The action of leukocytosis-promoting factor on the blood and bone marrow of the guinea pig

Ten normal guinea pigs each weighing approximately 400 Gm. were givenintraperitoneal injections of sterile pyrogen-free saline, and another ten weregiven injections of saline + 25 mg. of Leukocytosis-Promoting Factor (Menkin).The animals were killed 4 hours after the injection and quantitative studiesmade on the blood and the bone marrow.

LPF appears to give rise to a considerable discharge of granulocytes from thebone marrow, much greater than can be accounted for by the leukocytosis whichdevelops. The administration of LPF is also associated with a marked increasein the marrow lymphocytes.

Submitted on September 29, 1955 Accepted on October 28, 1955     

Eosinophilic leukemia; report of a case with autopsy confirmation; review of the literature

1. The data in a case of fatal leukemia with predominant eosinophilia in theperipheral blood and bone marrow are presented; we believe that this case was oneof eosinophilic leukemia.

2. During the period of observation, these eosinophils showed progesssiveimmaturity as the symptoms became more severe. Eventually this "left shift"became so marked that a large proportion of the cells were terminally myeloblastsin both the blood and the bone marrow.

3. Autopsy revealed invasion of many of the tissues and organs with thesemature and immature eosinophil granulocytes and with myeloblasts.

Note: ACKNOWLEDGMENTOur thanks for helpful criticism are particularly extended to Dr. Charles Doan and to Dr. WilliamDameshek.

     

Evaluation of Marrow Granulocytic Reserves in Normal and Disease States

Recent concepts of the relationship of the blood granulocyte mass to themarrow reserve of granulocytes have been reviewed. Evidence has beenpresented to show that the marrow is the chief area of granulocyte "reserves"or "stores." The development of acute leukocytosis in response to a stimulussuch as the intravenous injection of bacterial endotoxin depends upon releaseof cells from the intramedullary pool of granulocytes.

The turnover of the marrow granulocyte reserve (MGR) is an orderlyprocess in the steady state, and determines the form of the curve of DNA-labeled granulocytes in the peripheral blood. From estimates of the turnovertime of the MGR it appears that the granulocyte spends an average time ofabout 10 hours in the peripheral blood. Granulocytes do not appear to recirculate once they have left the peripheral blood and have entered the tissues.However, granulocytes may be sequestered within capillary beds for variableperiods, and may re-enter the circulating blood from such areas. Such cells arenot to be considered as having re-entered the blood from the tissues.

The intravenous injection of a purified bacterial lipopolysaccharide as astimulus to acute leukocytosis is described. The possbile usefulness of thisprocedure in assessing the MGR is discussed.

Submitted on July 28, 1958 Accepted on July 11, 1959     

The combined use of typhoid vaccine and P32 labeling to assess myelopoiesis

1. The intravenous administration of killed typhoid organisms to dogs resultsin the development of a severe leukopenia which is followed by a markedleukocytosis.

2. The myelopoietic response of dogs to typhoid injections is quite similar tothe response following leukopheresis.

3. Leukocyte DNA-P³² studies indicate that in response to typhoid stimulation, as with leukopheresis, the bone marrow constitutes the main reservoir forgranulocytes contributing to peripheral blood leukocytosis.

4. Evidence is presented that, under these experimental conditions, leukocytes, after once having left the vascular tree, are unable to re-enter in any significant numbers.

5. The use of typhoid vaccine to stimulate leukocytosis combined with the useof P³² to measure the fate of cells released from the marrow is presented as anaccurate and reasonably simple method to measure certain aspects of myelopoiesisin the experimental animal.

Submitted on August 20, 1956 Accepted on November 3, 1956     

The Histochemical Study of Zinc Content in Granulocytes in Normal Adults and in Hematologic Disorders

Zinc in granulocytes was determined using a dithizone histochemicalmethod. Investigations were performed in both bone marrow and in peripheralblood from ten healthy adults and in 62 patients with various hematologicdisorders. In bone marrow, zinc appears in metamyelocytes. The amount increases with maturation. In the granulocytes of peripheral blood, zinc valuesare about 30 per cent higher than in bone marrow.

Zinc is significantly decreased in granulocytes in chronic granulocytic leukemia, acute myeloblastic leukemia, multiple myeloma and Hodgkin’s andincreased in chronic lymphocytic leukemia and osteomyelosclerosis.

Submitted on September 16, 1963 Accepted on April 27, 1964     

Granulomatous lesions in the bone marrow in infectious mononucleosis; a comparison of the changes in the bone marrow in infectious mononucleosis with those in brucellosis,tuberculosis, sarcoidosis and lymphatic leukemia

1. The findings in the blood and in aspirated bone marrow in 23 cases of infectious mononucleosis have been described.

2. Unequivocal evidence of involvement of the bone marrow has been found in70 per cent of the cases.

3. Evidence of granulomatous inflammation of the marrow was found in 48 percent of the cases.

4. Epithelioid cells were found in the films of bone marrow in 48 per cent of thecases. These cells appear morphologically identical with those seen in imprints oflymph nodes from infectious mononucleosis and sarcoidosis and with the epithelioid cells seen in films of the marrow in brucellosis, sarcoidosis and tuberculosis.

5. The granulomatous lesions of infectious mononucleosis seem most similar tothose of brucellosis, but they also resemble the small granulomatous lesions ofsarcoidosis and tuberculosis.

6. Lymphocytosis of the marrow as well as of the blood was demonstrated in allcases. Evidence of formation of lymphocytes in the marrow was presented, and thealtered lymphocytes of infectious mononucleosis were found in films of the marrow.The degree of lymphocytosis of the marrow in infectious mononucleosis was shownto be less than that in lymphatic leukemia. Lymphocytosis of the marrow was notfound in brucellosis, sarcoidosis or tuberculosis. The lymphocytic reaction demonstrable in the marrow in infectious mononucleosis is believed to be of value in differential diagnosis.

Note: ACKNOWLEDGMENTSWe wish to acknowledge the generous cooperation of Dr. Ruth E. Boynton and the Staff of theStudent’s Health Service of the University of Minnesota throughout the course of this year long studyof infectious mononucleosis. We are indebted to Dr. T. Edward Bell and Dr. James Cardy for performingthe sternal aspirations. The photomicrographs were made by Mr. Henry Morris.

     

Leukokinetic Studies. VII. Morphology of the Bone Marrow and Blood of Dogs Given Vinblastine Sulfate

The effect of a single injection of vinblastine sulfate was studied in 50mongrel dogs. Nine of 34 dogs given 0.2 mg./Kg. of VLB died with gastrointestinal toxicity and the mortality rate increased as the dosage of VLB wasincreased. The morphologic pattern of leukocyte suppression and recovery inthe bone marrow and blood was studied in detail in surviving animals.

The cells of the bone marrow were markedly affected by VLB. Within 4hours there was an increase in the number of cells in metaphase and, by day1, virtually all proliferating leukocytes and erythrocytes had disappeared. Anorderly repopulation of the bone marrow followed.

The neutrophils, eosinophils, lymphocytes and monocytes of the blood wereall markedly altered in concentration after VLB. Each type of cell first decreased to abnormally small numbers and then increased to abnormally largenumbers in the blood. The curve of disappearance from and reappearance inthe blood differed for each cell type.

The changes in blood neutrophil number and morphology were correlatedwith changes in the blood neutrophil precursor cells of the marrow. The following conclusions were reached concerning the neutrophils and the assumptions implicit to these conclusions were detailed.

1. In the dog, the marrow contains enough post-mitotic granulocytes toreplace those lost from the blood for at least 3 to 4 days.

2. The release of mature neutrophils from the bone marrow is a functionof the rate at which blood neutrophils are lost and proceeds normally evenwhen the marrow granulocyte reserve is partially depleted.

Submitted on March 27, 1963 Accepted on August 20, 1963     

The Granulocyte Response to Endotoxin in Patients with Hematologic Disorders

Sixty patients with disorders involving the bone marrow were tested witha purified bacterial endotoxin given intravenously. Their leukocyte and granulocyte responses were evaluated based on criteria established in normal individuals and in patients with leukocytosis.

Results in patients with chronic myelocytic leukemia, untreated or in relapse, suggest that adequate granulocyte mobilization may still occur if thedisease has been of recent onset or if the count has recently started to rise inspite of therapy. Patients in remission demonstrated adequate granulocytereserves.

Most patients with chronic lymphocytic leukemia in this study respondedwell with an increase in the number of granulocytes. Patients with multiplemyeloma as a group showed inadequate granulocyte mobilization.

This study demonstrates that endotoxin testing is useful for the evaluationof bone marrow granulocyte reserves in patients with hematologic disorders.

Submitted on August 29, 1963 Accepted on November 11, 1963     

A Radioautographic Study of Hemopoietic Repopulation Using Irradiated Parabiotic Rats: Relation to the Stem Cell Problem

These radioautographic studies using parabiotic rats and partial marrowshielding showed that cells responsible for recovery of irradiated bone marrowhad their origin in the shielded marrow. Three morphologically distinct celltypes appeared in the blood of these parabionts, mature granulocytes, smalllymphocytes and monocytoid cells. The monocytoid was the major cell typewhich crossed from the shielded to nonshielded marrow, and the observationssuggested that it is this cell which served as a stem cell for both the erythrocytic and granulocytic cell lines.

Labeled erythroblasts and myeloblasts were observed in the recovering marrow, and the labeling intensity of these cells indicated that they were thesecond or third division products of labeled immigrant cells.

The effect of marrow shielding upon the recovery of lymphopoiesis inspleen, thymus, lymph nodes and bone marrow is also discussed.

Submitted on April 11, 1966 Accepted on June 10, 1966     

Radioautographic Study of Cellular Migration Using Parabiotic Rats

Leukocyte exchange between thehemopoietic tissues of parabiotic ratswas studied subsequent to giving multiple injections of ³H-thymidine to onemember of each pair while arrestingthe cross-circulation. Cell types thatmigrated from one parabiont to theother were segmented granulocytes,small, medium and large lymphocytes,immunoblasts, monocytoid cells, macrophages or their immediate precursors, and plasma cells. Evidence for thetransformation of circulating cells toother cell types was rarely seen. Thelong-lived small lymphocytes wereequilibrated between parabionts, suggesting that this is a single pool ofcells with respect to kinetic behaviorand recirculation. There was no evidence for a trephocytic function oflymphocytes. A small number of bonemarrow lymphocytes coursed directlyto lymph nodes and spleen. Evidenceis given for a limited recirculation ofshort-lived lymphocytes of thoracicduct lymph (TDL), as well as for long-lived cells. Only a few immunoblasts ofTDL recirculated. The majority of cellsthat entered the white pulp of thespleen were long-lived small lymphocytes, while the majority of immigrantcells to the red pulp were monocytoidcells and granulocytes. Many smalllymphocytes originated in splenic redpulp and entered the blood. No immigrant cells to the thymic cortex werenoted, although some small lymphocytes and monocytoid cells enteredthe medullary areas. Immigrant cells tothe bone marrow (less than 2% of thecells in marrow) included monocytoidcells, small lymphocytes, and plasmacells. Evidence for the direct transformation of a circulating cell into a committed blast, based on reduction ingrain count, was noted only in bonemarrow.

Submitted on April 14, 1971 Revised on August 9, 1971 Accepted on August 17, 1971     

The Effect of Etiocholanolone on Granulocyte Kinetics

The effect of etiocholanolone on granulocyte kinetics in 12 hematologicallynormal patients has been investigated using the technic of ³H-DFP labelingof autologous blood in vitro.

Baseline determinations of the total blood granulocyte pool (TBGP), thecirculating pool (CGP), and the marginated pool (MGP) were performed.The values for the total blood granulocyte pools were similar to those previously reported. Following the administration of etiocholanolone, there was a98 per cent increase in the TBGP, which was considered to be due to mobilization of granulocytes from the bone marrow reserve. There was no change inthe ratio of CGP to MGP.

These studies suggest that etiocholanolone may be a useful agent for theestimation of bone marrow reserve.

Submitted on November 21, 1966 Accepted on April 13, 1967     

Correlation of Granulocyte Mobilization with Etiocholanolone and the Subsequent Development of Myelosuppression in Patients with Acute Leukemia Receiving Therapy

1. Etiocholanolone was employed in the assessment of bone marrow granulocyte reserves in a group of patients with acute leukemia prior to theadministration of therapy.

2. Normal levels of circulating white blood cells and granulocytes and/orremission bone marrow status were often associated with abnormal testresponses.

3. Individuals having a positive test response experienced significantly lesshematologic toxicity following therapy than did those patients having a negative response.

4. Etiocholanolone proved to be a safe agent without significant side effects.

5. This test can be helpful in the prediction of toxicity following antileukemictherapy.

Submitted on July 10, 1967 Accepted on October 23, 1967     

Cell cycle kinetics of hematopoiesis before and after in vivo administration of GM-CSF in refractory anemia: Evidence for a shortening of the granulocyte release time

Summary GM-CSF administration to patients with refractory anemia (RA) induces an increase in neutrophils and eosinophils. We studied cell kinetic mechanisms underlying this observation using clonogenic assays and in vivo iododeoxyuridine labeling of bone marrow cells. Cell cycle kinetics were studied in three patients before and during GM-CSF administration (two daily subcutaneous injections of 54 or 108 g). No consistent effect on the relative number of bone marrow CFU-GM was noticed. The DNA synthesis time and potential doubling time of low-density bone marrow cells remained essentially the same. A slight decrease (1.5–3.7%) in labeling index was found, originating from the myelo(-mono)cytic lineage. In all three patients the release time of labeled granulocytes from the bone marrow into the peripheral blood was shortened (before GM-CSF treatment 5–7 days and during GM-CSF 3–4 days). Cell cycle kinetics of CD34⁺ cells were studied in order to obtain kinetic information on immature precursor and progenitor cells. The DNA synthesis time of the CD34⁺ cells was shortened during GM-CSF therapy, resulting in a shorter potential doubling time. GM-CSF administration to patients with RA results in a rise in granulocytes that might be due partly to an accelerated release of granulocytes from the bone marrow compartment into the circulating blood and partly to an increased proliferative activity of the immature precursor and progenitor cells.     

Reticulum Cell Sarcoma Terminating in Acute Leukemia

The records of 113 patients dying at the Francis Delafield Hospital withdocumented reticulum cell sarcoma revealed six cases whose course terminatedin a syndrome resembling acute leukemia. Their course was characterizedby weakness, pallor, petechiae, hemorrhages and hepatosplenomegaly. Theblood showed anemia, leukocytosis (white blood cell count 20,000 to 80,000/cu.mm.) and thrombocytopenia (platelet count [unknown] 100,000/cu.mm.). Differential count in the blood and the bone marrow revealed a high percentage ofimmature cells (35 to 96 per cent). These were identified as reticulum cellsin three patients, as myeloblasts in two and as monocytoid granulocytes in one.In all six patients, this explosive illness terminated in systemic infection orhemorrhage within two months. Therapy with 6-mercaptopurine, adrenalsteroids, or both, gave no benefit.

Submitted on April 2, 1959 Accepted on June 18, 1959     

Analytical Review: The Kinetics of Granulopoiesis in Normal Man

Present knowledge concerning the kinetics of granulopoiesis has been reviewed and quantitative data concerning granulokinetics in normal humansubjects are presented.

A. When granulocytes are labeled in vitro and returned to the circulationof the donor, the distribution of the cells in the circulation and the rate of disappearance of the cells from the circulation can be measured.

1. The total blood granulocyte pool (TBGP) consists of two compartmentswhich are in equilibrium with each other. These pools have been designatedthe circulating granulocyte pool (CGP) and the marginal granulocyte pool(MGP). The size of the pools has been measured in 109 normal male subjects.The mean values, expressed as numbers of cells x 10⁷ per Kg. of body weightwere as follows: TBGP, 70; CGP, 31; and MGP, 39. The mean ratio of the CGPto the TBGP was 0.44.

2. The labeled granulocytes leave the TBGP in an exponential fashion witha mean half-time disappearance (T) of 6.7 hours as determined in 56 normalmale subjects. No evidence has been obtained for a return of granulocytes tothe blood.

3. The mean value for the granulocyte turnover rate (GTR) in 56 normalmale subjects was 163 x 10⁷ granulocytes per Kg. of body weight per day.Thus, the TBGP turns over 2.3 times per day and the turnover time for theTBGP is 10.4 hours.

B. When granulocytes are labeled in vivo by the intravenous administrationof DFP³², the rate of disappearance of granulocytes from the circulation andthe time required for myelocytes to divide, mature and appear in the bloodcan be measured. In addition, the generation time of myelocytes can be approximated. From the time parameters and the GTR, the bone marrow poolsizes and turnover times can be calculated. These determinations and calculations have been made for a group of 21 normal male subjects.

1. The mean half-time disappearance (T) of in vivo labeled granulocytesfrom the circulation was 7.2 hours. This value agrees well with the valueof 6.7 hours obtained after the in vitro labeling of granulocytes.

2. The mean time required for myelocytes to divide, mature and appear inthe blood was 11.4 days.

3. The mean generation time of myelocytes was estimated to be not morethan 2.9 days.

4. The total granulocyte pool in the bone marrow (neutrophilic myelocytes,neutrophilic metamyelocytes and PMN neutrophils) was calculated to be186 x 10⁸ cells per Kg. of body weight with a mean turnover time of 11.4 days.The myelocyte pool was estimated to be 41 x 10⁸ cells per Kg. with a turnovertime of 2.5 days; the metamyelocyte pool consisted of about 76 x 10⁸ cells perKg. with a turnover time of 4.7 days; the average size of the mature marrowPMN neutrophil pool was 69 x 10⁸ cells per Kg. of body weight with a turnover time of 4.2 days.

C. A kinetic model for granulopoiesis, based on the studies with the DFP³²label, is presented. In this model, myelocytes are depicted as approaching aself-perpetuating population of cells. Some cells enter this population frompopulations which are less mature but this latter source of cells is small underconditions of normal steady state kinetics. One of the daughter cells of amyelocyte division remains in the myelocyte population to divide again. Theother daughter cell enters the metamyelocyte population. The metamyelocyteand PMN neutrophil population is incapable of division and cells move throughthis population in sequential fashion in the process of maturation. The cellsthen enter the blood where they equilibrate rapidly between the two bloodcompartments. The cells are removed from the total granulocyte pool in arandom fashion. There is no appreciable pool of granulocytes in the extramedullary tissues of normal subjects and granulocytes do not return from thetissues to the blood. The entire movement of granulocytes from marrow totissues is uni-directional.

Submitted on March 16, 1964 Accepted on April 9, 1964     

SERUM PROTEIN CHANGES IN MYELOGENOUS AND LYMPHOCYTIC LEUKEMIAS AND HODGKIN'S DISEASE

1. Using a methyl alcohol fractionation technic, the albumin, globulin, andtotal protein levels were determined in a series of normal adults and compared withcases of myelogenous and lymphocytic leukemias and Hodgkin’s disease.

2. Statistically significant decreases in albumin and increases in globulin werefound in the cases of Hodgkin’s disease and myelogenous leukemia, but withoutsignificant changes in total protein. Globulin levels above the highest normal valuewere found in 23 per cent of the former and 33 per cent of the latter group.

3. No apparent relationship was noted between the levels of the serum proteinfractions and (1) the hemoglobin level, (2) the erythrocyte count, (3) the peripheral white blood cell picture, and (4) the bone marrow smears.

Note: The authors wish to express their appreciation to Professor Ovid O. Meyer, Department of Medicine,for making available the clinical material in this study and for valuable suggestions. The authors arealso indebted to Professor J. A. E. Eyster, Department of Physiology, for suggestions as to statisticaltreatment of the data.

     

Some Factors Involved in the Effect of X-Irradiation on the Phosphatase Activity of Hematopoietic Tissues

Factors which modify lymphoid distribution of tissues were found to modifythe adenosine triphosphatase activity of these tissues. Starvation or cortisoneinjection, which produces destructive changes in lymphoid tissues, was foundto increase the enzyme activity of spleen and thymus tissues. The greater increment of enzyme activity of the thymus as compared to that of the spleenwas correlated with its normally higher content of lymphoid tissue.

The increase in adenosine triphosphatase activity of hematopoietic tissuesappears to be associated with the type of cells present in the assay medium.With respect to peripheral blood leukocytes of the rat, the cell type is confined largely to lymphocytes and granulocytes. The increase in adenosinetriphosphatase activity of the leukocytes after total-body x-ray was seen toparallel the increase in granulocytes present in the assay medium. The ratioof granulocytes to lymphocytes is not appreciably altered in dog peripheralblood after exposure to total-body x-ray; the adenosine triphosphatase activitysimilarly was not significantly altered. After total-body x-ray (390 r and 780 r),cells isolated from the rat bone marrow displayed a fivefold increase inadenosine triphosphatase activity. This increase was seen to correspond withan increase in the ratio of segmented leukocytes and reticuloendothelial cellsand a decrease in the immature forms of the erythroid and myeloid cells.

The heterogeneous cell mixtures used for our assay procedures permit theobservation that total-body x-irradiation results in an increased enzyme activityof the isolated cells of the peripheral blood, bone marrow and spleen tissue ofthe rat. The increased enzyme activity was associated with the increased ratioof cells with high enzyme activity present in the assay medium.

Submitted on September 22, 1958 Accepted on November 27, 1958     

Constant Post-Irradiation Repopulation Rates and Linear Relationship between Cellular Blood Response and Number of Transplanted Bone Marrow Cells in Inbread Mice

Graded doses of syngeneic bone marrow cells were transplanted into lethally irradiated mice. Repopulation curves of peripheral blood granulocytes and platelets were apparently exponential and parallel after doses larger than 5 times 10⁵ cells. The blood platelet T_d was reduced from 111 h to 53–57 h, and granulocyte T_d from 57 to 40 h in transplanted groups. The mean blood cell counts were reproducible enough to be used as a biological assay of the amount of bone marrow cells transplanted. Linear relationship between increment of blood cells up to day 16 and number of bone marrow cells transplanted on day 1 was demonstrated (1,200 granulocytes and 14,300 platelets/μl blood per 10⁵ bone marrow cells). The linearity suggested a mean T_d < 22.5 h of proliferating bone marrow cells, and allowed a rough estimation of mouse bone marrow stem cell radio-sensitivity (D_o 76 rad).     

G-CSF enhances the proliferation and mobilization, but not the maturation rate, of murine myeloid cells

Objectives: Whether G‐CSF enhances the maturation of neutrophilic granulocytes or just accelerates the mobilization of mature and maturing granulocytes from bone marrow to blood, or both, is not clear. Using an in vivo culture system where such mobilization cannot take place, we previously showed that G‐CSF did not accelerate maturation. To further clarify the role of G‐CSF, we now have examined its effect on murine granulopoiesis in situ. Methods: Murine bone marrow precursors in S‐phase were labeled with BrdU, and hematopoiesis stimulated by the long‐acting G‐CSF compound pegfilgrastim (peg‐G‐CSF). Performing flow cytometric analysis of incorporated BrdU and the granulocyte maturation antigen Gr1, we investigated the cell flux from the proliferative to the non‐proliferative granulocyte compartments in bone marrow and further from bone marrow to blood. Results: Peg‐G‐CSF mobilized neutrophils from bone marrow to blood and markedly increased their concentration in blood for several days. It also increased the proliferation of precursor cells. Newly produced, less mature granulocytes (Gr1⁺BrdU⁺) travelled faster to blood in treated mice than in controls. The flow cytometric and cell density analyses of the bone marrow cells showed that peg‐G‐CSF skewed the population toward less mature cells, mainly because of the mobilization of granulocytes to blood. Conclusions: Collectively, our data do not support the notion that G‐CSF accelerates murine granulocyte maturation per se.     

Two cases of unclassified chronic myeloproliferative disorders]

Two cases of unclassified chronic myeloproliferative disorders (UCMPD), diagnosed by hematological, cytogenetic and DNA analyses, are described. Case 1: a 63 year old female was admitted because of leukocytosis (96,800/microliters) and splenomegaly. Hematological examinations revealed an increase of the granulocytes in the peripheral blood and bone marrow. The neutrophil alkaline phosphatase (NAP) score was 121. The patient developed blast crisis after 12 months of the chronic phase. Case 2: a 48 year old male was presented with fever and leukocytosis (20,000/microliters). Hematological examinations revealed an increase of granulocytes in the peripheral blood and bone marrow. The NAP score was 33. Maturation-arrest in granulocytic series and morphological abnormalities of marrow cells were not observed in the two cases. Cytogenetic analysis of bone marrow cells disclosed 46, XX, i (17 q) in case 1 and 47, XY, +8 in case 2. Southern blot analysis using 3' bcr probe and TransProbe-1 showed no bcr rearrangement. These cases are thought to be valuable in order to clarify the relationship between UCMPD and CMPD such as Ph1 negative chronic myelocytic leukemia and myelodysplastic syndromes.