The oxydase reactions as applied to the megakaryocyte and blood platelet of the rat.код для вставкиСкачать
THE OXYDASE RESCTIONS A S APPLIED T O THE &fEGBKARYOCYTE AND BLOOD PLATEETAET O F THE RAT CHRISTIANNA S M I T I I , AZARGSRET ROBINSON AND REBECCA T YSON T h e Walter H . Nerriam Laboratory, Ikpartment of %~Olog?j, Xoiint HolyoXe College, South Hadlq4, 3fassnckv.sPtts Whether blood platelets originate from lympliocytes, monocytes, granular leucocytes or cells of the reticulo-endothelial system as well as from mcgalraryocytes is still an open question. The observations of Wright first made in 1906 and published again in 1910 regarding the latter as progenitors of platelets have been many times corroborated. The azurophilic granules of both platelets arid megakaryocytes as described by him have been the structural characteristics most often emphasized. Further investigation of the granules has brought little positive knowledge as to their nature. As is well known they stain supravitally with neutral red and brilliant crcsyl blue. Barta ('32) finds that after methyl alcohol fixation the granules are unstained in 1% eosin, erythrosiri o r acid violet but that they are colored in Loeffler's methylene blue, methyl violet and toluidin blue. Roskin and Grinbaum ('26) aiid Reams and King ('33) report that they do not react to Feulgen's nuclear stain, aiid Corradetti ('34) finds that they are the only structures of the blood cells to withstand treatment with hydrochloric acid. IGngsley ( '35, '37) has recently described them as specific granules by which he is able to differentiate even the early stages of the megakaryocytes from surrounding cells. Appreciative acknowledgmmt is made to E. Quainine and J. Hastings for certain technical details used in the various methods. 139 T H E ANATOMIOAL RECORD. VOL. 70, NO. 2 AND SUPPLEMENT NO. 2 JANU.4R.Y. 1938 140 C. SMITH, M. ROBINSON AND R. TYSON In the present study it was found that the azurophilic granules of both platelets and megakaryocytes were easily and clearly demonstrated with a combination of Wright's and Giemsa's stains using Isaac's technique for the marrow. The granules were uniformly negative in the oxydase and peroxydase tests. This last is contrary to findings reported jn 1935 by Smith and Hastings. METHODS After comparing ordinary smears of bone marrow on slides with others of cells suspended in plasma according to Isaacs ('28, '30) it was decided to use the latter method. By this procedure all of tlie cells of the marrow were better preserved and more deeply stained. And what was important for this study, it was possible by this method t o have both mcgakaryouytes and platelets in llie same medium giving a chance for more direct comparison of staining reactions. It was found that the most even and best results were obtained after the suspension had stood 20 minutes. As a further precaution the smears of bone marrow suspensions and peripheral blood were made on two halves of the same slide. Platelets were also studied in smears of human blood. In the examination of platelets in blood smears care was taken to make dccisioiis only on those which could be clearly seen. All glassware was cleaned according t o the methods ontlined for supravital technique (Sabin, ' 2 3 ) and pipettes and centrifuge tubes were coated with paraffin. I'Icpauin was used its the anticoagulant in obtaining tlie plasma. The oxydase reactions were always started xithin at least 2 hours aftcr making the preparations. OHRBRVATIOSS A S D DISCUSSION The azurophilic granules of the megakaryocytes arid blood platelets did not react in the oxydase and peroxydase tests which were used in this study (table 1) although tlie results were positive with the specific granules of the neutrophiles and eosiiiophiles and varying numbers of bodies within the OXYD.4SE REACTIOKS O F MEGAKARYOCYTES 141 monocybes. Whether these latter were exogenous or endogenous could not be determined. An examination of the basophiles was not made. Table 3 shows that while most of the previous investigators found these granules similarly inactive, TABLE 1 S?&wkmaryof methods used in t h e slzcdy of the oxydase and perozydase 1.eaclions of mpgakaryocytes and blood platelets RRAGENTR AUTHOP COUNTEBSTAIN Block and Perk Dopa Rafranin Imnbright Blplls naphthol, hydrogen perodde Bemidine, hydrogen peroxide Cnpper sulphatc, benzidinc, hydrogen perosidc Nitro prnsside, benzi dinr, hydrogen peroxide Methylene blue McJunkin Washburxi (Kolnicr and Boerner ) TiBLE COLOR O F OXYJJASE GRANULES Dark brown t o black Blue to red Wright 'R Saf ranin Blue grern Wright's Black 0, The nrydase uad peroxgdase t r s l s as a p p l i d l o the rr~eyakmyocyleand blood platelet METHOD HCYAN BLOOD PLATELETS RAT PERIPHEEAL BLOOD P U T H L S T B BAT BONE MARROW XEGAKARYOCYTES Block and Peck Lambright McJunkin Sato and Yoshimatsu Washburn (Kolmer and Roerner ) two report positive staining. Contrary to the findings of Roskin and Grinbaum ('26), with us a successful dopa reaction revealed unstained megakaryocytes surrounded by strikingly blackened cells of the granular series. Because of the precipitate the blood platelets were more difficult to see, 142 C. SMITH, &I. ROBINSON AND R. TYSON but with a safranin counterstain they could bc definitely ascertained. Katsunuma ( ’26) described a positive oxydase reaction in the blood platelets and megakaryocytes which was difficult to find in normal blood but quite clear in myeloicl leukaemia, fetal blood and in experimeiital anemia in rabbits. He believed the oxydase granules corresponded to the azurophilic ones and gave the positive reaction as a microchcinical support t o Wright’s findings. However, Barta ( ’32) helieved TABLE 3 Hislory of the oxyduuse and peroxyaase reactions applied l o mcgalaryocytcs and blood platelets INVE8TIQATOR YEAR G~alinm 1916 1918 Ttiehter Katsunuma 1923 1926 Eoskin and Gr inbsum Knoll 1926 Uarta 1932 METHOD bAGAAFARYOOYTE9 PLATESETS Alpha-naphthol, hydrogen peroxidc Benzidine, hydrogen peroxide MeJunkin Gierke, indophenol ox>dasc Dopa, Graham henzidine 1927 Blpha-naphthol, dimethylparaphenylendiamibase Schulze, Graham that the positive oxydase reactioiis of megakaryocytes in infectious diseases were due to phagocytosed material and therefore not specific differentiations of the cell. That blood platelets may be derived from cells other than the megakaryocytes is believed by many, though hard to prove. I n the study by Birch (’30) on the origin of pseudothrombocytes from leucocytes, she makes the statement that “the possibility exists that leukoplastids may be normal constituents of the human blood.’’ According to her thesc leukoplastids may be either granular in origin, arising from neutrophiles, eosinophiles, or basophiles, or non-granular, arising OXYDASE REACTIONS O F MEGAXARPOCYTES 143 from lymphocytes, lymphoblasts, or myeloblasts. Of these sources only the specific granules of neutrophiles and eosinophilcs were found to have clear-cut peroxydasc reactions in these studies and table 2 indicates that in normal blood (human and rat), at least, platelets are not so derived. I n the peripheral blood of the rat basophiles appear to be absent. In the case of two neutrophiles in fixed smears, pseudopods full of oxydase granules were seen and looked as though they were in the process of being pinched off. No such pieces with clear oxydase granules were seen free among the cells. SUMSIARY 1. Tlic azurophilic granules of the mcgakaryocyte stain morc deeply and easily in cells suspended in plasma (Tsaaes’ method) than in ordinary smears. 2. The aznrophilic granules of normal human blood platelets and of normal rat platelets and megakaryocytes give negative oxydase and peroxydase reactions. 3. In normal blood there is no evidence that any platelets arc derived from neutrophiles o r eosiaophiles. LITERATURE CITED 1932 Uber Xau und Punktion der Megakaryozyten. Folia Haemat., vol. 47, p. 168. BEANS, H. W., AND R. L. EING1933 Feulgen’s ‘nuclealreaktion’ and blood platelets. Anat. Rec., vol. 55 (suppl.), p. 46. BIRCH, C. L. 1930 The origin of pseudothrombocytes from leueocytes. Anat. Rec., vol. 46, p. 45. BLOCK, B., AND 8. M. PECK 1930 Der Xachweis der Oxydase i n den Zellen des myeloisehen Systems durch 3,4-Dioxyphenylalanin. Folia Haemat., vol. 41, p. 166. A. 1934 Ricerche inierocliimiche sui megacariociti e sullc piastrine. CORRADEWI, Hsemst., vol. 16, p. 207. GRAHAM, G. S. 1916 The oxidizing fcrmeiit of the myelocyte series of cells and its demonstration b y an alpha naphthol-pyroniii method. J. Ned. Research, vol. 35, p. 231. 1918-1919 Beiizidine as a peroxidase reagent f o r blood smears and tissues.. J. Med. Research, vol. 39, p. 15. ISAAC%, R. 1925 Alterations of tissue cells in the blood stream. Science, vol. 65, p. 547. 1930 The physiologic histology of the bone marrow. Polia Haemat., vol. 40, p. 395. BAttW, 1. 144 C. SMITH, M. ROBINSOX AND 1L TYSON KATSUNUMA,S. 1926 Genese der Blut-plLttehen im Lichte der Oxydssereaktiou. Folia Haemat., vol. 34, p. 29. KINGSLEY,D. M. 1935 The development of the megakaryocyte i n the pig. Anat. Bee., vol. 6 1 (suppl.), p. 29. 1937 A technic f o r megakaryocgtes and platelets in sections. Folia IIacmat., vol. 57, p. 87. KNOLL,1%'. 1927 Elut urid blutbildende Organc menschliclier Embryonen. Denkschriften der schweixcrischen naturforscliendeii Gescllschaft. M B m . de la SOC.Helvhtique dea Sci. Nat., vols. 62-64, p. 1. 1931 Approved laboratory technique, p. 89. KOLNER,J. A., AND F. BCYEENEE D. Appleton and Co., S e w York. LAMBRIGEIT, G. L. 1921 Leukemia: type diagnosis by oxydase method of blood staining. Am. J. Med. Sci., rol. 161, p. 209. MCJUNKIN, F. A. 1020 ,4 beiiziciin-polScliroriie stain for blood. J. Am. Xed. Assoe., vol. 74, p. 17. RICHTER,Jsf. N. 1925 Leukemia. The relative talucs of cell morphology and diagnostic aids. Arch. I n t . Med., vol. 36, thc peroxydasc reaction p. 13. ROSKIN,G. O., AND F. T. (+RllvBAUM 1926 A contribution t o the knowledge of blood platvlrts. Arb. hlicrohiol. Inst. (Mosrow), vol. 2, p. 1 3 7 . Riol, Abstracts, vol. 2, p. 1577. RUIN, .'E 1923 Stndies o f living human blood-cells. J o h n s Hopkiiis Hosp. Bull., vol. 34, p. 277. SATO,A., AND S. Y o s ~ i n r a ~ s u192.5 Thr perosidnse reirtion in epidemic encephalitis. Am. J. Dis. Child., vol. 29, p. 301. SMITH,C., AND J. H a s n l r ~ s 1933 A study of the mcgakaryocyte and blood platelet in the rat. Anat. Rcc., ool. 64 (suppl. no. l), p. 95. WRIGIIT,J. 11. 1910 The histogenesis of the blood platelets. J. Illorph., vol. 21, p. 263.