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Unstimulated whole saliva was collected from 21 HIV-positive men and women before and after dental treatment. The frequency of HIV detection did not increase after dental treatment. Infectious HIV was recovered from only one patient. Study findings raise the possibility that, in most cases, salivary inhibitors render the virus non-infectious.

In this chapter I will attempt to reconcile the existing data on such variables as platelet size, age, survival, cytoplasmic demarcations and megakaryocyte heterogeneity in order to construct a coherent whole.

Hematocrit,% Reticulocytes, % Erythrocyte porphobilinogen deaminase and uroporphyrinogen decarboxylase were measured as previously described (5, 6) by using porphobilinogen and pentacarboxylporphyrinogen I, respectively, as substrates. CV5 for these assays in this laboratory are 8.3% and 14.2%, respectively. rocytes,it is possible that detection of these diseases could be compromised when coexisting conditions or interventions stimulate erythropoiesis. It will be of interest, for example, to determine if erythrocyte uroporphyrinogen decarboxylase increases during therapeutic phlebotomy in patients with porphyria cutanea tarda. The intent of this therapy, which is effective in both inherited and acquired forms of porphyria cutanea tarda, is to stimulate erythropoiesis and thereby utilize and reduce iron stores. If phlebotomy does increase erythrocyte uroporphyrinogen decarboxylase, it might be recommended that the enzyme activity be measured before institution of phlebotomy.

The peripheral blood film and the red blood cell size distribution histogram were examined for evidence of red blood cell fragments in 2,350 subjects. To distinguish subjects with greater than or equal to 10 fragments/1,000 red blood cells (abnormal) from normal, examination of the blood film was 0.83 sensitive and 0.30 specific, whereas examination of the histogram was 0.97 sensitive and 0.87 specific. The most common causes of abnormal fragmentation were malignancy with cytotoxic chemotherapy and severe iron deficiency. In two subjects, an abnormal red blood cell fragmentation pattern was the clue to a spectrin mutant in subjects with an automated blood count previously evaluated as normal. The data suggest two conclusions: the red blood cell volume histogram appears more accurate than the peripheral blood film for routine identification of red blood cell fragments; and asymptomatic spectrin abnormality identifiable by abnormal histogram may be a relatively common disorder.

In the blood of normal subjects, the volumes of single erythrocytes are distributed with a coefficient of variation (CV) of 10.8 +/- 1.8%; while in hemoglobinopathies, CV increases proportionately to the degree of anemia produced. Using single cell Soret-band hemoglobinometry and focused-aperture impedance counting, we compared the distribution of red cell volume, area, hemoglobin content, and hemoglobin concentration in normals and subjects with anemic disorders. The CV, nondiscrete heterogeneity, is first, a general characteristic of biologic measurement, second, a sensitive indicator of abnormality of erythropoiesis, and third, consistently less for hemoglobin concentration than for volume, area, or hemoglobin content of the same cells.

In four patients with cold agglutinins, erythrocyte mean corpuscular volume was increased. Erythrocyte volume histograms showed that in each case macrocytosis was due entirely to doublet erythrocytes counted as single cells. For two of the four patients, macrocytosis was reported intermittently, when the blood had been allowed to cool to room temperature during laboratory processing. For these two patients, macrocytosis due to doublet erythrocytes could be elicited in vitro proportionate to the degree of cooling of the blood and could be abolished to rewarming to 37 C. Two other patients had macrocytosis constantly during the acute illness. Otherwise unexplained macrocytosis may reflect cold agglutinin activity without overt hemolysis, particularly if the macrocytosis is intermittent.

We measured the complete blood cell count, including the index of RBC size heterogeneity (RBC distribution width [RDW]), in 181 subjects without anemia to determine whether RDW became abnormal earlier in the development of iron deficiency than did other variables. In 163 subjects selected only for otherwise normal blood cell counts, an increased RDW was 66% specific (48/73) and 100% sensitive (48/48) for decreased serum iron saturation. Stool guaiac testing was equally specific but less sensitive. In 13 subjects with polycythemia, as iron deficiency developed, RDW increased a minimum of four weeks before mean cell volume changed. In one apparently normal young woman, an abrupt isolated rise in RDW revealed a newly developed low serum transferrin saturation; all values remained normal during serial testing of four other normal subjects. We conclude that RDW is the part of the routine blood cell count that first becomes abnormal during the development of iron deficiency.

New automated blood cell analyzers provide an index of red cell volume distribution width (RDW) or heterogeneity and a histogram display of red cell volume distribution. We have developed a classification of red cell disorders, based on mean corpuscular volume (MCV) or red cell size, heterogeneity, and histograms, to guide diagnosis from the peripheral blood analysis. The distinction of iron deficiency anemia from heterozygous thalassemia or the anemia of chronic disease and the detection of early iron and folate deficiency is improved. Red cell volume distribution histograms identify red cell fragmentation or agglutination, dimorphic populations, and artifactual counting of lymphocytes as red cells. We recommend the use of these new variables in the initial classification of anemia by the practicing physician.

An increasing number of kindreds with thrombocytopenia have been described in the past decade as platelet counts have been done regularly. These patients are delineated from ITP, recognizing that some patients with hereditary thrombocytopenia may benefit from splenectomy. Until more information on platelet biochemical information is available, these disorders have been categorized by platelet size, now quantifiable as mean platelet volume (MPV). The availability of rapid and quantitative measurement of platelet count and MPV allow a better understanding of megakaryocyte proliferation and platelet production. The evolving changes of MPV during treatment and recovery from chemotherapy will now allow a forecast of marrow hypoplasia, since the platelet count and increase in MPV follow the increase in megakaryocyte ploidy to herald marrow regeneration. It is anticipated that the careful plotting of the MPV relationship to the platelet count will allow the physician to evaluate marrow function indirectly, in many cases replacing repeated bone marrow studies.

Sixteen adults with chronic acquired aplastic anemia had abnormally large RBCs and abnormally small platelets before chemotherapy. During their therapy, transfusion initially obscured these macrocytic RBCs. In the eight who had erythropoietic recovery, endogenous RBCs again were macrocytic, and platelets remained small whether or not the platelet count increased. The percentage of cells containing hemoglobin F changed in only one of the eight subjects. In contrast, the eight who did not have erythropoietic recovery had no reappearance of macrocytes. Of 19 other previously treated patients whose hemoglobin level had recovered to normal for seven to 196 months, 16 had increased mean corpuscular volume (101 to 133 femtoliters) and abnormally small platelets. We conclude that in aplastic anemia the appearance of macrocytes reliably and easily predicts RBC recovery. Furthermore, even in treated, apparently recovered subjects, an abnormality of blood cell size remains.

To permit quantitative study of red cell volume heterogeneity, the coefficient of variation (CV) of erythrocyte volume was determined in blood samples from 868 subjects, including normal individuals and those with various hematologic disorders. In groups of subjects with various hemoglobinopathies, CV increased above normal in parallel with the presence and degree of anemia. However, all subjects with iron, folate, or vitamin B12 deficiency showed marked increase in CV (anisocytosis): increased CV accompanied even early iron deficiency not yet associated anemia or microcytosis. Effects of other conditions on CV also were studied.

We measured whole-blood mean platelet volume (MPV) and platelet count, determined by the Coulter Counter model S-Plus, in 683 normal subjects. There was nonlinear, inverse relation between MPV and platelet count throughout the normal range of platelet count: the change in MPV was most pronounced at the lower platelet counts. Because of this non-linear relation, "platelet-crit," the product of platelet count and MPV, was not constant, but varied directly with platelet count. Thus, the definition of "normal" values for MPV requires simultaneous reference to platelet count. In the whole blood of 13 patients, non-platelet cell fragments spuriously increased the automated platelet count. Seven patients with erythrocyte fragmentation had spuriously high MPV also: correct MPV and platelet count could be obtained from platelet rich plasma (PRP). In five patients with leukemic blast fragmentation, MPV remained spuriously low and platelet count spuriously high in PRP: only phase count allowed correct count. A patient with megakaryoblastic leukemia had megakaryoblast fragments confused for platelets.

We determined the platelet count and MPV in 100 normal subjects, in 147 subjects with thrombocytopenia or thrombocytosis due to other than primary hematologic disorders, and in smaller groups with immune or septic thrombocytopenia or iron deficiency. In these groups, the inverse, nonlinear relation between MPV and platelet count was the same as in a previous study of normal subjects. The same relation between platelet volume and count was found in individual patients as platelet counts rose during recovery from immune or septic thrombocytopenia. The concomitant progressive fall in MPV during recovery from thrombocytopenia, at which times rapidly rising platelets counts were necessarily associated with a population of young platelets, suggests that magnitude of stimulation of thrombopoiesis, not platelet age, is the major determinant of platelet volume. In contrast, as compared to normal persons with similar platelet counts, MPV was increased in subjects with heterozygous thalassemia but decreased in patients receiving chemotherapy for malignancy or renal transplantation. The undefined mechanism of regulation of platelet formation from megakaryocytes, reflected by the inverse relation of platelet size and count, thus seems altered in these disorders. Platelet volume is an easily obtained variable that appears to be useful in the evaluation of abnormal platelet production.

Presumptive distinction between iron deficiency and heterozygous thalassemia by analysis of the automated blood count and differential continues to be a challenge. We compared two proposed numerical discriminants (MCV2 x MCH, and MCV2 x RDW/100 x Hb) with an analytic microcomputer program (BCDE2 LeaFebiger). In 7114 subjects, the numerical discriminants and the BCDE2 program correctly identified greater than 90% of thalassemia. In subjects with iron deficiency, the BCDE program was greater than 90% sensitive and specific for positive identification, while the numerical discriminants were less than 75% sensitive and specific at inferential identification. The numerical discriminants, BCDE2, and 17 experts in blood counting were asked to interpret the blood-count data in 7 fully-defined actual cases. The mean experts' score was 5.65 cases correct out of 7. The BCDE program was correct in all cases. The numerical discriminants could not analyze all cases, and both were incorrect in at least one case. We conclude that for the task of analyzing blood counts for microcytic disorders, microcomputer analysis by BCDE outperforms both numerical discriminant functions and analysis by expert hematologists.

In normal persons, the mean platelet volume varies inversely but nonlinearly with the platelet count. Those with immune platelet destruction have a mean platelet volume that conforms to normal values. Nine patients with thrombocytopenia due to post-trauma sepsis without disseminated intravascular coagulation had a lower than normal mean platelet volume as the platelet count fell. In six patients who recovered, the mean platelet volume increased to the normal value before the platelet count began to rise. In contrast, three patients who did not recover from sepsis had no change from the low mean platelet volume and thrombocytopenia. Since mean platelet volume can now be part of the routine blood count, it is a useful variable to assay the degree of bone marrow suppression in the patient with thrombocytopenia and can be used as a predictor of recovery from thrombocytopenia.

Classification of platelet disorders has been based on the platelet count. Addition of a second variable, mean platelet volume (MPV), to the routine blood count allows classification of patients into 9 categories: high, low, or normal MPV, and high, low or normal platelet count. We studied 1,244 adult inpatients. 1,134 had both platelet values normal. 11 patients had high MPV and low platelet count: all had hyperdestructive causes. 15 patients had high MPV and normal platelet count: 12 had heterozygous thalassemia, and three had iron deficiency. Seven patients had high MPV and high platelet count: causes included myeloproliferative disorders, inflammation, iron deficiency, and splenectomy, 25 patients had high platelet counts and normal MPV: the causes were inflammation, infection, sickle cell anemia, iron deficiency, or chronic myelogenous leukemia. 52 patients had an MPV that was inappropriately low for the platelet count (high, normal, or low). All had sepsis, splenomegaly, aplastic anemia, chronic renal failure, or a disease being treated with myelosuppressive drugs. High MPV thus appears correlated with myeloproliferative disease or thalassemia; and low MPV, with cytotoxic drugs or marrow hypoplasia. Addition of MPV to the platelet count allows subtler disorders to be detected (when the platelet count is normal), and allows distinction of the cause of thrombocytopenia.

Coefficient of variation (C.V.) of red cell volume is inversely proportional to red cell mass in hemoglobinopathies and thalassemias. Even a subject with markedly reduced red cell survival and abnormal hemoglobin had a normal C.V. associated with normal hemoglobin level. In contrast are patients with nutritional deficiency, who have increased C.V. whether or not there is anemia, and patients with hypoplastic anemia, who have normal to near-normal C.V. independent of anemia.

Mononuclear cells from the peripheral blood of a patient with megakaryoblastic transformation of Philadelphia chromosome-positive chronic myelogenous leukemia were cultured. Morphological and cytochemical studies and cell ploidy determinations were done daily for 4 days. PAS staining of the cells increased progressively during culture. Ultrastructural study of circulating and cultured cells revealed demarcation membranes and alpha granules indicating the cells were micromegakaryocytes. Deoxyribonucleic acid synthesis, determined by 3H-thymidine uptake, peaked at 72 hours. The DNA content of cultured cells was diploid at all times. All 15 metaphases analyzed at 72 hours were Ph1-positive. Malignant (Ph1-positive) megakaryoblasts and micromegakaryocytes grown successfully were capable of partial cytoplasmic maturation as demonstrated by glycogen deposition and increase in subcellular organelles, while endoreduplication was impaired. Malignant megakaryoblasts and micromegakaryocytes can be grown successfully in short term liquid culture and have more complete maturation in vitro than observed in vivo.

We measured the complete blood cell count, including the index of RBC size heterogeneity (RBC distribution width [RDW]), in 181 subjects without anemia to determine whether RDW became abnormal earlier in the development of iron deficiency than did other variables. In 163 subjects selected only for otherwise normal blood cell counts, an increased RDW was 66% specific (48/73) and 100% sensitive (48/48) for decreased serum iron saturation. Stool guaiac testing was equally specific but less sensitive. In 13 subjects with polycythemia, as iron deficiency developed, RDW increased a minimum of four weeks before mean cell volume changed. In one apparently normal young woman, an abrupt isolated rise in RDW revealed a newly developed low serum transferrin saturation; all values remained normal during serial testing of four other normal subjects. We conclude that RDW is the part of the routine blood cell count that first becomes abnormal during the development of iron deficiency.

To the Editor. —The message of the article by Hansen et al1and Howe's2accompanying editorial in the January issue of theArchivesis sobering. During the last decade, not only have electronic counters made the traditional red blood cell counts and indices more reliable for detecting iron deficiency and thalassemia3,4but new indices, such as the red blood cell distribution width (RDW), are now widely available. The RDW improves the distinction between iron deficiency and heterozygous thalassemia when mean corpuscular volume (MCV) is low5-7and between iron-deficient and normal subjects when MCV is normal.8However, in view of the data of Hansen et al, it is unlikely that physicians who do not use the long-established MCV will do much better with the additional information given by the RDW. We share the view that the diagnostic approach to microcytic anemias needs to be improved. Incidentally