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The Hemolytic Anemia of the Graft-Versus-Host Reaction in the Chick, Before and After Hatching
The Hemolytic Anemia of the Graft-Fersus-Host Reaction in the Chick, Before and After Hatching C. R. MACPHERSON AND JANE C. DEAMER Department of Pathology, Ohio State University, Columbus (Received for publication July 12, 1965)
T
MATERIALS AND METHODS
The GVHR was produced in a total of 230 random, non-inbred White Leghorn embryos by injection of 0.1 ml. of adult whole blood intravenously. Injection on day 10 gave almost 100% mortality before day 21. All but a few of the embryos injected on day 18 hatched. Injection on intervening days produced intermediate mortality rates before hatching. Hemoglobin levels and microhematocrits were performed on peripheral blood specimens obtained by venepuncture (Macpherson and Deamer, 1964). In addition Romanowsky-stained smears were examined. Histologic sections of the spleen, liver and Bursa were stained by hematoxylin-
eosin, Giemsa and Prussian blue. Impression smears of spleen, liver and bone-marrow were also examined. RESULTS Although the earliest changes of GVHR in the embryo are seen in the peripheral blood, the progressive hemolytic anemia characteristically seen after hatching is not found. Table 1 compares the findings in normal and GVHR-affected birds, pre- and post-hatching. The peripheral blood. The most obvious effect of the GVHR in the embryo is to retard the normal progression of erythrocytes in the peripheral blood from Primary through Polychromatophilic to Mature Embryo Erythrocytes. The peripheral smear thus appears to be from a less mature embryo. Cells are also present, however, which are normally never seen after the 10th day of incubation (Figures 1 and 2). With practice therefore, knowing the age of the embryo one can diagnose GVHR with a high degree of accuracy. Intravenous injection of any material produces striking changes which may mimic those of the GVHR, but these non-specific changes disappear within 48 hours, and true GVHR changes begin to appear after about 72 hours. GVHR-induced changes in the peripheral smear after hatching are quite similar to those seen in the embryo, though only minimal changes are usually seen for the first few days. The time of appearance of marked changes is variable and may be related to the strength of the histocompati-
1594
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
HE hemolytic anemia associated with the Graft-i>em«-Host reaction (GVHR) in the chick is well-known. It was extensively studied by Simonsen (1957) who showed that in nearly all cases the chick red cells gave a strongly positive direct Coombs test when the anemia was rapidly progressive. He felt that this anemia was what killed the chick, usually in the first or second week after hatching. He assumed that the immunologically competent graft cells were reacting against the antigens of the host erythrocytes causing the anemia and direct Coombs test. During studies of the GVHR in the chick embryo, it became apparent that this sequence of events does not occur in the embryo dying of GVHR before hatching. A comparison of findings, before and after hatching, is the subject of this report.
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•
• •
••
•
VP •
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•
• • %
I
%
e FIG. 1.—A. Peripheral blood smear of a 12-day control embryo. 1. A primary erythrocyte. Secondary (polychromatophilic) erythrocytes predominate. There are no true mature embryo erythrocytes in this field. Wright-Giemsa stain. Final magnification X15S0. B. Peripheral blood smear of an 18-day control embryo. The cells are nearly all mature embryo erythrocytes, with the exception of the cell marked "A", which is a secondary (polychromatophilic) erythrocyte. Wright-Giemsa stain. Final magnification X15S0.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
®
•
1596
C. R.
MACPHERSON AND J. C.
DEAMER
TABLE 1.—Comparison of findings in normal and GVHR chicks, before and after hatching Hematocrit V, 1 Normal 20-day embryo
28-32
Direct Coombs test
Peripheral blooc! smear
deposits in spleen
Femoral bone marrow Moderate in amount, E ry th roid /myeloi d ratio 1/1.
2 20-day embryo, dying of GVHR
Predominant polychromatic cells, increased primary erythrocytes. Some to many primitive erythroid cells, reduction in thrombocytes.
Hyperplastic Erythroid/myeloid
3 Normal 5-day chick
Rare primary and polychromatic erythrocytes. No primitive cells.
4 5-day chick, 15-25 hemolytic anemia due to GVHR
Difficult to distinguish from 2 above, but more adult erythrocytes. Not so many primitive erythroid cells. Thrombocytes reduced.
5 12-day chick, dying of G V H R
Marked reduction in leucocytes and thrombocytes. Virtually no polychromatic or primitive cells seen.
H i
—
+ +to
— to +
Usually hyperplastic (depending on stage of anemia) Erythroid/ myeloid ration up to 10/1.
+ ~f- to —i—j—I—f-
Marrow " d r y " . Virtually no hematopoietic cells of any type on smear.
+ + ++ — to
+++ +
Reduced in amount from 1. Erythroid/ myeloid ratio 1/1.
• •
mm:
w
#
mm-
UH
JflK*'
FIG. 2. Peripheral blood smear of an 18-day GVHR embryo. The predominant cell is the mature embryo erythrocyte. 1. Secondary (polychromatophilic) erythrocytes. 2. Immature secondary erythrocytes. 3. More immature cell, a definite erythoblast, with very deep blue cytoplasm. Wright-Giemsa stain. Final magnification XI550.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
A few polychromatic erythrocytes. Rare primary erythrocytes, no cells of more primitive type.
GRAVT-Versus-KosT
Iron was also present in the bursae of birds with severe hemolytic anemia, but in smaller amounts. It wa& present in the connective tissues, and never in the follicles or epithelium of the bursa (Figure 4). DISCUSSION Our results in post-hatching chicks closely parallel those of Simonsen (1957). It seems quite clear that death in these cases is due to the anemia. The time of death correlates closely with the degree of anemia, recovery is reflected by a rise in hematocrit, and it is difficult to conceive of the birds surviving for any length of time with the aplastic marrows that are found at autopsy. By contrast, it seems equally clear that whatever the cause of death is before hatching, it is not hemolytic anemia. The absence of a positive direct Coombs could possibly be attributed to differences in antibody formation in the pre-hatching period. The presence of a hyperplastic rather than an aplastic marrow could be just a matter of the stage of the anemia. It could also be argued that the barely-reduced hematocrit, because of the immaturity of the embryo, is more significant than expected. Other studies however have shown that the embryo is capable of withstanding marked degrees of anemia produced either by venesection, the injection of distilled water, or of saponin (unpublished data). In view of all the differences between the pre- and post-hatching manifestations of the GVHR we feel that it is more reasonable to suppose that there is a difference in the mechanism of death at the two periods. In partial confirmation of this we have performed transfusion experiments, using irradiated blood from the donor whose blood was used to initiate the GVHR. If peripheral blood is irradiated with 10,000 r, all immunologically-competent cells will be destroyed, but there is no ob-
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
bility barrier. As the disease progresses, with falling hematocrit, there is a rapid increase in abnormal and primitive cells. As death approaches, with hematocrit levels of 5% or even less, the primitive cells disappear just as rapidly and all that is left in the smear is a thin suspension of mature erythrocytes, with marked diminution in leucocytes and thrombocytes also. Thrombocytopenia is a regular feature of GVHR both pre- and post-hatching. The bone-marrow. In the latter days of incubation there is regularly a definite increase in the amount of femoral marrow. The smear reflects this, showing marked erythroid hyperplasia. After hatching marrow hyperplasia is seen only in the earlier stages of the anemia. By the time of death femoral marrow has virtually disappeared and smears show few hematopoietic cells. Coombs testing. In spite of the characteristic peripheral blood changes pre-hatching, a positive direct Coombs test was found only once. Even after hatching the direct Coombs test usually does not become positive until the chick is 4-7 days of age. Two-thirds of the chicks developing a definite anemia will die, usually within a week of the appearance of the direct Coombs test. In some cases, as the anemia becomes more severe the Coombs test becomes weaker and may actually become negative before death. In those birds which recover, the Coombs test also becomes negative, about the same time that the hematocrit rises and primitive cells disappear from the peripheral smear. The presence of iron in the spleen is to be expected in hemolytic anemia, but it was not found in the spleen of any embryo, and even in chicks with severe anemia post-hatching it was not constant in amount. If present, it was always in the splenic tissue proper, and not in the necrotic nodules which are such a pathognomonic feature of the GVHR (Figure 3).
1597
REACTION
1598
C. R. MACPHERSON AND J. C. DEAMER
'•*» ": iff -
:'.*» *'.• •'•' \-
vious effect on the erythrocytes. If blood from the same donor as was used to initiate the GVHR is irradiated and transfused, any antibody produced by the graft cells will presumably not affect these erythrocytes. Using this principle, we transfused the affected embryos on days IS and 17 with sufficient amounts of washed erythrocytes to maintain the hematocrits at normal levels. There was no detectable retardation of the lethal effect of the GVHR, confirming the unimportance of the anemia as the cause of death. Just what is the cause of death we have been unable to ascertain as yet, but further studies are in progress. Another unexplained observation is that the positive direct Coombs test does not appear until the 3rd day after hatching, or later. Simonsen (1957) felt that this cor-
related well with the delay necessary for multiplication of immunologically competent cells in birds grafted on day 18. In our birds, however, grafted 8 days earlier than his, the positive direct Coombs tests also appeared after the 3rd post-hatching day. One observation which may be relevant is that the development of certain erythrocyte antigens is delayed until the time of hatching, or even beyond (McDermid, 1964). The delayed appearance of the positive Coombs test, and of the anemia might be due to the absence of the red cell antigens involved in the direct Coombs test and erythrocyte destruction. It does not explain why the changes already described are seen in the peripheral blood of the embryo, nor the death of these embryos.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
FIG. 3. Spleen of a 16-day post-hatching chick with severe anemia. There are numerous iron deposits in the splenic parenchyma (arrows) but none in the nodule ("N"). Prussian Blue stain. Final magnification X186.
GRAFT-Versus-HosT
REACTION
1599
SUMMARY
In the chick, death from GVHR in the post-hatching period is clearly due to severe hemolytic anemia going into an aplastic phase. In the pre-hatching period, anemia is much less prominent, though clearly present. It appears likely that different lethal mechanisms are operative in the two periods. ACKNOWLEDGMENT
This work was supported in toto by U. S. Public Health Service Research Grant A-
5031 from the Institute of Arthritis and Metabolic Diseases. REFERENCES Macpherson, C. R., and J. C. Deamer, 1964. Some observations on normal erythrocyte development in the chick embryo. Poultry Sci. 4 3 : 1587-1594. McDermid, E. M., 1964. Immunogenetics of the chicken. A review. Vox Sang. 9: 249-267. Simonsen, M., 1957. The impact on the developing embryo and newborn animal of adult homologous cells. Acta Path. Microbiol. Scand. 40: 480-500.
NEWS AND NOTES (Continued from page 1591) Winners in the Southeastern Youth Scholarship program were Rickey Lewis, Winston, Ga., F.F.A. winner, and Bill Farrington, Dadeville, Ala., 4-H Club winner. Each received a $500 scholarship.
Products Institute of Canada the following officers were elected: President—E. R. Hoover, Burlington, Ont.; Vice-Presidents—D. Brownlow, Regina, Sask., and W. H. Beaty, Thamesford, Ont.; Treasurer—M. McPhail, Toronto, Ont.; Member-atP.P.I. (CANADA) NOTES Large—C. Corkum, Port Williams, N.S.; French At the 14th annual meeting of the Poultry Secretary—R. Raynauld, Montreal, Que.; and Sec(Continued on page 1603)
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
FIG. 4. Bursa of the same chick as Figure 3. Note the connective tissue iron deposits (arrows), and absence of staining in the Bursa follicles (F). Prussian Blue stain. Final magnification X530,
T
MATERIALS AND METHODS
The GVHR was produced in a total of 230 random, non-inbred White Leghorn embryos by injection of 0.1 ml. of adult whole blood intravenously. Injection on day 10 gave almost 100% mortality before day 21. All but a few of the embryos injected on day 18 hatched. Injection on intervening days produced intermediate mortality rates before hatching. Hemoglobin levels and microhematocrits were performed on peripheral blood specimens obtained by venepuncture (Macpherson and Deamer, 1964). In addition Romanowsky-stained smears were examined. Histologic sections of the spleen, liver and Bursa were stained by hematoxylin-
eosin, Giemsa and Prussian blue. Impression smears of spleen, liver and bone-marrow were also examined. RESULTS Although the earliest changes of GVHR in the embryo are seen in the peripheral blood, the progressive hemolytic anemia characteristically seen after hatching is not found. Table 1 compares the findings in normal and GVHR-affected birds, pre- and post-hatching. The peripheral blood. The most obvious effect of the GVHR in the embryo is to retard the normal progression of erythrocytes in the peripheral blood from Primary through Polychromatophilic to Mature Embryo Erythrocytes. The peripheral smear thus appears to be from a less mature embryo. Cells are also present, however, which are normally never seen after the 10th day of incubation (Figures 1 and 2). With practice therefore, knowing the age of the embryo one can diagnose GVHR with a high degree of accuracy. Intravenous injection of any material produces striking changes which may mimic those of the GVHR, but these non-specific changes disappear within 48 hours, and true GVHR changes begin to appear after about 72 hours. GVHR-induced changes in the peripheral smear after hatching are quite similar to those seen in the embryo, though only minimal changes are usually seen for the first few days. The time of appearance of marked changes is variable and may be related to the strength of the histocompati-
1594
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
HE hemolytic anemia associated with the Graft-i>em«-Host reaction (GVHR) in the chick is well-known. It was extensively studied by Simonsen (1957) who showed that in nearly all cases the chick red cells gave a strongly positive direct Coombs test when the anemia was rapidly progressive. He felt that this anemia was what killed the chick, usually in the first or second week after hatching. He assumed that the immunologically competent graft cells were reacting against the antigens of the host erythrocytes causing the anemia and direct Coombs test. During studies of the GVHR in the chick embryo, it became apparent that this sequence of events does not occur in the embryo dying of GVHR before hatching. A comparison of findings, before and after hatching, is the subject of this report.
>
•
• •
••
•
VP •
<
•
• • %
I
%
e FIG. 1.—A. Peripheral blood smear of a 12-day control embryo. 1. A primary erythrocyte. Secondary (polychromatophilic) erythrocytes predominate. There are no true mature embryo erythrocytes in this field. Wright-Giemsa stain. Final magnification X15S0. B. Peripheral blood smear of an 18-day control embryo. The cells are nearly all mature embryo erythrocytes, with the exception of the cell marked "A", which is a secondary (polychromatophilic) erythrocyte. Wright-Giemsa stain. Final magnification X15S0.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
®
•
1596
C. R.
MACPHERSON AND J. C.
DEAMER
TABLE 1.—Comparison of findings in normal and GVHR chicks, before and after hatching Hematocrit V, 1 Normal 20-day embryo
28-32
Direct Coombs test
Peripheral blooc! smear
deposits in spleen
Femoral bone marrow Moderate in amount, E ry th roid /myeloi d ratio 1/1.
2 20-day embryo, dying of GVHR
Predominant polychromatic cells, increased primary erythrocytes. Some to many primitive erythroid cells, reduction in thrombocytes.
Hyperplastic Erythroid/myeloid
3 Normal 5-day chick
Rare primary and polychromatic erythrocytes. No primitive cells.
4 5-day chick, 15-25 hemolytic anemia due to GVHR
Difficult to distinguish from 2 above, but more adult erythrocytes. Not so many primitive erythroid cells. Thrombocytes reduced.
5 12-day chick, dying of G V H R
Marked reduction in leucocytes and thrombocytes. Virtually no polychromatic or primitive cells seen.
H i
—
+ +to
— to +
Usually hyperplastic (depending on stage of anemia) Erythroid/ myeloid ration up to 10/1.
+ ~f- to —i—j—I—f-
Marrow " d r y " . Virtually no hematopoietic cells of any type on smear.
+ + ++ — to
+++ +
Reduced in amount from 1. Erythroid/ myeloid ratio 1/1.
• •
mm:
w
#
mm-
UH
JflK*'
FIG. 2. Peripheral blood smear of an 18-day GVHR embryo. The predominant cell is the mature embryo erythrocyte. 1. Secondary (polychromatophilic) erythrocytes. 2. Immature secondary erythrocytes. 3. More immature cell, a definite erythoblast, with very deep blue cytoplasm. Wright-Giemsa stain. Final magnification XI550.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
A few polychromatic erythrocytes. Rare primary erythrocytes, no cells of more primitive type.
GRAVT-Versus-KosT
Iron was also present in the bursae of birds with severe hemolytic anemia, but in smaller amounts. It wa& present in the connective tissues, and never in the follicles or epithelium of the bursa (Figure 4). DISCUSSION Our results in post-hatching chicks closely parallel those of Simonsen (1957). It seems quite clear that death in these cases is due to the anemia. The time of death correlates closely with the degree of anemia, recovery is reflected by a rise in hematocrit, and it is difficult to conceive of the birds surviving for any length of time with the aplastic marrows that are found at autopsy. By contrast, it seems equally clear that whatever the cause of death is before hatching, it is not hemolytic anemia. The absence of a positive direct Coombs could possibly be attributed to differences in antibody formation in the pre-hatching period. The presence of a hyperplastic rather than an aplastic marrow could be just a matter of the stage of the anemia. It could also be argued that the barely-reduced hematocrit, because of the immaturity of the embryo, is more significant than expected. Other studies however have shown that the embryo is capable of withstanding marked degrees of anemia produced either by venesection, the injection of distilled water, or of saponin (unpublished data). In view of all the differences between the pre- and post-hatching manifestations of the GVHR we feel that it is more reasonable to suppose that there is a difference in the mechanism of death at the two periods. In partial confirmation of this we have performed transfusion experiments, using irradiated blood from the donor whose blood was used to initiate the GVHR. If peripheral blood is irradiated with 10,000 r, all immunologically-competent cells will be destroyed, but there is no ob-
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
bility barrier. As the disease progresses, with falling hematocrit, there is a rapid increase in abnormal and primitive cells. As death approaches, with hematocrit levels of 5% or even less, the primitive cells disappear just as rapidly and all that is left in the smear is a thin suspension of mature erythrocytes, with marked diminution in leucocytes and thrombocytes also. Thrombocytopenia is a regular feature of GVHR both pre- and post-hatching. The bone-marrow. In the latter days of incubation there is regularly a definite increase in the amount of femoral marrow. The smear reflects this, showing marked erythroid hyperplasia. After hatching marrow hyperplasia is seen only in the earlier stages of the anemia. By the time of death femoral marrow has virtually disappeared and smears show few hematopoietic cells. Coombs testing. In spite of the characteristic peripheral blood changes pre-hatching, a positive direct Coombs test was found only once. Even after hatching the direct Coombs test usually does not become positive until the chick is 4-7 days of age. Two-thirds of the chicks developing a definite anemia will die, usually within a week of the appearance of the direct Coombs test. In some cases, as the anemia becomes more severe the Coombs test becomes weaker and may actually become negative before death. In those birds which recover, the Coombs test also becomes negative, about the same time that the hematocrit rises and primitive cells disappear from the peripheral smear. The presence of iron in the spleen is to be expected in hemolytic anemia, but it was not found in the spleen of any embryo, and even in chicks with severe anemia post-hatching it was not constant in amount. If present, it was always in the splenic tissue proper, and not in the necrotic nodules which are such a pathognomonic feature of the GVHR (Figure 3).
1597
REACTION
1598
C. R. MACPHERSON AND J. C. DEAMER
'•*» ": iff -
:'.*» *'.• •'•' \-
vious effect on the erythrocytes. If blood from the same donor as was used to initiate the GVHR is irradiated and transfused, any antibody produced by the graft cells will presumably not affect these erythrocytes. Using this principle, we transfused the affected embryos on days IS and 17 with sufficient amounts of washed erythrocytes to maintain the hematocrits at normal levels. There was no detectable retardation of the lethal effect of the GVHR, confirming the unimportance of the anemia as the cause of death. Just what is the cause of death we have been unable to ascertain as yet, but further studies are in progress. Another unexplained observation is that the positive direct Coombs test does not appear until the 3rd day after hatching, or later. Simonsen (1957) felt that this cor-
related well with the delay necessary for multiplication of immunologically competent cells in birds grafted on day 18. In our birds, however, grafted 8 days earlier than his, the positive direct Coombs tests also appeared after the 3rd post-hatching day. One observation which may be relevant is that the development of certain erythrocyte antigens is delayed until the time of hatching, or even beyond (McDermid, 1964). The delayed appearance of the positive Coombs test, and of the anemia might be due to the absence of the red cell antigens involved in the direct Coombs test and erythrocyte destruction. It does not explain why the changes already described are seen in the peripheral blood of the embryo, nor the death of these embryos.
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
FIG. 3. Spleen of a 16-day post-hatching chick with severe anemia. There are numerous iron deposits in the splenic parenchyma (arrows) but none in the nodule ("N"). Prussian Blue stain. Final magnification X186.
GRAFT-Versus-HosT
REACTION
1599
SUMMARY
In the chick, death from GVHR in the post-hatching period is clearly due to severe hemolytic anemia going into an aplastic phase. In the pre-hatching period, anemia is much less prominent, though clearly present. It appears likely that different lethal mechanisms are operative in the two periods. ACKNOWLEDGMENT
This work was supported in toto by U. S. Public Health Service Research Grant A-
5031 from the Institute of Arthritis and Metabolic Diseases. REFERENCES Macpherson, C. R., and J. C. Deamer, 1964. Some observations on normal erythrocyte development in the chick embryo. Poultry Sci. 4 3 : 1587-1594. McDermid, E. M., 1964. Immunogenetics of the chicken. A review. Vox Sang. 9: 249-267. Simonsen, M., 1957. The impact on the developing embryo and newborn animal of adult homologous cells. Acta Path. Microbiol. Scand. 40: 480-500.
NEWS AND NOTES (Continued from page 1591) Winners in the Southeastern Youth Scholarship program were Rickey Lewis, Winston, Ga., F.F.A. winner, and Bill Farrington, Dadeville, Ala., 4-H Club winner. Each received a $500 scholarship.
Products Institute of Canada the following officers were elected: President—E. R. Hoover, Burlington, Ont.; Vice-Presidents—D. Brownlow, Regina, Sask., and W. H. Beaty, Thamesford, Ont.; Treasurer—M. McPhail, Toronto, Ont.; Member-atP.P.I. (CANADA) NOTES Large—C. Corkum, Port Williams, N.S.; French At the 14th annual meeting of the Poultry Secretary—R. Raynauld, Montreal, Que.; and Sec(Continued on page 1603)
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 13, 2015
FIG. 4. Bursa of the same chick as Figure 3. Note the connective tissue iron deposits (arrows), and absence of staining in the Bursa follicles (F). Prussian Blue stain. Final magnification X530,