SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Direct antiglobulin test (DAT)-negative (DAT-)autoimmune hemolytic anemia (AIHA) is empirically thought to show the same clinical conditions as DAT-positive (DAT+)AIHA, with the exception of an adequate amount of red blood cell (RBC)-bound immunoglobulin (Ig)G. We investigated the clinical characteristics of DAT−AIHA in comparison with DAT+AIHA. Of the 582 patients referred to our laboratory with undiagnosed hemolytic anemia, AIHA was clinically diagnosed in 216 patients (DAT−AIHA, n = 154; DAT+AIHA, n = 62). The percentage of reticulocytes, mean corpuscular volume, RBC-IgG levels, white blood cell count, and total protein (TP) levels were significantly higher in patients with DAT+AIHA than patients with DAT−AIHA. The hemoglobin level was significantly lower in patients with DAT+AIHA. No significant differences between patients with DAT−AIHA and DAT+AIHA existed with respect to age, gender, idiopathic/secondary nature, complications such as Evans syndrome, effectiveness of steroid treatment, or survival rate at 1 year following diagnosis. Patients with DAT−AIHA required significantly lower doses of steroids for maintenance therapy. Based on multivariate analysis of idiopathic DAT−AIHA (n = 110), TP and Evans syndrome were associated with the effectiveness of steroids (adjusted odds ratio [aOR], 1.36/[0.1 g/dl]; 95% confidence interval [CI], 1.01–1.84) and survival at the 1-year follow-up (aOR, 0.1; 95% CI, 0.01–0.88). Our results indicate that patients with DAT−AIHA generally suffer milder anemia and hemolysis than patients with DAT+AIHA, respond equally well to steroids, and have comparable survival at 1-year. Am. J. Hematol. 88:93–96, 2013. © 2012 Wiley Periodicals, Inc.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Steroid therapy is the first choice in treatment of patients with autoimmune hemolytic anemia (AIHA) with 80% effectiveness [1]. Without proper steroid therapy, patients with AIHA have a poor prognosis and a 31–53% mortality rate [2]. Since the 1950s, investigators have used steroids to treat AIHA and reported very high effectiveness through several case-series studies [3-5]. There are few case–control studies involving AIHA, which show higher evidence than case-series studies.

The detection of red blood cell-bound immunoglobulin G (RBC-IgG) and complement by a direct antiglobulin test (DAT) remains the main serologic assay in the diagnosis of AIHA [6]. Several methodologies have been investigated for the detection and evaluation of these autoantibodies. A DAT using the conventional tube technique (CTT) is the method most commonly used in blood centers and is still considered the gold standard [7]. A positive DAT almost always exists in association with AIHA [8] and forms the basis for the serologic diagnosis of AIHA [9]. However, it has also been shown that a negative DAT does not exclude the diagnosis of AIHA [9] and 1–10% of patients with AIHA have been reported to have a negative DAT [10-12]. These patients may carry a lower number of IgG molecules per RBC, yielding a negative tube DAT and in vivo hemolysis [13]. In fact, Petz and Garratty [14] reported that autoantibodies were detected in the eluates from 11 of 27 patients with acquired hemolytic anemia and a negative DAT, and concentrated elutes reacted with RBC of common Rh phenotypes, but did not react with Rh-null RBC. The immunoradiometric assay (IRMA) [15] for RBC-IgG is a representative method to quantitatively detect RBC-IgG. We previously reported [16] the value of RBG-IgG (33 ± 13) in 100 healthy Japanese adults and the RBC-IgG required for DAT-positivity (335 ± 72). We also proposed that the RBC-IgG level could be useful for the diagnosis of DAT-negative (DAT−) AIHA and that the cut-off value should be 78.5 if RBC-IgG is measured before treatment [16].

Whether or not the clinical entity of DAT-AIHA differs from DAT-positive (DAT+) AIHA in terms of differences in the underlying pathogenic mechanism, such as the IgG subclass of autoantibodies, is controversial [17, 18]. Analogous to the treatment of patients with DAT+AIHA, steroid therapy is empirically thought to be effective in DAT−AIHA on the basis of case-series studies, including 27 [19] and 9 cases [9].

Therefore, the aim of the study was to determine the characteristics of 154 DAT−AIHA in comparison with 62 DAT+AIHA and verify the effectiveness of steroid therapy in DAT−AIHA.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The study was conducted between 2003 and 2010 at the Laboratory of the Center for Community Medicine of Jichi Medical University in Tochigi, Japan after approval by the Institutional Ethics Panel Committee.

Patients

During the 8-year period, 605 samples from 582 patients throughout Japan were referred to our laboratory for quantification of RBC-IgG, based on the results of the DAT (mainly by the column method) in their laboratories. Of these samples, 78 (14%) were confirmed DAT-positive and 480 (86%) were DAT-negative in our laboratory, as shown by analysis of polyspecific DAT by CTT (Ortho Diagnostics, Rochester, NY) and monospecific DAT by CTT using anti-IgG and anti-C3d antibodies (Ortho Diagnostics), as per the manufacturer's instructions.

Sample preparation and immunoradiometric assay for RBC-IgG

Sample preparation and IRMA for RBC-IgG was performed according to a previous report [16]. Briefly, heparinized whole blood (10 ml) samples were collected. The RBC layer was prepared by centrifuging the whole blood at 1,000 rpm for 20 min. The supernatant plasma and buffy coat were discarded. One-milliliter samples of packed RBCs were diluted in 10 ml of phosphate-buffered saline (PBS; pH 7.0, 0.15 M). The diluted RBCs were passed through a cotton-wool column to exclude neutrophils and monocytes, according to the method by Jeje et al. [17]. The RBCs were washed four times with PBS, and the resulting RBCs (0.3 ml) were suspended in 0.4 ml of PBS.

The IRMA for RBC-IgG was performed according to the method by Jeje et al. [17], with some modifications. Samples of 125I-labeled goat anti-human IgG antisera derived (Du Pont, Wilmington, DE) were diluted in PBS containing 3% bovine serum albumin with a specific activity of 10,000 cpm/200 μl (Wakojunyaku, Osaka, Japan). A volume of 400 μl of the washed RBCs was incubated for 1 hr at 37°C with 200 μl of the diluted anti-human IgG. IgG beads were prepared using the methods described by Jeje et al. [17]. Human IgG and beads (Sephasorb™ HP) were purchased from Sigma Chemical (St. Louis, MO) and Pharmacia Fine Chemicals (Uppsala, Sweden), respectively. Two hundred microliter samples of IgG beads (2 × 106) were added to the mixture of RBCs and 125I-labeled anti-human IgG and incubated at 37°C for 30 min. The RBCs were lysed by the addition of 80 μl of 20% Triton X-100 (Sigma Chemical). The beads were washed four times with 20% Triton X-100-containing PBS, and the radioactivity was measured using a gamma counter (Aroka, Tokyo, Japan). A standard curve was generated using human IgG standards (10–10,000 ng IgG/ml; Sigma Chemical). The percent inhibition of binding was plotted against each concentration of IgG. Using the standard curve, RBC-IgG levels were calculated after counting the number of RBC. Each attending physician was informed of the RBC-IgG level within 3–10 days of ordering.

Clinical diagnosis and clinical course questionnaire

One year after referral to our laboratory, follow-up investigations were performed; the attending physician used a questionnaire to assess the clinical diagnosis and course. The bases for clinical diagnosis of AIHA were in vivo hemolysis (low hemoglobin [Hb] concentration, high percentage of reticulocyte (Retic%), high indirect serum bilirubin (IDBIL) level, high lactate dehydrogenase (LDH) level, low haptoglobin (Hp) level, and/or high erythropoiesis level in bone marrow), and exclusion of other anemic icteric diseases without hemolysis, such as megaloblastic anemia, myelodysplastic syndrome, erythroid leukemia, congenital dyserythropoietic anemia, hepatobiliary diseases, and constitutional jaundice. AIHA was diagnosed by means of the DAT, steroid-reactivity, and exclusion of alloimmune hemolytic anemia and drug-induced hemolytic anemia with respect to the RBC-IgG value [14].

Response criteria

There is no formal consensus on the definition of response criteria in patients with AIHA [20]. A response was defined as improvement in anemia (Hb > 10 mg/dl) and hemolysis after steroid treatment.

Statistical analysis

The distribution of categorical variables was compared among the groups using the X2 test or Fisher's exact test. The normality of the laboratory continuous variables was analyzed using the Kolmogorov–Smirnov test with Lilliefors significance correction. As most variables were not normally distributed, a Mann–Whitney U-test was used to determine the differences between patients with DAT−AIHA and DAT+AIHA. The backward multiple logistic regression method was used to select prognostic or therapeutic factors associated with effectiveness of steroids or survival at the 1-year follow-up evaluation in patients with idiopathic DAT−AIHA after forcing age and gender in the model; the statistical level of significance determined with the log-likelihood ratio test was set at P < 0.10. Statistical significance was defined as a two-sided P value < 0.05. All statistical computations were made using JMP statistical software 10.0.1 for Macintosh (SAS Institute, Inc., Cary, NC).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Four hundred fifteen surveys were returned for analysis, for a response rate of 71%. The mean age of the participants was 50.9 ± 25.3 years (range, 0.0–91 years [a lower age range of 0.0–0.1 contained four infants; suspected hemolysis with congenital TTP, with maternal SLE/APS, with maternal AIHA, and with CMV infection, there were no samples sent to evaluate maternal Rh Ig administration.]) and 55% of the participants were female. There were no significant differences in age and gender between the responders and non-responders (50.6 ± 25.6 or 50.7 ± 25.2 years of age and 54% or 55% female, respectively). Of the responders, 227 were diagnosed with AIHA. Sixty-four of the responders were DAT-positive; 62 had warm-type AIHA (DAT+AIHA) and 2 had cold-type AIHA. One hundred sixty-three responders were DAT-negative; 154 had warm-type AIHA (DAT−AIHA) and 9 had cold-type AIHA.

The clinical characteristics and laboratory measurements of patients with DAT−AIHA in comparison with DAT+AIHA are shown in Table 1. No significant differences between patients with DAT−AIHA and DAT+AIHA existed with respect to age, gender, idiopathic/secondary nature, or complications such as Evans syndrome. The percentage of reticulocytes, mean corpuscular volume, RBC-IgG level, white blood cell count, total protein (TP) level, and Hp were significantly higher in patients with DAT+AIHA than DAT−AIHA. Hemoglobin level was significantly lower in DAT+AIHA. No significant differences existed between patients with DAT−AIHA and DAT+AIHA with respect to the effectiveness of steroid treatment and survival rate 1 year following diagnosis (Table 2). Patients with DAT−AIHA required significantly lower doses of steroids for maintenance therapy. Patients with idiopathic warm AIHA had almost the similar pattern between DAT−AIHA (n = 110) and DAT+AIHA (n = 41; Table 3). Patients with secondary warm AIHA had also the same tendency, but a relatively poor response to treatments (Table 4). Other treatments had also been surveyed and the data of efficacy and 1-year survival following diagnosis are shown in Table 5. The sample sizes of these treatments were not large enough for statistical analyses. Treatments by intravenous gamma globulin and transfusion alone were mainly used for children (mean age, 5.2 ± 7.8 years) and young patients (mean age, 17 ± 11.8 years). In contrast, treatments with immunosuppressants were adapted for elderly patients (mean age, 71 ± 18.4 years), and treatments by splenectomy and observation were mainly used for adult patients (mean age, 57.9 ± 21.2 years) and (mean age, 56.5 ± 17.3 years), respectively. Rituximab had not been used for patients with idiopathic DAT−AIHA prior to 2010.

Table 1. Clinical Characteristics and Laboratory Measurements in Patients with DAT-Negative AIHA and DAT-Positive AIHA
VariableDAT-negative AIHADAT-positive AIHAP-value
Mean ± SDMean ± SD
  1. The P-value is the difference between the groups calculated with the Mann–Whitney U test.

  2. Hb, hemoglobin; % Retic, percentage of reticulocytes; MCV, mean cell volume; TP, total protein; LDH, lactate dehydrogenase; IDBIL, indirect bilirubin; Hp, haptoglobin; RBC-IgG, red blood cell-bound immunoglobulin G.

Number15462 
Age50.0 ± 25.850.5 ± 27.20.89
Female (%)54.658.10.64
Idiopathic (%)71.466.10.51
Evans syndrome (%)38.338.70.58
WBC (/μL)6401 ± 3638741 ± 5650.001
Hb (g/dL)8.0 ± 2.47.3 ± 2.60.02
%Retic (%)8.8 ± 11.311.2 ± 9.90.04
MCV (fL)100.2 ± 15.9106.3 ± 13.70.01
Platelets (103/μL)175 ± 139203 ± 1410.10
TP (g/dL)6.5 ± 0.96.9 ± 1.00.004
IgG (mg/dL)1361 ± 1741648 ± 2410.34
LDH (U/L)577 ± 560717 ± 8500.18
IDBIL (mg/dL)1.8 ± 1.81.8 ± 1.60.75
Hp (mg/dL)11.0 ± 2.817.3 ± 4.50.048
RBC-IgG179 ± 2881397 ± 1934<0.0001
Table 2. Efficacy of Treatments and 1-Year Survival Rate Following Diagnosis in Patients with DAT-Negative AIHA and DAT-Positive AIHA
 DAT-negative AIHADAT-positive AIHAP-value
Number15462 
Efficacy of treatments89%88%0.80
Treatment with steroids alone66%73%0.04
Remission within 4 weeks68%57%0.54
1-year survival rate following diagnosis83%88%0.50
Treatment with steroids alonen = 102n = 45 
Efficacy of steroids91%92%0.99
Remission within 4 weeks70%62%0.74
Maintenance steroids (< 15 mg/day)84%50%0.03
1-year survival rate following diagnosis84%92%0.27
Table 3. Efficacy of Treatments and 1-Year Survival Rate Following Diagnosis in Patients with Idiopathic DAT-Negative AIHA and Idiopathic DAT-Positive AIHA
 DAT-negative AIHADAT-positive AIHAP-value
Number11041 
Efficacy of treatments90%94%0.73
Treatment with steroids alone65%78%0.25
Remission within 4 weeks72%71%0.54
1-year survival rate following diagnosis84%87%0.98
Treatment with steroids alonen = 71n = 32 
Efficacy of steroids91%93%0.99
Remission within 4 weeks72%71%0.99
Maintenance steroids (< 15 mg/day)84%67%0.58
1-year survival rate following diagnosis84%93%0.34
Table 4. Efficacy of Treatments and 1-Year Survival Rate Following Diagnosis in Patients with Secondary DAT-Negative AIHA and Secondary DAT-Positive AIHA
 DAT-negative AIHADAT-positive AIHAP-value
Number4421 
Efficacy of treatments84%80%0.73
Treatment with steroids alone70%62%0.99
Remission within 4 weeks57%43%0.67
1 year survival rate following diagnosis83%80%0.74
Treatment with steroids alonen = 31n = 13 
Efficacy of steroids83%92%0.65
Remission within 4 weeks61%50%0.67
Maintenance steroids (< 15 mg/day)85%33%0.14
1 year survival rate following diagnosis83%82%0.74
Table 5. Efficacy of Treatments, Excluding Steroids Alone, and 1-Year Survival Rate Following Diagnosis in Patients with DAT-Negative AIHA
 Number/Effective cases/Survivor
TreatmentsIdiopathic DAT-AIHASecondary DAT-AIHA
No treatments22/–/173/–/3
Transfusion alone3/3/2 
Splenectomy alone4/3/4 
Splenectomy with steroids2/2/2 
Cyclophosphamide alone 1/1/1
Cyclophosphamide with steroids4/3/3 
Cyclosporine alone1/0/1 
Cyclosporine with steroids 1/0/0
Steroids+Cyclosporine+splenectomy1/0/1 
Steroids+Cyclosporine+Cyclophosphamide 1/1/1
Steroids+Cyclosporine+Mycophenolate mofetil 1/1/1
Steroids+Cyclophosphamide+Intravenous gamma globulin 1/1/1
Intravenous gamma globulin2/2/2 
Intravenous gamma globulin with steroids6/6/5 
Danazol with steroids1/1/1 
Rituximab with steroids 2/1/1
Plasmapheresis 1/1/1
Chemotherapy (unspecified) 2/2/0
R-CHOP 2/2/1
R-CHOP+Splenectomy 1/1/1
THP-CHOP 1/1/1
Others4/–/–5/–/–

Based on multivariate analysis of idiopathic DAT−AIHA (n = 110), TP and Evans syndrome were identified as the only significant factors associated with the effectiveness of steroids (adjusted odds ratio [aOR], 1.36/[0.1 g/dl]; 95% CI, 1.01–1.84) and survival at the 1-year follow-up (aOR, 0.1; 95% CI, 0.01–0.88).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Our results indicate that patients with DAT−AIHA generally have milder anemia and hemolysis than patients with DAT+AIHA, respond equally well to steroids, and show comparable survival at 1-year follow-up. These data verify the empiric image of DAT−AIHA to show the same clinical conditions as DAT+AIHA, with the exception of an adequate amount of RBC-IgG, and also renew our awareness of the significance of diagnosis of C-AIHA, as several serious side effects of steroids make clinicians hesitant to use steroids to treat DAT-negative hemolytic anemia patients without a diagnosis of AIHA [9].

The similarity in the clinical characteristics between patients with DAT−AIHA and DAT+AIHA suggests that the clinical entity of DAT−AIHA might be nearly equivalent to DAT+AIHA, with the exception of RBC-IgG. This clinical consanguinity may discourage efforts to investigate the basic mechanism underlying DAT−AIHA, but it is assumed there is a difference between DAT−AIHA and DAT+AIHA, such as cytokine pattern [21] or IgG subclass [17]. To elucidate the pathogenic mechanism of DAT-AIHA, one must have a thorough understanding of AIHA.

Multivariate analysis of patients with idiopathic DAT−AIHA revealed therapeutic and prognostic factors (TP and Evans syndrome, respectively) at the 1-year follow-up. In general, the natural history of AIHA cannot be predicted based on clinical features [22], but some early reports have suggested that a positive in DAT, reticulocytopenia or thrombocytopenia increased the mortality rate [23-25]. As these earlier reports were derived from data in the era of restricted available treatments, such as blood transfusion or splenectomy, whether or not thrombocytopenia affects prognosis using modern therapy is uncertain [22]. Our data showed 10 deaths in patients with Evans syndrome, one-half of whom were treated with steroids and the causes of death were pneumonia (n = 2), pulmonary bleeding (n = 1), acute myocardial infarction (n = 1), and unknown (n = 1); the remaining patients were untreated or treated with blood transfusion alone and the causes of death were hepatic failure (n = 2), myelodysplastic syndrome (n = 1), sepsis (n = 1), and unknown (n = 1). Evans syndrome remains a risk factor. Recently, diabetes mellitus was reported to be associated with high early mortality and poor prognosis in patients with AIHA [26]. We attempted to evaluate prognosis after intermediate or long intervals after diagnosis of DAT−AIHA with consideration for diabetes mellitus.

Our study had some limitations. First, because of the case–control study design, our data did not describe the prevalence or incidence of DAT−AIHA. Approximately 40% of undiagnosed hemolytic anemia referred to our laboratory was diagnosed with DAT−AIHA 1 year following the consult, which suggested that C-AIHA might be more common in AIHA than the rate previously assumed or that patients who were difficult to diagnose might merely accumulate in our laboratory. Secondly, the RBC-IgG value might affect the attending physicians in the diagnosis of DAT−AIHA more intensively than other clinical data. The RBC-IgG value was told to each attending physician within 1 week following the consult, because physicians struggled to diagnose their patients with unknown hemolytic anemia. The clinical diagnosis was assigned by attending physicians 1 year after the consult and 23% of untreated patients with high RBC-IgG values (>78.5 IgG/RBC) were diagnosed with non-AIHA because of other clinical data, and 28% of untreated patients with low RBC-IgG value (<78.5 IgG/RBC) were diagnosed with DAT−AIHA. Attending physicians appear to diagnose their patients using all their clinical data with respect to the RBC-IgG value.

In conclusion, our data characterized patients with DAT−AIHA in comparison with DAT+AIHA, verified the efficacy of steroid therapy in DAT−AIHA and identified significant factors for survival and effectiveness of steroids.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The authors sincerely thank Ms.Ohguri for her excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Petz LD, Garratty G. Warm antibody autoimmune hemolytic anemia. Corticosteroid therapy. In: Petz LD, Garratty G, editors.Immune Hemolytic Anemias. Philadelphia:Churchill Livingstone;2004. pp401407.
  • 2
    Murphy S, LoBuglio AF. Drug therapy of autoimmune hemolytic anemia. Semin Hematol 1976;13:323334.
  • 3
    Dameshek W, Komninos ZD. The present status of treatment of autoimmune hemolytic anemia with ACTH and cortisone. Blood 1956;11:648664.
  • 4
    Allgood JW, Chaplin HJr. Idiopathic acquired autoimmune hemolytic anemia. A review of forty-seven cases treated from 1955 through 1965. Am J Med 1967;43:254273.
  • 5
    Zupanska B, Sylwestrowicz T, Pawelski S. The results of prolonged treatment of autoimmune haemolytic anaemia. Haematologia (Budap) 1981;14:425433.
  • 6
    Engelfriet CP, Overbeeke MA, von dem Borne AE. Autoimmune hemolytic anemia. Semin Hematol 1992;29:312.
  • 7
    Roback JD, Barclay S, Hillyer CD. An automatable format for accurate immunohematology testing by flow cytometry. Transfusion 2003;43:918927.
  • 8
    Stroncek DF, Njoroge JM, Procter JL, et al. A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d. Transfus Med 2003;13:3541.
  • 9
    Gilliland BC. Coombs-negative immune hemolytic anemia. Semin Hematol 1976;13:267275.
  • 10
    Evans RS, Weiser RS. The serology of autoimmune hemolytic disease; observations on forty-one patients. AMA Arch Intern Med 1957;100:371399.
  • 11
    Worlledge SM, Blajchman MA. The autoimmune haemolytic anaemias. Br J Haematol 1972;23:6169.
  • 12
    Chaplin HJr. Clinical usefulness of specific antiglobulin reagents in autoimmune hemolytic anemias. Prog Hematol 1973;8:2549.
  • 13
    Issitt DP, Gutgseu NS. Clinically significant antibodies not detected by routine methods. In: Nance S, editor.Immune Destruction of Red Blood Cells. Arlington:American Association of Blood Banks;1989. pp9399.
  • 14
    Petz LD, Garratty G. Unusual aspects of acquired immune hemolytic anemias. A autoimmune hemolytic anemia with a negative direct antiglobulin test (DAT). In: Petz LD, Garratty G, editors.Immune Hemolytic Anemias. Philadelphia:Churchill Livingstone;2004. pp319334.
  • 15
    Jeje MO, Blajchman MA, Steeves K, et al. Quantitation of red cell-associated IgG using an immunoradiometric assay. Transfusion 1984;24:473476.
  • 16
    Kamesaki T, Oyamada T, Omine M, et al. Cut-off value of red-blood-cell-bound IgG for the diagnosis of Coombs-negative autoimmune hemolytic anemia. Am J Hematol 2009;84:98101.
  • 17
    Engelfriet CP, von dem Borne AEG, Beckers D, et al. Immune destruction of red cells. In: Bell CA, editor.A Seminar on Immune-Mediated Cell Destruction. Washington, DC:American Association of Blood Banks;1981. pp93130.
  • 18
    Zupanska B, Brojer E, Maslanka K, Hallberg T. A comparison between Fc receptors for IgG1 and IgG3 on human monocytes and lymphocytes using anti-Rh antibodies. Vox Sang 1985;49:6776.
  • 19
    Petz LD, Garratty G, editors.Acquired Immune Hemolytic Anemias, 1st ed. New York:Churchill Livingstone;1980.
  • 20
    Lechner K, Jäger U. How I treat autoimmune hemolytic anemias in adults. Blood 2010;116:18311838.
  • 21
    Toyotsuji T, Kamesaki T, Kajii E. Coombs-positive and negative autoimmune hemolytic anemia might immunologically differ. Rinsho Ketsueki (Japanese J Clin Hematol) 2011;52:461.
  • 22
    Petz LD, Garratty G. Prognosis and survival. Warm antibody AIHA. In: Petz LD, Garratty G, editors.Immune Hemolytic Anemias. Philadelphia:Churchill Livingstone;2004. pp7071.
  • 23
    Dausset J, Colombani J. The serology and the prognosis of 128 cases of autoimmune hemolytic anemia. Blood 1959;14:12801301.
  • 24
    Crosby WH, Rappaport H. Autoimmune hemolytic anemia. I. Analysis of hematologic observations with particular reference to their prognostic value; a survey of 57 cases. Blood 1957;12:4255.
  • 25
    Crosby WH. The clinical aspects of immunologic hemolytic anemia. Vox Sang 1955;26:36.
  • 26
    Nakasone H, Kako S, Endo H, et al. Diabetes mellitus is associated with high early-mortality and poor prognosis in patients with autoimmune hemolytic anemia. Hematology 2009;14:361365.