ALBERT

Description: A method is described for rapid and efficient separation of leukocytes and nucleated erythrocytes from blood or marrow. It is based on the rediscovery of a nontoxic hemagglutinin, isolated from common red or navy beans, which agglutinates all human erythrocytes and those of the animals which have been tested with it. The time from drawing the blood to complete separation of the cells is less than ten minutes. The cells remain in suspension in their own plasma and are countable. Negligible amounts of foreign material are introduced, a great advantage in chemical studies. The cells so isolated are living and suitable for culture studies. Any volume of blood from less than 1 ml. to over 500 ml. may be processed. In marrow nucleated erythrocytes are separated with the leukocytes and in their original porportions. The volume, motility, morphology and life span of the cells in cultures are not altered by addition of the bean extract.

Description: Analyses of the threshold dose of P32 to control 201 chronic lymphocytic leukemia patients and 100 chronic granulocytic leukemia patients and of the P32 dose to maintain 133 chronic lymphocytic leukemia patients for a mean period of 50.2 months and 49 chronic granulocytic leukemia patients for a mean period of 33.6 months are analyzed. These threshold doses fit the logarithmic probability distributions given in the tables and figures. See PDF for Figure See PDF for Figure When the dose in mc. to control was plotted against the leukocyte count, a straight line was obtained on log log paper. The equation for this curve for the median dose to control chronic granulocytic leukemia is: log mc. P32 per 12 weeks = 0.33 log L - 0.633, where L is the leukocyte count on the day the first P32 was given. The equation for the log log curve relating median dose to control chronic lymphocytic leukemia to initial counts above 15,000 is: log mc. P32 per 12 weeks = 0.47 log L - 1.420. But the 40 aleukemic or subleukemic cases with leukocyte counts below 15,000 had a median P32 requirement and standard deviation that did not differ significantly from that for the entire group, suggesting that this subleukemic group includes patients resembling the entire spectrum of cases with elevated initial leukocyte counts. See PDF for Figure See PDF for Figure The median dose of P32 per year to maintain chronic granulocytic leukemia cases fits the equation: log P32 per year = 0.546 log L - 1.506, and the corresponding equation for chronic leukemic lymphocytic leukemia is: log mc. P32 per year = 0.45 log L - 1.22. The group of lymphocytic leukemias with initial counts below 15,000 again showed a median and distribution similar to that for the entire leukemic group. The remarkable fact that the dose requirement years later is related to the initial leukocyte count is discussed. Revised recommendations on dosage schedule for titrated, regularly spaced P32 therapy of the chronic leukemias are presented. For each individual patient with chronic leukemia, there is a threshold dose of intravenously administered P32 below which no effect is observed. The dose and interval which will maintain a uniform leukocyte count of about 15,000 may remain unchanged for years or may show abrupt changes at any time to either a lower or higher requirement.

Description: The term "hiatus leukemicus" suggests a jump in cell development from an early stage to a late stage with nothing in between, analogous to changing from the appearance of a 10 year old to that of a 60 year old without any intermediate stages. The term "myelo-monocytic leukemia" suggests leukemia involving 2 distinct cell series. Better methods for identification of promonocytes indicate that both of these pictures are usually due to cases of acute monocytic leukemia misdiagnosed as chronic granulocytic leukemia. Monocytic leukemias have a greater tendency than other myelophthisic processes to chase normal immature cells of the granulocytic and erythrocytic series into the blood stream. The result is that blast and pro cells actually belonging to the monocytic series are frequently misinterpreted as myeloblasts and progranulocytes because of the association with normal progranulocytes and myelocytes. These misdiagnoses result in reports of too low a proportion of acute monocytic leukemias among the total leukemic population, account for a belief that leukemia is due to maturation arrest rather than early cell death or shortening of the n-cell life span, and for naming leukemias for cells more mature than the alpha-cell. Only the alpha-cell can maintain the necessary somatic genetic continuity.

Description: New culture strains containing humman hemic cells of monocytic, lymphocytic, granulocytic, plasmocytic and erythrocytic series are described with suggestions as to maintenance of the desired cell series in culture. The addition of cells from lymphocytic, plasmocytic and erythrocytic series to a previously well established culture has made possible long-term culture of these cell types, since this procedure aids in maintaining the necessary high gradient factor for their growth and multiplication. Although the life span of these cells has been considerably shortened in culture and immature forms predominate, the cells show recognizable characteristics of the cell series of origin. A high population density must be maintained in cultivation of the mixed cultures or the desired cell type may be lost.

Description: Study of our first 201 cases of polycythemia vera and of cases reported in the literature shows a remarkable age distribution for this disease. The age at onset, age at diagnosis, age at first treatment, and age at death, as well as survival times, each fits a normal distribution. In our cases, the median age at onset is 57 years with a standard deviation of 13 years, an entirely different distribution from that for the population of Oregon. This requires a logarithmic increase in relative age specific incidence from age 20 to 40, when there is little difference in the number of persons alive at each age, with a doubling of the proportion occurring in each 5-year interval every 7.5 years. After age 55, the proportion of new cases developing follows the slope of the number alive in the population. This means that the incidence remains almost constant after the peak age incidence is reached. Unfortunately, no data exist to transform these figures to absolute values, but if enough of the population could be studied to give absolute values at any one point, all other points could be determined. The implication is that polycythemia vera is due to a single cause which is highly correlated with age. The normal distribution of survival times means that polycythemia vera is not a malignant process since survival times in all malignancies studied fit a log normal distribution. The constant value of age at death means that age at first treatment is a most important prognostic factor. The mean age at death of patients with polycythemia vera, treated with P32, is 69 years ± 1, and treated without radiation therapy is 65 years ± 1. Apparently, if a patient lives to be treated with P32 previous treatment by other modalities or no treatment prior to that, will not affect the years to be gained by P32 treatment. However, the total survival time is highly correlated with age and the sooner after onset that the patient can be treated the more likely it is he will benefit from P32 therapy.

Description: Two methods of tissue culture based on gradient principles which have permitted continuous growth and multiplication of hemic cells for 6 months to 1 year are described. Three long-term cell strains were isolated from the bloods of patients with acute or chronic leukemias. Success with these methods depends on previous determination of the gradient factor for the particular cell type to be cultured. This can easily be accomplished by the gradient culture method, and these principles are in all probability applicable to all cell types grown in tissue culture. The isolation of these cells from blood excludes a fixed tissue cell as the only type capable of long term multiplication. The development of cells resembling tissue histiocytes, Dorothy Reed cells, and Langhan’s giant cells from cells cultured from the blood of acute monocytic leukemia lends further support to the view that the monocytic series and the histiocytic series (reticuloendothelial cells) are one and the same and can give rise by polyploidy and endomitosis to many types of giant cells.

Description: 1. Methods are described which make possible the calculation of the number and distribution of the hemic cells of the body. 2. The numbers of these cells in the various blood-forming organs, in the blood outside these areas, and in the total body are summarized in table 3, together with the ratios of the number in the blood to the number outside the hematopoietic organs for the standard 70 Kg. human male. Dividing these figures by 70 will give the values per Kg. 3. By similar methods the values for other age and sex groups in man and for other species may be calculated. 4. Leukocytes apparently are not primarily blood cells but are merely en route in the blood to the defense of body tissue generally. Only 1/40 to 1/400 of the total leukocytes outside the hematopoietic organs are present in the blood stream at any one time. 5. These somewhat surprising figures account for many previously made observations which were hitherto hard to explain.

Description: In cultures by the marrow culture technic of human marrow and leukemic blood containing concentrations of urethane from 1:200 to 1:40,000, marked changes in the morphology of the cells of the granulocyte series were noted. These changes were not noted in the control nor in duplicate cultures containing the methyl-bis (B-chloroethyl) amine hydrochloride form of nitrogen mustard in concentrations from 1:500,000 to 1:40,000,000, nor were they noted in previous studies of cultures containing colchicine or exposed to 200 kilovolt or million volt x-rays, neutron rays or radioactive phosphorus, nor in the bloods or marrows of patients with untreated chronic granulocytic leukemia, of healthy individuals or of persons with miscellaneous diseases. The changes consisted of an early increase in number of normal mitoses in the progranulocytes; a steadily rising percentage of granulocytes and progranulocytes showing condensation of the chromatin in the nucleus into dense fragments separated by clear spaces; a progressive increase in the number of cells of the granulocyte series with double nuclei, affecting all cells from the progranulocytes to the neutrophil lobocytes but appearing to be most numerous in the granulocyte stage; and the appearance in the cultures by 4 to 5 days of cells containing separated fragments of structureless material staining like basichromatin, which probably represents a karyorrhexis of the nucleus. Note: Nothing in this article is to be construed as a recommendation of urethane for the clinical treatment of leukemias. While many years must elapse before its place in therapy can be evaluated, it does seem worthwhile to give urethane a trial for metastatic malignant tumors. Our present impression is that either radioactive phosphorus or total body irradiation with x-rays given in small regularly spaced doses is far superior to urethane in the treatment of leukemias.17 When the cells become resistant to radiation therapy, urethane may be worthy of a trial.

Description: 1. The technic used to determine the average volume per cell of leukocytes from patients with leukemia has been described. 2. The distribution of the average volumes of leukocytes from 15 patients with granulocytic leukemia, based on 52 determinations, has a median value of 450 cu. µ and a mean of 460 cu. µ. 2. The 95 per cent range for this distribution is 340 to 570 cu. µ. 3. Sufficient data are not presently available to determine the Shape of the distribution for the volume of monocytes, but the average value for the leukocytes from 8 patients with monocytic leukemia is 470 cu. µ and the range is 360 to 580 cu. µ. 4. The average volumes of leukocytes from 27 patients with lymphocytic leukemia, based on 74 determinations, have a Poisson-like distribution with a median value of 230 cu. µ and a mean of 250 cu. µ. Ninety-five per cent of the values in this distribution fall in the range of 130 to 410 cu. µ. 5. Although leukocyte volumes from both granulocytic and lymphocytic leukemias follow Poisson-like distributions, the central values of these distributions are significantly different. However, there is an overlap of the distributions with approximately a 15 per cent chance that average lymphocytic volumes will fall into the range of the small granulocytic volumes. 6. For many purposes it may be assumed that two billion cells of the granulocytic or monocytic series or four billion cells of the lymphocytic series equal 1 ml.