In vitro effects of ciprofloxacin and pefloxacin on growth of normal human hematopoietic progenitor cells and on leukemic cell lines

The in vitro effect of ciprofloxacin and pefloxacin on growth of normal hematopoietic progenitor cells and on leukemic cell lines was investigated. Ciprofloxacin and pefloxacin caused dose-dependent inhibition of colony formation both from normal bone marrow cells and from the leukemic line K-562 cells. This inhibition exerted by ciprofloxacin and pefloxacin was statistically significant at concentrations of 25 micrograms/ml and above. Ciprofloxacin appeared to be the most potent inhibitor of colony formation among the antimicrobial agents tested. Although the inhibitory effect of pefloxacin on normal hematopoietic stem cells was similar to that of cefazolin and chloramphenicol, the inhibitory effect of pefloxacin on leukemic cells was more prominent than that of cefazolin and chloramphenicol. In a proliferation assay in liquid culture of the cell line HL-60, ciprofloxacin and pefloxacin caused a dose-dependent inhibition of cell proliferation. Both drugs failed to induce cellular differentiation, as assessed by the nitrogen blue tetrazolium dye reduction assay. In therapeutic concentrations no cumulative toxic effect of the combination of ciprofloxacin with cytosine-arabinoside, vincristine, actinomycin D and doxorubicin on colony formation by HL-60 cells was observed. It is concluded that ciprofloxacin does not exert in vitro inhibitory effect on human leukemic cells when assayed at concentrations of less than or equal to 5 micrograms/ml. However, at concentrations of 25 and 50 micrograms/ml of ciprofloxacin alone and in combination with several antineoplastic agents exerts an inhibitory effect on colony formation.     

Prognostic value of blood and bone marrow T-colony-forming cells (T-CFC) in patients with lymphadenopathy syndrome (LAS)

The in vitro proliferation capacity of peripheral blood committed T-cell precursors (T-CFC) from LAS patients was studied in order to define whether this parameter could be associated with transition to AIDS. In all patients a significantly (P less than 0.0001) decreased plating efficiency was detected. However, the lowest T-cell colony growth (less than 50 colonies/5 x 10(4) cells) was observed in the 23 patients who subsequently developed the full-blown disease within 150-570 days. Conversely, only one patient (n = 107) whose T-CFC generated greater than 50 colonies/5 x 10(4) cells developed AIDS after a mean follow-up of 867 days (range 120-1260 days). T-CFC from these patients displayed an impaired in vitro differentiation and self-renewal capacity which was independent of the clinical evolution of the disease. In 5 out of 12 AIDS patients, adherent cell-depletion of peripheral blood mononuclear cells (PBMC) enhanced the plating efficiency. Moreover, patients' but not normal adherent cells could inhibit normal T-cell colony growth in a dose-dependent manner. Media conditioned by patients' unstimulated adherent cells (LCM-A+p) also inhibited normal T-cell colony formation. In addition, LCM-A+p were capable of inhibiting interleukin 2-receptor (IL 2-R) expression and interleukin 2 (IL 2) production by normal mitogen-stimulated T-cells. These findings suggest that adherent cell-derived inhibitory activity(ies) could be responsible for the low T-cell colony formation observed in some AIDS patients.     

Relevance of the HPA-15 (Gov) polymorphism on CD109 in alloimmune thrombocytopenic syndromes

BACKGROUND: Alloantibodies against the human platelet (PLT) alloantigen (HPA)-15 system residing on CD109 can cause fetal and neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura, and PLT transfusion refractoriness. The detection of antibodies against HPA-15, however, is hampered by the variable low expression and instability of the CD109 molecule during preparation and storage. STUDY DESIGN AND METHODS: This study analyzed the occurrence of HPA-15 alloantibodies in 1403 patients: 930 FNAIT and 473 polytransfused (PT) patients by modified monoclonal antibody specific immobilization of PLT antigens (MAIPA) assay with well-defined phenotyped PLTs. A DNA typing technique was developed to confirm the phenotypes of PLT donors. B-cell lines were established as sources of reference DNA. RESULTS: Genotyping of 407 unrelated blood donors revealed the gene frequencies 0.512 and 0.488 for HPA-15a and -15b, respectively. Based on the selection of PLTs expressing high amounts of CD109 on the surface (mean fluorescence intensity ratio 4-5 on expression peak on Days 2-4 after apheresis) antibody screening by the MAIPA assay was performed. In total, 16 (1.1%) HPA-15 alloantibodies were found comprising four anti-HPA-15a and 12 anti-HPA-15b. Anti-HPA-15b without other PLT-reactive antibodies were detectable in three serum samples of PT patients. The incidence of HPA-15 alloimmunization in PT patients was significantly higher than in mothers with FNAIT (3.0% vs. 0.22%). In relation to all detected HPA-specific antibodies, HPA-15 is responsible for 6.2 percent of alloimmunizations. CONCLUSION: These observations indicate that alloimmunization against HPA-15 should be considered as a cause for immune thrombocytopenia, particularly in patients receiving multiple PLT transfusions.     

Clinical trial of tolerance of HPA-23 in patients with acquired immune deficiency syndrome.

An open-label, multicenter clinical trial assessed the tolerance of HPA-23 (ammonium-21-tungsto-9-antimoniate) in patients with acquired immune deficiency syndrome. Sixty-nine patients were sequentially assigned to receive 0.25, 0.5, 1.0, or 2.0 mg of HPA-23 per kg intravenously 5 days per week for 8 weeks. HPA-23 was fairly well tolerated at doses of 1.0 mg/kg or less; nearly 60% of patients given 2.0 mg/kg discontinued treatment. Twenty-six patients discontinued treatment because of adverse events or concurrent illness. HPA-23 produced dose-related decreases in platelet count and increases in serum glutamine oxalacetic transaminase. There were no changes in immune system function, as determined by total lymphocyte count, T4-cell count, T8-cell count, and T4/T8 ratio. The effects of HPA-23 seemed to be more closely related to the total dose than to the daily dose. No improvement in the clinical status of the patients was observed during the 8 weeks of treatment.     

Gene frequencies of the HPA-15 (Gov) platelet alloantigen system in Brazilians

The HPA-15 (Gov) alloantigen is a biallelic co-dominant system on human platelets, and its allele HPA-15a and HPA-15b differ by an A-->C single nucleotide polymorphism at nucleotide 2108 of the coding sequence resulting in a Tyr682Ser substitution in the mature CD109 glycoprotein. Employing the polymerase chain reaction-restriction fragment length polymorphism technique, we determined the HPA-15 gene frequencies among 276 subjects of distinct Brazilian ethnic groups including, 15 Caucasians, 15 African Brazilians, 15 Orientals, 106 Amazon Xikrin Indians, 31 Amazon Gavioes Indians and 94 blood donors. The calculated HPA-15a and HPA-15b allele frequencies found in Caucasians (0.53/0.47), African Brazilians (0.57/0.43), Orientals (0.57/0.43) and Brazilian blood donors (0.52/0.48) did not differ significantly. However, the HPA-15a and HPA-15b gene frequencies of Xikrin Indians (0.78/0.22) were significantly different from that of all other groups (P < 0.01). The HPA-15a/a, HPA-15a/b and HPA-15b/b genotype frequencies observed in Gavioes Indians were significantly different from those seen in African Brazilians (P = 0.04) and blood donors (P = 0.017). The present data showed that the distribution of the HPA-15 (Gov) system alleles observed among the Brazilian population is quite similar to the distributions already reported among Asian, Canadian and European populations. Moreover, the data indicated differences in the frequency of the HPA-15 system between Amazon Indians and other distinct Brazilian ethnic groups suggesting that Amerindians would be at higher risk of HPA-15 alloimmunization in the need of receiving blood components collected from blood donors of other ethnic groups.     

Frequency and specificity of platelet-specific alloantibodies in HLA-immunized haematologic–oncologic patients

The frequency and specificity of platelet-alloantibodies to human platelet antigens (HPA) -1, -3 and -5 was investigated in 59 multitransfused, HLA-immunized patients. Using the MAIPA test (monoclonal antibody specific immobilization of platelet antigens) platelet alloantibodies could be demonstrated in 10 (17%) patients.   In one patient the antibody was present prior to any transfusions and probably induced by multiple previous pregnancies. This antibody was directed to HPA-5b. The remaining nine antibodies were found in patients ( n  = 36) with HLA-antibodies reacting with over 95% of unselected lymphocytes. In these patients the target antigens were HPA-1b in six, HPA-3a in one and both antigens in two patients.   Our findings demonstrate platelet alloimmunization induced by transfusions to be restricted to patients with high HLA-immunization. 25% of these patients (9/36) show platelet-specific antibodies, primarily HPA-1b.     

The platelet-specific alloantigen PlA1 (HPA-1a): a comparison of flow cytometric immunophenotyping and genotyping using polymerase chain reaction and restriction fragment length polymorphism in a Swedish blood donor population

BACKGROUND: There is an increasing interest in the development of rapid and reliable techniques for platelet alloantigen typing. STUDY DESIGN AND METHODS: By use of standardized flow cytometry and a specific human alloantiserum, 236 Swedish blood donors were immunophenotyped for the platelet-specific alloantigen, PlA1 (HPA-1a). RESULTS: Ten individuals (4.2%) had low fluorescence intensities and were considered PlA1- negative (HPA-1a-negative); all of them also demonstrated a PlA2/PlA2 (HPA-1b/1b) genotype in a polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assay of the underlying DNA polymorphism. The remaining population had clear positive fluorescence and was regarded as PlA1-positive (HPA-1a-positive). The fluorescence distribution histogram among PlA1-positive (HPA-1a-positive) individuals was dome-shaped, and those individuals who were homozygous for PlA1 (HPA-1a) could not be distinguished from those who were heterozygous. This finding was further substantiated by PCR-RFLP analysis of the PlA1/PlA2 (HPA-1a/1b) genotype; a heterozygous genotype was found among those having a medium fluorescence intensity as well as among those having a strong fluorescence intensity. CONCLUSION: Flow cytometry is a valuable tool for large-scale detection of PlA1 (HPA- 1a). However, flow cytometry based on only one antiserum cannot distinguish between homozygous and heterozygous carriers of PlA1 (HPA- 1a). For zygosity testing and when platelets are difficult to obtain, the PCR-RFLP technique is the assay of choice.     

A naturally occurring LeuVal mutation in beta3-integrin impairs the HPA-1a epitope: the third allele of HPA-1

BACKGROUND: Single-amino-acid substitution Leu33Pro in the beta3-integrin is responsible for the formation of the human platelet antigen (HPA)-1. Alloimmunization against HPA-1a (beta3-Leu33) is the most frequent cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura. STUDY DESIGN AND METHODS: While HPA-1 genotyping a large cohort of patients with thromboembolic disease with a thermal cycler (LightCycler), one patient was identified with a unique HPA-1a melting curve. RESULTS: Sequence analysis revealed a C-to-G transversion at nucleotide 175 in the beta3-integrin (ITGB3) gene that alters the Leu33 codon to Val33. Further genotyping of healthy blood donors (n = 2950) identified one nonrelated Pro33Val33-positive individual. To examine whether the presence of Val33 affected the binding pattern of HPA-1 alloantibodies, transfectants were generated expressing recombinant beta3-Leu33 or beta3-Val33. Interestingly, differences in the reactivity of anti-HPA-1a were observed, with some HPA-1a alloantibodies showing diminished reactivity with beta3-Val33 compared to beta3-Leu33 and others reacting equally with both types. Similar findings were observed with recombinant human HPA-1a antibodies, with one of the three not binding to beta3-Val33. CONCLUSIONS: Our results demonstrate that the naturally occurring Leu33Val mutation in the beta3-integrin can disrupt some HPA-1a epitopes. These findings provide evidence for a heterogeneous humoral response against HPA-1a that may have potential clinical implications for alloimmune thrombocytopenia disorders.     

Inhibition of human erythroid colony-forming units by tumor necrosis factor requires beta interferon.

We have previously reported that inhibition of human CFU-erythroid (E) colony formation by tumor necrosis factor (TNF) is an indirect effect mediated by a soluble factor released from a fraction of marrow accessory cells which are predominantly stromal elements (Means, R. T., Jr., E. N. Dessypris, and S. B. Krantz. 1990. J. Clin. Invest. 86:538-541). Further studies reported here identify a mediator of this effect. The inhibitory effect of recombinant TNF on marrow CFU-E is ablated by neutralizing antibodies to human beta IFN, but not by antibodies to gamma IFN or IL-1. Anti-beta IFN also neutralizes the inhibitory effect of conditioned medium prepared from marrow cells exposed to TNF. Human beta IFN inhibits colony formation by unpurified marrow CFU-E as well as highly purified CFU-E generated from peripheral blood progenitors, and limiting dilution analysis shows that this is a direct inhibitory effect. TNF has been implicated in the pathogenesis of the anemia of chronic diseases since blood TNF levels are elevated in many patients with this syndrome, and since exposure to TNF produces a similar anemia in either humans or mice. The present study demonstrates that beta IFN is a required mediator of this inhibitory effect on erythropoiesis.     

Identification of Spermine as an Inhibitor of Erythropoiesis in Patients with Chronic Renal Failure

Fetal mouse liver and normal human bone marrow cell cultures were used for studies on the inhibition of erythroid colony formation (CFU-E) by sera from anemic patients with end-stage renal failure and the polyamine spermine. Sera from each of eight predialysis uremic anemic patients with end-stage renal failure produced a significant (P < 0.001) inhibition of erythroid colony formation in the fetal mouse liver cell cultures when compared to sera from normal human volunteers. In vivo or in vitro dialysis of the uremic sera with a 3,500-dalton exclusion limit membrane removed the inhibitor from uremic sera. The uremic serum dialysate provided by the membrane fractionation was significantly inhibitory in the erythroid cell cultures. When this dialysate was applied to gel filtration chromatography (Bio-Gel P-2) the inhibitor was found to be in the same molecular weight range as [¹⁴C]spermine. The polyamine spermine produced a dose-related inhibition of erythroid colony formation (CFU-E) in fetal mouse liver and normal human bone marrow cultures. Thus, the following evidence is provided that the in vitro inhibitor of erythropoiesis found in chronic renal failure patients' sera is identical with the polyamine spermine: (a) the inhibitor and radiolabeled spermine appeared in identical Bio-Gel P-2 effluent fractions; (b) when spermine was added to normal human sera at concentrations reported in sera of uremic patients, and studied in both the fetal mouse liver cell culture and normal human bone marrow cultures, a dose-related inhibition of erythroid colony (CFU-E) formation was noted; and (c) the inhibitory effects of crude uremic serum, uremic serum dialysate, and fractions of uremic serum dialysate from a Bio-Gel column, on erythroid colony formation were completely abolished by the addition of a specific rabbit antiserum to spermine.     

Gene frequencies of human platelet antigens on glycoprotein IIIa in Japanese

BACKGROUND: Polymorphism of glycoprotein IIIa on human platelets is one of the factors in alloimmunization that causes neonatal alloimmune thrombocytopenia and refractoriness to platelet transfusion. STUDY DESIGN AND METHODS: DNA typing methods were originally developed to determine the genotypes of five human platelet antigen (HPA) systems located on glycoprotein IIIa: HPA-1, HPA-4, HPA-6W, HPA-7W and HPA-8W. The gene frequencies of these platelet antigens were determined by DNA typing of 331 unrelated Japanese donors. RESULTS: The gene frequencies of the low-frequency antigens were 0.002, 0.011, and 0.027 for HPA-1b, HPA-4b, and HPA-6W(b), respectively. All 331 Japanese donors tested were HPA-7W(a/a) and HPA-8W(a/a). Moreover, in the present study, none of the donors tested had two or more of these low-frequency antigens. CONCLUSION: The risk of neonatal alloimmune thrombocytopenia and refractoriness to platelet transfusion induced by the antigens of the HPA-1, HPA-7W, and HPA-8W systems was extremely rare in Japanese. However, attention must be paid to the involvement of the HPA-4 and HPA- 6W systems in these clinical disorders.     

Suppression of normal human erythropoiesis by gamma interferon in vitro. Role of monocytes and T lymphocytes.

Interferons (IFN) have been shown to suppress the proliferation of human erythroid progenitors (erythroid burst-forming units [BFU-E] and colony-forming units [CFU-E]) in vitro. To examine the mechanism(s) underlying this inhibitory activity, the effect of different doses (50-10,000 U) of a highly purified preparation of recombinant DNA produced human gamma-IFN on erythroid colony formation by normal human bone marrow BFU-E and CFU-E in the presence and absence of monocytes and/or T lymphocytes was studied. The addition of gamma-IFN to whole marrow caused suppression of BFU-E (6-65%) and CFU-E (31-79%) in a dose-dependent fashion. This inhibition occurred both with the direct addition of gamma-IFN to the culture plates as well as by the preincubation of marrow cells with gamma-IFN followed by the washing of the cells; at the highest concentration of gamma-IFN (10,000 U), near-maximal inhibition of colony formation occurred with as little as 15 min of preexposure (BFU-E, 50%; CFU-E, 81%). Removal of monocytes and/or T lymphocytes before the addition of gamma-IFN significantly reduced the inhibitory effects of this lymphokine (BFU-E, -1 to 38%; CFU-E, -8 to 67%). Co-culture of purified autologous monocytes or T cells preexposed to gamma-IFN with monocyte and T cell-depleted marrow cells resulted in highly significant inhibition of erythroid colony formation even when these treated cells comprised less than 1% of the total nucleated cell populations in culture. The inhibitory action of gamma-IFN was not prevented or reversed by erythropoietin. These results demonstrate that the inhibitory effects of gamma-IFN on erythropoiesis are mediated to a significant degree through accessory cell populations, and suggest that gamma-IFN may represent a useful tool in the study of the role of immunocompetent cells in the regulation of erythropoiesis in vitro.     

Effect of weekly adefovir (PMEA) infusions on HIV-1 virus load: results of a phase I/II study

The compound 9-(2-phosphonylmethoxyethyl)adenine (adefovir; PMEA) is a potent inhibitor of a number of viruses in vitro, such as human immunodeficiency virus (HIV) type 1 and 2, herpes simplex virus (HSV) type 1 and 2, human papillomavirus virus (HBV) and Epstein-Barr virus (EBV). Adefovir also proved to be effective in vivo against feline immunodeficiency virus (FIV) in cats and simian immunodeficiency virus (SIV) in rhesus monkeys. In an open, non-placebo-controlled trial the antiviral activity of weekly doses of adefovir in nine patients with AIDS or AIDS-related complex was studied for a period of 11 weeks. CD4 cell counts at baseline were between 10 and 450 cells/mm3, HIV-1 RNA levels at baseline were between 24,210 copies/ml and 406,197 copies/ml. The drug was administered intravenously at a dose of 1000 mg every week and plasma viral load was assessed at multiple points during the study. Administration of adefovir was tolerated well and no severe side effects were seen. The response to adefovir treatment differed widely between patients. The increase in CD4 cell count at end point ranged from -40 to 120 cell/mm3. The lowest HIV RNA levels were measured after 3-5 days, showing an increase thereafter. The nadir in viral load was achieved after 2 weeks, with a mean viral load decline of 0.7 from baseline. The decrease of the HIV RNA level at end point ranged from -0.3 log10 to 1.8 log10 with a mean decrease of 0.4 log10. Our results indicate that adefovir given intravenously once weekly has a short-lasting initial antiviral effect. The effect of more frequent dosing requires further evaluation. If adefovir is to be useful clinically, it needs to be combined with other antiviral agents.     

NH2-terminal globular domain of human platelet glycoprotein Ib alpha has a methionine 145/threonine145 amino acid polymorphism, which is associated with the HPA-2 (Ko) alloantigens.

The glycoprotein (GP) Ib/IX complex, a prominent platelet GP complex, is the primary receptor for vWF. Previously, we have established that an antigenic polymorphism of platelets, the HPA-2 or Ko alloantigen system, is located on the 45-kD amino-terminal globular domain of GPIb alpha. With the polymerase chain reaction, we have amplified two segments of the GPIb alpha gene coding for the first 382 amino acids of two HPA-2a and two HPA-2b homozygous individuals. Nucleotide sequence analysis revealed as the only difference a C-T polymorphism at position 434 of the coding region for the mature protein. This base change results in a substitution of threonine (ACG) in HPA-2a (Kob) to methionine (ATG) in HPA-2b (Koa) at amino acid position 145. The C-T polymorphism is reflected in a difference in restriction enzyme recognition, resulting in an Aha 2-site in the HPA-2b allele and a SfaN1 site in the HPA-2a allele. Restriction fragment length polymorphism analysis of the amplified DNA of 3 HPA-2(a-,b+), 2 HPA-2(a+,b+), and 11 HPA-2(a+,b-) donors showed that these restriction sites were associated with the HPA-2 alleles. DNA-typing for the HPA-2 alloantigen system on genomic DNA obtained from a small number of cells may be applied for determining the genotype of a fetus from an immunized mother or of severely thrombocytopenic patients.     

Lymphokine-Activated Killer (LAK) Cell Phenomenon in Cluster Headache. "In Vitro" Activation by Recombinant Interleukin-2

Previous studies showed that the Natural Killer (NK) activity of peripheral blood lymphocytes (PBL) from cluster headache (CH) patients is lower than that of controls. This decreased activity seems to be independent of the cluster period. beta-interferon has been shown to be more effective in increasing NK activity when incubated with PBL from CH patients, than with PBL from control donors. Lymphokine-Activated Killer (LAK) cells can be generated by incubation of human PBL in recombinant Interleukin-2 (rIL-2). This phenomenon was studied in 10 CH patients and 8 healthy volunteers. PBL were activated to LAK cells by "in vitro" incubation for 72 hours in Control Medium containing rIL-2 (1000 I.U./ml). A four hour Chromium 51 release was used to measure LAK Cell Killing of K562 target cells. The released radioactivity was measured in a gamma scintillation counter. The CH patients showed a marked increase of LAK generation compared to control subjects. This effect seems to be augmented during the cluster period.     

Suppression of scrapie infection in mice by heteropolyanion 23, dextran sulfate, and some other polyanions.

Studies of polyanions that suppress scrapie have been done to pinpoint the cell types in the lymphoreticular system which are important in pathogenesis and to suggest possible prophylactic or therapeutic strategies for the unconventional slow viruses. A regime of three daily injections of the inorganic heteropolyanion HPA-23 reduced the effective scrapie dose by more than 99%; i.e., some mice survived peripherally injected doses of 100 50% lethal dose units. The effect was greatest when the first dose of HPA-23 was given 4 h after injecting scrapie, but it declined rapidly as this interval was increased, and there was virtually no effect 2 days after infection. A single dose of high-molecular-weight organic polyanions such as carrageenan or dextran sulfate (DS-500) greatly reduced (i.e., greater than 99%) the efficiency of scrapie infection. In contrast to HPA-23, DS-500 was equally effective whether given 4 days before or 8 h after the time of infection. The antiscrapie effect of DS-500 appeared to be independent of its activity as a B-cell mitogen and of its ability to produce a cytotoxic blockade of phagocytic cells. DS-500 probably caused the aggregation and loss from blood of scrapie inoculum which was present immediately after injection, but it had additional effects on scrapie at later times.     

Effects of oxidative stress on human erythroid colony formation: Modulation by gamma-interferon

Evidence of increased oxidative stress is a hallmark of many chronic diseases associated with anemia. The current study was undertaken to evaluate the effects of oxidative stress on erythroid colony formation in vitro by bone marrow light density mononuclear cells (LDMN) and by peripheral blood derived cells enriched for erythroid colony forming units (CFU-E), and how these effects can be modified by a cytokine implicated in the anemia of chronic disease. When blood-derived and marrow cells were cultured with 50 microM H(2)O(2), CFU-E colony formation by blood-derived cells but not by marrow cells was significantly inhibited, suggesting a protective effect of marrow accessory cells. This inhibitory effect on peripheral blood-derived CFU-E was shown to be caspase-dependent. rhgammaIFN at concentrations which did not inhibit CFU-E colony formation sensitized LDMN marrow cells to inhibition by H(2)O(2). Exposure of LDMN marrow cells to rhgammaIFN at concentrations of 10 U/mL or higher significantly decreased the concentration of thioredoxin (Trx) in cell supernatant. Addition of recombinant Trx to LDMN marrow cells cultured with H(2)O(2) and rhgammaIFN partially (although not completely) reversed inhibition of CFU-E colony formation. These findings suggest that inflammatory cytokines implicated in the pathogenesis of the anemia of chronic disease may exert their effects at least in part through modulation of oxidative stress.     

Lower hemoglobin levels in human immunodeficiency virus-infected patients with a positive direct antiglobulin test (DAT): relationship with DAT strength and clinical stages

BACKGROUND: There are conflicting opinions regarding the effect of positive direct antiglobulin test (DAT) on hemoglobin (Hb) levels in human immunodeficiency virus-infected (HIV+) patients. STUDY DESIGN AND METHODS: A total of 166 samples from HIV+ outpatients were studied. The DAT was performed with the tube test and column agglutination technology (CAT). RESULTS: The DAT was positive in 18.67 percent with the tube method and 33.73 percent with the CAT. Patients with DAT-positive results showed lower Hb levels than DAT-negative patients, 12.3 g per dL versus 14.3 g per dL (p = 0.0002). The univariate logistic regression enabled us to study the phenomenon better and fit the probability of having a DAT-positive result on the basis of the Hb levels. The relationship between the CAT and the tube test when washing the red blood cells (RBC) at 4 degrees C was stronger than when washing these at room temperature (phi = 0.8156; p = 0.000). The Hb levels were significantly lower in the positive DATs of Stage C (acquired immune deficiency syndrome [AIDS]) and Stage B (symptomatic non-AIDS patients), which showed decreasing Hb values for increasing agglutination strengths (p = 0.000). Anemia was related with the DAT results (odds ratio [OR], 8.005; p = 0.000) but not to the AIDS condition (OR, 1.741; p = 0.221). DISCUSSION: Our study indicates that the DAT-positive results may be specifically related to lower Hb levels in HIV+ patients. The immunologic RBC clearance could be part of the anemic multifactorial condition in HIV+ patients.