Erythrokinetics in Cooley''s anemia

Blood production and destructions have been measured in four patients withCooley’s anemia. Methods employed included the determination of erythroid/myeloid ratio of the marrow, reticulocyte count, plasma iron turnover and redcell utilization, Cr⁵¹ survival and fecal urobilinogen. Rates of production obtainedby these measurements have been compared to normal.

Patients with Cooley’s anemia have been shown to have an increased turnover of hemoglobin constituents comparable to the maximal response seen inother hemolytic anemias. There is, howvever, a marked decrease in maximaldelivery of erythrocytes to the peripheral blood amounting to about 50 per centin the mildly anemic patients and 85 per cent in severely anemic patients. Therate of destruction of circulating erythrocytes was similar in the three patientsstudied. The severity of anemia was therefore largely related to the productiondefect.

It was concluded that the defect in Cooley’s anemia is not in total hemoglobin synthesis, but in the fabrications of circulating erythrocytes, which inturn have the associated manifestations of hypochromia, increased percentageof fetal hemoglobin and shortened survival time.

Submitted on May 23, 1956 Accepted on June 25, 1956     

Hematologic observations on patients with sickle cell anemia sustained at normal hemoglobin levels by multiple transfusions

(1) Three sickle cell patients were sustained at normal hemoglobin levels for3-4 months by means of repeated transfusions of fresh blood.

(2) In response to transfusions, there was a decline in reticulocytes to normallevels during the first 5-7 days of observation. During periods in which the hemoglobin was maintained at high-normal or super-normal levels, the reticulocytevalues were depressed below the normal range. A distinct reticulocyte responsewas observed when the hemoglobin declined to approximately 11.0 Gm. per centfollowing cessation of transfusions.

(3) Employing anti-M differential agglutination, a simple exponential declinewas observed in the number of sickle cells in each patient’s circulation duringthe period of sustained normal hemoglobin concentration.

(4) The continued production of new sickle cells during the first week ofobservation complicated the interpretation of the differential agglutinationdata, but provided indirect evidence for the presence of an especially short-livedproportion of the patient’s cells. Support for this concept was derived from theradioactive iron utilization studies performed in Case 1.

Submitted on November 14, 1955 Accepted on March 9, 1956     

THE EXCRETION OF UROBILINOGEN IN THE STOOLS AND URINE DURING MALARIAL INFECTION

In 10 cases of malaria (6 benign tertian, 4 malignant tertian), the excretion ofurobilinogen in the stools and in the urine was studied. In all 10 cases the amountof urobilinogen excreted in the stools was found to be increased. After defervescence and disappearance of parasites from the blood the excretion gradually declined. The increased excretion of urobilinogen in the stools was the constant andsometimes the only evidence of increased blood destruction occurring at times in the complete absence of jaundice and reticulocytosis. Increased excretion of urobilinogenin the urine was not a constant feature.

It is suggested that the development of jaundice and of urobiligenuria is duenot only to the liberation of pigments by the hemolysis, but to a disturbance in theliver function.

This study lends further confirmation to the concept that the only unequivocalevidence of increased blood destruction is shown in an increased output of urobilinogen in the feces.

Note: The author is indebted to Dr. M. Rachmilewitz for his suggestions and criticisms.

     

Hookworm Anemia: Iron Metabolism and Erythrokinetics

Iron metabolism, balance of red cell production and destruction and ironabsorption from hemoglobin were determined in 11 patients with heavy hookworm infection and severe anemia.

The plasma iron, total iron binding capacity, bone marrow hemosiderinand plasma Fe⁵⁹ clearance are in agreement with the idea that the anemia associated with hookworm infection is of the iron deficiency type.

The rate of red cell production measured by the E/M ratio, absolute reticulocyte count and plasma iron turnover showed an increase to about twicenormal, while the rate of destruction estimated by the T erythrocytesurvival showed a destruction about 5 times normal. This unbalance betweenproduction and destruction could explain the severity of the anemia.

The increase of fecal urobilinogen output to twice normal was interpretedas due to the metabolism of the hemoglobin lost into the intestine rather than toan increase of hemolysis.

The estimation of fecal blood loss in the patients whose red cells weretagged with Cr⁵¹ and Fe⁵⁹, showed that the radioactivity counted with Fe⁵⁹was only about 63 per cent of the radioactivity counted with Cr⁵¹. This difference was interpreted as due to iron absorption from the hemoglobin lostinto the intestine.

The mean daily fecal excretion of iron reaches 4.7 mg. Since the ironmetabolism in these patients is in equilibrium, we have concluded that theiron loss is replaced by the iron from food; this is in addition to the 3 mg.hemoglobin iron which is reabsorbed from the blood lost into the gut.

Submitted on January 9, 1961 Accepted on April 2, 1961     

Pyridoxine-Responsive Anemia

1. Two patients with microcytic hypochromic anemia, hyperferremia, andhemosiderosis have been described .A partial hematologic remission was observed in both patients following the administration of pyridoxine.

2. Interruption of pyridoxine therapy resulted in reticulocytopenia, a decline in the concentration of hemoglobin and a decrease in free erythrocyteprotoporphyrin. Increased excretion of kynurenine and kynurenic acid, butnot xanthurenic acid, was observed in the urine of one of the patients following administration of tryptophane. The excretion of tryptophane metaboliteswas within normal limits in the second patient.

3. Reinstitution of pyridoxine therapy was followed by reticulocytosis, anincrease in free erythrocyte protoporphyrin and an increase in the concentration of hemoglobin. The excretion of tryptophane metabolites in the urinefollowing the administration of tryptophane was within normal limits in bothpatients. However, microcytosis, hypochromia, anemia and hyperferremiapersisted.

4. The administration of brewer’s yeast, Valentine’s liver extract, calciumdisodium ethylenediamine tetra-acetic acid, ethinyl estradiol, pantothenicacid, pyridoxal, pyridoxamine, ascorbic acid, niacin, riboflavin, glutamic acidand tryptophane failed to elicit a further hematologic response.

5. These patients have been compared with the seven other reported casesof "pyridoxine-responsive" anemia.

Submitted on April 20, 1961 Accepted on May 13, 1961     

Unbound Amino Acids in the Plasma and Erythrocytes of Children with Various Anemias and with Erythroblastosis Fetalis

The unbound amino acid concentrations in the plasma and erythrocytesof 24 children with anemias of various types have been determined by themethod of salt-saturated paper chromatography (iron deficiency anemia—8 subjects; sickle cell anemia—3 subjects; hereditary spherocytosis—3 subjects; erythroblastosis fetalis—4 subjects; and 1 subject each for thalassemiaminor, thalassemia major, unstable reduced glutathione, acquired hemolyticanemia, autoimmune hemolytic anemia, S & F hemoglobinopathy). Anemiaper se was found to have little effect on the plasma amino acid concentrations,although a marked increase in the erythrocyte concentrations of every aminoacid determined was found. In multiple regression and correlation analyses,the unbound amino acid concentrations in the erythrocytes were found tobe a function of both the reticulocyte counts and the blood hemoglobin content, with a somewhat greater dependence on the latter. No recognizabledistinction as to the disease category was evident in the unbound aminoacid patterns of the anemia patients. The extent of the unbound aminoacid elevations in the erythrocytes was largely a reflection of the severity ofthe anemia.

The subjects with erythroblastosis fetalis were in a category different fromthose with anemia. Their erythrocyte amino acid concentrations were probably the same as those of normal babies during the first few hours after birth.

Submitted on October 2, 1964 Accepted on November 20, 1964     

Studies on abnormal hemoglobins. III. The interrelationship of type S (sickle cell) hemoglobin and type F (alkali resistant) hemoglobin in sickle cell anemia

1. It could be demonstrated that of the three tested types of human hemoglobin—N (normal adult), S (sickle cell) and F (fetal)—only the reduced S compound shows tactoid and gel formation in sufficiently concentrated solutions.These physico-chemical phenomena may be used for the qualitative identificationof S hemoglobin.

2. The alkali resistant hemoglobin fraction, present in sickle cell anemiaerythrocytes (but not in trait red cells), was concentrated in purified form. Notactoid or gel formation could be elicited. Therefore, this alkali resistant pigment does not appear to be a variant of S hemoglobin. It seems probable thatsickle cell anemia erythrocytes contain two separate types of pathologic hemoglobin (S and F) which are not directly related to each other.

Submitted on June 21, 1951 Accepted on July 23, 1951     

Pernicious anemia and related anemias treated with vitamin B12

Eleven cases treated with vitamin B₁₂ have been presented. Eight patients withpernicious anemia in relapse responded hematologically. Two patients with mildneurologic involvement were relieved by therapy with B₁₂ alone.

Consideration of the quantities of the crystalline vitamin required to promotemaximal erythropoiesis in pernicious anemia indicates that less than about 0.75µg. daily in doses at intervals of several days will not suffice to establish and maintain blood values as high as does adequate treatment with liver extract. Parenteraldaily doses of 1.0 µg. promoted good erythropoiesis in one patient, although itappears that the maximum rate of hemopoiesis may require the initial averagedaily dose of approximately 3.0 µg.

The reticulocyte count is an unreliable quantitative criterion of activity or adequacy of therapy.

It is suggested that hemopoietic factors in addition to PGA and B₁₂ may berequired by some patients to obtain maximal erythrocyte levels.

Vitamin B₁₂, as well as PGA, effects a reduction in the fecal urobilinogen output of patients with pernicious anemia. The significance of this finding is discussed.

No change in urinary excretion of pteroylglutamate or of porphyrin was detected in patients treated with vitamin B₁₂.

     

Idiopathic Dyserythropoietic Jaundice

1. The term "dyserythropoietic jaundice" is used to designate a peculiarabnormality characterized by marked overproduction of bilirubin and associated with unconjugated hyperbilirubinemia and great excesses of fecalurobilinogen. Labeling with N¹⁵ glycine revealed that these excesses weremainly unrelated to destruction of mature circulating red cells, which hadonly a slightly shortened life span.

2. The hyperplastic, normoblastic bone marrow exhibited considerablephagocytosis of red cells and normoblasts and marked hemosiderosis withmany iron-laden phagocytes. Siderocytes were relatively rare in the peripheralblood, which also exhibited consistent reticulocytosis, 3-10 per cent, butnormal erythrocyte porphyrin concentrations. There was increased plasmairon turnover but decreased appearance of Fe⁵⁹ in the circulating red cells.

3. The possibility is considered that there is a basic abnormality in hemoglobin synthesis in the normoblasts, with excessive production of hemoglobin or heme which is then converted to bilirubin either within thenormoblast or after excretion into the blood. The observed phagocytosis ofnormoblasts and erythrocytes may be a secondary rather than basic disturbance of the disease.

4. This case appears to represent a unique form of chronic jaundice dueto a remarkable overproduction of bilirubin without evidence of hemolysisin the ordinary sense. The extent to which this is related to intramedullarydestruction of young red cells or to a basic abnormality in hemoglobin synthesis in the normoblasts, as above, cannot be determined. It is consideredless likely that the overproduction is due to a true shunt—that is, bilirubinformation from porphyrin precursors. The marked dyserythropoiesis suggeststhat the overproduction of bilirubin was medullary rather than hepatic,although the latter is not excluded.

Submitted on December 26, 1963 Accepted on March 3, 1964     

Studies on the serum haptoglobin level in hemoglobinemia and its influence on renal excretion of hemoglobin

A short survey is given of the literature on haptoglobin, the hemoglobin-binding serum protein, its properties and biologic variations. The principles of anelectrophoretic method for quantitative determination of the serum haptoglobinare described.

Electrophoretic studies showed that haptoglobin has a high affinity for hemoglobin at physiologic pH and that every haptoglobin molecule can bind at least2 hemoglobin molecules.

Observations made following the intravenous injection of hemoglobin showed:

that hemoglobin administered intravenously is bound by the haptoglobin;

that free hemoglobin is not demonstrable until more hemoglobin has beeninjected than can be bound by the haptoglobin;

that the complex hemoglobin-haptoglobin is eliminated from the plasma afterintravascular hemolysis or intravenous administration of hemoglobin withoutbeing excreted in the urine;

that the hemoglobin-haptoglobin complex is removed from the plasma at aconstant rate during the major part of the elimination period;

that the haptoglobin level will fall to nil within 24 hours, if the amount ofhemoglobin injected is sufficient to bind all the haptoglobin available. Duringthe following days the rate of formation of haptoglobin can be studied.

From the data available it can be concluded that hemoglobinuria cannotappear until the amount of hemoglobin administered intravenously or the amountliberated intravascularly exceeds the binding power of the haptoglobin and thereabsorption capacity of the tubules. The variation observed by earlier authorsin the so-called renal threshold for hemoglobin on intravenous injection of hemoglobin can be explained among other things by the variation in the haptoglobincontent in one and the same subject, i.e., if the haptoglobin level is low, thethreshold value will also be low, and vice versa.

Submitted on August 21, 1956 Accepted on November 28, 1956     

Hemolytic reactions produced in dogs by transfusion of incompatible dog blood and plasma; serologic and hematologic aspects

1. Dogs injected intravenously with dog erythrocytes containing one or moreantigenic factors lacking in their own red cells developed iso-hemagglutinins andhemolysins exhibiting characteristics of immune antibodies.

2. Transfusions of incompatible whole dog blood and plasma were carried outunder controlled conditions. Pretransfusion observations were made and followedby closely spaced post-transfusion measurements of serologic and hematologicalterations.

3. The rate of destruction of incompatible donated corpuscles was determinedby tagging the cells with radioactive iron and also by employing the technique ofdifferential agglutination of erythrocytes. It was thereby shown that all of theincompatible donated cells disappeared from the recipient’s circulation withinthe first thirty to ninety minutes following transfusion. The probable mechanismsand relative importance of intra- and extravascular destruction of erythrocytes arebriefly discussed.

4. Destruction of recipient dogs’ corpuscles by donated immune plasma wasrelatively slow, and spherocytosis and increased osmotic fragility of the recipients’ cells were evident for periods as long as twenty days. These observationsare compared with those made in human beings after transfusions of plasma and ofblood from dangerous universal donors.

5. The titer of complement in the sera of recipient dogs was sharply reducedfor at least five hours after all transfusions of incompatible whole blood, but isoagglutinin titers were less regularly reduced after such transfusions.

6. Other notations of interest included estimates of the concentrations of serumbilirubin, sodium and potassium, determinations of clotting time, prothrombinconcentration, and observations on red cell morphology, intravascular erythrophagocytosis, and shifts in distribution of leukocytes and in the electrophoreticpatterns of plasma.

Note: ACKNOWLEDGMENTSIt is a pleasure to acknowledge the technical assistance of Mrs. Jane Peters, Miss Mary Jane Izzo andMiss Shirley Deshon.

     

THE ANEMIA OF INFECTION, VI. THE INFLUENCE OF COBALT ON THE ANEMIA ASSOCIATED WITH INFLAMMATION

The influence of cobalt on the anemia associated with inflammation has beenstudied in three experiments involving observations in 108 rats.

It was found that by the simultaneous administration of cobalt the anemia associated with inflammation, as produced by the injection of turpentine, could beprevented from developing and polycythemia appeared instead.

This effect was accompanied by hypoferremia and an increase in erythrocyteprotoporphyrin values similar to those encountered when anemia develops in association with inflammation.

Similar, though less marked, chemical changes were observed when only cobaltwas given and polycythemia was produced.

A decrease in plasma albumin was noted in rats injected with cobalt or turpentine, or both, but this was not accompanied by an increased excretion of urinarynitrogen as measured by the urine urea and ammonia.

The observations cited are consistent with the hypothesis that cobalt favorablyinfluences the utilization of iron for the synthesis of hemoglobin.

     

Experimental production of grape cells and their relation to the serum gamma globulin and seromucoids

Hypenimmunization in rabbits with three different antigens produced anelevation of the serum gamma globulin, a plasma cell proliferation and anincrease of the seromucoid content of the serum.

Grape cells and intracellular crystals were found in hyperimmune animals,but not in controls. The globules of the grape cells stained positive withperiodic acid Schiff and with osmium tetroxide dissolved in a non-polar solution, indicating the presence of a mucopolysaccharide.

It is suggested that there is a relationship between the process of immunization—producing hyperglobulinemia, plasma cell hyperplasia and an elevation of the serum seromucoid content—and the presence of grape cells.

Submitted on January 24, 1958 Accepted on March 25, 1958     

Hemoglobin Affinity for Oxygen in Chronic Renal Disease: The Effect of Hemodialysis

The affinity of hemoglobin for oxygen mayincrease significantly in subjects who arehypophosphatemic and alkalotic. Westudied the organic phosphate content andoxygen binding by hemoglobin of red cellsin subjects undergoing hemodialysis, during which time a decrease in plasma inorganic phosphate and an increase inblood pH may occur. Red cell 2,3-DPGwas not correlated with plasma inorganicphosphorus, whereas red cell ATP washighly correlated with plasma inorganicphosphorus when analyses were made onpredialysis samples. Predialysis red cellinorganic phosphorus was highly correlated with plasma inorganic phosphorus,supporting the concept that intraerythrocytic inorganic phosphorus is maintainedby a gradient from plasma to cell. Plasmainorganic phosphorus decreased by 45%during the period of hemodialysis, whereasred cell inorganic phosphorus did notchange. Red cell 2,3-DPG, ATP, and oxygen binding by hemoglobin at standardconditions of temperature, pH, and pCO₂were not altered after 6 hr of hemodialysis. Plasma pH and base excess increasedduring dialysis. The increase in base excess, an estimate of the non-pH-dependenteffect of CO₂ on oxygen binding by hemoglobin, counterbalanced a portion of theeffect of elevated pH on hemoglobin—oxygen affinity under in vivo conditions.Hence, only a slight increase in oxygenbinding by hemoglobin occurred. Moreover, late dialysis symptoms were notassociated with the degree of alkalosis orwith the extent of change in hemoglobin’saffinity for oxygen. Red cell 2,3-DPG content was lower and hemoglobin’s affinityfor oxygen was higher in subjects withchronic renal disease than in nonazotemicsubjects with similar hemoglobin deficits.Moreover, increased red cell ATP in chronicrenal disease patients did not influenceoxygen binding by hemoglobin.

Submitted on June 11, 1973 Revised on July 30, 1973 Accepted on September 13, 1973     

Starch Block Electrophoresis of Plasma and Serum Clotting Factors. Separation of Activated PTC (PTC')

1. Plasma clotting factors separate into two groups on starch block electrophoresis. The contact activation factors—Hageman factor, PTA, and activatedPTA—remain around the origin, whereas the vitamin K-dependent factors—prothrombin, proconvertin, Stuart factor, and PTC—migrate between the-globulins and albumin. AHG, proaccelerin, and thrombin are not recovered.

2. The electrophoretic pattern of serum differs from that of plasma mainly inthe absence of prothrombin and in the presence of activated PTC (PTC’).

3. The electrophoretic mobility of PTC’ is found to differ from that ofnative PTC. This difference may be exploited to separate PTC’ from its nativeform and from Stuart factor.

Submitted on May 12, 1964 Accepted on July 26, 1964     

L.E. Cells in Lymphoma

Two elderly women suffering from lymphoma—lymphosarcoma and Hodgkin’s disease respectively—had positive L.E. cell tests.

In one case the L.E. phenomenon was strongly and consistently positive,as were tests for thyroglobulin antibody and rheumatoid factor; treatmentwith radioactive phosphorus was beneficial.

The origin of antinuclear and other autoantibodies in lymphoma could beattributed to (a) the development of self-reactivity by the neoplasticlymphoid cells themselves, (b) to weakness of homeostatic control over otherself-reactive cells in neoplastic lymphoid tissue, or (c) to the release of arange of abnormal clones, possibly as a consequence of a combination ofgerminal and somatic mutation.

Submitted on December 18, 1962 Accepted on April 3, 1962     

Inhibiting Effects of Human Plasma and Serum on Neutrophil Random Migration and Chemotaxis

Various authors have associated increasedsusceptibility to infectious diseases in certain patients to inhibitors of neutrophilchemotaxis demonstrable in serum or diluted plasma of these patients. The present experiments showed, however, thatserum and diluted plasma but not undiluted plasma from normal human donorsconsistently inhibited chemotactic migration of autologous human neutrophilgranulocytes. Therefore, the presence ofsuch inhibitors in the circulating bloodcan only be assessed by the evaluation ofundiluted plasma. The findings suggestthat the experimental conditions whichhave been used in the past to demonstratesuch inhibitors in the circulating blood ofpatients with increased susceptibility toinfections are inadequate, and resultsneed reexamination. The inhibitors affectrandom locomotion and chemotaxis ofneutrophil granulocytes but not phagocytosis or the metabolic burst resulting innitroblue—tetrazolium reduction. On theother hand, phagocytosis of Staphylococcus albus rendered neutrophils chemotactically unresponsive. The significanceof so-called cellular defects of neutrophilchemotaxis in such patients is also considered.

Submitted on March 6, 1974 Accepted on June 17, 1974     

Sickle Hemoglobin: A Specific Radioimmunoassay

For the quantitation of hemoglobin S, aradioimmunoassay has been developedwhich is specific and highly sensitive.Hemoglobin S was purified by columnchromatography and injected with complete Freund’s adjuvant into goats. Eachgoat serum was tested for reactivityagainst hemoglobins A and S by immunodiffusion and by quantitative precipitation. Hemoglobin A reactivity was removed by absorption with hemoglobin A.One serum so treated was specific forhemoglobin S; it reacted negligibly withhemoglobins A or F. Hemoglobin S waslabeled with ¹²⁵I by the chloramine Tmethod. In the radioimmunoassay, complete precipitation of the antigen—antibody complex was insured by the additionof rabbit antigoat gamma globulin. Thisassay offers reliable and specific quantitation of as little as 1 ng of hemoglobin S.Assuming that 10% of the hemoglobin infetal blood at 16 wk gestation is of adulttype, this assay is capable of detecting theamount of hemoglobin S in 10^-7 ml ofhomozygous hemoglobin S blood. The prenatal diagnosis of sickle cell anemia byradioimmunoassay will require, in addition, a method for demonstrating theabsence of hemoglobin A and a safemethod for obtaining fetal erythrocyteswithout significant contamination by maternal erythrocytes.

Submitted on June 28, 1973 Revised on October 11, 1973 Accepted on November 1, 1973     

Normal Human Lymph Node Cells: An Electron Microscopic Study

Lymph nodes, from 15 patients undergoing surgery for conditions not related to lymphoid tissue disease, have been examined with the electron microscope. The human lymph node cell types—including lymphocytic, reticularand plasma cells—have been described at low and medium electron microscopic magnifications, and the criteria for their identification are discussed. Thecharacteristic features outlined for identification of these cell types provide abasis for comparison with pathologically altered lymph node cells.

Submitted on May 4, 1965 Accepted on September 25, 1965     

The Effect of Amelioration of Anemia on the Synthesis of Fetal Hemoglobin in Sickle Cell Anemia

Two adult patients with sickle cell anemia of blood group A and A₂Brespectively, received sufficient transfusions of group O blood to maintainnearly normal hemoglobin concentrations for 4 months or longer.

Serial samples of the erythrocytes of each recipient were obtained by agglutination with anti-A (and anti-B) serum. The proportion of Hb F in theagglutinated erythrocytes was determined. Early in the transfusion period, amarked rise in the proportion of Hb F was noted. This rise was attributed toprolonged survival of erythrocytes which contained larger proportions of Hb F.In the later part of the transfusion period, the proportion of fetal hemoglobindeclined to pre-transfusion levels or below. However, significant amounts offetal hemoglobin in the erythrocytes of each patient were demonstratedthroughout the period of study, and Fe⁵⁹ incorporation into Hb F in vivo wasdemonstrated in one patient after 4 months of transfusion therapy. Under theconditions of these studies, synthesis of Hb F continued despite prolongedcorrection of the anemia. A decline in the proportion of Hb F in the erythrocytes of one patient after 5 months of transfusions suggested that Hb F synthesis may ultimately be depressed by transfusions. It was suggested that theproportion of fetal hemoglobin observed in the erythrocytes might in certaindiseases reflect the degree of anemia present many months before.

Submitted on February 26, 1964 Accepted on April 5, 1964