Hemolytic Episodes Research Articles

Impaired CD163 Mediated Hemoglobin-Scavenging and Hemolytic Crisis in Patients Treated with CD33 Targeted Chemotherapy (Gemtuzumab Ozogamicin, Mylotarg™).

Hemoglobin liberated to plasma during intravascular hemolyses is rapidly bound to haptoglobin. The hemoglobin-haptoglobin complexes undergo endocytosis through the monocyte/macrophage specific scavenger receptor for hemoglobin (CD163). This mechanism protects against oxidative and NO-scavenging adverse effects of free hemoglobin. In this study, we describe a novel syndrome of severe intravascular hemolysis and serious hemolytic crisis due to impaired hemoglobin scavenging in three acute myeloid leukemia patients following CD33-directed therapy with the immunotoxin gemtuzumab ozogamicin (GO, Mylotarg™). A synchronous high free hemoglobin, haptoglobin, and low bilirubin after septicemia-induced intravascular hemolysis indicated abrogated clearance of haptoglobin-hemoglobin complexes. This was further supported by low levels of plasma soluble CD163 and a concordant low number of CD163-expressing monocytes. We show that CD163 positive monocytes and bone marrow macrophages coexpress CD33 thus suggesting that the GO-induced cellular cytotoxicity of CD33 positive cells eradicates a significant part of the CD163 positive monocytes and macrophages. The patients had severe inflammation and serious organ failure symptoms that may be a direct effect of the persistent high level of free hemoglobin. One of the patients had a fatal outcome whereas, the other two recovered from the hemolytic episode, and the peripheral blood CD163 expression returned to normal. The risk of a serious hemolytic crisis should be considered following CD33-targeted chemotherapy. Furthermore, the cases provide circumstantial evidence of a key role of CD163 plasma hemoglobin clearance in vivo.

Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice

Increased generation of reactive oxygen species (ROS) leads to oxidative stress in diabetes. Catalase is a highly conserved heme-containing protein that reduces hydrogen peroxide to water and oxygen and is an important factor decreasing cellular injury owing to oxidative stress. Hyperglycemic conditions increase oxidative stress and angiotensinogen gene expression. Angiotensinogen conversion to angiotensin II leads to a furtherance in oxidative stress through increased generation of reactive oxygen species. In this study, we utilized mice transgenically overexpressing rat catalase in a kidney-specific manner to determine the impact on ROS, angiotensinogen and apoptotic gene expression in proximal tubule cells of diabetic animals. Proximal tubules isolated from wild-type and transgenic animals without or with streptozotocin-induced diabetes were incubated in low glucose media in the absence or presence of angiotensin II or in a high-glucose media. Our results show that the overexpression of catalase prevents the stimulation of ROS and angiotensinogen mRNA in tubules owing to elevated glucose or angiotensin II in vitro. Additionally, overexpression of catalase attenuated ROS generation, angiotensinogen and proapoptotic gene expression and apoptosis in the kidneys of diabetic mice in vivo. Our studies point to an important role of ROS in the pathophysiology of diabetic nephropathy.

Paroxysmal Nocturnal Hemoglobinuria (PNH): Long Term Follow up of 48 Patients in a Single Institution.

Background: PNH is a clonal acquired disease characterized by poor expression of CD55 and CD59, adding higher lytic activity to the complement pathway, ending up in hemolysis, pancytopenia and thrombosis. The clinical variability and the lack of effective therapeutics make the treatment of PNH a real challenge.Material and methods: A retrospective analysis of 48 patients diagnosed with PNH between feb/1997 and march/2006 admitted to our center was performed. Before 1999, diagnosis was made by HAM and SACAROSIS tests. From this point immunophenotyping analysis was used to establish diagnosis. Bone marrow aspirate and biopsy, cytogenetic and biochemistry analysis were performed for exclusion of comorbities. Patients were divided in two groups: 1) de novo PNH associated with marrow failure (MF) and 2) PNH as an evolution of severe aplastic anemia (SAA) or mielodysplastic syndrome (MDS). Response was defined as reduction at hemolytic episodes as well as improvement of CBC and transfusion requirements.Results: Median age was 24 years (range 9–75); male patients constituted 58% of the sample (male/female: 1,4:1). De novo PNH with associated marrow failure occurred in 22 patients (group 1) and evolutive PNH in 15, being 13 from previous SAA and 2 from previous hypoplastic MDS (group 2). Thirty seven patients were followed at our clinic during this time: 25 (67,5%) received immunesupression with cyclosporine and steroids. Overall response (partial or complete) after one year of therapy was 84% (31/37). The remaining 11 patients (32,5%) received steroids alone, androgens or only observation, four of them remained with stable disease. HLA match hematopoietic stem cell transplantation (HSCT) was performed in 18/48 (38%), being 11 from group 1 and 7 from group 2. Main indications were: pancytopenia (77%), thrombosis (17%) and AML (6%).Only nine patients died (19%): five after HSCT and 4 of those followed at our clinic (massive thrombosis and hemolysis). Overall survival after HSCT was 72% with median follow-up of 890 days. Overall survival among outpatient clinic patients was 86% with a median follow-up of 2340 days. Most frequent outpatient events were: hemolysis (48%), pancytopenia (13%), thrombosis (13 %), renal failure (9%) and infection (8%).Fourteen patients (37%) remained without symptoms.Conclusions:Immunosuppressive therapy with cyclosporine and corticosteroids is an excellent strategy in patients with PNH and associated marrow failure, leading to durable clinical responses.The present data supports previous published indications for HSCT: ASS and thrombosis.Overall survival of PNH patients who received HSCT achieved 72%.

Endogenous inhibitors of death proteins inhibit the progression of hemolytic injury

Priming dose of butoxyethanol (BE, 500 mg/kg, po) protects female SD rats against LD90 dose of BE administered 7 d later. Incubation of naïve RBCs with the plasma of LD90 BE treated rats obtained 8 h later (with < 1 mM butoxyacetic acid, BAA, hematotoxic metabolite of BE, t1/2(BAA) = 2.5 h, t1/2(BE) = 0.2 h) led to cytotoxicity. Priming dose causes hemolysis from 3 to 24 h. Why BE-initiated hemolysis continues beyond 4 half lives of BAA is unknown. We hypothesized that death proteins viz. calpain, cytosolic phospholipase A2 (CAP, cPLA2) released from lysed RBCs, upon activation by plasma Ca2+ mediate the progression of BAA-initiated hemolysis. Plasma CAP activity and incubation of naïve RBCs with CAP confirmed the role of CAP in the progression of hemolysis. Rats recovered from the priming dose hemolytic episode, exhibit lower hemolysis after challenge with LD90 BE dose on 7 d. We hypothesized that overexpression of survival factors such as inhibitors of CAP and cPLA2 viz. calpastatin and annexin I (CAST and ANX) in the new RBCs imparts resistance against progression of hemolysis mediated by these death proteins. Presence of higher CAST levels in the RBCs of primed rats till only 1 d suggests that CAST alone may protect the new RBCs initially. Overexpression of ANX in primed and 90% reticulocytosis-induced rat RBCs implicates its role in the resiliency of new RBCs against progression of hemolysis. Moreover, protein patterns in Coomassie stained gels of control and primed rat RBCs imply involvement of other survival factors (ankyrin, band 3, adducin) in the resiliency of new RBCs. The roles of these survival factors in resiliency of new RBCs and cPLA2 in the progression of hemolysis remain to be investigated.

Cholelithiasis associated with haemolytic-uraemic syndrome

Cholelithiasis occurs infrequently in the paediatric age group. Hereditary spherocytosis, sickle cell anaemia and thalassemia are the haemolytic disorders most commonly associated with development of gall stones in paediatric age group. The question is whether an isolated episode of haemolysis can cause gallstones.

Cefotetan-induced hemolytic anemia after perioperative prophylaxis

Cephalosporin-induced hemolytic anemia is an acquired form of hemolytic anemia caused by interaction of drug with the immune system. Drug adsorption, drug-dependent antibody, and autoimmune induction are the three mechanisms of hemolysis. Cefotetan-induced hemolytic anemia (CIHA) has been described to occur through all three mechanisms. We report four cases of CIHA that occurred after appropriate perioperative use of cefotetan. All of our patients developed an acute and severe hemolytic episode that caused significant symptoms and led to hospitalization within 1-2 weeks after exposure to cefotetan. The hemolytic process was self-limited, and all our patients responded to supportive measures and blood transfusion. This report adds to our knowledge of CIHA, a rare complication of cefotetan use. Our cases suggest that cefotetan-induced acute severe hemolysis is caused by membrane modification (nonimmunologic protein adsorption) in addition to immune complex formation. Prompt diagnosis and aggressive supportive measures are essential in minimizing morbidity and mortality from hemolysis. Physicians should warn their patients about this complication. Given that hemolysis occurs when the subject is no longer under direct clinical supervision, patient awareness on how to recognize signs and symptoms of hemolysis is paramount to reducing the likelihood of this potentially lethal side effect. Finally, physicians might consider restricting cefotetan use.

Recurrent acute hemolytic transfusion reactions by antibodies against Doa antigens, not detected by cross‐matching

An 81-year-old male patient suffered from recurrent acute hemolytic transfusion reactions after transfusion with phenotyped cross-match-negative red blood cells (RBCs). Extensive posttransfusion workup eventually revealed Dombrock (a) (Do(a)) antibodies. Because commercially available cell panels do not allow for identification of anti-Do(a) and owing to the lack of Do(a) typing serum samples, selection of matched units of RBCs is dependent on negative cross-match results. In this case, selection of Do(a-) units by cross-matching failed, indicating that serologic methods were not reliable. A polymerase chain reaction with sequence-specific priming assay was used to detect DOA and DOB alleles, which encode Do(a) and Do(b) antigens, respectively. The patient was confirmed to be DOB/DOB by DNA sequencing. Furthermore, the involved mismatched units in each of the three hemolytic episodes were shown to be Do(a+). In the presenting case, DNA typing appeared to be superior to serologic methods in selecting matched RBC units in the presence of anti-Do(a).

Soluble CD30 (sCD30), a Marker of Th2 Polarization, Is Elevated in Patients with Autoimmune Hemolytic Anemia (AIHA) but Not with Idiopathic Thrombocytopenia (ITP).

Introduction. CD30, member of the TNF-receptor family, is expressed on activated T and B cells. Upon CD30 signaling and the downstream event of cytokine release of IL-4 and IL-13, CD30 is cleaved by a metalloprotease, resulting in soluble CD30 (sCD30). sCD30 has been shown to be a reliable proxy for Th2 activity and is elevated in Th2-polarized disorders such as atopic dermatitis, rheumatoid arthritis, and systemic lupus erythematosus (SLE) but is subnormal in patients with Th1-polarized disease such as X-linked agammaglobulinemia (Eur J Immunol 2001; 31:1927–1934). Elevated sCD30 levels were also found in patients with Hodgkin's lymphoma or anaplastic large cell lymphoma. We investigated sCD30 levels in patients with chronic idiopathic thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA).Method. We measured plasma sCD30 levels in 35 patients with ITP, 12 with AIHA, 8 with SLE, 9 with lymphoma and 18 healthy controls. sCD30 was measured by ELISA (Bender Medsystems) in platelet poor plasma from citrated blood.Results. Compared to controls (19.0 ± 6.2 units/mL), sCD30 levels were significantly higher in AIHA (mean 145.6, p=0.03) but not in ITP (22.7, p=0.1). sCD30 levels were also elevated in SLE (mean 73.6, p=0.001) and lymphoma (mean 107.4, p=0.04), confirming previously published studies. In AIHA, sCD30 levels were not significantly different in patients with active/chronic disease versus those in clinical remission, although levels trended higher during the acute episodes of hemolysis. In ITP, sCD30 levels were also similar between active disease versus remission and splenectomized versus unsplenectomized patients.Conclusion. Our results support the hypothesis that AIHA and SLE are Th2-polarized disorders, whereas ITP is not. Lack of significant differences in sCD30 between active disease and remission in AIHA and ITP may suggest that imbalances of Th1/Th2 immunity persist regardless of clinical course, consistent with Panitsas et al (BMC Blood Disord 2004; 4:4).Discussion. Prior studies have examined serum cytokine concentrations such as IFN-γ and IL-4 in patients with ITP and reached inconclusive results. But recent studies using more sensitive techniques such as RT-PCR indicate that ITP is a Th1-polarized disorder (Blood 2004; 103:2645–2647). Our findings of elevated sCD30 levels in AIHA but not ITP lead to a provocative question: what is it about the immunological destruction of platelets that makes it associated with a markedly different Th1/Th2 milieu in comparison to the destruction of red blood cells? We propose that the key differences are 1) platelets are more potent in modulating immunity via mechanisms including CD40L (CD154) release upon platelet activation (Immunity 2003; 19:9–19) and 2) platelets express MHC I molecules whereas red blood cells do not and hence they are processed by macrophages in a different manner (Transfusion 2002; 24:958–961).

Heme and innate immunity: new insights for an old molecule

Hemolytic episodes such as sickle cell disease, malaria and ischemia-reperfusion occurrence are often associated to the statement of an inflammatory response which may develop or not to a chronic inflammatory status. Although these pathological states are triggered by distinct etiological agents, all of them are associated to high levels of free heme in circulation. In this review, we aim to focus the very recent achievements that have led to the statement of free heme as a proinflammatory molecule, which may play a central role during the onset and/or persistence of inflammation during these pathologies.

Delayed and acute hemolytic transfusion reactions resulting from red cell antibodies and red cell–reactive HLA antibodies

It has been controversial whether HLA antibodies cause hemolytic transfusion reactions (HTR) or shortened red blood cell (RBC) survival. A patient is reported who had two episodes of HTR, the latter of which was likely due to RBC-reactive HLA antibodies. A 77-year-old woman, admitted for gastric varix rupture, had no RBC-irregular antibodies detected before transfusion. On Hospital Day 12, after transfusion of 2 units of RBCs and 2 units of fresh-frozen plasma, the first delayed hemolytic episode occurred and anti-E, anti-c, anti-Jk(a), and unidentified RBC-reactive antibodies were detected in a serum sample from Day 14. Two additional units of matched RBCs were transfused with a leukoreduction filter on Days 19 and 22. After 4 hours of starting a transfusion on Day 22, the patient had fever, and a second hemolytic episode was recorded. Multireactive HLA antibodies (reactive against 20 of 20 donor panel lymphocytes) were detected in serum samples from Day 15 to Day 21. These HLA antibodies reacted strongly with HLA-A2 and HLA-B7 antigens, corresponding to Bg(c) and Bg(a) antigens on RBCs, respectively. RBCs transfused on Day 22 were found to be HLA-A2 by genotyping. Strong HLA alloantibodies in this recipient appear to have caused a HTR. It is suggested that HLA antibodies be considered in patients with unexplained HTRs.

Plástica valvar mitral pela técnica de "Duplo Teflon" em pacientes com anel valvar calcificado e degeneração mixomatosa

OBJETIVO: O objetivo deste estudo é apresentar resultados imediatos e tardios da plástica da valva mitral pela técnica de Duplo Teflon, em pacientes com insuficiência mitral importante que apresentam anel valvar gravemente calcificado e degeneração mixomatosa. MÉTODO: Entre 1985 e 2002, 162 pacientes com insuficiência mitral devido à degeneração mixomatosa foram submetidos a plástica da valva mitral no Instituto do Coração da Faculdade de Medicina da Universidade de São Paulo. Deste total, 13 pacientes que apresentavam anel valvar gravemente calcificado foram submetidos à plástica da valva com ressecção quadrangular e anuloplastia pela técnica de Duplo Teflon. A idade média dos pacientes foi 65,4 +/- 12,9 anos, e 69,2% dos pacientes eram do sexo masculino. Um (7,7%) paciente tinha doença coronariana associada e foi submetido à cirurgia de revascularização do miocárdio. No período pré-operatório, 15,4% dos pacientes estavam em classe funcional IV, 69,2% em classe III e 15,4% em classe II. RESULTADOS: Não houve óbito no período pós-operatório imediato. No período pós-operatório tardio, 90,9% dos pacientes vivos estavam em classe funcional I. Não houve complicações como hemólise ou endocardite. Um paciente foi reoperado no segundo mês do período pós-operatório sendo realizada troca da valva mitral. A sobrevida atuarial em 14 anos foi 71,4 +/- 17,1%. CONCLUSÃO: A plástica da valva mitral com a técnica de Duplo Teflon, em pacientes com insuficiência mitral importante devido à degeneração mixomatosa e anel valvar gravemente calcificado, apresentou taxa de sobrevida satisfatória e boa evolução clínica, sugerindo ser melhor alternativa em relação à troca valvar.

Hb Geldrop St. Anna [β94(FG1)Asp→Tyr]: a New Hemoglobin Variant Observed in a Diabetic Patient

An abnormal hemoglobin (Hb) fraction was observed during a high performance liquidchromatographic (HPLC) Hb A1c control for diabetes mellitus in a 56-year-old north Europeanwoman. Family analyses revealed the abnormal fraction in three of her five siblings and in her son.Elevated Hb and packed cell volume (PCV) values and red blood cell (RBC) counts were present in allcarriers. No histories of anemia, hemolytic or circulatory episodes were reported. The abnormal Hbfraction estimated at 40%, migrated just below Hb F on alkaline electrophoresis and overlapped the HbA2 peak on cation exchange HPLC. Direct sequencing of the β-globin genes revealed a newGAC→TAC transversion in heterozygous form at codon 94 of the β-globin gene. Based on thehematological/biochemical data and the decreased P50 value, we conclude that the new variant is astable Hb associated with a slightly elevated oxygen affinity.

GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY AND GILBERT SYNDROME

The authors describe the paradoxical clinical phenotype of an undetected severe hemolysis in parallel with the development of severe jaundice in a 13-year-old male suffering from a confirmed interaction of glucose-6-phosphate dehydrogenase deficiency (Mediterranean variant, 563 C/T) and Gilbert syndrome [variant (TA)7/(TA)7]. The child had 2 acute hemolytic episodes at the age of 10 and 13 years following infections of unknown origin. Both episodes were characterized by considerably high bilirubin levels (1st episode: 10.8 mg/dL, 2nd episode: 17.8 mg/dL) associated with unexpectably mild hemolysis indices (1st episode hemoglobin levels, 11.1 g/dL; reticulocyte counts, 2.5%; 2nd episode hemoglobin values, 12.7 g/dL; reticulocyte counts, 2.5%). During the steady-state condition of the child, hemoglobin values were within the normal ranges for his age (14.2 g/dL) and bilirubin levels were slightly elevated (1.70 mg/dL, indirect 1.5 mg/dL). The interaction of the two genetic abnormalities in the causation of this odd clinical phenotype is discussed.

ＲｈＤ陰性血の赤血球輸血にもかかわらず抗Ｄを産生したＲｈＤ陰性の１例

We experienced a case in which an anti-D antibody was developed after transfusion of RBCs from 19 apparently D-negative donors over a half-year period beginning in July 2000. The patient was 67-year-old. B-negative female with a history of sensitization by transfusion of D-positive RBCs. Antibody screening became positive and an anti-D was detected by indirect antiglobulin testing. However, no hemolytic episodes due to the anti-D were found in her clinical course. Specificity of the antibody was confirmed as an allo-anti-D by serological studies using differential absorption-elution or dithiothreitol-treated RBCs. We initially suspected the possibility of transfusion from D-positive or weak D donors and the Rh phenotypes of all 19 donors were back-checked. However, none was positive for D antigen by the indirect antiglobulin test and 5 donors included Ce haplotype. Genomic analysis to detect a Del, allele will demonstrate whether RBCs from a donor carrying a Del allele with Ce haplotype induced an allo-anti-D immune response.

Transient anti rhesus alloantibody produced by graft after non-myeloablative allogeneic stem cell transplant

Background: We report the case of a patient who received an allogeneic transplant with peripheral blood compatible ABO, Rhesus mismatched progenitor cells and who developed an asymptomatic transient anti Rhesus alloimmunisation. Case report: A 56-year-old man with renal cell carcinoma received a non-myeloablative allogeneic PBPC ABO compatible graft from his HLA-identical brother. Graft-versus-host disease prophylaxis consisted of cyclosporine alone. On day + 59, prior to any transfusion, a positive direct antiglobulin test (IgG++, C3d−) was detected. The indirect antiglobulin test (IAT) was considered doubtful, and IAT identification revealed the presence of an active anti Rhesus antibody (anti D specificity) in the patient’s serum. This immunisation had no clinical consequence, with no acute hemolytic episode. Further monitoring showed negative antibody screening tests on day + 78. Conclusion: To our knowledge this is the first reported case of transient anti Rh (D) allo-immunisation after non-myeloablative allogeneic peripheral blood progenitor cell (PBPC) transplant. The period of occurrence and the specificity of this antibody strongly suggest a donor cell origin.

Two novel ADAMTS13 gene mutations in thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome (TTP/HUS)

Thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS) are now considered to be variants of one single syndrome called thrombotic thrombocytopenic purpura/hemolytic-uremic syndrome (TTP/HUS). Key features are thrombocytopenia, hemolytic anemia, and subsequently impaired function of different organs, especially the kidneys and the central nervous system (CNS). One possible reason is the deficiency of von Willebrand factor-cleaving protease (vWF-CP) resulting in persistence of uncleaved, ultralarge von Willebrand factor multimers (ULvWFM). We report a patient who was initially diagnosed with Evans syndrome (hemolytic anemia and autoimmune thrombocytopenia) as infant. At 10 years of age he developed HUS-like disease with gastrointestinal tract infection, hemolytic anemia, thrombocytopenia,and acute renal failure. However, enteropathogenic Escherichia coli-like or Shiga-like toxins were not detected. Further investigations revealed severe deficiency (<3%; normal >40%) of vWF-CP activity caused by compound heterozygosity of two novel ADAMTS13 gene mutations (1170 G>C [W390C] and 3735 G>A [W1245X]. vWF-CP autoantibodies were not detected. Periodic (every 2 weeks) treatment with fresh frozen plasma (FFP) maintained both platelet level and kidney function within normal range and prevented new episodes of TTP/HUS. Enteropathogenic E. coli- and Shiga-like toxin-negative patients who present with hemolytic or thrombocytopenic episodes and HUS like symptoms should be tested for vWF-CP deficiency and other noninfectious reasons for TTP/HUS since plasma substitution possibly provides an efficient therapeutic option for this subgroup of patients.

Heme inhibits human neutrophil apoptosis: involvement of phosphoinositide 3-kinase, MAPK, and NF-kappaB.

High levels of free heme are found in pathological states of increased hemolysis, such as sickle cell disease, malaria, and ischemia reperfusion. The hemolytic events are often associated with an inflammatory response that usually turns into chronic inflammation. We recently reported that heme is a proinflammatory molecule, able to induce neutrophil migration, reactive oxygen species generation, and IL-8 expression. In this study, we show that heme (1-50 microM) delays human neutrophil spontaneous apoptosis in vitro. This effect requires heme oxygenase activity, and depends on reactive oxygen species production and on de novo protein synthesis. Inhibition of ERK and PI3K pathways abolished heme-protective effects upon human neutrophils, suggesting the involvement of the Ras/Raf/MAPK and PI3K pathway on this effect. Confirming the involvement of these pathways in the modulation of the antiapoptotic effect, heme induces Akt phosphorylation and ERK-2 nuclear translocation in neutrophils. Futhermore, inhibition of NF-kappa B translocation reversed heme antiapoptotic effect. NF-kappa B (p65 subunit) nuclear translocation and I kappa B degradation were also observed in heme-treated cells, indicating that free heme may regulate neutrophil life span modulating signaling pathways involved in cell survival. Our data suggest that free heme associated with hemolytic episodes might play an important role in the development of chronic inflammation by interfering with the longevity of neutrophils.

Haptoglobin gene subtyping by restriction enzyme analysis.

Haptoglobin is an acute-phase protein synthesized by the liver in response to inflammatory cytokines (1)(2). Its major function is to form a stable complex with free hemoglobin released from old red blood cells or during episodes of hemolysis, thus preventing iron loss and renal damage (1)(2). Haptoglobin consists of two chains, the α-chain and the β-chain, which are derived from a single polypeptide after proteolytic cleavage (3). The α-chain exists in two major forms, the α1-chain and the α2-chain, which are characterized by the presence (α2-chain) or absence (α1-chain) of a direct repeat of 63 amino acids (1)(2)(3). The structural heterogeneity of the α-chain is the result of an intragenic duplication of ∼1700 bp (3). The 1700-bp sequence of the Hp 1 allele and the upstream 1700-bp sequence of the Hp 2 allele contain exons 3 and 4, and the downstream 1700-bp sequence of the Hp 2 allele carries exons 5 and 6 (3). Three principal genotypes, Hp 1-1, Hp 2-1, and Hp 2-2, result from the haptoglobin gene polymorphism, and we have previously established a technique, based on PCR, for genotype determination (4). Two common sequence versions of the 1700-bp unit exist, which are correlated with the presence of amino acids Asp-Lys (version F) or Asn-Glu (version S) at amino acid positions 52 and 53 in the α1- and α2-chains of haptoglobin and amino acid positions 111 and 112 in the α2-chain (3)(5). Additional differences between the nucleotide sequences of the F-specific and S-specific 1700-bp units occur, including point mutations and small insertions or deletions; however, these differences have no effect on the amino acid sequence (3)(5). Conventional assays for haptoglobin subtyping …

Component therapy.

The dramatic advances that have taken place in recent years in the care of sick and premature infants also have been matched by a similar increase in the use of blood transfusion therapy. Haematological features indicate that a newborn has a blood volume of 85-125 ml/kg the foetal haemoglobin is 60-85% and average Hb in full term infant is 18 gm/dl. By 2-3 months it falls to 11-12 g/dl the main cause of anemia are iron poor diet, weaning diets recurrent or chronic infections and hemolytic episodes in malarious areas. The red cells transfusions are usually top up transfusions, exchange transfusions, partial exchange transfusions. Top up- are for investigational losses and correction of mild degrees of anemias, upto to 5-15 ml/kg. They comprise 90% of all neonatal transfusions and are used in low birth babies in special care units for a maximum of 9-10 episodes. The walk in donor programs once popular are not much in vogue. The threshold for transfusion is 8-10 g/dl Hb for upto 5 weeks. Exchange transfusions are done for correction of anemia, removal of bilirubin, removal of antibodies and replacement of red cells. Ideally plasma reduced red cells that are not older than 5 days are used. It is prepared by removal of 120 ml of standard whole blood donation. The advantage of fresh cells is that hyperkalemia is avoided and good post transfusion survival acceptable red cell oxygen affinity. However it has to be screened for sickle cell disease and G6PD deficiency. Indications for exchange transfusion are kernicterus, neonatal hemolysis, G6PD deficiency, ARDS, neonatal sepsis, DIC and neonatal isoimmune thrombocytopaenia. Complications include over transfusion, perforation of major vessels, hypocalcaemia, citrate toxicity, hypothermia, hypoglycaemia, thrombocytopenia, necrotizing enterocolitis, GVHD, bacterial, viral infections. Partial exchange transfusions are done for symptomatic anemia, where Hb<10 g/dl, it is indicated in polycythemia and hyperviscosity syndromes. Exchange volume = Blood volume x (observed Hct-Desired HCt) divided observed Hct. Points to consider-there is weak expression of ABO antigens so particular care while grouping. Transfusing volumes should be 2-5 ml/kg/hour in paediatric bags of 50-100 ml with infusion devices. Platelet transfusion are indicated in neonatal throbocytopaenia, thrombocytopaenia due to sepsis, DIC, bacterial pathogens, CMV, TORCHS, Obstetric conditions such as pre eclampsia, intrauterine death abruption placenta birth injury hypoxia schock neonatal iso immune thrombocytopaenia and maternal ITP. Administration 1 RDE/pack per 2.5 kg single dose of fresh platelets less than 24hrs which contains 55 x 10(9) cells. This also contributes fresh plasma so is useful for coagulation defects also, though there is a risk of CMV and GVHD due to leucocyte contamination. Granulocyte concentrate; Gravity leucopheresis-1:8 ratio of 60 ml of 6% HES made to stand for 1hr.

Blood versus marrow hematopoietic allogeneic graft

Allogeneic G-CSF-mobilized blood cell transplantation (BCT), an alternative to allogeneic bone marrow transplantation (BMT), is associated with enhanced engraftment and accelerated hematopoietic recovery. In addition, immune reconstitution and overall alloreactivity after BCT versus BMT differ significantly. Indeed, despite an increased number of donor T cells infused, the incidence of acute graft-versus-host disease (GvHD) after BCT appears to remain identical or lesser than after BMT. On the other hand, a higher risk of chronic GvHD has been reported after BCT. In a SFGM phase III trial, 101 patients with early leukemia and an HLA-matched sibling donor randomly received a BCT or BMT. BCT was associated with a higher number of infused CD34+ cells, accelerated platelet and neutrophil reconstitution, fewer platelet transfusions and similar acute GvHD incidence. However, chronic GvHD occurred more frequently after BCT. With a median follow-up of 20 months, relapse, survival and leukemia-free survival were not different. In the course of this study, immune parameters related to the graft as well as to early reconstitution were prospectively examined. T cells subsets, B cells, NK cells and monocytes numbers were significantly higher in BC grafts (versus BM). T cells in BC grafts were less activated than in BM grafts. Frequency of IFN-γ, IL-2- and TNF-α-secreting cells and single-cell IFN-γ production potential was reduced in BC graft. One month after BCT, blood T-cell counts were 3-fold higher than after BMT. Moreover, post-BCT T cells were less activated and counts correlated with the number of T cells infused with the graft, which was not the case after BMT. Several acute hemolysis episodes, resulting from anti-A and/or -B donor-derived Ab directed at Ag present on recipient red blood cells (minor ABO mismatch), have been described after BCT. Recipients indeed exhibited significantly increased anti-A and/or -B Ab titers after BCT, particularly in the setting of a “minor” ABO mismatch. Furthermore, the frequency of anti-HLA Ab early after BCT was significantly increased (despite the reduction in platelet transfusion requirements). The higher number of activated B cells and/or CD4 T cells and monocytes in a BCT graft and/or the higher number of circulating CD4 T- and B-cells after BCT could be associated with the enhanced alloAb production. G-CSF-induced TH2 cytokine profile of the T cells present in the graft could also be contributive. Recent studies have determined that BC grafts contained a higher number of type 2 dendritic cells (DC2), themselves associated with high frequencies of TH2 CD4+ cells. Since chronic GvHD is associated with the occurrence of Ab-mediated auto-immune-like syndromes, it is tempting to speculate that a higher incidence of chronic GvHD may result from these findings. In conclusion, BCT results in clinically relevant distinct hematopoietic and immune reconstitution patterns.