Tohoku J. exp. Med ., 1966, 89, 387-399

Histochemical Studies on Leukocyte Alkaline Phosphatase Activity with Special Reference to Various Types of Hemolytic Disorders

Shinobu Sakamoto Departmentof Internal Medicine(Prof. T. Torikai), TohokuUniversity School of Medicine,Sendai

Alkaline phosphatase activity of neutrophils was estimated histochemically with special reference to various types of hemolytic disorders. Enzyme activity

tended to be lower than normal in all but one of the cases studied . Leukocytes incubated for one hour at 37•Ž in plasma obtained from a patient with paroxysmal nocturnal hemoglobinuria and from a patient with congenital spherocytosis showed a decrease in enzyme activity. When leukocytes were incubated in

normal plasma containing bovine hemoglobin, enzyme activity also decreased . Plasma obtained from patients with hyperbilirubinemia caused by bile duct obstruction did not affect enzyme activity in vitro at all. It was concluded from these observations that the low enzyme activity seen in various hemolytic disorders was due to the inhibitory effect of hemoglobin in plasma and not due to indirect bilirubin. Differences in enzyme activity in these hemolytic states were considered to be due to differences in plasma hemoglobin concentration.

It was Kaplow1 who first devised a fairly satisfactory, clinically applicable cytochemical staining method for the demonstration of leukocyte alkaline phosphatase activity in blood smears. This method, however, had disadvantages of low sensitivity and ill-defined localization of enzymatic activity. With this method, it is difficult to detect low levels of activity of this enzyme in leukocytes and it is impossible to determine minute differences in enzyme activity. Recently it was disclosed that certain substituted naphthol AS phosphate derivatives2-5 offered most satisfactory staining results because of their sensitivity, specificity and sharp topographical localization of enzymatic activity in cells. Its diagnostic value in certain hematological disorders is well established.5-12 However, little is known about what influence the strength of the activity of this enzyme has on the leukocyte and in what way this enzyme activity is regulated in leukocytes. It is a well known fact that enzyme activity is very low in paroxysmal nocturnal hemoglobinuria.6,10,13-16 However, only a few studies have been presented concerning this enzyme in hemolytic disorders other than paroxysmal nocturnal hemoglobinuria. Tanaka et al.13 reported depressed activity of the

Received for publication, April 27, 1966. 387 388 S. Sakamoto

enzyme in some cases of acquired hemolytic anemia and sickle-cell anemia. Martinez-Maldonado et al.8 found the same tendency in a case of sickle-cell anemia. On the other hand, Hayhoe and Quaglino7 reported normal or high enzymatic activity in four cases of idiopathic acquired hemolytic anemia. Mitus et al.10 reported normal activity in a case of acquired hemolytic anemia. Hashimoto10 observed normal activity in four cases of congenital spherocytosis and in a case of congenital non-spherocytic hemolytic anemia; and slightly elevated activity in a case of acquired hemolytic anemia with leukemoid reaction. The same observations were made by Leonard et al.9 in acquired auto-immune hemolytic anemia with leukemoid reactions. As cited above, quite different findings have been reported by different investigators. This led the present author to examine leukocyte alkaline phosphatase activity in various types of hemolytic disorders including paroxysmal nocturnal hemoglobinuria and to determine whether any influence of hemolysis on this enzyme activity in leukocytes can be seen.

MATERIALS AND METHODS Sources: Thin blood films on glass slides were obtained from 40 healthy subjects (20 males and 20 females), consisting of doctors, nurses and others, ranging from 18 to 53 years in age. The various hemolytic diseases studied in this report are as follows: a) 2 cases of acquired auto-immune hemolytic anemia, b) 3 cases of congenital spherocytosis, c) I case of congenital non-spherocytic hemolytic anemia, d) 3 cases of paroxysmal nocturnal hemoglobinuria, e) 1 case of methemoglobinemia, and f) 1 case of sulfhemoglobinemia. Histochemical demonstration of leukocyte alkaline phosphatase activity: Leu kocyte alkaline phosphatase activity was demonstrated histochemically by Tomonaga's method in which naphthol AS-MX phosphate was used as the substrate and fast blue RR salt as the azo dye.5 Scoring was done according to Tomonaga's modified criteria and the sum of the ratings of 100 successive neutrophils was designated as the alkaline phos phatase score, "AP" scores, for a given blood smear. Examination of in vitro effect of plasma obtained from patients with hemolytic disorders on leukocyte alkaline phosphatase activity: By venipuncture with a heparinized syringe, 10 ml of blood were obtained respectively from a patient with paroxysmal nocturnal hemoglobinuria and from a patient with congenital spherocytosis and with a low "AP" score. These blood samples were placed separately in test tubes and centrifuged at 1,500 r.p.m . for 5 minutes. Three milliliters of plasma were taken from each of these samples and kept aside for a while. Leukocyte Alkaline Phosphatase in Hemolytic Disorders 389

In the same way 10 ml of blood were drawn from healthy persons with the same blood type as the above-mentioned patients and poured into test tubes. After standing one hour at room temperature, these samples were centrifuged at 1,500 r.p.m. for 5 minutes.

The buffy coat was collected by capillary pipette and poured into the test tubes containing previously prepared plasma. The contents were then mixed

gently, but well. The rest of the blood, from which the buffy coat was taken, was used as control during incubation. After incubation of these mixtures and

control samples for one hour at 37•Ž, the buffy coat was collected into Wintrobe

tubes by capillary pipette and centrifuged at 1,000 r.p.m. for 3 minutes. This

last procedure was omitted when white cells proved to be sedimented sufficiently

as a white layer after incubation. The in vitro effect on leukocyte alkaline

phosphatase of plasma from a patient with chronic myelogenous leukemia was also examined in a similar way.

Examination of in vitro effect of hemoglobin on leukocyte alkaline phosphatase activity: As described previously, 2 ml of normal plasma were prepared in a test

tube. The rest of the blood from which plasma was taken was used as a leukocyte

source and as control during incubation. Eight milligrams of bovine hemoglobin

(Tokyo Kasei, commercially available) were dissolved in this plasma. The huffy coat of the same blood sample was placed into this hemoglobin dissolved plasma

and mixed gently and well. Blood films were made in exactly the same way from the leukocyte layer after one hour incubation at 37•Ž. Examination of in vitro effect of plasma obtained from patients with hyper bilirubinemia on leukocyte alkaline phosphatase activity: Plasma obtained from two patients with obstructive jaundice was used in this experiment. The procedure was the same as described above.

RESULTS 1. Normal subjects

The results obtained from normal subjects are summarized in Table 1. "AP"

scores for healthy men were distributed in the range from 156 to 271 and those for healthy women ranged from 183 to 334. "AP" scores were higher for women

than for men. The mean and standard deviation for both sexes were 233.9 •} 60.4. Women were all premenopausal and were not menorrheic when examined.

2. Hemolytic diseases It is obvious from Fig. 1 that all but one case among the hemolytic disorders showed lower values than normal. In acquired auto-immune hemolytic anemia, one case showed a remarkably low value which approximates the value seen in chronic myelogenous leukemia, while the other showed a subnormal value. The course of this case with low 390 S. Sakamoto

TABLE 1. Neutrophil alkaline phosphatase scores in healthy subjects

* Standard deviation

Fig. 1. Neutrophil alkaline phosphatase activity in hemolytic disorders. enzyme activity is illustrated in Fig. 2. This case was the so-called "Lederer type" with leukemoid reaction. When first studied at the time of admission, its "AP" score was found to be near the upper limits of the normal range, and leukocytosis was observed. As leukocyte counts returned to normal and anemia improved by the administration of steroid hormone, its "AP" score became remarkably low. The "AP" score remained low until the second leukemoid reaction and marked anemia occurred. Unfortunately, leukocyte alkaline phosphatase activity was not determined at the time of the highest leukocyte count, but the "AP" score rose to normal when leukocyte counts became normal. Afterwards, contrary to the increase in erythrocytes and leukocytes, the "AP" score fell below normal. The probable implication of this phenomenon will be discussed later. Relatively low values were obtained in two of three cases of congenital apherocytosis; the other case showed normal values. A case of congenital non spherocytic hemolytic anemia which showed no hitherto known erythrocyte Leukocyte Alkaline Phosphatase in Hemolytic Disorders 391

Fig. 2. Acquired auto-immune hemolytic anemia (male, 40 y.).

Fig. 3. Paroxysmal nocturnal hemoglobinuria following aplastic anemia (male 28 y.). Note gradual fall of "AP" score and its return to normal after disappearance of hemoglobinuria. 392 S. Sakamoto

Fig. 4. Relationship between serum indirect bilirubin and neutrophil "AP" score. enzyme defects had moderately low enzymatic activity. Remarkably low values were obtained in two cases of paroxysmal nocturnal hemoglobinuria, and a fairly low value was obtained in another case. Aplastic anemia was first diagnosed in this last case, but clinical pictures turned to those of paroxysmal nocturnal hemoglobinuria in the course of the disease. As shown in Fig. 3, this patient presented very high "AP" scores when he was treated as having aplastic anemia. A gradual fall of "AP" scores was noted but its implication was rather obscure until hemoglobinuria occurred. The "AP" score returned to normal when complete remission ensued. From Fig. 1, it is also apparent that enzyme activity was fairly low in sulfhemoglobinemia. The cause of this sulfhemoglobinemia was considered to be habitual use of magnesium sulfate in doses of 60 to 70 g daily for several years because of constipation. The "AP" score was subnormal in a patient with methemoglobinemia caused by accidentally drinking petroleum. The relationship between serum indirect bilirubin level and leukocyte alkaline phosphatase activity is illustrated in Fig. 4. Although there exists no apparent negative correlation between the two, it can be inferred that there is a tendency for enzyme activity to become lower with increased serum indirect bilirubin level . 3. In vitro studies a) Effect of plasma from patients with hemolytic diseases In order to ascertain whether it was due to a factor (or factors) in plasma Leukocyte Alkaline Phosphatase in Hemolytic Disorders 393

Fig. 5. In vitro effect of plasma from hemolytic patients on alkaline phosphatase activity of normal neutrophils.

Fig. 6. In vitro effect of plasma from a patient with chronic myelogenous leukemia on alkaline phosphatase activity of normal neutrophils.

that leukocyte alkaline phosphatase activity was low in hemolytic states, in vitro studies on the effect of plasma on this enzyme in leukocytes were conducted using the previously described methods. It is apparent that plasma obtained from paroxysmal nocturnal hemoglo binuria and congenital spherocytosis markedly depressed the enzyme activity (Fig. 394 S. Sakamoto

Fig. 7. In vitro effect of bovine hemoglobin on alkaline phosphatase activity of normal neutrophils.

Fig. 8. In vitro effect of plasma from hyperbilirubinemic patients on alkaline phosphatase activity of normal neutrophils.

5). On the contrary when, normal leukocytes were incubated in plasma obtained from a patient with chronic myelogenous leukemia and who showed a low "AP" score, no decrease of enzyme activity was observed (Fig. 6). Therefore, inhibitory action on enzyme activity seemed to be specific to plasma from patients in hemolytic states with low "AP" scores. b) Effect of hemoglobin on leukocyte alkaline phosphatase activity Bovine hemoglobin was chosen for this experiment because it is commercially available. As for the doses of hemoglobin, 4 mg per ml was adopted because this value is sufficiently above the renal threshold for hemoglobin, as in paroxysmal nocturnal hemoglobinuria. Bovine hemoglobin inhibited leukocyte alkaline phosphatase activity to a considerable extent (Fig. 7). Leukocyte Alkaline Phosphatase in Hemolytic Disorders 395 c) Effect of plasma from patients with hyperbilirubinemia The estimated values of plasma bilirubin in one case of hyperbilirubinemia were 24.6 mg/100 ml in total, 18.9 mg/100 ml for direct bilirubin, and 5.7 mg/100 ml for indirect bilirubin; and those in another case were 20.1 mg/100 nil in total, 14.2 mg/100 ml for direct bilirubin, and 5.9 mg/100 ml for indirect bilirubin.

There was no inhibitory effect on leukocyte alkaline phosphatase activity in these plasmas (Fig. 8). This result was expected because the patients from whom plasma had been drawn showed high enzyme activity in leukoƒÃytes.

DISCUSSION

Although leukocyte alkaline phosphatase has been studied by both biochemical and histochemical methods, the advent of substituted naphothol AS phosphate derivatives has made it easier, more satisfactory and reliable to demonstrate this enzyme activity histochemically in routine examinations. Many investigators have worked with keen interest to disclose the mechanism by which leukocyte alkaline phosphatase activity is controlled. They have attempted this by studying enzymatic activity in association with the etiology of certain hematologic disorders and from the standpoint of its function in neutrophils. Nevertheless, it is as yet only poorly understood. Valentine and his co-workersle18,19 demonstrated that pituitary-adrenal hyperfunction increased peripheral leukocyte alkaline phosphatase activity. This system was considered partly as a common denominator in various stressful sit uations such as infection, surgery, myocardial infarction, etc., where elevated enzyme activity was observed. They proved this theory experimentally in human beings by administration of ACTH, hydrocortisone and cortisone. In the present study women showed higher "AP" score than men. Recently, Rosner and Lee20 claimed that different leukocyte alkaline phosphatase activity in men and women was due to differences in endocrine function and suggested that androgenic hormones might act inhibitorily on this enzyme. It is evident that alkaline phosphatase activity of neutrophils shows a tendency to be lower than normal in all but one of these hemolytic states. This seems to be a very interesting and significant finding. In the light of the influence of certain substances" on enzyme activity in vitro, great interest is aroused on the problem as to whether this is due to some defect in the leukocyte itself or is due to some factors in plasma. Tanaka and his co-workers13 considered that the low enzyme activity in paroxysmal nocturnal hemoglobinuria might be an indication of biochemical defect in leukocytes. In the present experiment, when leukocytes were incubated at 37•Ž for one hour in plasma obtained from a patient with paroxysmal nocturnal hemoglobinuria and from a patient with congenital spherocytosis, neutrophil alkaline phosphatase was markedly inhibited as compared with those incubated in normal plasma. As 396 S. Sakamoto it is generally accepted that enzyme activity is increased when leukocytes are incubated in their own serum for one hour,11,13 this suggests that certain inhibitors of this enzyme really exist in the plasma of these two diseases. On the other hand, no inhibitor exists in plasma of chronic myelogenous leukemia in which whitout exception low enzyme activity is observed. Weissel22 reported the same observation that there was no inhibitor in plasma of patients with chronic myelogenous leukemia. This indicates that the similarly low enzyme activity demonstrated in paroxysmal nocturnal hemoglo binuria and chronic myelogenous leukmia is a reflection of quite different mechanisms. What factors in plasma are responsible for this phenomenon? Does marked reduction in leukocyte alkaline phosphatase in paroxysmal nocturnal hemoglobinuria suggest that hemoglobin or its metabolites in plasma may play an important role in reducing this enzyme activity? The above inference was proved to be true by the present study in which normal leukocytes were incubated in plasma containing bovine hemoglobin. Enzyme activity was apparently suppressed by this manipulation. Although the direct effect in vitro of indirect bilirubin itself on neutrophil alkaline phosphatase could not be examined in the present experiments because of technical difficulties, plasma obtained from two patients with hyperbilirubinemia did not affect enzyme activity at all. Total bilirubin was markedly elevated in these two samples of plasma, and indirect bilirubin levels approximated those found in hemolytic anemias. Therefore, it may be said that neither direct bilirubin nor indirect bilirubin in plasma contributed to lower enzyme activity in hemolytic disorders. Thus, it might be hemoglobin in plasma that was responsible for the suppression of enzyme activity. As reported by Crosby,23,24 plasma hemoglobin concentration is elevated in various types of hemolytic disorders. Plasma hemoglobin levels were not determined in the patients examined in the present study. It can, however, be inferred that the relatively wide distribution of "AP" scores in hemolytic states shown in this study might be due to differences in plasma hemoglobin concentration. In other words, larger increase in plasma hemoglobin level may act more inhibitorily on this enzyme; the few cases which showed normal or slightly lower values than normal might have normal or slightly elevated hemoglobin levels insufficient to depress enzyme activity. This is supported by the fact that without exception low values are found in paroxysmal nocturnal hemoglobinuria6,13-16 and in sickle-cell anemia8,13 where plasma hemoglobin levels are highly increased. Further support for this hypothesis is the present observation of a case of paroxysmal nocturnal hemoglobinuria following aplastic anemia. As mentioned previously, this case showed remarkably high values when it was treated as aplastic anemia for a few years. Then, "AP" score gradually fell and hemoglobinuria occurred. Leukocyte Alkaline Phosphatase in Hemolytic Disorders 397

Enzyme activity returned to normal in step-wise fashion, as complete remission occurred. Tanaka et al.13 stated that alkaline phosphatase activity returned to normal in cases of paroxysmal nocturnal hemoglobinuria in the phase of complete remission. This interesting phenomenon can be explained as follows: It is probable that a gradual increase of hemoglobin occurred to such an extent as to suppress enzyme activity when the "AP" score began to fall . With the disappearance of hemoglobinemia, the enzyme activity returned to normal, because the inhibitory effect of hemoglobin was removed . If this hemoglobin theory is applied to the explanation of the low enzyme activity in one of the present cases of acquired auto-immune hemolytic anemia, it seems to take some interval for the manifestation of the inhibitory effect on the enzyme . Not only hemoglobin but also sulfhemoglobin and methemoglobin might inhibit this enzyme activity, although only one case of each was examined in the present study. Very recently, Lewis and Dacie25 reported low alkaline phosphatase activity of neutrophils in severe cases of paroxysmal nocturnal hemoglobinuria, in contrast with normal or above normal activity in less serious cases. Furthermore, they observed an inverse correlation between the "AP" score and the degree of lysis of erythrocytes in acidified serum test. They tried to explain this different enzyme activity by the double population theory. However, if the degree of lysis in acidified serum is considered as showing susceptibility of erythrocytes to intravascular hemolysis, there would be no objection to considering that plasma hemoglobin is responsible for the different enzyme activity. A hypothesis was recently proposed that leukocyte alkaline phosphatase activity might be governed by a gene on chromosome 21.26 Karyotype studies were not done in all cases studied in the present experiments. No chromosomal abnormalities, however, were discovered in any of the three present cases of paroxysmal nocturnal hemoglobinuria. Further, no paroxysmal nocturnal hemoglobinuria with chromosomal abnormalities has been reported in the literature.27,28 From the present observation, it is rather unlikely that low alkaline phosphatase activity in this disease is a reflection of chromosomal abnor malities. As mentioned above, neutrophil alkaline phosphatase activity was increased after one hour incubation in its own serum, but no remarkable increase occurred in neutrophils of paroxysmal nocturnal hemoglobinuria incubated in normal plasma. Rebuck et al. found no increase in the enzyme activity in the exudate cells from a patient having paroxysmal nocturnal hemoglobinuria.29 Examination was not carried out as to whether neutrophil enzyme activity in paroxysmal nocturnal hemoglobinuria was not influenced by those stimuli known to increase enzyme activity. The variability of this enzyme in leukocytes may 398 S. Sakamoto be due to multiple factors which are not fully understood as yet, but at least the problem as to what causes the hitherto reported low enzyme activity in some cases of hemolytic disorders seems to be solved by the present study. Hemoglobin, be it in any form, suppresses leukocyte alkaline phosphatase activity both in vivo and in vitro, and it can become a common inhibitor in various types of hemolytic states when it exists in plasma in such a concentration as to suppress enzyme activity.

Acknowledgment

The author wishes to express his sincere gratitude to Prof. T. Torikai and Dr. A. Shibata for their guidances and to Dr. S. Takase and Dr. A. Suzuki for their assistances throughout this study.

References

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