T H E PLATELET I N LEUKEMIC RETICULOENDOTHELIOSIS

Functional und Mo?-phological Evidence of a Qualitative Disol-de r

PETER H. LEVINE, MD, AND ISAO KATAYAMA, MD

Platelets were studied in a group of 10 patients with typical clinical coum, morphological findings, and specific histochemical criteria for leukemic reticu- loendotheliosis. In 8 of these, marked qualitative abnormalities were found. These included lack of aggregation following epinephrine stimulation (6 pa- tients), and decreased platelet factor 3 availability following ADP stimulation (4 patients). In addition, platelets in 4 of the 10 patients were studied by elec- tron microscopy. All had granular abnormality, and 1 caw showed the presence of rough-surfaced endoplasmic reticulum. The functional and ultrastructural abnormalities of platelets reported here may be responsible for the clinically important bleeding episodes which were not attributable to thrombocytopenia in 2 of our patients. The findings also provide a clue to the basic nature of this histogenetically controversial malignancy.

Cancer 36:1353-1558,1975.

EUKEMIC RETICULOENDOTHELIOSIS IS CHAR- L acterized by an insidious onset, usually indolent course, splenomegaly, and the pres- ence of “atypical reticulum cells” or “hairy cells” in the spleen, bone marrow, and blood.29 The atypical reticulum cells can be identified by a specific histochemical method, since they contain an isoenzyme of acid phosphatase which resists inhibition by tartrate.16 The pre- cise origin of the “atypical reticulum cells” remains unknown.

While isolated cytopenia or pancytopenia is not unusual in this disorder, it has generally been ascribed to the often massive spleno- megaly found. Indeed, splenectomy has been reported to produce marked improvement in cytopenias in a number of ~ases.2~ In the course of studying the response of thrombocy- topenia to splenectomy in one of our patients, we observed that the bleeding time remained

From the Blood Coagulation Laboratory, Hema- tology Service, New England Medical Center Hospital, and Department of Medicine, Tufts University School of Medicine, Boston, MA., and the Department of Pathology, St. Vincent Hospital, and University of Massachusetts School of Medicine, Worcester, MA.

Address for reprints: Dr. P. H. Levine, 171 Harrison Ave., Boston, MA. 02111.

T h e authors are indebted to Drs. Harvey Finkel and Lung Yam for their helpful advice and assistance in the completion of this work.

Received for publication August 28, 1974.

markedly prolonged in spite of a return to normal of the platelet count. For this reason, a prospective study of platelet function and ultrastructure was undertaken in a group of 10 patients with welldocumented leukemic reticuloendotheliosis. The qualitative platelet defects which were found form the basis for this report.

MATERIALS AND METHODS

Patient Population The 10 patients ranged in age between 41

and 69 years, and included 8 males and 2 fe- males. The clinical features of these cases have been outlined by our group in previous re- p o r t ~ . ~ ~ ~ ~ ~ In each case, diagnosis was con- firmed by classical light and electron micro- scope findings, as well as the histochemical demonstration of characteristic cells contain- ing the acid phosphatase isoenzyme resistant to tartrate.

No patients gave histories suggestive of life- long bleeding tendencies. Several had noted excessive bruising. Patient 4 had had excessive hemorrhage at the time of splenectomy, re- quiring blood transfusions, in the absence of severe thrombocytopenia. Patient 9 had noted excessive bleeding following dental extractions in recent months, and had had several episodes

1353

1354 CANCER October 1975 Vol. 36

of rectal bleeding, for which complete gastro- enterologic studies revealed no cause.

In each case, blood coagulation and platelet function studies were performed after 10 days of abstaining from all medications. None of these patients had received chemotherapy. A p propriate studies were performed in each case in order to exclude azotemia, liver dysfunc- tion, and abnormalities of serum proteins.

Technique Venous blood was collected in plastic sy-

ringes and immediately mixed (9: 1) with acid-citrate anticoagulent (6 parts 0.1 M so- dium citrate to 4 parts of 0.1 M citric acid) in plastic tubes. The blood was centrifuged at 200 g for 6 minutes at 22°C and the platelet- rich plasma (PRP) was removed. The remain- ing blood was then centrifuged at 850 g for 15 minutes to yield platelet-poor plasma (PPP). When necessary, the platelet count of the PRP was adjusted to a final count of 200,000-300,000/mm3, by either recentrifuga- tion or by addition of PPP.

Aggregation studies were completed on PRP within 90 minutes of its collection, using the turbidometric method of Born,l as modified by Mustard et a1.21 One and one-half milli- liters PRP were placed in the cuvette of a Chrono-Log aggregometer (Chrono-Log Corp., Broomal, PA) and the light transmittance was recorded on a moving-strip chart recorder. The blank for each study was a similarly treated sample of PPP. When the addition of an aggregating agent caused the platelets to form aggregates, the light transmittance in- creased, proportional to the size of the aggregates.

Adenosine diphosphate (ADP) (Sigma Chem- icals) was dissolved in veronal buffer (pH 7.35) at a concentration of 10 mg/100 ml, adjusted to pH 6.8, and frozen in aliquots at -40°C. Addition of 0.1 ml of this solution to the PRP during aggregation studies yielded a final concentration of 1.3 x l V M ADP. For some studies, the stock ADP was further diluted with veronal buffer to yield a final concentration of 2.6 x IO-UM ADP.

Dessicated bovine Achilles tendon (Sigma Chemicals) was used to make a collagen sus- pension by the method of Hovig.14 Aliquots of 5 mg collagen/ml of 0.9% NaCl were kept frozen at -40°C. Addition of 0.1 ml of this suspension to the PRP during aggregation studies yielded a final concentration of 0.33 mg collagen/ml PRP.

Epinephrine HCl (Parke-Davis), 1 mg/ml was stored in the dark until use; 0.2 ml was added to the PRP during aggregation studies, yielding a final concentration of 0.13 mg epi- nephrine per ml PRP.

Bleeding time was measured using the stan- dardized template technique of Mielke et aLZ0 Platelet adhesiveness studies were performed by the in vivo method of S a l ~ m a n . ~ ~

Platelet factor 3 (PF3) availability was de- termined by a modification of the method of Zucker and Peterson.30 T w o tenths milliliter of diluted Russell's viper venom (Stypven, Burroughs Wellcome) plus calcium chloride, 0.0125 M was added to the 0.1 ml PRP pre- warmed to 37"C, and clot formation timed (Stypven time). This measurement was made on aliquots of PRP taken from the aggrego- meter cuvette immediately before addition of the aggregating agent (Sample 1) and at 7

TABLE 1. Parameters of Platelet Function

Bleeding time Platelet ( Miel ke adhesiveness % Maximum platelet Platelet factor 3

template) Platelet count (Salzman) aggregation availability (seconds) Patient (minutes) ( x 103/mm8) (%) A B C D ADP Epinephrine

Normals 2.5-9.0 200-400 20-60 31-78 82-96 78-92 69-94 5.6-12.6 1.65.0 1 6 204 4 70 88 92 97 19.0 8.5 2 3.0 450 - 100 100 95 100 13.0 9.5 3 >20 56 36 18 25 0 0 7.4 0 4 18 89 29 13 30 36 0 4.0 0 5 9.5 67 85 15 19 46 0 0 0 6 6.0 196 79 61 62 76 32 13.1 9.0 7 18.5 110 51 29 59 39 0 13.0 0 8 3.5 211 - 36 85 90 71 6.0 2.5 9 7.0 120 - 2 14 32 0 4.0 0 10 > 50 38 12 20 27 0 0 0 0

Aggregating agents used were: A, ADP, 2.6 x 10+M; B, ADP, 1.3 X 10-bM; C, collagen; D, epinephrine.

No. 4 PLATELET IN LEUKEMIC RETICULOENDOTHELIOSIS * Leuine and Katayama 1355

z v) P

t a t- I- I -1

I- z W 0 W

52

a a

I CONTROL

PATIENT

m.

PATIENT

CONTROL

PATIENT

PATIENT

0 1 2 3 4 5 6 7 TIME IN MINUTES

FIG. 1. Platelet aggregation curves in Patient 4, compared with a normal control.

minutes afterward (Sample 2). Results were expressed as the number of seconds by which the Stypven time is shortened following aggre- gation (Sample 1 minus Sample 2), which is proportional to PF3 availability.

The quick prothrombin time, partial throm- boplasm time, and thrombin time were de- termined using standard methods,28 as were the Stypven time,'B clot solubility,*B and colorimetric fibrinogen measurement.1l Plate- let counts were performed by phase contrast microscopy.3

Sixty healthy volunteers abstained from all medications for 10 days, following which plate- let aggregation studies and PF3 availability measurements were performed by the methods noted above. Calculation of the mean result f 2 standard deviations was made for each test.

For electron microscopy, the buffy coat of heparinized blood was processed in the man- ner described previously.l7

RESULTS Half of the 10 patients exhibited prolonga-

tion of the template bleeding time. In 4 of these, the bleeding time was longer than would have been predicted for the given platelet count.13 Platelet adhesiveness measure- ments were made in 7 patients, and were nor- mal in all but 1. All of these data are sum- marized in the first three columns of Table 1.

Platelet aggregation studies revealed abnor- malities in 7 of the 10 patients. These ab- normalities usually took the form of lack of secondary wave (release reaction) with epi- nephrine, ADP, and collagen. In 6 cases, epinephrine caused no aggregation whatever. Platelet factor 3 availability was decreased in most of those cases which demonstrated abnormal aggregation. All of these data are summarized in the last six columns of Table 1. A typical set of platelet aggregation curves is shown in Fig. 1.

The following studies were normal in all cases: one-stage prothrombin time, partial thromboplastin time, thrombin time, clot solubility test, and fibrinogen level.

Electron microscopy revealed the majority of platelets from all four patients to have essentially normal ultrastructure. Most plate- lets contained the usual number and size of organelles: azurophilic granules with occa- sional electron-dense bodies, mitochondria, vacuoles, and vesicles, Golgi apparatus, micro- tubules, and glycogen. However, there was in each patient a variable number of platelets which had granules of giant size or unusual shape (Fig. 2). Furthermore, in one patient, some platelets contained profiles of ribosome- studded endoplasmic reticulum (Fig. 3).

DISCUSSION Our results indicate that the blood platelet

is involved in LRE in a qualitative manner, aside from the well-known quantitative deficit in this illness. In some cases, the functional and morphological abnormalities were strik- ing. For example, abnormality of the second- ary wave of platelet aggregation (release re- action) is common in a variety of settings,2 but total lack of aggregation on exposure to epinephrine is unusual. Unusual also is the presence, in occasional platelets from one pa- tient, of ribosome-studded endoplasmic retic- ulum. Rough-surfaced endoplasmic reticulum in circulating human platelets has only rarely been described previously, in gray platelet

1356 CANCER October 1975 Vol. 36

FIG. 2. Electron mi- crograph of a platelet showing granules of giant size and unusual shape (Uranyl and lead, ~29,900).

FIG. 3. Electron mi- crograph of a platelet showing profiles of ri- bosome-studded endo- p lasmic r e t i c u l u m (Uranyl a n d lead, x 23,600).

No. 4 PLATELET IN LEUKEMIC RETICULOENDOTHELIOSIS * Lm’ne and KQ~QYQWUI 1357

syndrome,m in a case of acute myelofibrosis,4 and most recently in chronic myeloprolifera- tive disorders.10

In our experience, patients with thrombo- cytopenia do not begin to exhibit prolonga- tion of the bleeding time until the platelet count falls to levels below 75,000 per cubic mm, unless a qualitative defect is present. This observation is in accord with published studies by Shulman et al.24 A precise relation- ship between platelet count and bleeding time has recently been put forth.13 Patient 3, 4, 7, and 10 all had bleeding times of 18 minutes or more, at platelet counts ranging between 50,000 and 110,000/mm3. This severe pro- longation of bleeding time alerted us to the possibility of a qualitative platelet defect.

In the present study, 8 of 10 patients demon- strated thrombopathy, and 2 had histories in- dicative of significant hemorrhagic tendency. Next to infection, hemorrhage is the second most frequent cause of death among patients with LRE.l6 Thrombopathy may contribute to this statistic.

Leukemias and lymphomas are now under- going reappraisal as to their histogenesis be- cause of newly available parameters such as immunologic surface receptors and membrane anatomy by scanning electron microxopy.6822 In this field, LRE is drawing particular atten- tion among the investigators.7 So far, the data are conflicting, e.g. immunologic and scanning electron microscopic studies have suggested B-lymphocyte origin to sorne,e,B and monocyte origin to others.’* Similarly, positive phago- cytic capacity of LRE cells was demonstrated as indicative of their macrophagic nature by one group,10 while a negative result for phago- cytic function in LRE cells was one of the bases for the lymphocyte theory of another group.0 The platelet defects demonstrated in this study should be appraised in this context. The high incidence and type of platelet de- fects in LRE are quite comparable to those of various myeloproliferative disorders.*~26 On this basis, LRE is more identifiable with mye- loproliferative than with lymphoproliferative disorders.

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