Figure 1 -A graphic illustration of the cyclic variation of the neutrophil count in Patient M.J.and Patient R.J. ANC, absolute neutrophil count used synonomously ...
Cyclic Ultrastructural Abnormalities in
Human Cyclic Neutropenia
RICHARD T. PARMLEY, MD, GERALD J. PRESBURY, MD, WINFRED C. WANG, MD, and
JUDITH WILIMAS, MD The ultrastructure of neutrophils from two brothers with cyclic neutropenia was studied at various time intervals of the neutrophil cycle, which varied from 15 to 21 days. During the nonneutropenic portion (middle neutrophil counts third) of the cycle, when the blood approached normal, the majority of blood and marrow
segmented neutrophils appeared morphologically normal, whereas marrow promyelocytes demonstrated increased autophagy with decreased condensation of primary granules. During the early neutropenic portion of the cycle (first third), increased autophagy and granule abnormalities persisted in promyelocytes, myeand the few segmented neutrophils present. locytes, the During late neutropenic portion (last third) of the CYCLIC NEUTROPENIA is a hereditary disorder in humans and grey collie dogs and is characterized by a cyclic depression in the blood neutrophil count every 12-21 days.'-5 Other marrow elements appear to be involved to a limited extent as evidenced by cyclic changes in numbers of erythroid cells, platelets, monocytes, and colony-stimulating activity.6-8 The involvement of several cell lines suggests the presence of a stem cell defect. This is corroborated by the observation that cyclic neutropenia in grey collie dogs can be cured by marrow transplant,6 and marrow from a patient with cyclic neutropenia has been successfully transplanted as curative therapy for acute leukemia to a nonneutropenic sibling who subse-
From the
Department of Pediatrics,
Division
of Pediatric Hematology/
Oncology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas; and the Department of Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee
cycle, which preceded neutrophil recovery, most promyelocytes appeared morphologically normal, whereas the few segmented neutrophils present frequently contained increased autophagic and/or heterophagic vacuoles and decreased cytoplasmic granules. Abundant neutrophil debris was present in marrow macrophages consistent with dysgranulopoiesis and intramedullary destruction of abnormal myeloid cells. Similar changes were not observed in monocytes, eosinophils, or basophils. These results demonstrate a cyclic insult which has its initial morphologic impact on the promyelocyte of patients with cyclic neutropenia. (Am J Pathol 1984, 116:279-288)
granule genesis.1213 These findings, however, are absent in many children with presumed congenital neutropenia and a benign clinical course. 4 Increased autophagy and hypergranularity of neutrophils have been observed in patients with neutropenia secondary to vitamin B,2 deficiency.'5 The purpose of the present study was to determine whether distinct ultrastructural abnormalities could be identified in humans with cyclic neutropenia and whether these abnormalities could be related to the fluctuations in blood neutrophil counts.
Materials and Methods Two brothers aged 13 ½/ (M.J.) and 15
quently developed cyclic neutropenia.9 Several investigators have demonstrated ultrastructural abnormalities, including autophagy and abnormal granule genesis, in neutrophils from grey collie dogs with cyclic neutropenia.2'0 These findings
2
(R.J.) with
cyclic neutropenia were studied after we obtained inSupported in part by National Institutes of Health Grant Accepted for publication February 28, 1984. Address reprint requests to Richard T. Parmley, MD, Department of Pediatrics, Division of Pediatric Hematology/Oncology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78284.
are consistent with the demonstration of abnormal
DE-02670.
neutrophil function and metabolism in grey collie dogs." Neutrophils from some children with severe congenital neutropenia similarly demonstrate autophagy, hypogranularity, and distinct abnormalities in 279
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PARMLEY ET AL
AJP · August 1984
formed consent. Previous observations had documented cyclic episodes of neutropenia approximately every 15-21 days (Figure 1). The father was known to have chronic neutropenia but was not available for study. The patients were relatively asymptomatic and were not receiving medication at the time of the study. Blood and marrow samples were collected on 3 or 4 occasions from each patient during neutropenic and nonneutropenic phases of the cycle. For comparison, two normal marrow samples were obtained from a volunteer and a patient being staged for possible solid tumor involvement of the marrow. Marrow and blood samples were respectively collected by needle aspiration of the posterior iliac crest and venipuncture in heparinized syringes. The samples (0.2-1.0 ml) were immediately centrifuged at 15001700g for 3 minutes in 1-ml glass tubes. The plasma was removed and the buffy coat (0.5-1.0 mm thick) was overlaid with cold 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.35. After 10 minutes' fixation the buffy coat was removed and minced with a razor blade in cold fixative. The specimens were allowed to fix an additional 50 minutes at 4 C and then rinsed in 0.1 M cacodylate, 7%o sucrose buffer. The specimens were then processed for morphology and cytochemistry. Peroxidase staining of primary granules was performed according to a modified method of Graham and Karnovsky.16 The specimens were incubated for 30 minutes in a solution obtained by dissolving 5 mg of 3,3'-diaminobenzidine in 10 ml Tris-HCl buffer, pH 7.6, and adding 3 drops of 3%o H202 immediately before use. Specimens processed for staining of vicinal glycol-containing complex carTable 1 -Cytoplasmic Vacuoles in
bohydrates, a positive stain for secondary granules,17 were incubated in a-amylase for removal of glycogen staining as described previously.17 Morphologic and peroxidase-stained specimens were postfixed for 1 hour at 22 C in 1%o OsO4 in 0.1 M cacodylate buffer, pH 7.35; whereas those specimens processed for vicinal glycol staining were not postosmicated. The specimens were then routinely dehydrated in graded alcohols and propylene oxide and embedded in Spurr low-viscosity medium. Thin sections, 50-70 nm thick, of morphologic preparations were collected on copper grids and counterstained with uranyl acetate and lead citrate, whereas cytochemical preparations were not counterstained. Thin sections of specimens for vicinal glycol staining were collected on stainless steel grids and stained with Thiery's periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method as described previously.7 The specimens were examined in a Philips 300 electron microscope at an accelerating voltage of 60kv. Results Blood and marrow findings could be grouped into three phases of the neutrophil cycle, which lasted approximately 15-21 days for each patient. The nonneutropenic phase of the cycle was that period when the absolute blood neutrophil count was more than 500 cells/cu mm. This generally lasted 5-7 days. An early neutropenic period lasting approximately 5-7 days followed the nonneutropenic period and was characterized by severe neutropenia with the predominant myeloid marrow cell being the promyelocyte,
Cyclic Neutropenia* Promyelocytes
With >3 vacuoles Marrow
specimen
Nonneutropenic Patient R.J. Patient M.J.
phaset
Early neutropenic phase Patient R.J. Patient M.J. Late neutropenic Patient R.J. Normal marrows Sample 1 Sample 2
phase
(n
=
25)
Average number of vacuoles per cell (mean
+
SD)
84% 84%
5.2 ± 2.4 5.0 ± 2.7
68% 32%
3.6 + 2.1 2.2 ± 1.6
12%
1.0 + 1.4
8% 4%
0.9 ± 1.1 0.6 ± 0.8
Segmented neutrophils Average number of vacuoles per cell With >3 vacuoles (mean ± SD) (n = 25) 16% 20%
1.1 ± 1.6 1.2 ± 1.5
4% 4%
0.6 ± 0.8 0.6 + 0.9
* The vacuoles or granules with heterogeneous material were presumed to be autophagic because of the presence of cytoplasmic organelles in some vacuoles; however, some vacuoles could conceivably be heterophagic in origin. The vacuoles scored did not include condensing vacuole precursors of primary granules. Data obtained by consecutively scoring 25 promyelocytes or segmented neutrophils in ultrastructural preparations. Inadequate numbers of segmented neutrophils were present in the neutropenic phase of the cycle to allow for comparative statistical evaluation. t Vacuoles in patient promyelocytes were significantly greater than in segmented neutrophils in the nonneutropenic phase of the cycle (P < 0.001), as well as normal marrow promyelocytes and segmented neutrophils (P < 0.001).
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·
No. 2
with only rare late neutrophils. The early neutropenic period was followed by the late neutropenic period, which lasted approximately 5-7 days and represented early marrow recovery with numerous promyelocytes, moderate numbers of myelocytes, and a few segmented neutrophils in the marrow, but blood neutrophil counts remained less than 500 cells/cu mm. On the basis of these criteria, samples from patient R.J. were obtained once in the nonneutropenic phase of the cycle, once in the early neutropenic phase, and twice in the late neutropenic phase. For patient M.J., samples were obtained twice in the nonneutropenic phase and once in the early neutropenic phase of the cycle.
Nonneutropenic Phase of the Cycle Promyelocytes in the nonneutropenic phase of the cycle appeared grossly abnormal. These cells contained numerous cytoplasmic vacuoles, most of which were presumed to be autophagic in origin (Table 1, Figure 2). The vacuoles were membrane-limited organelles containing heterogeneous material and/or myelinlike figures or lamellated membrane material. Mitochondria often appeared disrupted and were occasionally incorporated into autophagic vacuoles. Segments of endoplasmic reticulum contained variable dense material and occasional intracisternal
myelinlike figures. Fully condensed primary granules appeared decreased, whereas a relative increase in condensing vacuoles was apparent. Several primary granules also contained heterogeneous membrane
cn
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281
material. Relatively normal and grossly abnormal myelocytes were observed with similar frequency. The abnormal cells contained increased autophagic vacuoles and variable decreases in cytoplasmic granules, consistent with a maturation product of the abnormal promyelocytes. Segmented neutrophils demonstrated surprisingly normal morphologic features during the nonneutropenic phase of the cycle (Table 1, Figure 2). Although the number of autophagic vacuoles was decreased, compared with promyelocytes (P
