j observations haemopoieticresponse critical illness

J Clin Pathol 1990;43:850-56

Observations on the haemopoietic response tocritical illness

R J Amos, M Deane, C Ferguson, G Jeffries, C J Hinds, J A L Amess

Department ofHaematology,St Bartholomew'sHospital,West Smithfield,London EC1R J AmosM DeaneJ A L AmessIntensive Care UnitC FergusonG JeffriesC J HindsCorrespondence to:Dr R J Amos.Accepted for publication11 June 1990

AbstractPeripheral blood cytopenias are com-mon in patients receiving intensive care,particularly in those with multiple organfailure. To assess the contribution ofbone marrow hypoplasia in such patients44 bone marrow samples from 24patients under intensive care werestudied by standard morphological tech-niques and by the granulocyte-macro-phage colony forming cell (GM-CFC)assay. Frequently observed mor-phological abnormalities in the bonemarrow included the following: (i) areduction in overall cellularity in sevenpatients, with a progressive decrease inmost patients studied sequentially; (ii)an increase in the number of activelyphagocytic macrophages; and (iii) a dis-ruption of normal bone marrowarchitecture with the accumulation ofintercellular hyaluronic acid glyco-saminoglycan. Mean GM-CFC growthwas significantly reduced when com-pared with that in a group of normalcontrols. In four of five patientsstudied sequentially GM-CFC growthbecame subnormal in association with areduction in bone marrow cellularity.Inhibitory serum factors were not iden-tified. These morphological abnor-malities are similar to the changesobserved in gelatinous degeneration ofthe bone marrow.In both situations disruption of the

haemopoietic microenvironment, withthe accumulation of hyaluronic acidproteoglycan, may be an important fac-tor in the inhibition of haemopoieticprogenitor cell growth. The proliferationof macrophages, by the release of avariety of cytokines or reactive oxygenintermediates, may also be implicated inimpaired haemopoiesis and the develop-ment of disordered erythropoiesis.

Persistent or recurrent sepsis, often associatedwith multiple organ failure, is a commonclinical finding in patients receiving intensivecare.' The haematological response in thissituation often seems to be inadequate;anaemia is alrmost universal and variabledegrees of thrombocytopenia are common.The neutrophil count may be inappropriatelylow and overt neutropenia may supervene. Itis clearly very difficult in such complex situa-tions to define causative factors; several

elements may be involved in the developmentof anaemia, including blood loss, the"anaemia of chronic disorder,"2 3 andhaemolysis.4 The presence of throm-bocytopenia and neutropenia is oftenexplained on the basis of an increase in theperipheral consumption of mature cells due,for example, to infection or disseminatedintravascular coagulation. From previousobservations, however, it is our impressionthat bone marrow hypoplasia and the sub-sequent failure of blood cell production is animportant additional factor in the develop-ment of these cytopenias.Of particular interest is the possible associa-

tion between multiple organ failure andimpaired haemopoiesis. The syndrome ofsequential/progressive multiple organ failurecame to prominence in the 1970s when itbecame possible to resuscitate and supportpatients with previously lethal critical ill-ness.' No generally agreed definition of thisclinical syndrome is available but it is charac-terised by a hypermetabolic state, usually inassociation with infection, together withfailure of the lungs, kidneys, heart and cir-culation, liver and gastrointestinal tract.8 Con-siderable interest has recently been focused ona group of peptide mediators derived fromactivated monocytes and lymphocytes, such asinterleukin 1 (IL-1), interleukin 2 (IL-2), andtumour necrosis factor (TNF), which may beimplicated in the pathogenesis of multipleorgan failure."'0 It has been suggested thatcontinued, uncontrolled production of thesemediators of acute inflammation, in response,for example, to overwhelming infection ortissue necrosis, may be one of the mechanismsunderlying damage to vital organs.8 Thesepeptides also have a wide range of effects onthe haemopoietic system, including themediation of acute changes in iron metab-olism,2 direct effects on haemopoietic pro-genitor cell proliferation,""1-3 regulation ofhaemopoietic colony stimulating factor geneexpression,'4'7 and modulation of the res-ponse of haemopoietic progenitor cells tocolony stimulating factors.'8

MethodsObservations were made on 24 patients (18men and six women, mean age 52 years, range18-78 years) admitted to the intensive careunit (ICU) at this hospital. Seventeen died,giving a mortality of 64%. Brief clinicaldetails are provided in table 1. Patients withprimary haematological disorders and those

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Haemopoietic response to critical illness

Table I Brief clinical details and peripheral blood data of 24 patients studied

Mean (range) peripheral bloodfindings

Exposure to Haemoglobin Neutrophils PlateletsCase No Clinical details nitrous oxide Outcome (gldl) ( x 109/1) ( x 1O'/l)

1 M/58 Cardiac arrest No D 14-6 (13-4-16-2) 14-0 (9-3-16-0) 169 (121-296)2 M/71 Aortic aneurysm repair Yes D 9-6 (8-6-10-9) 143 (52-197) 250(130-444)3 F/70 CAVBG/AVR Yes D 10-7 (6-5-13-2) 116 (31-290) 57(16-128)4 M/19 Multiple injuries Yes A 121 (10-5-13-9) 13-0 (5-5-25-7) 181 (103-380)5 F/22 Head injury/IC surgery Yes A 101 (6-8-12-3) 112(86-157) 244 (107-446)6 M/32 Septicaemia/debridement Yes A 10-5 (8-3-11-4) 10-9(5-6-11-4) 242(17-467)7 M/56 CAVBG Yes D 12-1 (10-0-180) 146(76-28-3) 66(11-210)8 M/29 Proctocolectomy Yes A 11-3 (9-0-16-6) 11-7 (5-9-455) 393(239-529)9 M/50 Head injury/subdural Yes A 10-6 (9-4-11-2) 8-0 (5-6-11-6) 266(108-544)10 M/23 Stab wound Yes A 10-5(83-12-6) 14-3 (8-0-22-0) 337(106-545)11 M/68 Subdural haematoma Yes D 10-7 (8-5-15-6) 6-7 (6 3-9 6) 174(124-249)12 M/34 Pancreatitis No D 12-1 (8-3-15-2) 14 6(3-4-26-2) 111(47-242)13 M/70 Aortic aneurysm Yes D 11-3 (9-7-125) 117 (5-1-14-4) 46(16-95)14 F/39 Abdominal sepsis No A 10-0 (9-8-10-2) 150(13-5-16-5) 556(498-614)15 F/67 Laparotomy/septicaemia Yes D 9-9 (7-7-11-7) 6-4 (20-12-0) 142 (67-247)16 M/62 Septicaemia/tracheostomy Yes D 9-2 (7-0-14-9) 8-2 (52-12-2) 156(57-167)17 M/73 Aortic aneurysm Yes D 10-2 (96-10-8) 12-4(8-5-15-2) 105(72-261)18 M/61 Aortic aneurysm Yes D 116(8-4-16-0) 158(3-0-30-0) 79(16-168)19 F/52 Septicaemia No D 11-6(78-14-5) 114(2-9-34-0) 121(16-221)20 F/77 MVR/AVR Yes D 9-7 (78-12-4) 17-6(10-0-288) 133(21-228)21 M/63 Laparotomy/pneumonia Yes D 10-2 (74-11-6) 11-1 (54-21-6) 141 (57-306)22 M/60 Pancreatitis No D 10-2(9-5-11-7) 156(10-6-18-5) 127(54-266)23 M/31 Cardiac arrest No D 102 (7-6-13-4) 13-0 (8-1-26-7) 451 (221-610)24 M/74 Bowelperforation Yes D 11 3(8-8-13-0) 8-5 (3-0-11-6) 88(15-273)

D-Died on the ICU.A-Discharged alive from the ICU.CAVBG-Coronary artery vein bypass graft.AVR-Aortic valve replacement.MVR-Mitral valve replacement.

Table 2 Bone marrowfindings in 24 patients studied

Intercellular GM-CFC/Case No Cellularity Erythropoiesis Macrophages material Fat atrophy 105 cells

1.1 NC NC MB + +1.2 NC NC MB + -1.3 NC NCMB + + + 1461.4 NC NCMB + + + + + 301.5 NC NCMB +++ ++ ++ 482.1 NC REDMB + + + -

2.2 NC NC + + + + + -

2.3 NC/RED NC + + + -2.4 RED NC + + + + + + -3.1 NC/RED NC + + + 703.2 NC/RED NC + + +. 414.1 NC NC MB + 164.2 RED RED + + + + 1205.1 RED RED MB + + + -5.2 RED RED + + + + -6.1 NC NC MB + + + -6.2 NC/RED NCMB + + + + + -6.3 NC/RED NC MB + + + + + + 867.1 NC/RED RED + + + + -7.2 RED RED + + + + + +8 NC NC + + 49.1 NC NC MB + + -9.2 NC NCMB + + + + + 97

10.1 NC NC + + + -10.2 NC NC + + +10.3 NC NC + + + + ++11.1 RED NC MB + + + + + 8311.2 RED RED + + + + + + 2612 NC RED + -13 RED NC MB + + + + + + 2514 NC RED + + 6615 NC RED + + 3016 NC RED MB + + + 7417 NC RED + + + 3518 NC RED + + 3419 NC RED + + 1820 RED RED + 4121.1 NC RED -21.2 NC NC + +22 NC NC + + + +23.1 NC RED + ++ +23.2 NC RED + + + + 9024.1 NC RED + + + 22324.2 NC REDMB + + + + + + 22

Trephine biopies were performed in patients 1-11. In patients studied on more than one occasion the bone marrows arenumbered consecutively. Semiquantitative estimates (indicated by +) are provided for dyserythropoiesis (under erythropoiesis),macrophage numbers, intercellular material and fat atrophy.NC-Normocellular.RED-Reduced cellularity.NC/RED-Areas of both normal and reduced cellularity.MB-Megaloblastic.

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Amos, Deane, Ferguson, Jeffries, Hinds, Amess

Figure I Bone marrowtrephine biopsy specimen(case 7.2) showing earlymorphological changeswith areas offocalhypoplasia, loss ofhaemopoietic cells, and theaccumulation ofintercellular material(haematoxylin and eosin).

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who had received cytotoxic chemotherapy orradiotherapy were excluded. Eighteen patientswere admitted within 24 hours of surgery andanaesthesia with nitrous oxide. None of thepatients was exposed to nitrous oxide afteradmission to the Unit during the period ofstudy. All patients received parenteral folatesupplementation during admission (1 mg/day). They remained in the ICU for a mean of20 days (range seven to 49 days).The study was approved by the local ethical

committee. Whenever possible, informed con-sent for participation in the study wasobtained from the patients; otherwise it wasobtained from their next of kin.

BONE MARROW MORPHOLOGYForty four bone marrow samples wereobtained from the 24 patients in the study; 28samples from 11 patients included a bonemarrow trephine biopsy specimen. Elevenpatients were studied on only one occasion, onaverage nine days after admission (range oneto 30 days); 13 patients were studied sequen-tially, on two or more occasions, every threedays for periods of up to 14 days.

All samples were processed using standardtechniques. Aspirate smears were fixed inmethanol and stained by May-Gruniwald-Giemsa and Perls's reaction for iron. Tre-phine biopsy specimens were decalcified,

Figure 2 Central area offig 1, enlarged, showingdisruption and loss ofnormalfat spaces.

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Figure 3 Bone marrowtrephine biopsy specimen(case 2.4) showing anextensive area of marrowhypoplasia. Hyaluronicacid glycosaminoglycan ispresent in the spacesbetween the fat cells(haematoxylin and eosin).

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paraffin wax embedded, sectioned and stainedwith haematoxylin and eosin and by silverimpregnation for reticulin. Selected trephinebiopsy specimens were studied by additionalhistochemical techniques including periodicacid Schiff (PAS) (neutral polysaccharides),PAS with diastase (glycogen), alcian blue atpH 2-5 + /- hyaluronidase (hyaluronic acid)and alcian blue at pH 1-0 (sulphatedmucopolysaccharides). Bone marrow slideswere reviewed independently by threeobservers (RJA, JALA, and MD) and thefindings collated.A blood count, including examination of a

peripheral blood film, was performed on a

daily basis. Serum vitamin B12 and serumand red cell folate were estimated bymicrobiological assay before starting treat-ment with prophylactic folic acid. Otherinvestigations were performed as clinicallyindicated.

BONE MARROW CULTURE STUDIESBone marrow culture studies were performedon 17 patients; in 12 patients on one occasionand in five patients sequentially.Myeloid progenitor cells were assayed in

the granulocyte-macrophage colony formingcell assay (GM-CFC).'9 Briefly, bone marrowcells were collected into 10 ml Hanks's balan-

Figure 4 Bone marrowaspirate (case 11.2)showing increased numbersof macrophages withfoamy cytoplasm; somecontain necrotic cellulardebris (May-GrunwaldGiemsa).

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ced salt solution (HBSS) (Flow Laboratories)containing 50 units of preservative free

o heparin. A mononuclear cell enriched fractionwas prepared by density gradient centrifuga-tion over Histopaque (density 1 077; Sigma)

o and 105 mononuclear cells were cultured in 2ml 0-3% agar in Alpha medium (Gibco) con-

§ taining 10% fetal calf serum, 10% human AB0 serum, and 500 placental conditioned

medium20 as a source of colony stimulating800 activity. Cultures were incubated at 370C in a

fully humidified atmosphere of 5% carbondioxide in air and colonies counted on day 10.All assays were performed in duplicate.

rmal Using the method described, bone marrowatients cultures from 31 haematologically normal

subjects gave a mean GM-CFC growth of 108colonies/105 mononuclear cells (range 40-215; SD 44). Results were compared usingStudent's t test.

0 10 15 20

Days after adWssion to intesve core

Figure 6 GM-CFCgrowth in five patientsstudied sequentially afteradmission to the ICU. Thebroken line indicates thelower limit of colonygrowth in 31haematologically normalsubjects.A case I cardiac arrest,bronchopneumonia; died* case 3 coronary arteryvein bypass graft, aorticvalve replacement; diedO case 4 multipleinjuries; survived0 case 1I subduralhaematoma; died* case 24 bowelperforation; died

ResultsPERIPHERAL BLOOD HAEMATOLOGYPeripheral blood findings are summarised intable 1. Anaemia was almost universal. Anumber of red cell abnormalities were com-monly observed, including rouleaux forma-tion, target cells, burr cells and irregularlycontracted cells, together with occasional redcell fragments and spherocytes. When tested,the direct antiglobulin test was uniformlynegative.

Overt neutropenia (neutrophils <2-5 x109/1) only developed in one patient, but in sixpatients the neutrophil count was <3-5 x109/l in the context of severe infection.Characteristically, peripheral blood neutro-phils showed toxic granulation and cytoplas-mic vacuolation. Immature granulocytes wereoften present.Thrombocytopenia (platelets < 140 x 109/1)

developed in 21 (83%) patients usually inassociation with massive blood transfusionor documented disseminated intravascularcoagulation.

BONE MARROW MORPHOLOGYBone marrow morphological findings aresummarised in table 2 and shown in figs 1-4.

CellularityA hypocellular bone marrow was found inseven (29%) patients at some point duringadmission to the ICU. Ofthe 13 patients whosebone marrow was studied sequentially, aprogressive reduction in bone marrowcellularity was noted in 10 (77%). Trephinebiopsy specimens showed both a diffuse reduc-tion in cellularity and focal, intertrabecularareas of hypoplasia, usually in association withstromal changes and a disruption of normalbone marrow architecture.

ErythropoiesisAlthough megakaryocyte numbers and gran-ulopoiesis seemed to be normal in mostpatients, erythropoiesis was reduced in 16(66%) patients at some point during admissionto the ICU. Dyserythropoiesis, such as binu-

cleate erythroblasts, abnormal nuclear con-figurations, cytoplasmic vacuolation and cyto-plasmic bridging was also observed in 17 (71 %)patients. Megaloblastic change was noted in 10(42%) patients.

Megaloblastic erythropoiesis was observedduring the first week of admission in sevenpatients (cases 1, 2, 4-6, 11, and 13), six ofwhom had been admitted following anaesthesiawith nitrous oxide. In the six patients in whomfollow up studies were available erythropoiesisbecame normoblastic in four (cases 2, 4, 5, and11) and in these, the original megaloblasticchange is likely to have been related to theinactivation of vitamin B12 by nitrous oxide.2'In two patients, however (cases 1 and 6),megaloblastic change persisted for periods ofup to 18 days and, in addition, a further threepatients were identified (cases 9, 16, and 24) inwhom megaloblastic change developed sevento 20 days after admission. These five patientswith unexplained megaloblastic change all hadnormal pre-treatment serum vitamin B,2,serum and red cell folate concentrations, andthey had all received prophylactic folate sup-plements.

Abnormal cellsA moderate increase in the number of matureplasma cells (up to 10%) was noted in 13 (54%)patients. An almost universal observation in 22(9200) patients was a variable and sometimesstriking increase in the number of foamy,actively phagocytic macrophages. Many of themacrophages contained phagocytosed red cells,granulocytes or platelets. They occurred singlyor in clumps and in the latter case weresometimes associated with fibrillary, pink-staining material. There was a progressiveincrease in the number ofmacrophages in 11 ofthe 13 (85O ) patients studied sequentially.

Stromal changesStromal changes were most clearly appreciatedin the 11 patients in whom trephine biopsyspecimens were obtained. Typically, there wasa localised disruption of normal bone marrowarchitecture, usually associated with theaccumulation of intercellular ground substancetogether with varying degrees of fat atrophyand loss ofhaemopoietic cells. In some patientsbone marrow necrosis (one out of 11) and focal,fibrinous exudates (six out of 11) were alsoidentified. A mild, diffuse increase in reticulinwas noted in five of 11 patients.A focal increase in pink-staining ground

substance was seen in 10 of 11 patients andthere was a progressive increase in the sevenpatients with follow up trephine biopsies. Thematerial was associated with varying degrees offat atrophy and loss of haemopoietic cells. Inmarrow aspirates it was identified in 13 (540%)patients as pink-staining, fibrillary materialassociated with bone marrow fragments orclumps of macrophages. The material stainedweakly with PAS; pre-digestion with diastasehad no effect. It also stained with alcian blue,much more strongly at pH 2 5 than at pH 1 -0;staining was prevented by pretreatment with

Number 31Meon (SEM) 108(8-4)

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Figure S GM-CFgrowth in 31haematologically nosubjects and in 17 ptfrom the ICU.

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hyaluronidase. Negative results were obtainedwith Congo red and silver impregnation. Thehistochemical reactions suggest that the extra-cellular material consists of acidic muco-polysaccharides, predominantly hyaluronicacid-that is, hyaluronic acid glyco-saminoglycan, one of the proteoglycans widelydistributed in the extra-cellular environment.22

BONE MARROW CULTUREBone marrow was cultured from 17 patients.The mean GM-CFC growth was 67 colonies/105 mononuclear cells (range 4-222). Thiscompares with results from a group of normalsubjects where mean GM-CFC growth was108 colonies/ 105 mononuclear cells (range 40-215) (fig 5). The difference between the twogroups was significant (p < 0-01). In six (38%)patients colony growth was less than the rangefound in normal subjects.

In five patients GM-CFC assays were per-formed on more than one occasion. In four offive patients GM-CFC growth decreased andbecame subnormal during admission to theICU; in each case this was associated mor-phologically with a reduction in bone marrowcellularity (fig 6). All four patients died.The addition ofautologous serum to cultures

of patients' bone marrow produced no inhibi-tion of colony growth, and similarly, the addi-tion of patients' serum to cultures of bonemarrow from normal subjects did not inhibitcolony growth. There was therefore noevidence of any serum factors inhibitory tohaemopoietic progenitor cell growth.

DiscussionWe have described a constellation of mor-phological abnormalities in the bone marrow ofa group of patients with critical illness. Inessence they consist of: (1) hypoplasia, whichwas also reflected by a reduction inhaemopoietic progenitor cell growth in vitro;(2) disruption ofnormal bone marrow architec-ture, with the accumulation of abnormal inter-cellular material, identified as hyaluronic acidglycosaminoglycan, variable degrees of fatatrophy, fibrinous exudates and bone marrownecrosis; and (3) proliferation of activelyphagocytic macrophages. A reduction in eryth-ropoiesis was also almost universal and wasoften associated with dyserythropoietic andmegaloblastic change, the latter apparentlyunrelated to vitamin B12 or folate deficiency. Ina proportion of patients, particularly in thosewho developed multiple organ failure and whosubsequently died, these changes seemed tohave been progressive.There was no clear association between the

presence or severity of these changes andperipheral blood variables, an observationwhich is perhaps not surprising in view of thecomplex, interacting factors that determine theperipheral blood count in such patients. Thesemorphological abnormalities, however, doprobably limit the capacity of the bone marrowto respond appropriately. Indeed, we havedirect evidence in vitro :of- impairedhaemopoietic progenitor cell proliferation.

There are some similarities between theabnormalities we have noted and the changesseen in gelatinous degeneration of the bonemarrow."24 In this condition bone marrow

hypoplasia, fat atrophy, and the accumulationof acidic mucopolysaccharide ground sub-stance, identified as predominantly hyaluronicacid,2425 develop in patients chronically mal-nourished for prolonged periods of time. It hasbeen observed in tuberculosis and other longstanding infections,23 disseminated malig-nancy,23 24 anorexia nervosa and in onepatient maintained on chronic intravenousnutrition.31 In the latter two situations gelatin-ous degeneration has been associated withperipheral blood cytopenias or pancytopenia.Interestingly, in some patients refeeding andclinical improvement have been associatedwith resolution of the bone marrow abnor-malities or the peripheral blood cytopenias.2730It has been emphasised that gelatinous degen-eration is a feature of conditions characterisedby energy or carbohydrate deprivation and isnot a feature of protein malnutrition.26 Theseconsiderations may have some relevance to theabnormalities noted in patients receiving inten-sive care, but a striking dissimilarity is therapidity with which the changes we have des-cribed develop and progress.Whatever the precise association is between

the abnormalities in ICU patients and ingelatinous degeneration it is clear that in bothsituations major disruption ofthe haemopoieticmicroenvironment occurs. This is reflectedparticularly by changes in the extra-cellularmatrix, which may be directly implicated in thedevelopment of impaired haemopoiesis. Thereis now considerable evidence that the bonemarrow microenvironment has an importantrole in haemopoietic cell regulation anddifferentiation.32 Of particular importanceare the proteoglycans of the extra-cellularmatrix3435 which may regulate haemopoieticactivity by influencing the diffusion of largemacromolecules, including haemopoieticgrowth factors,22 by direct binding ofhaemopoietic growth factors36 37 and by effectson cell adhesion and detachment.38 In rabbitbone marrow hyaluronic acid is only a minorcomponent (16%) of the proteoglycans in theextra-cellular matrix.39 An increase in thequantity of hyaluronic acid, because of its largemolecular domain, may contribute to theinhibition of haemopoietic activity by restrict-ing the diffusion of haemopoietic growth fac-tors22 or by preventing the adhesion ofhaemopoietic progenitor cells to stromal com-ponents.'The accumulation of actively phagocytic

macrophages was a further striking abnor-mality in many of the patients studied. Macro-phages have emerged as a key element incurrent concepts about the pathogenesis ofmultiple organ failure,4142 and their presence inthe bone marrow may have important implica-tions for haemopoiesis. Macrophages producea wide variety of secretory products,43 includ-iing the cytokines IL-1, IL-2, and TNF,together with reactive oxyrgen intermediates.TNF, in addition to its possible-role in thetsposs e-r..

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pathogenesis of multiple organ failure,42 alsoinhibits haemopoietic progenitor cell growth invitro4445 and may contribute to the anaemia ofmalaria by promoting dyserythropoiesis anderythrophagocytosis,46 both of which werefeatures commonly observed in this study.Reactive oxygen intermediates also inhibithaemopoietic progenitor cell growth in vitro,both in short47 and long term48 bone marrow

culture systems, and may therefore be a furtherfactor implicated in the development ofimpaired haemopoiesis. Finally, it is of interestthat oxygen radicals, released by stimulatedhuman macrophages, have been shown directlyto damageDNA in cultured mammalian cells.49Furthermore, megaloblastic change is a featureof erythroid colonies grown in vitro in thepresence of monocytes.50 It may be, therefore,that the megaloblastic change noted in some ofour patients is also related to macrophageproliferation and DNA damage by releasedreactive oxygen intermediates.

We thank the medical and nursing staff of the intensive care unitat St Bartholomew's Hospital for their help and co-operationand the technical staff of the Department of Histopathology fortheir expert assistance.

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