blood cells was transfused. In addition, the patient received intravenous steroids (methylprednisolone, 50 increased to 100 mg every 8 hours) and two courses of immune globulin (1 g/Kg) (Fig. 2). The low reticulocyte count prompted a bone marrow examination to rule out erythroid aplasia. The marrow was normocellular, with active erythropoiesis, complete maturation, and a normal erythroid/myeloid ratio (1.0/1.3). The post-transfusion hematocrit value of 0.24 gradually decreased during the next 24 hours and the patient remained reticulocytopenic. The hematocrit level stabilized at 0.15 on the third hospital day and was followed by a gradual increase in reticulocyte count. The patient was discharged to the care of his primary physician on the fourth hospital day.
A.J.C.P.-January 1992
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The patient was typed as group 0/Rh 0 (D) negative. The direct antiglobulin (Coombs') test was positive with antisera specific for the C3b and C3d complement components, but negative with antisera specific for IgG. Patient serum agglutinated the patient's own erythrocytes and all panel cells tested, including adult I and cord i positive cells. This agglutination was uniformly weak at room temperature and absent at 37 °C. At 4 °C, the antibody agglutinated group 0/Rh 0 (D) positive test erythrocytes to a titer of 32. We think that antibody loss due to improper specimen handling accounted for this relatively low titer (i.e., the whole blood sample used for this study was refrigerated for 7 hours before serum separation, allowing the CA to absorb onto the patient's erythrocytes). The proteases ficin and papain significantly damaged the target antigen (Table 1, rows 3 and 4). The target antigen also appeared to be susceptible to sialidase. Testing of sialidase susceptibility is complicated by the fact that this enzyme not only destroys the sialic groups of erythrocytic sialoproteins but also enzymatically exposes an antigen substance called the T antigen.6 Our patient, like most individuals, had strong, naturally occurring anti-T CAs (Table 1, row 2, column 1), which strongly agglutinate sialidase-treated erythrocytes and interfere with the interpretation of antigen susceptibility testing, especially when the CA titer is low. The anti-T antibodies therefore were absorbed out of the patient's serum by an excess of sialidase-treated adult erythrocytes (Table 1, row 2, column 2). Persistence of the CA at a titer of 8, after absorption (Table 1, row 1, column 2), suggests that the target antigen was sialidase sensitive6 {i.e., the CA was not adsorbed onto the sialidase-treated erythrocytes because its antigenic target was destroyed). The agglutinating ability of the antibody was lost after treatment with the reducing agent dithiothreitol. No biphasic (Donath-Landsteiner) hemolysin was present. Immunofixation electrophoresis showed polyclonal immunoglobulin in the
WBC absolute Neutrophils
FRIEDMAN ET AL. Cold Agglutinin Disease After Chicken Pox Follow-up
Examinations
The reticulocytes peaked at 860 X 109/L (reticulocyte production index of 9.6) 5 days after discharge, when the hematocrit value was 0.29. The steroid therapy was reduced gradually, so that the last dose of methylprednisolone was administered on the 59th day after hospital admission. DISCUSSION
(one was the patient reported here) received treatment in the form of either ACTH 10 or glucocorticosteroids. 9 " In all five cases, the hemolytic anemia was transient. Because CAD usually occurs during the late acute or convalescent phase of infection,7 the temporal association was stronger in the first three cases listed on Table 2. Northoif and co-workers9 reported a West German woman with CAD that occurred during the acute phase of disease. The CA was monophenotypic IgG-kappa and also was directed against the Pr antigen. Borbolla and associates10 described a Cuban boy with Coombs' positive autoimmune hemolytic anemia that also occurred in the convalescent period. The association with chicken pox appears less strong in the last two cases of Table 2. Parashar and colleagues1' reported an Indian boy who developed persistent fever with progressive jaundice 1 month after having chicken pox. After 1 month of these symptoms, he was admitted with severe Coombs' positive autoimmune hemolytic anemia and reticulocytosis. The fact that this secondary illness began at least 1 month after chicken pox raises the question of whether another infection had been contracted. Although the patient was stated to have had both cold and warm active antibodies, the specificity of the antiglobulin reagent used in testing was not stated. Therefore, the positive antiglobulin reaction at 37 °C may have been due to C3b/C3d-complement-coated red blood cells rather than to a warm-reactive antibody. Kaiser and associates8 reported the case of an American boy with paroxysmal cold hemoglobinuria whose hemolytic crisis occurred after several days of cold-like symptoms. On the sixth hospital day, the patient developed the classic exanthema of chicken pox. Because hemolysis occurred at a time in the incubation period of chicken pox, before seroconversion or detectable viremia,12 a second infection may have elicited the AIHA. The Pr antigen is found on all human and many mammalian erythrocytes, leukocytes, platelets, as well as on certain somatic cells. As a sialoprotein, the Pr antigen is digestible by both sialidase and protease.6 Although antiPr CAs are somewhat unusual, the Pr antigen is the next
TABLE 2. CHICKEN POX WITH ASSOCIATED AUTOIMMUNE HEMOLYTIC ANEMIA Age (years) 1sex 43/F 8/M 6/M 14/M 2.5/M
Onset of Hemolysis
i,Hb 9/L
"Several days"
111 42 30* 40 24*
+7 + 13 +30 -6
Cold Agglutinin
+ +
(+)t -
jHb = lowest measured hemoglobin; Onset of hemolysis = days after the appearance of the exanthema, negative signifies a time before the exanthema. * Extrapolated from original measurements by relative colorimetry.
Antibody/specificity IgG-kappa/Pr IgM/Pr "Complete antibody" "Warm and cold reactive" IgG/P*
Author Northoff9 Current case Borbolla10 Parashar1' Kaiser8
t Very low titer that may have represented a benign naturally occurring anti-1 cold agglutinin. X Donath-Landsteiner antibody.
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Our patient first became pallid during convalescence, 9 days after the appearance of the exanthema of chicken pox. This temporal association of the CAD with chicken pox strongly suggests a causal relationship.7 We surmise that exposure to the cold, before and during the patient's trip to our facility, may have exacerbated the hemolysis. The initial loss of CA, due to improper specimen handling (see Immunohematologic Findings above), hampered the CA analysis. The fact that our patient's serum agglutinated all test cells examined indicates a specificity to a high-incidence antigen. The equally strong agglutination of adult 1+ and cord i+ test erythrocytes and susceptibility of the CA target antigen to protease digestion rules out I/i and Gd specificity.2 The presence of the CA in the serum after adsorption by sialidase-treated red blood cells suggests that the antibody is directed against a sialidase-sensitive antigen. These three findings, (1) panagglutination, (2) sensitivity to protease, and (3) sensitivity to sialidase, suggest that this CA was specific for the Pr antigen.2 The low temperature range of the agglutinin, its sensitivity to dithiothreitol (a reducing agent capable of dissociating the IgM pentamer into its multimeric units), and the immunofixation electrophoresis indicate that the antibody was a polyclonal IgM-type antibody. Although chicken pox is common, we are aware of only four other cases with associated AIHA (Table 2). The predominant autoimmune antibody was cold reactive in all five cases. A Donath-Landsteiner antibody was present in one of the four cases tested for biphasic hemolysis.8 A CA was responsible for two of the five (one was the case reported here) hemolytic anemias.9 Four of the patients
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COAGULATION AND TRANSFUSION MEDICINE Single Case Report extensive erythrophagocytosis in a routinely processed peripheral blood sample is unusual and knowledge of its significance may be important for clinical management. Acknowledgments. The authors thank Ronald L. Dubowy, M.D., and the pediatric department for the excellent care of their patient; Karla J. Lauenstein, M.S., MT(ASCP), SBB, Douglas A. Nelson, M.D., W. Laurence Marsh, Ph.D., Matthew R. Pincus, M.D., Ph.D., and Frederick R. Davey, M.D., for their invaluable suggestions; and Linda DeHority and Harry Ludke for their technical help.
REFERENCES 1. Warren RW, Collins ML. Immune hemolytic anemia in children. CRC Crit Rev Oncol Hematol 1988;8:65-73. 2. Roelcke D. Cold agglutination. Transfusion Medicine Reviews 1989;3:140-166. 3. GieBler RG, Kobberling J. Kalteagglutininkrankheit. Klin Wochenschr 1988;66:277-283. 4. Walker RH, Hoppe PA, Judd WJ, et al. Technical Manual, 10th Edition. Arlington, VA: American Association of Blood Banks, 1990. 5. Judd JW. Methods in Immunohematology. Miami, FL: Montgomery Scientific Publications, 1988. 6. Roelcke D, Ebert W, Anstee DJ. Demonstration of low-titer antiPr cold agglutinins. Vox Sang 1974;27:429-441. 7. Dacie JV. The Haemolytic Anaemias—Part II: The Auto-immune Haemolytic Anaemias. New York: Grune and Stratton, 1962, pp 526-540. 8. Kaiser AD, Bradford WL. Severe hemoglobinuria in a child occurring in the prodromal stage of chickenpox. Arch Pediatr 1929,46:571577. 9. Northoff H, Martin A, Roelcke D. An IgG kappa-monotypic antiPr,h associated with fresh varicella infection. Eur J Haematol 1987;38:85-88. 10. Borbolla L, Barquet Chediak A. Anemia hemolitica de etiologia viral revision de la literature: Presentacion de un caso en la convalescencia de una varicela con Coombs positivo. Rev Cubana Pediat 1953;25:557-564. 11. Parashar S, Bhandary S, Menezes S, et al. Auto immune haemolytic anaemia. J Assoc Physicians India 1981;29:679-682. 12. Asano Y, Itakura N, Hiroishi Y, et al. Viral replication and immunologic responses in children naturally infected with varicellazoster virus and in varicella vaccine recipients. J Infect Dis 1985;152:863-868. 13. Liesveld JL, Rowe JM, Lichtman MA. Variability of the erythropoietic response in autoimmune hemolytic anemia: Analysis of 109 cases. Blood 1987;69:820-826. 14. Hauke G, Fauser AA, Weber S, Mass D. Reticulocytopenia in severe autoimmune hemolytic anemia of the warm antibody type. Blut 1983;46:321-327. 15. Conley CL, Lippman SM, Ness PM, et al. Autoimmune hemolytic anemia with reticulocytopenia and erythroid marrow. N Engl J Med 1982;306:281-286. 16. Zinkham WH, Diamond LK. In vitro erythrophagocytosis in acquired hemolytic anemia. Blood 1952;7:592-601. 17. Friedman HD, Dracker RS. Intravascular erythrophagocytosis. JAMA 1991;265:1082. 18. Friedman HD, Dracker RS. Intravascular erythrophagocytosis (reply to letter to the editor by SL Lee). JAMA 1991;266:217-218.
A.J.C.P.-January 1992
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most common CA specificity, after the I/i system.2 The immunoglobulin classes of anti-Pr CAs resemble those of anti-I/i CAs, with the exception that virtually all IgA class CAs have anti-Pr specificity.2 As a postinfectious complication, anti-Pr CAD has been associated with rubella2 and varicella.9 Low reticulocyte counts are not uncommon at the time of diagnosis of AIHA.13 Bone marrow examination usually shows normal to increased erythroid precursors. In most cases, the reticulocytopenia is transient and chiefly due to a lag in reticulocyte production. Other cases appear to be due to inadequate erythropoiesis.13'4 The transient reticulocytopenia in this case probably is the result of substantial reticulocyte loss through indiscriminate hemolysis of young and mature erythrocytes and a lag in reticulocyte production; preferential destruction of reticulocytes also may have played a role, although this is speculation.15 The erythrocyte agglutination, and most of the erythrophagocytosis and erythrocyte-rosette formation around leukocytes (Fig. 1), probably occurred while the anticoagulated blood sample stood and cooled, during a 60minute interval between the blood draw and processing. In a standing tube of blood, the leukocyte and opsonizederythrocyte have much more opportunity to interact. Zinkham and Diamond16 described this effect in four children with Coombs' positive AIHA and few erythrophagocytes on direct blood films. By incubating anticoagulated blood for 1 hour at 37 °C, they significantly augmented the amount of erythrophagocytosis in three of the four cases; in one case, more than 80% of the peripheral leukocytes contained phagocytized erythrocytes. The circumstance surrounding our observation differed from that of the previously mentioned study in two possibly important aspects: (1) our patient was examined after an apparent complement-mediated hemolytic crisis (indicated by a 7% decrease in hematocrit, hemoglobinuria, and the immunohematologic findings) and (2) our blood sample cooled to room temperature (at which leukocytes are less active). Therefore we speculate that some erythrophagocytosis may have occurred in vivo (i.e., intravascular). Intravascular erythrophagocytosis has been described in conditions and diseases that favor opsonization of erythrocytes. In most of these cases, only rare erythrophagocytic leukocytes are present on direct blood films1718; acute complement-mediated hemolysis (as in incompatible blood transfusions) may be an exception. In any event,
Cold Agglutinin Disease After Chicken Pox An Uncommon Complication of a Common Disease HENRY D. FRIEDMAN, M.D.,1 AND ROBERT A. DRACKER, M.D.1'2
fifth reported case of autoimmune hemolytic anemia and the second reported case of cold agglutinin disease with anti-Pr specificity to be associated with chicken pox. (Key words: Chicken pox; Varicella; Autoimmune hemolytic anemia; Cold agglutinin disease; Anti-Pr antibody; Erythrophagocytosis) Am J Clin Pathol 1992;97:92-96
Common infections may be associated with autoimmune hemolytic anemia (AIHA), especially in children. Although the hemolytic antibody may be either warm or cold reactive, the cold-reactive type is more common. Two types of cold-reactive hemolytic antibodies occur: those that only lyse erythrocytes and those that lyse and agglutinate erythrocytes. Most pure cold-reactive hemolysins are Donath-Landsteiner antibodies. These are of immunoglobulin (Ig) G class, specific for the P antigen, and exhibit biphasic hemolysis. They sometimes cause an explosive hemolysis that is referred to as paroxysmal cold hemoglobinuria. Historically, most cases of paroxysmal cold hemoglobinuria have been associated with chronic syphilitic infection.' Today it is a relatively common form of childhood AIHA and accompanies a variety of nonsyphilitic infections and vaccinations.' Cold agglutinins (CA) are typically of the IgM class. Those associated with infection are usually polyclonal. Immunoglobulin G and A class CAs are uncommon but also occur.2 Immunoglobulin M is a powerful complement-fixing and -activating class of immunoglobulin and causes intravascular hemolysis by classical complement activation. Most CAs react with the polysaccharide antigen "I" that is present on all adult erythrocytes. The next
most common CA specificity is directed against the i antigen that is strongly expressed on fetal red blood cells and belongs to the same antigenic family as I. Less common target antigens are Pr and Gd. Most infection-associated cold agglutinin disease (CAD) is transient. The association with certain infectious agents is particularly strong and these tend to elicit CAs with a particular specificity. Mycoplasma pneumoniae is the most commonly associated microbe and elicits anti-I CAs. Epstein-Barr viral disease is associated with anti-i CAs. Although less common, CAD has been observed in a variety of other viral, bacterial, and protozoal disease.3 We present a case of CAD that was associated with chicken pox. As far as we are aware, this is the fifth reported case of AIHA associated with chicken pox. CASE REPORT Preadmission History
An 8-year-old boy was referred to our institution for suspected hemolytic anemia. He had been in his usual state of good health until 10 days before admission when he developed chicken pox. This illness was worst 7 or 8 days before admission, with disseminated pox, fever, catarrh, and general discomfort. His only medication was aspirin. The patient had been feeling well for approximately 3 days and his pox were healing when he became pale, jaundiced, lethargic, and thirsty within a period of 24 hours. His pediatrician measured a hematocrit value of 0.19 and referred the patient to our facility for further evaluation. 1 2 From the Departments of Pathology and Pediatrics, State University of New York-Health Science Center at Syracuse. Syracuse, New York.
Physical Examination
Received February 25, 1991; received revised manuscript and accepted for publication May 21, 1991. Address reprint requests to Dr. Friedman: Department of Pathology, SUNY Health Science Center; 750 East Adams Street, Syracuse, New York 13210.
When he came to our institution, the patient was pale and jaundiced. His oral temperature was 38.1 °C, with a pulse of 150 bpm. Blood pressure, respiration frequency, and auscultation of lungs and heart were unremarkable. There was no hepatosplenomegaly or peripheral lymph-
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Chicken pox, although common, is rarely associated with autoimmune hemolytic anemia. Reported here is the case of an 8year-old boy who was found to have cold agglutinin disease and severe anemia several days after he contracted chicken pox. The cold agglutinin appeared to be a polyclonal immunoglobulin M antibody with anti-Pr specificity. To our knowledge, this is the
FRIEDMAN ET AL. Cold Agglutinin Disease After Chicken Pox adenopathy. He had no complaints of nausea, vomiting, or diarrhea and was not of Mediterranean descent. His initial hemoglobin level was 42 g/L, with a hematocrit value of 0.12 and a relative reticulocyte count of 30 X 10"3 (calculated reticulocyte production index was about 0.3; the normal value in a nonanemic state is 1.0). The white blood cell count was 26 X 109/L, with 85% neutrophils, 11% lymphocytes, 2% monocytes, and 2% bands. The peripheral blood film was made from an anticoagulated blood sample (ethylenediaminetetraacetic acid) that was incubated for approximately 60 minutes at room temperature. It was remarkable for erythrocyte agglutination and stunning erythrophagocytosis by 9% of the segmented neutrophils (Fig. 1). The erythrocytes were normocytic; anisocytosis and spherocytosis were minimal. The serum lactate dehydrogenase value was 1,430 U/L and the bilirubin level was 120 Mmol/L (7.1 mg/dL), of which 80% was unconjugated. Urine was brown tinged and contained 4+ protein and hemoglobin. Liver enzymes were normal and neither heterophil nor antinuclear antibodies were detected.
Blood group testing was performed by accepted tube test techniques.4 The direct and indirect antiglobulin tests were performed using polyspecific anti-IgG and anti-C3b/ C3d complement anti-human globulin reagent (OrthoDiagnostic Systems, Raritan, NJ), according to accepted blood banking procedures.4 The antibody detection test was performed using albumin (two drops of 22% bovine albumin [OrthoDiagnostic Systems]) with incubation at 37 °C followed by the indirect antiglobulin test.4 The CA specificity was determined using a commercial reagent red blood cell panel (OrthoDiagnostic Systems) and cord
FIG. 1. Photomicrograph of the peripheral blood smear at admission. This film was made within 60 minutes of the blood draw; ethylene diaminetetracetic acid as the anticoagulant and the specimen was left at room temperature. There is striking erythrophagocytosis by neutrophilic granulocytes; one neutrophil contains three engulfed erythrocytes (arrow). Note the erythrocyte agglutination and the rosette-like pattern with which red blood cells surround neutrophils. (Wright Giemsa; 870x; reproduced with permission from the American Medical Association. Friedman HD, Dracker RA. Intravascular erythrophagocytosis. JAMA 1991 ;265:1082).
erythrocytes. The serum/test cell mixture was incubated for 30 minutes at room temperature, centrifuged, and observed for agglutination and hemolysis. Positive reactions were graded using a standard protocol.4 After the addition of two drops of 22% bovine albumin, the reaction mixture was incubated for 30 minutes at 37 °C. After centrifugation and grading for agglutination and hemolysis, the specimen was tested in the "Coombs' phase" (by washing the serum/test cell mixture three times with normal saline and adding two drops of polyspecific antihuman-globulin reagent, centrifuging, and grading reactions). The CA titer was determined using fresh adult 0/Rh 0 (D) positive red cells. Two drops of a 3% red blood cell saline suspension were washed with normal saline. After centrifugation, the saline was decanted and the tubes were blotted to yield a dry cell button. The cells were resuspended in two drops of serum or serum/saline dilution, incubated for 18 hours at 4 °C, centrifuged, and read at 4 °C. Two-step protease (ficin [Organon Teknika Corp., Durham, NC] and papain [Sigma Chemical Co., St. Louis, MO]) and sialidase (neuraminidase from Vibrio cholera source, Calibiochem, San Diego, CA) enzyme treatments were performed on 0.3-mL aliquots of fresh packed O/ Rh 0 (D) positive adult red blood cells by the method of Judd. 5 A method for the detection of low-titer anti-Pr agglutinins6 was used in an adapted form to evaluate target antigen sensitivity to sialidase. Briefly, equal volumes of packed sialidase-treated adult 0/Rh 0 (D) positive erythrocytes and patient serum were mixed and incubated for 1 hour at 0 °C to absorb out anti-T antibodies and CA (if its antigenic target is sialidase resistant). The mixture was centrifuged in the cold and the supernatant serum was tested for the presence of residual agglutinating antibody, as previously described. Biphasic hemolysis was evaluated with and without addition of complement (in the form of fresh serum from a healthy volunteer) according to the method of Judd. 5 Serum was treated with the reducing agent dithiothreitol according to the method of Judd 5 and subsequently tested for (retained) agglutinating ability at 4 °C, as previously described. Serum immunonxation electrophoresis was performed on both untreated serum and eluted agglutinin. The elution procedure was as follows: 0.5 mL of packed washed fresh adult 0/Rh 0 (D) positive erythrocytes were mixed with 0.5 mL of untreated patient serum. The mixture was incubated for 5 hours at 4 °C; the red blood cells were washed twice with saline cooled to 4 °C. The washed cells were resuspended in 0.5 mL saline and incubated for 4 hours at 37 °C, with periodic agitation. The mixture was centrifuged and the supernatant was immediately recovered for analysis. The serum immunonxation electrophoresis procedure used was a kit that employed antisera directed against IgG, IgA, IgM, kappa, and lambda (Paragon IFE, Beckman, Brea, CA).
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TABLE 1. COLD AGGLUTININ TITER AND TARGET ANTIGEN ENZYME SUSCEPTIBILITY Cold Agglutinin Test RBC
Preabsorption
Untreated Sialidase treated Ficin treated Papain treated
32 128 0 1
Titer Postabsorption*
* Absorption was performed using an equal volume of sialidase-treated adult erythrocytes. Table adapted from NorthofT et al.9 RBC = red blood cell.
absolute Lymphocytes
20
30
40
50
60
immun* globulin
Day of Illness FIG. 2. Clinical summary and medical treatment. The first day of hospitalization is counted as the first day of illness. Leukocyte and reticulocyte counts are in 109/L.
untreated serum and a predominance of polyclonal IgM in the eluted antibody. Hospital Treatment The clinical presentation and laboratory findings strongly suggested cold agglutinin disease with recent acute hemolysis. To prevent further hemolysis, the patient was kept warm. Because of tachycardia and diaphoresis, oxygen was administered and one unit of red blood cells was transfused. In addition, the patient received intravenous steroids (methylprednisolone, 50 increased to 100 mg every 8 hours) and two courses of immune globulin (1 g/Kg) (Fig. 2). The low reticulocyte count prompted a bone marrow examination to rule out erythroid aplasia. The marrow was normocellular, with active erythropoiesis, complete maturation, and a normal erythroid/myeloid ratio (1.0/1.3). The post-transfusion hematocrit value of 0.24 gradually decreased during the next 24 hours and the patient remained reticulocytopenic. The hematocrit level stabilized at 0.15 on the third hospital day and was followed by a gradual increase in reticulocyte count. The patient was discharged to the care of his primary physician on the fourth hospital day.
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The patient was typed as group 0/Rh 0 (D) negative. The direct antiglobulin (Coombs') test was positive with antisera specific for the C3b and C3d complement components, but negative with antisera specific for IgG. Patient serum agglutinated the patient's own erythrocytes and all panel cells tested, including adult I and cord i positive cells. This agglutination was uniformly weak at room temperature and absent at 37 °C. At 4 °C, the antibody agglutinated group 0/Rh 0 (D) positive test erythrocytes to a titer of 32. We think that antibody loss due to improper specimen handling accounted for this relatively low titer (i.e., the whole blood sample used for this study was refrigerated for 7 hours before serum separation, allowing the CA to absorb onto the patient's erythrocytes). The proteases ficin and papain significantly damaged the target antigen (Table 1, rows 3 and 4). The target antigen also appeared to be susceptible to sialidase. Testing of sialidase susceptibility is complicated by the fact that this enzyme not only destroys the sialic groups of erythrocytic sialoproteins but also enzymatically exposes an antigen substance called the T antigen.6 Our patient, like most individuals, had strong, naturally occurring anti-T CAs (Table 1, row 2, column 1), which strongly agglutinate sialidase-treated erythrocytes and interfere with the interpretation of antigen susceptibility testing, especially when the CA titer is low. The anti-T antibodies therefore were absorbed out of the patient's serum by an excess of sialidase-treated adult erythrocytes (Table 1, row 2, column 2). Persistence of the CA at a titer of 8, after absorption (Table 1, row 1, column 2), suggests that the target antigen was sialidase sensitive6 {i.e., the CA was not adsorbed onto the sialidase-treated erythrocytes because its antigenic target was destroyed). The agglutinating ability of the antibody was lost after treatment with the reducing agent dithiothreitol. No biphasic (Donath-Landsteiner) hemolysin was present. Immunofixation electrophoresis showed polyclonal immunoglobulin in the
WBC absolute Neutrophils
FRIEDMAN ET AL. Cold Agglutinin Disease After Chicken Pox Follow-up
Examinations
The reticulocytes peaked at 860 X 109/L (reticulocyte production index of 9.6) 5 days after discharge, when the hematocrit value was 0.29. The steroid therapy was reduced gradually, so that the last dose of methylprednisolone was administered on the 59th day after hospital admission. DISCUSSION
(one was the patient reported here) received treatment in the form of either ACTH 10 or glucocorticosteroids. 9 " In all five cases, the hemolytic anemia was transient. Because CAD usually occurs during the late acute or convalescent phase of infection,7 the temporal association was stronger in the first three cases listed on Table 2. Northoif and co-workers9 reported a West German woman with CAD that occurred during the acute phase of disease. The CA was monophenotypic IgG-kappa and also was directed against the Pr antigen. Borbolla and associates10 described a Cuban boy with Coombs' positive autoimmune hemolytic anemia that also occurred in the convalescent period. The association with chicken pox appears less strong in the last two cases of Table 2. Parashar and colleagues1' reported an Indian boy who developed persistent fever with progressive jaundice 1 month after having chicken pox. After 1 month of these symptoms, he was admitted with severe Coombs' positive autoimmune hemolytic anemia and reticulocytosis. The fact that this secondary illness began at least 1 month after chicken pox raises the question of whether another infection had been contracted. Although the patient was stated to have had both cold and warm active antibodies, the specificity of the antiglobulin reagent used in testing was not stated. Therefore, the positive antiglobulin reaction at 37 °C may have been due to C3b/C3d-complement-coated red blood cells rather than to a warm-reactive antibody. Kaiser and associates8 reported the case of an American boy with paroxysmal cold hemoglobinuria whose hemolytic crisis occurred after several days of cold-like symptoms. On the sixth hospital day, the patient developed the classic exanthema of chicken pox. Because hemolysis occurred at a time in the incubation period of chicken pox, before seroconversion or detectable viremia,12 a second infection may have elicited the AIHA. The Pr antigen is found on all human and many mammalian erythrocytes, leukocytes, platelets, as well as on certain somatic cells. As a sialoprotein, the Pr antigen is digestible by both sialidase and protease.6 Although antiPr CAs are somewhat unusual, the Pr antigen is the next
TABLE 2. CHICKEN POX WITH ASSOCIATED AUTOIMMUNE HEMOLYTIC ANEMIA Age (years) 1sex 43/F 8/M 6/M 14/M 2.5/M
Onset of Hemolysis
i,Hb 9/L
"Several days"
111 42 30* 40 24*
+7 + 13 +30 -6
Cold Agglutinin
+ +
(+)t -
jHb = lowest measured hemoglobin; Onset of hemolysis = days after the appearance of the exanthema, negative signifies a time before the exanthema. * Extrapolated from original measurements by relative colorimetry.
Antibody/specificity IgG-kappa/Pr IgM/Pr "Complete antibody" "Warm and cold reactive" IgG/P*
Author Northoff9 Current case Borbolla10 Parashar1' Kaiser8
t Very low titer that may have represented a benign naturally occurring anti-1 cold agglutinin. X Donath-Landsteiner antibody.
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Our patient first became pallid during convalescence, 9 days after the appearance of the exanthema of chicken pox. This temporal association of the CAD with chicken pox strongly suggests a causal relationship.7 We surmise that exposure to the cold, before and during the patient's trip to our facility, may have exacerbated the hemolysis. The initial loss of CA, due to improper specimen handling (see Immunohematologic Findings above), hampered the CA analysis. The fact that our patient's serum agglutinated all test cells examined indicates a specificity to a high-incidence antigen. The equally strong agglutination of adult 1+ and cord i+ test erythrocytes and susceptibility of the CA target antigen to protease digestion rules out I/i and Gd specificity.2 The presence of the CA in the serum after adsorption by sialidase-treated red blood cells suggests that the antibody is directed against a sialidase-sensitive antigen. These three findings, (1) panagglutination, (2) sensitivity to protease, and (3) sensitivity to sialidase, suggest that this CA was specific for the Pr antigen.2 The low temperature range of the agglutinin, its sensitivity to dithiothreitol (a reducing agent capable of dissociating the IgM pentamer into its multimeric units), and the immunofixation electrophoresis indicate that the antibody was a polyclonal IgM-type antibody. Although chicken pox is common, we are aware of only four other cases with associated AIHA (Table 2). The predominant autoimmune antibody was cold reactive in all five cases. A Donath-Landsteiner antibody was present in one of the four cases tested for biphasic hemolysis.8 A CA was responsible for two of the five (one was the case reported here) hemolytic anemias.9 Four of the patients
95
96
COAGULATION AND TRANSFUSION MEDICINE Single Case Report extensive erythrophagocytosis in a routinely processed peripheral blood sample is unusual and knowledge of its significance may be important for clinical management. Acknowledgments. The authors thank Ronald L. Dubowy, M.D., and the pediatric department for the excellent care of their patient; Karla J. Lauenstein, M.S., MT(ASCP), SBB, Douglas A. Nelson, M.D., W. Laurence Marsh, Ph.D., Matthew R. Pincus, M.D., Ph.D., and Frederick R. Davey, M.D., for their invaluable suggestions; and Linda DeHority and Harry Ludke for their technical help.
REFERENCES 1. Warren RW, Collins ML. Immune hemolytic anemia in children. CRC Crit Rev Oncol Hematol 1988;8:65-73. 2. Roelcke D. Cold agglutination. Transfusion Medicine Reviews 1989;3:140-166. 3. GieBler RG, Kobberling J. Kalteagglutininkrankheit. Klin Wochenschr 1988;66:277-283. 4. Walker RH, Hoppe PA, Judd WJ, et al. Technical Manual, 10th Edition. Arlington, VA: American Association of Blood Banks, 1990. 5. Judd JW. Methods in Immunohematology. Miami, FL: Montgomery Scientific Publications, 1988. 6. Roelcke D, Ebert W, Anstee DJ. Demonstration of low-titer antiPr cold agglutinins. Vox Sang 1974;27:429-441. 7. Dacie JV. The Haemolytic Anaemias—Part II: The Auto-immune Haemolytic Anaemias. New York: Grune and Stratton, 1962, pp 526-540. 8. Kaiser AD, Bradford WL. Severe hemoglobinuria in a child occurring in the prodromal stage of chickenpox. Arch Pediatr 1929,46:571577. 9. Northoff H, Martin A, Roelcke D. An IgG kappa-monotypic antiPr,h associated with fresh varicella infection. Eur J Haematol 1987;38:85-88. 10. Borbolla L, Barquet Chediak A. Anemia hemolitica de etiologia viral revision de la literature: Presentacion de un caso en la convalescencia de una varicela con Coombs positivo. Rev Cubana Pediat 1953;25:557-564. 11. Parashar S, Bhandary S, Menezes S, et al. Auto immune haemolytic anaemia. J Assoc Physicians India 1981;29:679-682. 12. Asano Y, Itakura N, Hiroishi Y, et al. Viral replication and immunologic responses in children naturally infected with varicellazoster virus and in varicella vaccine recipients. J Infect Dis 1985;152:863-868. 13. Liesveld JL, Rowe JM, Lichtman MA. Variability of the erythropoietic response in autoimmune hemolytic anemia: Analysis of 109 cases. Blood 1987;69:820-826. 14. Hauke G, Fauser AA, Weber S, Mass D. Reticulocytopenia in severe autoimmune hemolytic anemia of the warm antibody type. Blut 1983;46:321-327. 15. Conley CL, Lippman SM, Ness PM, et al. Autoimmune hemolytic anemia with reticulocytopenia and erythroid marrow. N Engl J Med 1982;306:281-286. 16. Zinkham WH, Diamond LK. In vitro erythrophagocytosis in acquired hemolytic anemia. Blood 1952;7:592-601. 17. Friedman HD, Dracker RS. Intravascular erythrophagocytosis. JAMA 1991;265:1082. 18. Friedman HD, Dracker RS. Intravascular erythrophagocytosis (reply to letter to the editor by SL Lee). JAMA 1991;266:217-218.
A.J.C.P.-January 1992
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most common CA specificity, after the I/i system.2 The immunoglobulin classes of anti-Pr CAs resemble those of anti-I/i CAs, with the exception that virtually all IgA class CAs have anti-Pr specificity.2 As a postinfectious complication, anti-Pr CAD has been associated with rubella2 and varicella.9 Low reticulocyte counts are not uncommon at the time of diagnosis of AIHA.13 Bone marrow examination usually shows normal to increased erythroid precursors. In most cases, the reticulocytopenia is transient and chiefly due to a lag in reticulocyte production. Other cases appear to be due to inadequate erythropoiesis.13'4 The transient reticulocytopenia in this case probably is the result of substantial reticulocyte loss through indiscriminate hemolysis of young and mature erythrocytes and a lag in reticulocyte production; preferential destruction of reticulocytes also may have played a role, although this is speculation.15 The erythrocyte agglutination, and most of the erythrophagocytosis and erythrocyte-rosette formation around leukocytes (Fig. 1), probably occurred while the anticoagulated blood sample stood and cooled, during a 60minute interval between the blood draw and processing. In a standing tube of blood, the leukocyte and opsonizederythrocyte have much more opportunity to interact. Zinkham and Diamond16 described this effect in four children with Coombs' positive AIHA and few erythrophagocytes on direct blood films. By incubating anticoagulated blood for 1 hour at 37 °C, they significantly augmented the amount of erythrophagocytosis in three of the four cases; in one case, more than 80% of the peripheral leukocytes contained phagocytized erythrocytes. The circumstance surrounding our observation differed from that of the previously mentioned study in two possibly important aspects: (1) our patient was examined after an apparent complement-mediated hemolytic crisis (indicated by a 7% decrease in hematocrit, hemoglobinuria, and the immunohematologic findings) and (2) our blood sample cooled to room temperature (at which leukocytes are less active). Therefore we speculate that some erythrophagocytosis may have occurred in vivo (i.e., intravascular). Intravascular erythrophagocytosis has been described in conditions and diseases that favor opsonization of erythrocytes. In most of these cases, only rare erythrophagocytic leukocytes are present on direct blood films1718; acute complement-mediated hemolysis (as in incompatible blood transfusions) may be an exception. In any event,
