Heinz Body Anemia--An Ultrastructural Study. I. Heinz Body Formation

The ultrastructural changes in red blood cells exposed to phenylhydrazine,either in vivo or in vitro, are described. There is an age-dependent gradientof red cell sensitivity to this drug which includes the more mature reticulocytes as well as the population of circulating erythrocytes. Oxidative denaturation of hemoglobin and the formation of Heinz bodies, which constitute themajor drug-induced lesion, are accompanied by a regular sequence of structural changes commencing in the central cytoplasm of erythrocytes and thedrug-sensitive reticulocytes. These early changes often appear in close associaion with clusters of mitochondria. The initial morphologic lesion has anapparently crystalline structure and the significance of this stage is discussed.Heinz bodies grow by coalescence and condensation and finally come tolie just beneath the cell surface. Here they result in considerable distortionof cell shape and deformation of the plasma membrane. Thus, phenylhydrazine administration produces in red blood cells extensive ultrastructural alterations both in hemoglobin and in the cell membrane which may have considerable bearing on the fate of these cells in the circulation.

Submitted on September 29, 1964 Accepted on October 9, 1964     

Association between HL-A and Red Cell Antigens. An AutoAnalyzer Study

Abstract. Sera containing cytotoxic HL-A antibodies were examined in order to detect red cell antibodies, using an AutoAnalyzer. In sera with cytotoxic anti-HL-A2 antibodies, we were able to demonstrate haemagglutinins that reacted mainly with red cells from HL-A28 positive donors. In sera with cytotoxic anti-HL-A8 antibodies, we found antibodies that reacted with red cells from some, although not all, HL-A8 positive donors. The haemagglutinins gave indentical and highly reproducible reaction patterns, but there were marked variations in antigenic strength.     

The Use of FE59 and CR51 for Estimating Red Cell Production and Destruction: An Interpretive Review

In surveying some of the limitations of studies with Cr⁵¹ and Fe⁵⁹ my purposehas been not to deny their usefulness but to put them in perspective. Thesetechnics have made possible many extensions of the fundamental understanding of red cell physiology and iron metabolism. They will continue to be valuable experimental tools. It is hoped that if some of the limitations of isotopetechnics are considered, the non-experimental use of these isotopes will beconfined to situations in which otherwise unavailable information of diagnosticor therapeutic importance can be obtained. Unfortunately isotope technicsare used when more conventional means would be adequate or even preferable. An extreme example is the suggestion²⁴ that repeated Fe⁵⁹ turnoverstudies might be used to determine the total dose of parenteral iron (as Imferonor saccharated iron oxide) to be given in iron deficiency anemia, pointing outthat in so doing the possibility of iatrogenic hemochromatosis could be avoided.The usual calculations for determining dose²⁵ however are not only safer butmore accurate.

The use of Fe⁵⁹ and Cr⁵¹ entails some risk, the main hazards being hepatitis,with the use of donor plasma or cells, and the possibility of untoward effectsfrom radiation. An estimate of the risk of hepatitis can be gained from itsincidence after transfusion. The radiation hazard is more difficult to assess.Leukemia has occurred after large doses of radiation but the extent of the radiation hazard is unknown from the much smaller doses of radiation employedin the usual isotopes studies. Certainly the risk is not such as to preclude theuse of isotopes to obtain information essential for diagnosis. However, whensuch information can be obtained by other means or when results cannot beadequately interpreted, the use of isotopes in clinical medicine appears unwarranted. In considering the use of isotopes in the doubtful case, the dose ofradiation to be delivered should not be thought of as an isolated event butrather as adding to a total radiation dose, which as shown by the British survey²⁸ may be appreciable.

Submitted on December 6, 1960 Accepted on April 3, 1961     

Haemolysis in Cold Agglutinin Disease: The Role of C''and Cell Age in Red Cell Destruction

The mechanism of red-cell destruction was studied in a patient with cold-agglutinin disease. It was confirmed that the patient's red cells were abnormally resistant to lysis by the anti-I cold agglutinin in the serum. This increased resistance is probably due to the accumulation of C'on the red-cell membrane. It was found that red cells were only protected if C'was brought on to the red cell by anti-I antibodies and not if this was the result of treatment with normal serum at low ionic strength. The resistance was also found only to apply to lysis by anti-I antibodies and not to a haemolytic rabbit anti-human-red-cell serum. These results can be explained by assuming that inactive C'complexes around the I-antigen were blocking receptor sites for new haemolytic C'factors.
It was shown in normal individuals that red-cell age does not effect the susceptibility of the cells to lysis by anti-I serum. In the patient with cold-agglutinin disease, however, the young cells were preferentially lysed, probably because these cells had not yet accumulated C'to the same extent as the more mature cells and were, as a result, less resistant to haemolysis.     

Congenital Dyserythropoietic Anemia Type II: Ultrastructural and Radioautographic Studies of Blood and Bone Marrow

A 12-yr-old white girl with congenitaldyserythropoietic anemia (CDA) type IIwas studied. Excessive cytoplasmicmembranes (appearing like "doublemembranes") were found in the majority of the normoblasts. There wasmarked decrease in the uptake of tritiated thymidine in the binucleatednormoblasts as demonstrated by radioautography. The results suggest thatthe cells with more severe structuralcytoplasmic abnormalities and/or decreased DNA synthesis are destroyedwithin the bone marrow, and the circulating red cells are derived from a lessabnormal population of precursors.

Submitted on May 28, 1971 Revised on August 9, 1971 Accepted on August 10, 1971     

Studies on the Formation of Heinz Bodies. I. Methemoglobin Production and Oxyhemoglobin Destruction

A study of the changes in hemoglobin of erythrocytes incubated with varioustest substances has been reported. The concentrations of oxyhemoglobin andmethemoglobin in these erythrocytes were measured and the term "intact"hemoglobin was introduced, in order to distinguish these pigments from laterdegradation products of hemoglobin. Certain agents which are known to causeHeinz body formation and erythrocyte destruction in vivo have been shown tocause methemoglobin formation and "intact" hemoglobin destruction in vitro,and some of these agents were also shown to produce these changes in vivo.The possible significance of these findings in relation to the role of methemoglobin formation in Heinz body production has been discussed.

Submitted on May 26, 1960 Accepted on September 13, 1960     

Cell Antigens and Cell Specialization. II. Demonstration of Some Red Cell Antigens of Human Normoblasts

Evidence is presented for the presence of the antigens of nine bloodgroup systems on normoblasts. The antigen receptors are detected on all stagesof normoblasts. The in vivo demonstration of gamma globulin coating thereticulocytes and the normoblasts of a patient with erythroblastosis fetalis andthe normoblasts of a patient with autoimmune hemolytic anemia are discussedin relationship to the pathogenesis of the anemia in both conditions.

Submitted on December 24, 1963 Accepted on March 23, 1964     

东亚钳蝎毒诱发大肠癌细胞凋亡的电镜观察

东亚钳蝎毒素是东亚钳蝎毒腺细胞分泌的一种具有多种生物活性的小分子多肽类物质。我们应用体外细胞培养的方法,已证实蝎毒素对大肠癌细胞具有显著的抑杀作用。为进一步探讨蝎毒素抑杀肿瘤细胞的机理,我们用东亚钳蝎毒素灌胃治疗大鼠实验性大肠癌,详细研究了用药后大肠肿瘤细胞超微结构的变化,结果表明,蝎毒素能诱发大肠癌细胞程序化死亡,本实验详尽地观察了大肠癌肿瘤细胞程序化死亡的基本过程,这种作用可能是蝎毒抗肿瘤的重要途径之一。     

Studies on the Formation of Heinz Bodies. II. The Nature and Significance of Heinz Bodies

Simultaneous studies were made as to the degrees of Heinz body formation,of change in hemoglobin and of change in osmotic fragility in erythrocytes incubated with certain drugs which are known to produce Heinz bodies in vivo.Certain factors that modify the pattern of effect of drugs on normal adult erythrocytes were also studied.

A close relationship was found between the degrees of Heinz body production and "intact" hemoglobin destruction. Factors that caused variation in thedegree of "intact" hemoglobin destruction caused similar variation in thedegree of Heinz body production. Staining studies suggested that Heinzbodies are protein in nature and derived from hemoglobin. These observationswere taken to indicate that Heinz bodies probably result from the direct effectof certain drugs on intra-erythrocytic hemoglobin.

-naphthol and primaquine were found to cause marked increase in osmoticfragility relative to the degrees of Heinz body production and of "intact" hemoglobin destruction, whereas acetyl phenylhydrazine produced marked Heinzbody production and "intact" hemoglobin destruction with relatively littleincrease in osmotic fragility. These observations were regarded as evidencethat Heinz body production is not the sole determinant of instability of erythrocytes exposed to these various drugs.

Under no experimental conditions was Heinz body production or "intact"hemoglobin destruction found without methemoglobin formation. Evidencewas presented to indicate that Heinz body production could be inhibited bytrapping methemoglobin as methemoglobin cyanide or methemoglobin azide.The suggestion was advanced that methemoglobin is in fact an essential stagein the destruction of "intact" hemoglobin and the formation of Heinz bodies.

Submitted on October 6, 1960 Accepted on January 12, 1961     

Post-Transfusion Hyperhaemolysis in a Patient with Sickle Cell Disease: Use of Steroids and Intravenous Immunoglobulin to Prevent Further Red Cell Destruction

Delayed haemolytic transfusion reactions (DHTRs) are seen more frequently in patients with sickle cell disease (SCD) than in other groups of patients, and are characterised by a positive direct antiglobulin test and the appearance of previously undetected red blood cell (RBC) alloantibodies in the patient's serum. Recently a syndrome of post-transfusion hyperhaemolysis has been described in children with SCD, characterised by destruction of both autologous and transfused RBCs with negative serological findings: continuation of RBC transfusion exacerbated haemolysis further. We describe a case of life-threatening posttransfusion hyperhaemolysis in an adult patient with SCD in whom severe anaemia necessitated further RBC transfusion, which was successfully performed in conjunction with intravenous immunoglobulin. This approach may be useful in the management of post-transfusion hyperhaemolysis in SCD as well as in the management of severe DHTRs.     

Shear Rate Dependency of Red Cell Sequestration in Skin Capillaries in Sickle Cell Disease and Its Variation with Vasoocclusive Crisis

Objective: To develop techniques for assessing the sequestration of red blood cells (RBCs) in skin capillaries of sickle cell disease (SCD) patients due to RBC adhesion to endothelium (EC) and RBC aggregation, and to determine the extent to which these processes correlate with onset of painful vasoocclusive crisis. Methods: Video recordings of nailfold capillaries in the skin of patients with SCD were made during steady-state periods and episodes of painful crisis. A transient low-flow state was induced with a pressure cuff, and reductions in RBC velocity were measured by spatial cross-correlation of light intensity along arterial and venous capillary limbs. An RBC accumulation index (AI) was calculated from RBC flow to represent the percentage of arterial in-flow sequestered in a capillary. An index of hematocrit (HI) was derived from axial distributions of light intensity, and a sequestration index (SI) was calculated to represent the relative increase of venous limb HI relative to that in the arterial limb with onset of stasis. Results: Both AI and SI increased dramatically from zero at steady flow to a maximum as shear rates within the capillary were reduced to zero. The increase was small until shear rates (γ) fell below a transition value (γ_T), following which both AI and SI increased sharply with onset of stasis. For 20 ≤ γ < γ_T the transient increase in AI was significantly elevated in the order AI_CRISIS > AI_{STEADY STATE} > AI_CONTROL, thus reflecting increasing RBC sequestration in the venous limb due to either adhesion or aggregation. Compaction of RBCs in the venous limb was evidenced by increased SI that was greater than control for both steady-state and crisis subjects, but insignificantly elevated during crisis compared to steady state, thus supporting a lesser role of RBC aggregation. Conclusions: Transient sequestration of sickle RBCs in the low-flow state appears to be dominated by RBC-EC adhesion, which becomes enhanced during crisis. Although aggregation may enhance adhesive contact of RBCs with EC, it does not increase to the same extent as the rate of sequestration, thus reflecting a greater role of RBC-EC adhesion.     

Automated Red Cell Antibody Analysis. A Parallel Study

Abstract. A parallel study of red cell antibodies was undertaken using two well-known AutoAnalyzer techniques: the bromelin-methylcellulose (BMC) method as described by Marsh, and the low-ionic strength-Polybrene (LISP) technique of Lalezari. In the present study, minor modifications were made to both methods, without changing the basic principle of antibody detection. The results obtained in this parallel study were compared to standard manual techniques. Screening of 13,135 sera gave 8.7% positive reactions. Of these, 22% were identified manually as being caused by red cell antibodies. Further antibodies could be identified by the machine [described elsewhere].
The sensitivity of the automated methods was generally higher than manual techniques, about 12 times for the BMC and 70 times for the LISP method. Anti-s,-Fy^a,-Jk^a,-Jk^b, and-K were less sensitively detected by the BMC than the most appropriate manual method in a test of 14 commercial typing sera. In no case was the LISP less sensitive than manual techniques. However, hemolyzing antibodies can be missed in rare instances. Anti-D was found at a lower sensitivity level of 20–50 ng/ml with the indirect antiglobulin technique, 5–15 ng/ml with the two-stage papain technique, 24 ng/ml with the BMC method and 0.2–0.4 ng/ml with the LISP method. Some weak anti-D's (5%) reacted more strongly in the BMC than LISP. When anti-D preparations used for Rh prophylaxis were quantitated with both methods, the results agreed well.     

Automated Red Cell Antibody Analysis. A Parallel Study

Abstract. The AutoAnalyzer method of red blood cell antibody detection can be adapted to identify the serological specificity of these same antibodies. In the present study, 1,152 AA-positive sera obtained during AA screening were investigated. In 217 (19%) of these sera blood group antibodies were identified by manual techniques. Since the AA techniques are generally more sensitive than manual methods an attempt was made to identify antibodies by the AA methods in 823 sera which were negative manually. The low-ionic strength-Polybrene (LISP) method was found to be suitable for antibody identification because of its sensitivity in the detection of most red cell antibodies and the small technical deviations of OD from the base-line. The bromeline-methyl cellulose (BMC) technique is less sensitive and often more difficult to interpret in antibody identification. In some cases, however, this method was the only one that gave positive reactions. In 214 instances antibodies were identified by the AA techniques, about half of which were anti-D's, the others being RBC antibodies of more or less rare varieties. The relative frequency of these rare antibodies was higher in the group of manual-negative sera, 47 versus 21%, normally found. Positive identification but no clear-cut blood type antibody was found in 155 cases (19%). Indications were obtained that some of the reactions were due to HL-A antibodies. Antibodies to HL-A7 and HL-A5 were found to react with red cells. In addition the AA techniques seemed to be more sensitive to detect RBC autoantibodies than standard manual methods. However, the usefulness of the AA techniques for routine antibody identification is diminished by the relative slowness of the machine.     

Effect of a Red Blood Cell Transfusion on Biological Markers Used to Determine the Cause of Anemia: A Prospective Study

Background

Blood test results required for the evaluation of anemia are considered difficult to interpret after red blood cell transfusion. However, this hypothesis is neither supported by a strong physiological rationale nor is it evidence based.

Studies on Human Erythrocyte Nucleotide Metabolism. II. Nonspherocytic Hemolytic Anemia, High Red Cell ATP, and Ribosephosphate Pyrophosphokinase (RPK, E.C. 2.7.6.1) Deficiency

A 29-yr-old black woman was found tohave a long-standing, nonspherocytichemolytic disorder associated with amarked reduction in the activity oferythrocyte ribosephosphate pyrophosphokinase (RPK, PRPP synthetase, E.C. 2.7.6.1). Although the patient’s erythrocytes had about 50% ofthe average RPK activity of normalmature human erythrocytes, this levelrepresented only about 20-30% of theactivity in comparable reticulocyte-richblood samples from patients with othertypes of hemolytic anemias. The concentrations of adenosine triphosphateadenosine diphosphate, adenosinemonophosphate and, therefore, of totaladenine nucleotides in her erythrocytes were markedly increased, evenwell above the levels in extracts ofcomparable reticulocyte-rich bloodsamples. ATPase activity was increased three- to fourfold, consistentwith the reticulocytosis. Adenylate kinase and adenine phosphoribosyltransferase activities were normal. The activities of all enzymes of the Embden-Meyerhof and hexose monophosphateshunt pathways and enzymes relatedto glutathione metabolism were normal or increased, consistent with thereticulocytosis. The concentrations ofglycolytic intermediates, other thanadenine nucleotides, were normal. Theconversion of glucose, adenosine, andinosine to lactate was normal or increased. Autohemolysis was of theDacie Type II. The concentrations oferythrocyte-reduced glutathione werehigh normal or elevated. The stainedblood film showed a striking degree ofbasophilic stippling of the erythrocytes. Studies of the erythrocytes ofthe patient’s only known relative, ason, have failed to reveal any hematologic or enzymatic abnormalities. Adirect causal relationship betweenRPK deficiency, high ATP concentrations, and nonspherocytic hemolyticanemia could not be derived from datanow available. The final decision as towhether the deficiency is primary andcausative or is an epiphenomenon requires investigation of additionalcases.

Submitted on August 30, 1971 Revised on November 15, 1971 Accepted on November 16, 1971     

Studies in Red Blood Cell Preservation: 9. The Role of Glutamine in Red Cell Preservation

Previous studies have demonstrated that an additive solution containing ammonium chloride (NH₄+) and phosphate (Pi) in addition to adenine, glucose and mannitol would support red blood cell (RBC) in vitro characteristics and in vivo 24-hour viability after storage for 9 weeks. The purpose of the present study was to determine if NH₄+generated by the action of glutaminase on glutamine could be substituted for added NH₄+salts. Packed RBCs were stored with equal volumes of adenine, glucose, mannitol, and citrate containing additive solutions with 10 m M glutamine (EAS 31) or with 10 m M glutamine and either 10 (EAS 36) or 20 m M (EAS 37) Pi. One aliquot was stored with Adsol®. The mean ATP levels of the RBCs stored in the glutamine plus phosphate EASs were 132 (10 m M Pi) and 144% (20 m M Pi) of the initial levels at 28 days, and at 84 days remained at 48 and 56%, respectively. The ATP levels of the RBC stored in Adsol were 105 and 25% at 28 and 84 days of storage, respectively. Percentage hemolysis and vesiculation was significantly lower (p<0.01) for RBCs stored in glutamine and glutamine plus phosphate as compared to RBCs stored in Adsol. The levels of NH₄+were 22 to 34% higher in the EASs than in Adsol at the end of 84 days of storage, suggesting that glutamine is broken down by glutaminase to generate NH₄+. The mean corpuscular volumes (MCVs) of RBCs in EASs 36 and 37 were substantially higher than in Adsol throughout the course of storage (p<0.01). However, the MCVs in the additive containing glutamine and no Pi were higher only for 42 days.     

Erythropoiesis: Splenic Immunoglobulin Variable Region Genes Encoding Red Blood Cell Binding Fab Fragments in Autoimmune Hemolytic Anemia

An absolute requirement for the V(H(4-34) ) immunoglobulin (Ig) variable (V) region heavy chain (V(H) ) gene has been demonstrated in pathogenic cold agglutinin autoantibodies. Investigation of IgG binding anti-Rhesus (Rh) alloantibodies provides further evidence of V gene restriction in red blood cell (RBC) binding antibodies and demonstrates that the V(H(4-34) ) gene used to form cold agglutinins may also encode RBC antibodies of varied specificities. We reasoned that a similar V gene restriction may be evident in the gene segments encoding IgG anti-RBC autoantibodies mediating autoimmune hemolytic anemia (AIHA). To further examine this question IgG Fab fragment phage display libraries were constructed from the spleen of a patient with AIHA. The index autoantibody appeared to have incomplete anti-C specificity and bound all panel RBCs except Rh null. The Fab fragment phage display libraries were therefore panned twice on CDE/CDe RBCs and binding phage were eluted. Binding of the phage displayed Fab fragments to RBCs was confirmed by immunoflourescence and flow cytometry. Specificity was confirmed by the absence of binding to Rh null cells, murine RBCs and to human peripheral blood lymphocytes. Molecular analysis of Ig V genes encoding the pan RBC binding Fab fragments revealed a relative V(H) gene restriction and evidence of somatic mutation. The V(H(3) ) family member V(H(26) ) was prominent in RBC binding Fabs. The V(H(3) ) family member hV3005 and the V(H(4) ) family DP-65 gene segments also encoded RBC binding Fabs. The J(H(4) ) gene segment was present in all binding clones. Varied kappa and lambda light chain (V(L) ) genes were identified by sequencing and no single light chain was prevalent. Three of the ten V(L) and two of the three V(H) identified by sequencing appeared to derive from germline genes previously noted to have RBC binding specificity. We conclude that splenic Ig V genes can encode pan RBC binding antibodies with specificities similar to autoantibodies found in AIHA and that V(H) gene segment utilization by these antibodies is derived from a limited pool of somatically mutated V(H) gene segments.     

Congenital Erythropoietic Protoporphyria: II. An Experimental Study

Investigations into the pathophysiology of erythropoietic protoporphyriasuggest that the basic defect is an inborn error of metabolism resulting inthe overproduction of protoporphyrin IX by the bone marrow not in responseto a failure of heme synthesis. A method is described for evaluating the invitro synthesis of protoporphyrin and heme by human bone marrow.

Submitted on January 10, 1963 Accepted on July 3, 1963