24
Tron.~/usion Medicine. 1992. 2, 7 1-94 Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16-1 7 August 199 1 S1 HEPATITIS C VIRUS INFECTION IN BLOOD DONORS: RESULTS OF THE FIRST YEAR OF SCREENING AT THE MONTREAL CENTRE. G. Delaae, F. Aubin, A. Masson, F. Decarv. Canadian Red Cross, Blood Services, Uontreal Centre. Donor screening for antibodies against hepatitis C virus (HCV) began in May 1990. As of the March 31, 1991, 171 601 units have been screened: 830 were found to be anti-HCV reactive by ELISA, for a prevalence rate of 0.48%. This prevalence rate remained fairly stable during the study period. The 830 positive specimens belonged to 756 donors: 174 (23%) of these were confirmed positive by first generation RIBA (performed at NRL). The 174 confirmed positive donors were compared to a cohort of 45 103 donors who gave blood between December 2, 1990 and February 23, 1991. The confirmed positive donors were more often male (76% vs 65%), aged between 30 and 39 years (52% vs 30%), and first time donors (36% vs 20%); their place of residence was similar to the general donor population. We further analysed a subset of 326 ELISA-reactive donors: 23% were RIBA positive, 23% were RIBA indeterminate, and 53% were RIBA negative. The median ELISA optical density ratios (interquartile interval) in the three groups were the following: RIBA-positive, 3,27 (3.086-3.392); RIBA-indeterminate, 2.24 (1.248-3.315); RIBA-negative, 1.26 (0.73-1-53). A sizeable proportion (40%) of RIBA-indeterminates had ratios in the range of the RIBA-positives. CONCLUSION: the prevalence rate of ELISA reactive units in our center is comparable to those found in other North American studies. The age and sex distribution in our confirmed positive donors is not surprising in light of the fact that acute non A non B hepatitis (most of which is due to HCV) is a disease with male predominance and more frequent in young adults. The low rate of RIBA confirmation of ELISA-reactive units and the sizeable proportion of RIBA indeterminates illustrate the need for a more specific screening test and a more effective confirmatory assay (both of which will soon be marketed). Finally, a strategy for reintegration of ELISA-positive, RIBA-negative donor should be developed.

Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

  • View
    229

  • Download
    12

Embed Size (px)

Citation preview

Page 1: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

Tron.~/usion Medicine. 1992. 2, 7 1-94

Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16-1 7 August 199 1

S1 HEPATITIS C VIRUS INFECTION IN BLOOD DONORS: RESULTS OF THE FIRST YEAR OF SCREENING AT THE MONTREAL CENTRE. G. Delaae, F. Aubin, A. Masson, F. Decarv. Canadian Red Cross, Blood Services, Uontreal Centre.

Donor screening for antibodies against hepatitis C virus (HCV) began in May 1990. As of the March 31, 1991, 171 601 units have been screened: 830 were found to be anti-HCV reactive by ELISA, for a prevalence rate of 0 . 4 8 % . This prevalence rate remained fairly stable during the study period. The 830 positive specimens belonged to 756 donors: 174 (23%) of these were confirmed positive by first generation RIBA (performed at NRL).

The 174 confirmed positive donors were compared to a cohort of 45 103 donors who gave blood between December 2, 1990 and February 23, 1991. The confirmed positive donors were more often male (76% vs 65%), aged between 30 and 39 years (52% vs 30%), and first time donors (36% vs 20%); their place of residence was similar to the general donor population.

We further analysed a subset of 326 ELISA-reactive donors: 23% were RIBA positive, 23% were RIBA indeterminate, and 53% were RIBA negative. The median ELISA optical density ratios (interquartile interval) in the three groups were the following: RIBA-positive, 3,27 (3.086-3.392); RIBA-indeterminate, 2.24 (1.248-3.315); RIBA-negative, 1.26 (0.73-1-53). A sizeable proportion (40%) of RIBA-indeterminates had ratios in the range of the RIBA-positives.

CONCLUSION: the prevalence rate of ELISA reactive units in our center is comparable to those found in other North American studies. The age and sex distribution in our confirmed positive donors is not surprising in light of the fact that acute non A non B hepatitis (most of which is due to HCV) is a disease with male predominance and more frequent in young adults. The low rate of RIBA confirmation of ELISA-reactive units and the sizeable proportion of RIBA indeterminates illustrate the need for a more specific screening test and a more effective confirmatory assay (both of which will soon be marketed). Finally, a strategy for reintegration of ELISA-positive, RIBA-negative donor should be developed.

Page 2: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

72 CRCS Ahstracls

S2 ANTI-HCV SCREENING AT A CANADIAN RED CROSS CENTRE: SIGNIFICANCE OF A POSITIVE HCV C-100 ELISA SCREENING TEST A. Giulivi, M.T. Aye, G. Chena. E. Gray. and m. Canadian Red Cross Society. Blood Transfusion Service, Ottawa Ccnm and *National Office, Onawa. Ontario.

Non-A. non-B hcpatitis (NANBH) rcprcscnrs more than 90% of musfusion-associatd hcpatitis in Nonh America. The cloning of the Hepatitis C virus (HCV) genome led to the devcloprncnt of an enzyme l i e d immunoabsorknt assay (ELISA) for antibodies against the HCV C- 100 antigen. The HCV C-100 ELISA test has bcen uscd to screen blood donors to prevent transfusion-associatcd NANBH. However. this screening test is not spccific and only 17-25% of HCV C-100 ELISA positive blood samples transmit HCV. Rccombinant immunoblot assay with the C-100 antigens (RIBA) improves specificity, but this assay is merely a supplementary test rather than a confirmatory test. About 3040% of HCV C-100 ELISA positive samples are RIBA positivc. Detection of HCV RNA by polymerase chain reaction is more specific. but this test is not rcadily avdlablc at most blood c e n m . In this papcr. we report the usage of the HCV C-100 ELISA test and the RIBA tcst to evaluate volunteer b i d donors at thc Ottawa Red Cross Centre. The clinical significance of a positivc HCV ELISA lcst is discussed. Blood samples for 19.983 voluntecr blood donors at a Canadian Red Cross Centrc were scrcencd for antibodies to hcpatitis C virus with an enzyme linked immunoabsorbcnt assay (ELISA). Fifty-nine (0.3%) of the donors were rcpcatably ELISA rcactive. Whcn the rcpcatably ELISA reactive sera wcrc tcstcd with thc radioimmunoblot assay (RIBA), 26 (44%) of the samples were also positive. Thirty-onc of the 59 ELISA rcactivc donors had OD reading grcater than 2.0. Among thcsc 31 donors, 29 (93.5%) had clevatcd serum ALT. 22 (70%) wcre RIBA positive and 22 (70%) had risk factors for hcpatitis. Whilc Lhc HCV ELISA screening kst may lack specificity. it may identify a subgroup of donors (those with OD 2 2) that arc at high risk of hcpatitis transmission. The irnplcrnentation of this scrcening test should funhcr rcducc thc incidcnce of mfusion-associate non-A. non- B hcpatitis.

S3 ANTIBODY TO HUMAN T-CELL LYHPHOTROPIC VIRUS TYPE I (ANTI- HTLV-I) IN BLOOD DONORS IN TORONTO, ONTARIO. Lau, W., Chiavetta, J.A., Wall, A., Wright, I , . , and Ilcrst, R. C.R.C. Blood Transfusion Service, Toronto Centre.

HTLV-I positive donors in relation to our donor base. This information will assist us to evaluate our donor screening methods. Since the onset of anti-HTLV-I oerologic screening on Hay 15, 1990 through February 28, 1991, 34 ( 0 . 0 2 % ) of 162,556 donations were found to be anti-IITLV-I positive confirmed by Western blot/RIPA. This rate is similar to that found in the 1990 CRC anti-HTLV-I pilot study, but is substantially lower than the 2.28% found in our survey of Caribbean immigrants in Toronto. Three (8.8%) of the 34 anti-HTLV-I positive blood donors were RIDA positive for antibody to hepatitis C. No other markers were found in the anti-HTLV-I positive donors. None excluded their unit from transfusion using the confidential unit exclusion form.

Our objective was to identify characteristics of anti-

The distribution of our donors is as follows: ANTI-KTLV-I POSITIVE DONOR BnsE NUHBER (PERCENT) (PERCENT)

SEX HEN 11 (32.41) (57.2%) *

AGE 17-29 5 (14.7%) (38.1%)'

1 DONATION 12 (35.31) (17.8%)

WOHEN 23 (67.61) (42.8%)

30-39 9 (26.5%) (27.9%) > 4 0 20 ( 58.8%) (34.0%)

>2 DONATIONS 22 (64.7\) (82.29) *X2 comparison with distribution of donor base ~ 5 0 . 0 5

When the anti-HTLV-I seropositive donors were compared with the donor base, there were more women, first-time donors, and donors over the age of 40. The highest sero- prevalence was observed in an industrial composite clinic in which 3 (0.67%) of 446 donors were seropositive. When the distribution of the anti-HTLV-I positive donors was plotted, there were no residential clusters or clusters in known Caribbean areas of Toronto. ~nforrnation volunteered by one- half of the seropositive donors who communicated with the Centre suggested that the majority had a HTLV-I r i sk back- ground. A formal study of the backgrounds of newly identi- fied anti-HTLV-I positive donors and a comparison group is planned.

Page 3: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Abstracts 73

S4 TREATHENT OF THROMBOTIC THRoMBOCYTOPENIC PURPURA (TTP). N. Buskard. for the Canadian Apheresis Study Group (CASG).

We have studied, treated and followed 129 patients with a diagnosis of TTP over the past 8 years. Patients with this diagnosis (102) were randomized to treatment with either plasma exchange (PE) or plasma infusion (PI) using frozen plasma on 7 of 9 days following entry into the trial. All randomized patients also received aspirin and dipyridamole. Another 27 patients were ineligible or refused to be random- ized into the study and were treated by PE alone and 14 had oliguria at presentation. At the end of the first cycle of treatment, the patients randomized to receive PE had a higher rate of response (24/51) than patients who received PI (31/51) (p-0.025). Two patients receiving PE died whereas 8 receiving PI died during the first cycle of treatment (p-0.035). At 6 months, the response rate in the PE group was higher (40/51) compared to the PI (25/51), (p-0.002). Overall mortality was 11 patients in the PE group and 19 patients in the PI group (p-0.036). Of the 27 patients who were not randomized 7 died, one during the first cycle of PE. Of the 23 patients who responded to PE, all but three survived. A factor VIII RAg level was elevated at presenta- tion in responders and nonresponders but levels of other circulating factors which may be related to the pathogenesis of TTP were not constantly abnormal. PE appears to be the treatment of choice for TTP whether or not there is evidence of renal failure at presentation. Our studies identified no consistent biochemical markers that can be correlated with disease severity or predict treatment outcome. Although levels of vWF were elevated in most patients the heterogeneity of multimeric patterns made it impossible to determine the specific role if any of VUF in TTP.

S5 THROKBOCYTOPENIA CAUSED BY THE PASSIVE TRANSFUSION OF PLATELET ALLOANTIBODY ANTI-HPA-5b (ANTI-Zav', Era, HCa). T.E. Warkentin, J.W. Smith, C.P.H. Havward, 3 .G. Kelton. XcMaster University, and the Canadian Red Cross Blood Transfusion Service, Hamilton, Ontario.

Platelet .alloantibodies can cause neonatal alloimmune thrombocytopenia and post-transfusion purpura. Rarely, severe transient thrombocytopenia and bleeding can occur following transfusion of blood products containing anti-PIA' alloantibodies. We investigated whether plasma transfusion was the cause of unexpected, moderately severe (platelet count nadir 35 x 109/L), transient (total duration 60 hours), but asymptomatic thrombocytopenia that occurred 18 hours following surgery. We employed the novel technique of direct radioimmunoprecipitation (RIP) to show that the patient's platelets possessed IgG bound specifically to the glycoprotein (GP) Ia/IIa complex, detectable during, but not follpwing, resolution of thethrombocytopenia. Since GPIa/IIa is the site of the HPA-5 (=Zav=Er=Hc) alloantigen system, we used indirect RIP to confirm serologically that serum obtained from the plasma donor contained anti-tav', and that the patient's platelets typed as Z~V"~. No anti-PlA' or other alloantibodies were found. We conclude that (a) passive transfusion of anti-Zav'can causetransientthrombocytopenia; (b) passive transfusion of anti-Zav' appears to cause less severe thrombocytopenia compared with the passive transfusion of anti-PIA1; this is analogous to neonatal alloimmune thrombocytopenia, where Zav system alloantibodies usually cause less severe thrombocytopenia than PIA system alloantibodies (presumably because of the markedly lower number of target antigens); (c) direct RIP is a powerful technique for identifying unknown alloantibodies as a cause Of unexpected thrombocytopenia following transfusion.

Page 4: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

74 CRCS Abstracts

S6 COXPARISON OP P W o w c e OF NO secom CEWUUTION AHTI-IICV KITS IN NOWAL BLOOD DONORS AND u s e S U P U S . v . scolh ; -; t4uL; J . v e t ;

r. I l s r sn: w: tf. 0 u s w ; m. The Canadian Red Cross Society Blood Transfusion Centre.

of t w o second generatloo oncl-HCV enzyme imunoassays. A total of 1500 routine blood donor sampler. I11 scored samples from donations lmpllcatcd in HAN0H. and from recipients of blood transfuslons vhlch vere suspected t o be NAN0 cases. verc tested usin& t w o second generation HCV assays. samples. obtalned by e l thcr assay. vere conflmcd by A peptide confirmatory assay or second seneration recombirunr i m n o b l o c assay. summerlres the results obtalned:

me objectlvc of thlr study vas t o evaluere the sensltlvlty and specificity

Repeat Reactlve (RR)

The followlng table

I [ POST-TRANSNSION NON-A NON-B HEPATITIS I

/I RECIPIEKfS I IMPLICATED DONORS I 1 ROUTINE DONORS I I

I I I I ASSAY II I

I-I-I-II-I-I-I-I-I-I I I I I I 1 I I

1 I I I I I 1 I I

I f NO. 1 RR I CONF [I NO. I RR I CONF !-NO. I RR I CONF I

1KAhVFACT 11 1500 I 5 I I ) ) 69 I 18 1 35 I 372 I 2 8 I 28 I

jKANUFACT 21 1500 1 8 I I 1 ) 69 I 38 I 32 I 372 I 2 8 I 28 I

I

It I appear. ! ~ r ~ ~ ~ ! k & ~ ~ ~ ~ ~ ! ~ ~ ~ l specificity coiiparrd to liccnced First Ccncratlon HCV assays. Second Ceneretlon essays would result In the detectlon of additional lndlvlduals with antlbodies LO HCV. of routine donors shoved that 19 ( 3 . 3 t ) of the donors had reactlvicy to a t leasc one rurropte marker. donors had a Positive surrogate marker.

The U S + Of

Surrogate testing (ALT and anti-HBc)

Three of the four conflmed Posiclve routine

S7 THE CANADIAN RED CROSS SOCIETY (CRCS) UNRELATED BONE MARROW DONOR RECISTRY(UBMDR) . N. Buskard for the CRCS UBMDR and the Canadian Bone Marrow Transplant Study Group, Vancouver, B.C.

Up to April 30, 1992, 90 unrelated BMT's have been per- formed. There has been an annual incremental activity in Canada since 1988. Countries of origin of the donors were: Canada - 39, USA - 3 6 , U.K.- 16, France - 02, Netherlands - 01. Male donors predominate. At the A.B,DR locl, 70 patients have been matched, 20 mismatched. The MU: has been variably reactive and not performed in all cases. Standard protocols have been used for most rransplants. Canada has supplied 9 bone marrows to the USA and 1 to Sweden. The diagnoses of the Canadian transplanted patients were as

- 1, SCIDS - 1. and WA Syndrome - 1. There are over 21,000 donors in the UBKDR with a target of 50,000 by April 1, 1992. About 500.000 donors are available worldwide.

Unfortunately, only 90 (12.1%) out of 744 Canadian patients for whom UBMDR searches were carried out received a transplant from Feb. 1988 to June 30, 1991 and only 38 5.1%) of eligible Canadian patients would have received an unrelated BMT if Canadian donors, only were available. The average search time for Canadian donors, 7.6 months, is significantly shorter than France. 9.0 months, USA, 9.3 months U.K., 13.0 months. The average survival of all patients at one year is 50% and the incidence of graft-vs- host disease exceeds that seen with related donors. Survival is related to degree of tissue match.

Summary. There is no relationship between search and survival times. Less than 15% of eligible Canadian patients are receiving a transplant. Incidence of graft-vs-host disease is higher and survival times are shorter when unre- lated bone marrow donors are used. New strategies to improve the outlook for Canadian patients will be outlined.

fOllOWS: CML - 42. AKL - 16, ALL - 18, SAA - 8, MDS - 7 , LBL

Page 5: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Abstracts 75

S8 BLOOD DONATION BY PEOPLE AGED 70 AND OVER. p. Benoit, S . Marchand, and F. D b r y . Canadian Red Cross Society, Blood Services, Montreal. Can people aged 70 and over donate blood safely? In order to

answer this question, we have reviewed autologous donations made by such people at the Montreal Centre in the last three years, from April 1988 to March 1991. Out of 108 consecutive aged patients referred by their physicians to donate their own blood, only six were excluded after review of their medical record, mostly for cardiovascular problems. Of the 102 accepted in the program (40 M, 62 F), 54 did not have any other health problem that would have excluded them from giving blood. Forty-eight were accepted in spite of such problems, usually well controlled high blood pressure (36) or antecedents of cardiac problems (8). Those 102 patients made between one and five donations in preparation for an elective surgery (mostly orthopedic). Of 293 units of blood requested by surgeons, 282 were collected (96%). Reasons for not collecting were: low hematwrit (4), venous access (3), cancellation by patient (4). Only five minor reactions were experienced, aI1 very mild and none requiring cessation of present or future donations. Of aI1 units collected, 75% were actually transfused. Eighty-two percent of all surgical procedures were done using autologous blood only. This autologous donation program has shown us that people aged 70 and over can be safely accepted as blood donors, even with heaIth problems, if their medical condition is carefully evaluated beforehand.

s9 HLA TYPING BY I'CR-ASO 0 L I G O T " G ME"H0D EVALUATION.

Gilles Roy, Lvrie Tremblav, MichEle Paauin, and Francine Decirv. Canadian Red Cross Blood Services, Montreal Centre.

In the context of the 11" International Hiistocompatibility Workshop, HLA typing by Polymerase Chain Reaction (PCR) followed by allele specific oligonucleotides (ASO) oligotyping was developed. One hundred samples derived from 2 different populations were studied: 1) unrelated individuals referred for HLA typing, 2) HPA-lb homozygous women referred to our laboratory for investigation. Following PCR amplification with speciiic primers for class II region genes DR& DQa, DQB, D P a and DPB, DNA was dot blotted on nitrocellulose and hybridization was performed using "P radiolabelled A S 0 in presence of tetramethylammonium chloride ("MAC) to increase specificity and precision.

Our results indicated that a minimal amount of genetic material is needed. DP typing, difficult by serological methods, was clearly resolved for both alleles. DQ and DR typing can be more precisely determined than by serology and a large number of specimens (100) can be processed at once. Finally the DNA spots can be stored, reused for future investigations and the autoradiograms can be consulted at any the . We have found the method to be laborious, expensive and requiring manipulation of chemical products and radioactivity.

Nevertheless, HLA typing by PCR-AS0 is the method of choice because of its extraordinary accuracy. Because the preparation of probes and hybridization is similar whether applied to 1 or 100 samples, the method remains impracticable for the study of a smdI number of cases.

Page 6: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

76 CRCS Abstracts

S10 I N C R E A S E D CIRCULATING CSF-1 (M-CSF) IN PATIENTS W I T H PRELEUKEMIA, LEUKEMIA, AND LYMPHOID MALIGNANCIES. h a Janowska-Wieczorck, A.R. Belch, A. Jacobs, D. Bowen. &. Padua. E. Paictta.&d - E . R . W . CRC BTS Edmonton Centre. Departments of Mcdiane, Cross Cancer Institute, and the University of Alberta; Department of Haematology, University of Wales Collcgc of Medicine, Cardiff, U.K.; and the Montefiore Medical Center and Department of Dcvelopmental Biology & Cancer, Albert Einstein College of Medicine, B r o w N.Y.

Recently, several malignant cell types have been reported to express colony- stimulating factor-1 (CFU-1) transcripts; however, the clinical significance of CSF-1 in malignancy has not been investigated. Using a CSF-1 radioimmunoassay, we surveyed concentration of biologically active CSF-1 in the peripheral blood of 316 patients with malignant and prcmalignant hematological disorders; 75 had a myclodysplastic syndrome (MDS) , I2 acute myelogenous leukemia (AML), 7 chronic myelogenous leukcmia, 21 chronic lymphocytic leukemia (CLL), 106 non-Hcdgkin’s lymphoma (NHL; of low-, intermediate;, and high-grade malignancy), 46 Hodgkin’s disease (HD), 46 rnultiplc myeloma (MM), and 3 monoclonal gammopathy of undetermined significance. Controls wcre 64 healthy subjects. T h e CSF-1 concentration was correlated with the type of disease, status of the disease, treatment status, and hematologic parameters. CSF-1 concentration was significantly elevated in 83.5% of the patients with active disease, and for each active disease group it was sigdicantly greater (P < .OOO1) than in the control. Thus, the high circulating CSF-1 concentration was not associated with a particular malignant phenotype or MDS subtype, but did correlate with the disease activity of both NHL and HD. and the tumor burden in MM, AML and CLL. There was no correlation of the CSF-1 lcvcls with total counts of monocytes or ncutrophils in patients with MDS or other malignancies. T h e cellular basis for the elevated circulating CSF-1 was not investigated. Howevcr, the results are consistent with the possibility that t h e prcmalignant or malignant cells themselves produce CSF-1 or regulate its production by normal cclb.

S11 RECOMBlTlANT HuMlw ERYIXROPOlI3IN ( r h - m ) FOR A JEHOVAH’S WXTNESS WITH ANEhilA OF TIiJ3MA.L INJURY: RESPONSE TO IIICH DOSE rh-EPO DESPITE ENDOGENOUS ELEVATED ERYTHROPOIETIN LEVEfS L. K. Doshkov. E. E. Tredret, and Anna Janowska-Wieczorek. Depts. of Medicine and Surgery, University of Alberta Hospital and the Canadian Red Cross Blood Transfusion Service, Edmonton Centre, Edmonton AB.

W e used recombinant human crythropoietin (rh-EPO) as an alternative to transfusion therapy in a burn victim who was a Jchovah’s Witness and refused blood transfusion. Despite endogenous elevated erythropoietin levels of 105 mU/rnl (normal 10-20 mU/ml) the patient was reticulocytopenic and administration of rh-EPO a t 300 U/kg daily for seven days then 150 mU three times weekly for 3 weeks was accompanied by a 10-fold increase in reticulocyte response and a rise in hemoglobin from 7.4 t o 10.4 over a 12-day period. The exogenously administered erythropoietin apparently overcame a clinically inadequate endogenous erythropoietin response. Preliminary data from ourselves and others suggest that endogenous levels of erythropoietin a re elevated in bum patients consistent with their degree of anemia. Despite this burn patients remain reticulocytopenic and frequently require multiple red cell transfusions. We postulate there is inhibition of erythropoietin or i ts action by inflammatory mediators such as tumor necrosis factor (TNF) and interleukin I (IL-I), which antagonism can b e partially overcome by administration of pharmacological doses of erythropoietin. The ability of patients with physiologically elevated endogenous erythropoietin levels to respond to pharmacological doses of rh-EPO i sa f potential importance in evaluating erythropoietin as an alternative t o transfusion therapy in anemia accompanying surgery and trauma as well as in the anemia of malignancy and chronic disease.

Page 7: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Abstracts 77

S12 KELt HEm3LYTIC DISEASE(KHDN) .J.Bawman. J.Pallock. C . m , F.MWUU.lW * , s.Mentiooslou. Rh LabarataJry & Dept.Okskt., university of Manitoba and CRCS BTS, Wpg.(=entre.

ollr T i e n c e with KHDN & the acQILacy of A.F.AOD 450 rea- in KHDN was reviewed for 1948 to 1990. ?he mi- ence in patients refaxed fran cutside of Manitoba was dLS0 r&&.In 43 years,311 warnen w i t h arrti-K had 458 p r e p a y cies,63 of wkich erded in abortion or SB,unrelitkd to ank- K.Of the 395 babies,18 were affected,lO i n 1948-1970,8 in 1971-199O.m 1971-1990 the k i d - of a€f- babies was 2.5%. l2 of the affect& babies did not require tr=bmt,2 needed @&therapy,1 a sinple trarrsfusian,l exhange trans- fusion,2 - hydropic =(in the f i r s t 2 w m x ~ farrd to haw

prqmncies) were referred frcm artside of Manitoba. 9 had a prior history of hydmpic -;the other 5 had hydropic fe- tuses at referral. In the 6 ~nreg~ncies(5 w~men)More PUBS, 4 ended in hydmpic SB,1 with a prior hydrqx was shown to be K-ve by serial fi00 450,- 5th,a hydmps, was rwersed by 3 m.In the 10 pregnancies(9 wnnren)after HIBs,1 enled in hydropic SB a t 20 wks;5(4 hrampS and 1 hb 65 g/l )survived a f t e r 5 to 7 IVT;4,all w i t h a prior history of hydrops, had K-ve febses,pmven in 3 by PUBS and in the other by serial A00 450.KWN,althougfi rare,because of the frquenq of K- hununkition by transfusion and the >95% incidenz of K-ve fetuses,when it does ocaa,may be as severe as wl(D) HCN. Serial AOD 450 are 90% a m t e in predictin3. K-ve f e w ard are 80 to 85% a m t e in predicting the pmsence and severity of ~~~N.Life-threatenhg AOD 450 inacr=uracies are

sive measures may b= preven- by a t 20-22 wks gesta- tion when there is a prior history of severe KHDN or when AOD 450 r-diq's reach the 65% level of filey's zone 2, d f i e d <24 wks gestat ion.

mti-K in Manitoba in 1948-1950). 14 K -ized (16

in ear ly and mid second t r i m e s t e r . SE or inapprop?ziate h-

S13 DEVELOPMENT OF ANTI-HUMAN GLOBULIN AND ANTI-D REAGENTS BASED ON MONOCLONAL ANTIBODIES. S. Perron, M.J. S irois. U Ch, M. St-Laurent. G. Mo r&, S. V e r r u and R., Canadian Red Cross BTS, Quebec City.

Monoclonal antibodies (mAbs) are now routinely used in the preparation of several blood grouping reagents and we have been interested in the last years to develop new mAb-based reagents in addition of the ABO reagents. Using culture supernatants of murine B-cell hybridomas isolated in our laboratory, we have developped a polyspecific anti-human globulin reagent composed of a blend of four mAbs. The two anti-human IgG mAbs (8D2-8 and 5H4) have been shown to recognize all four human IgG subclasses and to cooperate in agglutination tests. The two mAbs are of the IgG isotype and the 5H4 mAb is a IgM to IgG switch variant of the IgM-secreting 8A8 hybridoma cell line. The two anti-C3 mAbs were shown to recognize the red cell bound C3c (5F12) and C3d (5G5) fragments. Serological testing of the reagent showed a reactivity similar to the ones of several commercial AHG reagents both in terms of potency and detection of weak blood goup antibodies. The developped reagent will now be tested in field trials in collaboration with the Serology Section staff of the NRL prior to its larger scale production for routine use.

The use of human-mouse heterohybridoma cell lines secreting human anti-D and available in our centre through a collaborative agreement with the CRTS, Lille (H. Broly) has permitted us to define the composition of an anti-D reagent to be used in several techniques. The reagent is a blend of one IgM (HM10) and 2 IgG (43F10 and P3X83) anti-D mAbs and is reactive in both saline and antiglobulin tests with a potency similar or higher than the ones of commercial reagents. The use of the P3X83 mAb permits the detection of the DVI category cell. Further work on this reagent will include the definition of optimal conditions (culture, additives) for its production and additional testing in independent laboratories.

Page 8: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

78 CRCS Abstracts

s14 CHANGES IN LMMUNOGLOBULIN AND TOTAL PROTEIN LEVELS IN FREQUENT PLASMA DONORS. A. Giulivi. M.T. Ave. G. Chenz!, E. Senack. and S. Wasi*. Canadian Red Cross Society. Blood Transfusion Service, Onawa Centre and *National Oflice. Ottawa. Ontario.

Due to a pcruption that some of our plasma donors may have a high incidence of allergy, a pilot rcmspective study was done to determinc the effect of biweekly plasmaphcresis on different immunoglobulin (Ig) levels. Five groups of donors were defined: (1) donors who donated for one year [n=20] (2) donors who donated for two years (n=22]; (3) donors who donated for three ycars [n=26]; (4) donors who had high normal IgG or IgA levels on entry to the program [n=13]; and (5) those who had low normal IgG or IgA levels on entry ID the program [n=12]. Plasma donations were collected using thc Autophcnsis-C cell separator (Baxtcr Corp., Fenwal Div.). Donors' Ig and protein levels wen: done every 3 months (Nor-Panigen. Behring). Groups 1. 2. 3 and 5 showed a mean reduction in IgG and IgA levels at 9 months rcspcctively of 0.3 g/L and 0.4 g/L In group 4. upper limit valucs wcrc observed for IgA in 6 donors and for IgG in 7 donors aftcr 10 months in the program. They were deferred for 6 months and then mntercd into thc program for phcrcsis on a monthly basis. However. persistently raised Ig lcvels led to their pcrmancnt dcfcrral. lmmunofixation was donc and none of the donors showed monoclonaI gammopathy. Group 5 showed intercsting rcsulu: Seven donors had lower limits of IgG and five had lower limits of IgA. Thcse donors wen: deferred at 6 months bccausc Ig levels fell bclow plasma regulations. Thcy were reinstated aftcr 4 months but donatcd once per month only. Their Ig Ievels remained normal. In conclusion. plasmaphercsis tended to causc a mild increase in Ig levels in donors who had initial values at the uppcr limit. Howcvcr. those with low normal values showed no furthcr dccrcase when donating once pcr month and wen retained in thc program. Further, no specific changes wcrc obsewcd in thc differcnt classes of Ig as a rcsult of plasmaphcrcsis.

S ,15 IDENTIFICATION OF HLA-A11 SUBTYPES. E. Yang, K . H . Wong and 11. Mervart . Nat ional Reference Labora tory , The Canadian Red Cross S o c i e t y , Ottawa, Canada.

Caucasian and O r i e n t a l popula t ions . O u r l a b o r a t o r y h a s been H L A - A l l is a common s p e c i f i c i t y f r e q u e n t l y observed i n t h e

a b l e t o demonstrate t h e e x i s t e n c e of 2 s e r o l o g i c a l v a r i a n t s of t h i s a n t i g e n u s i n g reagents obtained from t h e HLA a n t i b o d 9 s c r e e n i n g programme. The most common subtype i s d e s i g n a t e d A l l . l and h a s been observed i n a l l e t h n i c groups w h i l e t h e much more r a r e v a r i a n t , A11.2, has been d e t e c t e d only i n O r i e n t a l s .

i n t o 3 groups. The f i r s t group produces s t r o n g , p o s i t i v e r e a c t i o n s wi th a l l A l l panel cel ls whi le t h e second and t h i r d s e t s s e l e c t i v e l y r e a c t wi th e i t h e r t h e A l l . l o r A11.2 sub- types r e s p e c t i v e l y .

l a b o r a t o r i e s p a r t i c i p a t i n g i n t h e 1 1 t h I n t e r n a t i o n a l Work- shop. However, i t appears t h a t on ly t h e broad A l l and t h e A11.2 r e a g e n t s a r e a v a i l a b l e f o r a n a l y s i s i n t h i s workshop.

The e x i s t e n c e of biochemical v a r i a n t s of t h e A l l a n t i g e n in O r i e n t a l s was d e s c r i b e d i n t h e 10th I n t e r n a t i o n a l Work- shop. The a n a l y s i s of our s e r o l o g i c a l l y d e f i n e d All.l and A11.2 subtypes w a s performed by i s o e l e c t r i c f o c u s i n g i n o r d e r t o de te rmine whether o r n o t biochemical correlates f o r t h e s e v a r i a n t s e x i s t e d . Our r e s u l t s i n d i c a t e t h a t t h e A11.2 a n t i g e n s h a r e s a n i d e n t i c a l i s o e l e c t r i c p o i n t as t h e common form o f t h e A l l . l molecule.

We c a n d i f f e r e n t i a t e our local, monospecif ic A l l r e a g e n t s

Confirmation of our f i n d i n g h a s been r e p o r t e d by o t h e r

Page 9: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Abstracts 79

516 THE ISOZATION AND CEARACTERIZATION OF A MJRINE ANTITBROHBIN-111 cDNA. John Wu. William Sheffield, Morris Blaichman, CRCS BTS Hamilton Center, Ontario, Canada.

Congenital antithrombin-111 (AT-111) deficiency is the com- monest inherited deficiency of the naturally occurring antico- agulants in blood. The reported incidence of 1 in 2000 is pro- bably an underestimate of the incidence because up to 509 of affected individuals do not manifest clinical thromboembolism. AT-111 deficient children are only infrequently affected pos- sibly because of increased levels of a -macroglobulin. The studies of the natural history and the effect of therapeutic interventions on PT-I11 deficiency have been hampered by the lack of a good animal model. With advances in molecular techno- logy, the creation of transgenic mice with targeted gene dele- tion is now possible. We now report the isolation and charac- terization of mouse AT-I11 cDNA. A mouse liver Lambda Zap@ cDNA library was screened using rabbit AT-111 cDNA as the probe. The longest clone obtained measured 1.5 kilobases. This was sequen- ced and confirmed by sequencing of the complementary strand. The cDNA was found to be 1514 nucleotides long coding for 433 amino acids with a 32 amino acid signal sequence. It includes a 20 nucleotide S'-non-translated region and a 75 nucleotide 3 ' -nontranslated region with a 20 nucleotide polyadenine tract. It is highly homologous to its human counterpart with 84% homo- logy at the nucleotide level and 89% homology at the amino acid level. Proof that this cDNA clone was indeed mouse AT-I11 was confirmed by its in vitro expression in a rabbit reticulocyte lysate system which generated a protein of relative molecular mass of 50 ma. This corresponds to the unglycosylated precur- sor form of AT-111. It was cleaved at the reactive site by thrombin and formed thrombin-antithrombin-ILI complex. This reaction was enhanced by the presence of heparin indicating that a functional protein was expressed. Currently, effort is being undertaken to use these data in the generation of trans- genic AT-I11 deficient mice, Attempts to do so entail either homologous recombination or generation of antisense RNA. Thus the knowledge obtained in this study represents a critical first step towards the generation of a novel animal model for evaluating the role of AT-111 in hemostasis.

5317 CHARACI-ERIZATION OF NOVEL PLATELET AND ENDOTIIELXAL CELL TARGET ANTIGENS IN A FAhlILY WmI GENFnC SUSCEPTIBILlTY TO A U T O I h M U " . &fia Hashemi Jeanne Drouin. Else Trudel. M.T. Avc and Petcr R. Ganz Ottawa Blood Outre , Catudian Red Cross Society and the University of Ottawq Ontario.

This repon dcsaibcs a French Canadian family whose mcmbcn &%it a high inadencc of d o and autoantibodies to antigens present on both platelets and cndorhciial cck (ECS). This is carrelated with various HZA spcdfiatics horn to bc d t c d with autoimmunity such as Al, B8, DR3. and in some cases with dinical disorders including nephritis and thrornbccytopcnia Inmunoblot analysis using platelet and EC lysatcs showed wrum antibodies to a 75 k D a EC surface polypeptide and to polypeptides with apparent mass of 1s kDa and 26 kDa found on both platelets and ECs. This lL5 kDa internal platelet protein was also found in a variety of other cell types such as mononudear cells, and increased following cell aaivation Monodonal anfibody immobilization assays were used to charactcrizc the 26 kDa polypeptide: ki h c c of the four patients tested, an antibody to leukocyte differenciation antigen CD9 was identified. The asymptomatic child of the propositus aLr0 exhibited an autoantibody aga;.st an 80 kDa platelet protein which was sensitive to thrombin digcstion s q e s t i n g that this polypeptide may be platelet gIycnprotein V. In addition, PL aIIoantibody was identified in one sister who had given b i to a sevcrcly thrombocytopenic boy, and who herself had a severe vasahr rejcaion to cadaver kidney nvo yean prior to Lhis study. The propositus aIS0 dcvclopcd hrpcrtensive renal diseasc and h e dialysis-dependent. Thus, members of this family have dcvcloped a k c t y of antibodiq particularly to platelet and EC antigens Some subjeas haw remained asymptomatic in spite of having au toan t i i c s . Howcvcr, 0 t h ~ ~ have been scr iody iIl, and their immune rcsponsc to these anrigens is believed to have played a role in the pathogenesis of their neonatal alloimmunc thrombcqopeonic purpura, r e d hypertension disease, renal gaft rejection and hombocycopcnia.

Page 10: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

80 CRCS Ahstructs

S18 CHARACTERIZATION OF A GROWTH-INHIBITORY FACTOR ACTIVE ON NEWLY-FORMED B-CELL HYBRIDOMAS. S, BBrub6. D. Robichaud, R. Bazin and R . Lemieux. Canadian Red Cross Transfusion Service and Lava1 University, Quebec City.

Previous work from this and other laboratories has shown that newly-formed 6-cell hybridomas are sensitive to various cytokines, the most extensively studied being interleukin-6. Using IL-6-containing P388D1 cell conditioned medium (CM), we have observed that the growth of some B-cell hybridomas was strongly inhibited by a factor (named TO) present in the CM. Biochemical characterization experiments using sensitive hybridoma cell lines showed that the murine TO factor is a protein with an apparent M.W. of 25-30 kDa and a p l of 7,O-7,4. The high stability of the TO factor during various chemical treatments suggested that it may be a member of the TGF-8 family. However the use of neutralizing antibodies revealed that it was different from TGF-81 and 02. Analysis of the DNA integrity in sensitive cells showed that the TO factor could induce apoptosis in these cells. We are now in the process of cloning this factor by direct expression in COS-1 cells. The availability of the cloned factor will facilitate the further study of its biological activity on transformed and normal cells.

THE RELATIVE ROLE OF ENDOCENOUS TNF PR0I)UCTIOII IN HURINE HACROPHAGES STIHULATED WITH UACROPHAGE GROWTH FACTORS - -- D.R. Branch and L.J. Guilbert. C.R.C.S. Edmonton, Alberta and C.R.C.S. Toronto, Ontario.

Based on our observation that the macrophage secretory product tumor necrosis factor-alpha (TNF) synergistically cooperates with the macrophage-specific growth factor CSF-1 to stimulate macrophage proliferatlon (Blood 73: 307, 1989), we propose that endogenous production of TNF may be essen- tial to CSF-1-stimulated macrophage proliferation. This hypothesis was examined by determining whether mitogenically stimulated macrophages produce TNF and, if so, what the effect of blocking TNF production is on proliferation. CSF-1 increased expression of TNF mRNA in both primary cells (bone marrow derived macrophages, BHH) and a growth factor-depend- ent macrophage cell line (Sl). Quantitation of TNF protein production was possible only after deveLopment of an extremely sensitive (lower detection limit 200 fg/mL) bio- assay. Levels of both secreted and cell associated TNF from cells derived from LPS hyporesponsive C3H/HeJ mice (BHH and Sl) were increased by CSF-1 and decreased by GH-CSF (which does not cooperate with TNF). Hore TNF was found associated with HeJ cells than secreted. In contrast TNF levels from LPS responsive C3H/HeN BXM were constitutively high and were decreased by both CSF-1 and GH-CSF. The importance of TNF secretion to macrophage proliferation was aseessed by adding neutralizing anti-TNF IgC to both CSF-1 and GH-CSF-stimu- lated cultures. Anti-TNF inhibited CSF-1-stimulated prOli- feration by 10-209 only with HeN BHH, and as expected, had no effect on GH-CSF stimulated proliferation. In order to evaluate the role of cell-associated TNF, synthesie Was . speclfically blocked with 22mer phosphothioate deoxyoligo- nucleotideb (S-oligos) made antisense to TNF mRNA (synthesis was not blocked with base-matched nonsense controls). HOW- ever, both control and antisense s-oligos strongly inhibited CSF-1-stimulated proliferation. Thus, the importance Of cell-aeaociated TNF to CSF-1-stimulated proliferation cannot yet be assessed, but these data demonstrate that endogen- ously secreted TNF significantly contributes to CSF-1, but not GH-CSF. stimulated macrophage proliferation, and that TNF production can be specifically blocked with antisense s-01 igos .

Page 11: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Abstracts 8 I

s20 In v i t ro PRODUCTION OF THE ERYTHROID SPECIFIC TRANSCRIPTION FACTOR GATA-1. P. Trudel, S. Provost, *L. Wall, +B. Massie, R. Lemieux, F. Mca P. Chartrand. Canadian Red Cross Society, Services, Montreal and Quebec City, *Institut du Cancer, Montreal, +Biotechnology Research Institute, Montreal.

GATA-1 (also known as NF-El, GF-1 and Ery-fl) is believed to be a major regulator of transcription of most, if not all erythroid specific genes. In vitro the protein was shown to be a positive activator of transcription. GATA-1 is expressed at all stages of red cell development and deletion of the gene coding for GATA-1 has shown this factor to be absolutely required for erythroid development. In order to further characterize the physical and biological properties of GATA-1, we have set out to develop an expression system for its large-scale production. Three different expression systems were tested: a plasmid vector that produces a fusion protein in bacteria, an adenovirus vector which is used in mammalian cells and a baculovirus vector which is used in insect cells. The cDNA coding for the mouse GATA-1 was cloned into the three vectors. Pure clones were obtained with the bacterial and insect systems. Polypeptides produced. by these two systems have the expected size and were shown to specifically bind oligonucleotides carying GATA-1 DNA binding sites, as detected by electrophoretic mobility shift assays. Impure adenovirus vector clones produced detectable quantities of GATA-1 In 293 cells, but for unknown reasons we were unable to isolate pure GATA-1 producing clones. We are currently in the process of overproducing GATA-1 with the bacterial and insect system.

S21 TEMPORAL SPECIFICITY OF IMMUNOGLOBULIN GENE TRANSCRIPTION IN TRANSGENIC MICE IS GOVERNED BY ENHANCER AND PROMOTER SEQUENCES. A. Darveaul, C. Van Genderen, A. Travis and R. Grosschedl. The Howard Hughes Medical Institute and Dept of Microbiology, U. of California, San Francisco; 1CRC BTS, Quebec City Centre. Elements controlling the expression of Immunoglobulin (lg) genes are crucial for the normal function of the immune system. lg p and K genes are expressed in a tissue-specific and temporally-ordered manner during mouse development and B lymphocyte differentiation. Expression of the p gene is established in pre-B cells representing the early stage of the B cell lineage, whereas the K gene is not expressed until later stages. In this study, we examined the role of enhancer and promoter sequences in defining the temporal and cell type- specificity of lg gene transcription. We introduced into the mouse germ line mutated p genes that lack the intragenic enhancer and chimeric genes that have the enhancer or promoter replaced with the corresponding regulatory element from the K gene. Analysis of developmental expression pattern of the transgenes indicated that enhancer sequences are essential for establishment of p gene transcription. Second, the K enhancer confers most, but not all, of the K-specific expression pattern; low activity of the K enhancer is observed in early stage cells in which the endogenous I; gene is transcriptionally silent, whereas high activity is detected in later stages of differentiation. Third, the K promoter also confers part of the K-specific expression program. From these data, we suggest a model for the temporal regulation of lg genes in which the proper developmental expression pattern of the K gene is dependent upon the combination of the K enhancer with the K promoter.

Page 12: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

82 CRCS Abstracts

s22

S23

TOWARD THE DEVELOPMENT OF A NOVEL APPROACH FOR GENE THERAPY. A. Belmaaza, J.C. Wallenbure, S. Rrouillette, & Gusew and P. Chartrand. Canadian Red Cross, Blood Services, Montreal Centre.

The repetitive LINE (LI) elements of the mouse, which are present at about 10' copies per genome and share over 80% of sequence homology, were examined for their ability to undergo genetic exchange with exogenous L1 sequences. The exogenous L1 sequences, carried by a shuttle vector, consisted of an internal fragment from LlMd-AIL, a previously described member of the L1 family of the mouse. Using an assay that does not require the reconstitution of a selectable marker we found that this vector, in either circular or linear form, acquired DNA sequences from endogenous L1 elements at a frequency of 10" to lo4 per rescued vector. Physical analysis of the acquired L1 sequences revealed that distinct endogenous L1 elements acted as donors and that different subfamilies participated. These results demonstrate that L1 elements are readily capable of homologous interaction with other L1 sequences. This suggested to us the possibility of using LINE-1 sequences to develop a novel approach for gene therapy. The basis of the approach would be to integrate genetic information by homologous recombination into endogenous LINE-1. Based on the results presented above, we would expect the frequency of targeting in these sequences to be much higher than that obtained for singlecopy genes.

Z I N C B I N D I N G BY TETANUS TOXIN LIGHT CHAIN. F. Id r igh t . 21. Rcboul , , F l - P e r n o l l c t , and M . Colornb. L a b o r a t o i r e d ' i m u n o - ch imie . C e n t r e d ' E t u d e s N u c l e a i r e s de Crenob le , F r a n r e .

Examina t ion of t h e p r i m a r y s t r u c t u r e of t h e f c t a n t i s t o x i n shows t h e p r e s e n c e of a p o t e n t i a l m e t a l b i n d i n g s i t e (HExxH) c o n t a i n e d w i t h i n a h i s t i d i n e r i c h r e g i o n of t h e l i g h t c h a i n sequence . I t has b e e n shown by sequence comparison t h a t t h i s p u t a t i v e metal b i n d i n g m o t i f a p p e a r s to be ana logous t o t h e Zn-binding c a t a l y t i c c e n t r e i n Zn-dependent e n d o p e p t i d a s e s ( Jongenee l e t a l . , E B S L e t t s 2 4 2 , 2 1 1 , 1989) . Hence the p r e s e n c e o € t h i s m o t i f may have i m p o r t a n t s t r u c t u r a l and f u n c t i o n a l i m p l i c a t i o n s . In o r d e r t o b e t t e r c h a r a c t e r i z e t h e t e t a n u s t o x i n , which i s u s e d as a model a n t i g e n i n t h e s t u d y of a n t i g e n p r o c e s s i n g and p r e s e n t a t i o n in o u r l a b o r a - t o r y , w e have examined Z n b i n d i n g by t h e p u r i f i e d t o x i n molecu le . and t e s t e d t h e t o x i n f o r c o l l a g e n a s e and thermo- l y s i n - t y p e p r o t e o l y t i c a c t i v i t i e s . Using (65)Zn i n c u b a t e d w i t h t e t a n u s t o x i n heavy and l i g h t c h a i n s s e p a r a t e d by SDS- PAGE and t r a n s f e r r e d t o n i t r o c e l l u l o s e , t h e t o x i n l i g h t c h a i n showed a n a b i l i t y t o b i n d Zn. The b i n d i n g a f f i n i t y of the i n t a c t t o x i n m o l e c u l e unde r non-dena tu r ing c o n d i t i o n s gave a v a l u e of Kd = 10dl; t h e s e b i n d i n g d a t a i n d i c a t e d 1 t o 2 Zn b i n d i n g s i tes p e r t o x i n molec'ule. The i n h i b i t i o n of a coppe r i n d u c e d o x i d a t i v e c l e a v a g e o f t h e l i g h t c h a i n a p p r o x i - m a t e l y midway a l o n g t h e l i g h t c h a i n sequence by Zn s u p p o r t s t h e h y p o t h e s i s t h a t Zn b i n d s t o the h i s t i d i n e r i c h r e g i o n . To i n v e s t i g a t e t h e f u n c t i o n a l i m p l i c a t i o n s o f t h i s Zn b i n d - i n g , p r o t e o l y t i c a c t i v i t y u s i n g t h e t h e r m o l y s i n / n e u t r a l m e t a l l o p r o t e a s e s u b s t r a t e e n d o t h e l i n , and c o l l a g e n a s e sub- strates t y p e I c o l l a g e n and g e l a t i n were s e a r c h e d f o r . N O p r o t e o l y t i c a c t i v i t y o f e i t h e r of t h e s e t y p e s c o u l d b e a t t r i - b u t e d t o t h e i n t a c t heavy o r l i g h t c h a i n s o f t h e t e t a n u s t o x i n .

Page 13: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Ahstructs 83

s24 STAUROSPORINE CLAMPS CYTOPLASMIC FREE CALCIUM IN NEUTROPHILS. K. Wonq and L. Kwan-Yeunq. Canadian Red Cross Society, Calgary Centre, Calgary, AB.

Staurosporine, (STA) , 50 n ~ - 0 pM, increased cytosolic free Ca2+ levels, [ca li, of fura-2 loaded neutrophils in a nonlinear, step-wise manner. The rise in [Ca2+]i was rapid, plateaued within 30 s and was maintained for more than 2 0 min. A similar response occurred with pertussis toxin-treated cells. The elevation of [Ca2+] i was due entirel3+to mobilization of intracellular Ca2+ stores. Mn quenc%+studies confirmed the absence of Ca2+ influx. Ca efflux was absent in STA- treated cells. In combination studies, STA potentiated Ca2+ influx induced by n- formylmethiony3;leucyl-phenylalanine (FMLP) and did not block Ca efflux associated with peptide stimulation of neutrophils. Studies with permeabilized cells showed that STA did not directly release intracellular Ca2+ stores nor id it affect the sequestration of Ca2+ by a Ca'+/ ATPase pump. A radioimmunoassay failed to detect changes in the level of inositol 1,4,5- trisphosphate in neutrophils incubated with -< 1 pM STA; however in cells treated with 10 pM STA, the assay recorded a transient elevation of this second messenger similar to that induced by FMLP. Finally lysozyme but not 6-glucuronidase was released from STA-treated cells. Present results suggest that

99 STA increased [ Ca2+] i by indirectly mobilizi internal ca2+ stores. STA suppression of ca efflux and generation of a persistent signal may account for maintained elevation of [Ca2+] i-

a+

s25 INDUCTION OF NON-RESPONSIVENESS TO HUMAN RED BLQOD CELL ANTIGENS IN BALB/C MICE FOLLOWING MULTIPLE IMMUNIZATIONS. S.Neron and R.Lemieux, Canadian Red Cross Transfusion Service and Lava1 University, Quebec (Que . )

In hybridoma technology, it is well known that the myeloma partner fuse efficiently only with antigen-activated B lymphocytes at the proper differentiation state. The common procedure used to obtain the activated lymphocytes is to immunize a group of mice and select for the final boost and the fusion experiment the animal showing the higest antibody titre. We have previously observed that the use of mice immunized with human red blood cells failed to yield a high number of monoclonal antibodies in fusion experiment despite the presence of a high serum anti-human red blood cell titre. In order to study this phenomenon, we have studied the effect of the number of immunizations on the efficiency of the fusion experiment.The results showed that under similar experimental conditions, the maximal yields of mAb were obtained using the mice that had received only 1 or 2 antigen injections while the mice immunized with 3 antigen injections consistently yielded a much reduced number of monoclonal antibodies. The negative effect could not be reversed by prolonged resting of the animals and suggests the induction after multiple immunizations of B-lymphocytes non- responsiveness preventing the final activation step. These results point out to the importance of avoiding repeated immunizations of mice for the preparation of a high number of monoclonal antibodies by cell fusion technique.

Page 14: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

84 CRCS Abstracts

S26 ANTITHROMBIN-111-STOCKHOLM: A POINT MUTATION IN CODON 392 (GLYCINE T O ASPARTIC ACID) CAUSING IMPAIRED SERINE PROTEASE REACTIVITY. W.P. Sheffield, MA. Blaichman, F. Fernandez- Rachubinski, R. Austin. and S. Schulman. The Canadian Red Cross Society BTS, Hamitton, Ontario and the Karolinska Hospital. Stockholm, Sweden.

Antithrombin-111-Stockholm is a new structural variant of antithrombin 111 (AT- 111) with normal heparin affinity but defective serine proteasc inhibitory activity. The proposita, a Caucasian female born in 1966, developed a pulmonary cmbolism while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-I11 deficiency as they had normal levels of kmunoreactive AT-111 associated with d c c r w e d (-60%) functional AT-I11 levels when measured with either a-thrombin or human factor Xa as the substrate; either in the presence or abscnce of heparin. Furthermore, there was no evidence of abnormal electrophoretic mobility of the AT-I11 from the proposita. either in the presence or absence of heparin. Genornic DNA was prepared and all 7 AT-I11 exons PCR-amplified and scqucnced in both directions, using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G-A substitution. Such a mutation would cause the substitution of aspartic acid (GAC) at the site of the normally appearing g1ycine (GGC) residue in the translated product. This mutation is also associated with the destruction of an & I11 restriction site at this point in the AT-111 gene. The abolition of this- Ill site was confirmed using PCR-amplified material from the proposita. Experiments with AT-111 from the proposita. together with experiments from 'cell-free derived translated AT-111-Stockholm, provide evidence that the mutant AT-I11 protein has normal heparin affinity; is poorly cleaved by .-thrombin; and does not efficiently form a stable covalent inhibitor complex with its cognate protease. This study indicates that the Gly 392 residue is important for the recognition of the reactive centre of AT-I11 by its cognate serine proteases. Furthermore, the available data from this and other naturally-occurring AT-I11 mutanK continue to providc important new information a b u t the mechanism Of action of antithrombin 111.

S27 PROTEOLYSIS OF FACTOR VIII BY a-THROMBIN AND FACTOR Xa: CONSEQUENCES ON FACTOR VIII COFACTOR ACTIVITY. Linq Yin, F.A. Blaichman and F.A. Ofosu. Canadian Red C r o s s Society and Departments of Pathology and Medicine, McMaster University, Hamilton, Ontario.

Factor VIII, after limited proteolysis by thrombin, factor Xa, or plasmin, provides the protein cofactor for factor X activation by factor IXa. Factor VIII is absent or defective in hemophilia A. Factor VIIIa consists of a 50 and 43 kD heavy chain-derived and a 73kD light chain-derived polypeptides. The. cofactor,. activity of factor VIIIa is unstable, and loss of cofactor activity does not apparently require additional proteolysis. Nonetheless, the generation of a 45kD from the 50kD polypeptide by activated protein C leaas to the rapid loss of cofactor activity. We immunized rabbits with synthetic peptides (17- to 20- mers) of factor VIII, beginning and ending at the reported heavy and light chain cleavage sites, and obtained high titer antibodies reactive with the polypeptides generated from recombinant factor VIII by lOnM thrombin. and lOnM factor Xa. We also determined the cofactor activity generated when factor VIII reacted with thrombin, factor Xa, and a combination o f two enzymes. Thrombin proteolyzed recombinant factor VIII more rapidly and effectively than factor Xa. In addition, thrombin was equally effective in generating the 73, 50 and 43kD fragments required for optimal cofactor activity, while the 45kD fragment, i.e. that generated by activated protein C, was a predominant fragment generated by factor Xa. Consistent with these results, the combined proteolysis of factor VIII by factor Xa and thrombin resulted in a more rapid loss of factor VIIIa cofactor activity than either thrombin or factor Xa alone. Thus, factor Xa, the principal product of factor VIIIa action, can potentially inhibit its own generation by proteolytical ly inactivating factor VII la.

Page 15: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Abstracts 85

S28 SCREENING O F FACTOR IX MUTATIONS BY SINGLE STRAND CONFORMATION VARIATION (SSCV) ANALYSIS. bl-C. Poon. G.D. Sinclair, B, Fraser. $. Anand. and Q.I. Hoar. Calgary Red Cross Centre, University of Calgary. Foothills Hospital, Alberta Children's Hospital. and Southern Alberta Hemophilia Clinic. Calgary, Alberta.

SSCV analysis is based on the identification of s ingle stranded D N A f r a g m e n t s with var iant e lec t rophore t ic mobi l i ty ( in n o n - denaturing gels) due to con formational changes induced by sequence variations. This relatively rapid technique is capable of detecting v a r i a n t f r a g m e n t s w i t h s i n g l e b a s e c h a n g e s . W e h a v e used hemophilia B as a model system to investigate the feasibility of this SSCV analysis for the localization of point mutations. W e studied 15 well character ized hemophil ia B kindreds using S S C V a n a l y s i s combined with PCR using primers spanning the 8 exons of the factor IX gene. In 2 kindreds with known mutations in separate TaqI sites in exon 8 (factor I X Calgary 1 and Calgary 2). t h e var ian t e x o n 8 fragments could be detected by their electrophoretic mobility changes, and t h e point mutat ions verified by sequencing the PCR amplied variant fragments. In the remaining 13 kindreds. the variant SSCV fragments f rom 12 could be unambiguously ident i f ied us ing th i s PCR/SSCV technology. Sequence analysis of these PCR amplified f r a g m e n t s i s in p r o g r e s s t o p r e c i s e l y c h a r a c t e r i z e t h e defects responsible for hemophilia and to relate this information to the altered factor IX gene expression in these individuals. We are also using this technique in gene tracking studies to directly determine carrier status in f e m a l e f a m i l y m e m b e r s ; t h e c a r r i e r s h o u l d c a r r y t h e S a m e hemophilic family specific SSCV fragments. Unlike s tandard RFLP analysis . th i s car r ic r de tec t ion technique is not compl ica ted by nonpaternity. RFLP homozygosiry of key females. recombination, or a lack o f family his tory o f the disease. This typc of s tudy can b e completed with DNA from less than I mL of blood.

S29 STUDIES ON AN UNIDENTIFIED FACTOR Va-BINDING PROTEIN. E.L.C. Prvzdial . Protein Chemistry Sect ion, National Reference Laboratory, Ottawa.

Factor Va is an e s sen t i a l cofactor f o r the f a c t o r Xa- mediated physiological generation of thrombin. In the course of def ining procedures t o study the f lu id -phase associat ion of f ac to r s Xa and Va, an i n t e r a c t i o n was indicated between f ac to r V a and an un iden t i f i ed t r a c e component (apparent 4 - 105 kDa) common t o f a c t o r X a preparations. The crosslinking reagent d i th iob i s ( succ in - imidyl propionate) w a s used to t rap equilibrium complexes formed between bovine f ac to r V a and a c t i v e s i t e blocked '251-factor X a . A high molecular weight covalent adduct corresponding t o f ac to r Va/'=I-Xa could not be demonstrated by SDS-PAGE and autoradiography. However, prolonged exposure of the autoradiogram enabled the v i s u a l i z a t i o n of a complex between f ac to r V a and the 105 kDa t r a c e p r o t e i n . Reve r s ib i l i t y o f t h i s i n t e rac t ion was demonstrated by the lack of detectable complex i n the presence of excess unlabelled a c t i v e s i t e blocked factor X a (which included the 105 kDa spec ie s ) . The f ac to r Xa zymogen ( f a c t o r X) w a s unable t o i n h i b i t complex formation. S p e c i f i c i t y w a s indicated by the absence o f crosslinked adducts when the 105 kDa p ro te in was incubated with bovine serum albumin or the inac t ive precursor of factor V a ( f a c t o r V). The unident i f ied p ro te in w a s able t o incorporate a dansyl- o r rhodaminyl-tripeptidylchloromethylketone, i n d i c a t i n g t h a t it is a func t iona l s e r ine protease. Chloromethyl ketone was not incorporated i n t o the factor X a s t a r t i n g ma te r i a l ( f ac to r X I . This suggests t h a t the 105 kDa species c i r c u - l a t e s a s a zymogen and t h a t events leading t o the gener- a t i o n of f ac to r X a a l s o ac t iva t e t h i s un iden t i f i ed p r o t e i n . Amino-terminal sequencing is i n progress.

Page 16: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

86 CRCS Abstracts

s30 CLONING AND CELL-FREE EXPRESSION OF F~ABBIT ANTI- THROMBIN 111 AND DERIVATIVES. W.P.Sheffield, A.B. Brothers, M.W.C. Hatton, B.J. Clarke and M.A. Blaichman. Canadian Red Cross Society, and McMaster University, Hamilton, Ontario. Antithrombin I11 (AT-111) is the principal thrombin inhibitor in plasma. We have cloned rabbit AT-111. A cDNA containing the complete AT-I11 open reading frame of 465 amino acids was isolated from a rabbit liver cDNA expression li- brary. Sequence analysis showed 84% homology with human AT-I11 at the deduced amino acid level. In v i t r o transcription of the cDNA, fol- lowed by cell-free translation of the resultant -A in a rabbit reticulocyte lysate system sup- plemented with 35S-methionine was performed. SDS -PAGE showed the synthesis of a 51 Kd protein capable of forming covalent complexes with a- thrombin. Identical treatment of a 4 7 Kd re- combinant rabbit AT-I11 lacking the signal pep- tide resulted in significantly enhanced complex formation. Deletion mutants lacking the carbow- terminal 64 and 216 amino acids of the protein were shown to retain heparin-binding capability. Cell-free expression plasmids encoding the human and rabbit AT-I11 polypeptides were manipulated to produce an interspecies fusion protein con- taining human codons 1-367 fused in frame to rabbit codons 369-433. This translation product demonstrated an impaired ability to form com- plexes with a-thrombin, indicating that portions of the two AT-111s are not interchangeable. Use of the rabbit AT-I11 cDNA will add to our under- standing of AT-I11 function in living systems.

S31 MOLECULAR CIIARXCTERlZATlON OF PLASMINOCEN IIINDING SITES ON lIUhlAK ENDOTHELIAL CELL?. Anil K. Dudnni. Sofia Hashcmi. M.T. Avc and I’ctcr R. Gm7- Ottawa Blood Centre. Canadian Rcd Cross Socicly, SS Plyrnourh Street, Ottawa, Ontario, Canada

We have prcviously shoun (Biochcm. Ccll Biol. @: 554-560, 1301) ih3t human cndothclial cells (ECs) isolatcd from umbilical vcins, umbiliul artcrics and apillaries diffcr in their capacity to bind plasrninogcn and that thcsc binding diffcrcnccs may signal functional diffcrenccs relating to fibrinolytic activity on the surface of thcsc cells. We havc also shown thal umbilical vcin ECs cxprcss a 45 kDa cell surfacc protcin that binds plasrninqcn. To furthcr characterize thc intcraction of plasminogen with ECs, wc studicd binding of plasrninogcn to intact human umbiliul vcin ECs as well as to EC cxtracts. Equilibrium binding and Scatchard analyscs revealed that venous ECs cxprcss two C ~ ~ S S C S of binding sites for plasminogcn (Kds 0.07 p M and 0.40 p M) as compared to ECs from xtcr ies and capillaries that have only one class of binding sites (Kds 0.3 - 0 . 4 ~ M). Analyses of venous EC extracts using ligand- blotting indicated that rhc bindmg of plasminogen to the 45 kDa EC polypeptidc was kringlc depcndent and specific, as the binding could bc inhibited by both excess cold lysinc (>&I%) and Plasminogcn (9% inhibition at 50-fold cxccss) but not by unrelated proteins. Analogous to plasminogen binding to ECg plasminogcn binding to the EC 45 kDa polypeptide was also reversible. Binding of plasminogcn to the above polypeptide on ECs pretreated with proteinax K was significantly reduced (>!XI%) indicating that h e above polypeptide is a cell-surface protein. Moreover, a 45 kDa EC protein could be cluted from plasminogcn affinity columns but not from columns containing unrclated proteins. Since plasmhogen has ako been reported to bind to platelet membrane glycoprotein (GP) IXb/IIIa integrins, we also bvestigatcd whethcr plasminogen could bind to similar rcczptors OD ECs. It was observed that plasminogen binding to ECs was inhibited (25 - 9%) by a peptide containing Arginine-Glydnc-Aspatic acid, and by fibrinogen, but not by fibronectin, suggesting that EC CP IIb/IIIa-like i n t e e may also contribute to binding of plzsminogea In summary our results suggest that plasminogen interacts with more than one component on venous EG. In addition to E C intcgrins, a 45 kDa surface protein constitutes one of thc major cellular rcccptors for plasminogcn on these cells.

Page 17: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Abstracts 87

s32 INCREASED REACTlVlrY OF MONOCLONAL IGM ANTI-A CAUSED BY A SINGLE AMINO ACID SUBSTITUTION IN THE CONSTANT REGION OF THE HEAVY CHAIN. R. Razin, A. Darvw. A. Pelle ti!X*L€!iChi*EJdi3&lMarfel, M. 3-1 aumlt T h i W , l L ! a u m x . and E , , Canadian Red Cross BTS and Lava1 University, Quebec City.

We have previously described the isolation from the E l l hybridoma cell line. of two hybridoma variants (05 and 7F5) secreting monoclonal anti-A of increased avidity in agglutination tests. Determination of the association constants of the E l 1 (2,1x107 Ilm) and D5 (7,1x107 Ilm) mAbs confirmed the higher avidity of the D5 variant. In order to determine the molecular modification(s) responsible for the increased reactivity, the cDNAs e n d i n g the H and L chains of the three mAbs were sequenced. Comparison of the nucleotide sequences showed a single point mutation in each of the two mAbs produced by the hybridoma variants. The mutations were both located in the constant region of the heavy chain Q genes and caused a Ser to Phe substitution at position 565 in the D5 mAb and a Asn to Tyr substitution at postion 563 in the 7F5 mAb. Both substitutions modified the consensus glycosylation sequence Asn-X-Ser/Thr located in the CH4 domain of the p chain. The absence of glycosylation at this site was confirmed by CNBr cleavage of the 1%-mannose labeled mAbs. The two single point mutations, and not other undetected modifications, were actually responsible for the increased avidity of the antibodies, as confirmed by site-directed rnutagenesis of the E l l mu chain and serological analysis of the mutated E l l antibodies. We conclude that the absence of glycosylation at position 563 is responsible for the increased avidity possibly by altering the quaternary structure of the IgM polymer. To our knowledge, this is the first report that point mutations in the constant region can influence the avidity of monoclonal IgM antibodies.

S33 MOLECULAR CHARACTERIZATION OF HUMAN MONOCLO- NAL ANTIBODIES REACTING WfTH THE Rho (D) ANTIGEN. G. Boucher , H. Brolv and P. Lemieux .Canadian Red Cross BTS ,Lava1 University , Quebec city and Centre R6gional de Transfusion Sanguine, Lille , France.

The D antigen of the Rhesus blood group system is one of the most important red cell antigens in transfusion medecine. Routine red cell phenotyping and the prevention of the hemolytic disease of the newborn require large amounts of human Rh(D) antibodies. In the last five years, a number of laboratories have described the preparation of human anti-D mAbs which could be used in replacement of the polyclonal anti-D sera. In order to better characterize the human anti-D mAbs and the human immune response to this antigen ,we have studied a library of 7 anti-D IgG mAbs secreted by mouse-human heterohybridomas and prepared using lymphocytes of a single donor. The nucleotide sequences coding for the H and L chain V regions were determined after PCR amplification of the V regions using human IgG-specific probes. Comparison of the sequences indicated a restriction of V gene segment usage in these antibodies since six out of the seven H chains used similar VHi, D and J gene segments with a percentage of homology higher than 95%. The VL gene segments used showed more variability suggesting a predominant role of t he H chain in the reactivity of these mAbs. Complete serological testing of these mAbs will permit to better define the important structural characteristics associated with the anti-D specificity of these mAbs.

Page 18: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

88 CRCS Abstracts

S34 COMPLEMENT AND COAGULATION ACTIVATION IN PLATELFT CONCENTRATES. Maria I.C. Gvonavossv-lssa and Dana V. Devine, CRCS BTS, Vancouver Centre, British Columbia

We have shown an increase in levels of the complemenr activation fragments C4d and Bb in stored platelet concentrates (PC) or in platelet poor plasma (PPP) stored in CLX bags. C1 inhibitor levels declined in stored PPP but not in stored PC. Because the C4d fragment can be generated by the action of plasmin on C4, we measured SC5b-9, the membrane attack complex of complement, as a measure of physiologically relevant complement activation. SC5b-9 levels rose steadily with days of storage; the rate of SC5b-9 formation was more rapid in PPP. Complement activation detected in PC occurs mainly via the classical pathway which can be initiated by factor Xlla. Xlla was measured in PPP and PC using an APlT based assay. The factor Xlla activity measured by this assay indicated that there was an overall increase in Xlla activity in both PC and PPP compared to reference plasma (RP). In PPP, Xlla activity was significantly greater by day 4 of storage. Because the A P l 7 is affected by activated factors other than XII, we measured factor Xlla activity, both as total Xlla (with actin FS) and previously activated XI1 (without actin FS), using a chromogenic assay for Xlla (S-2222). The chromogenic assay reported no change in Xlla with time in storage. However, the total Xlla activity in PC was significantly greater than in RP or stored PPP. Measurement of activated XI1 without addition of actin FS indicated that PPP contained a greater proportion of Xlla than PC. These observations suggest that the activation of complement and factor XI1 is modulated by the presence of platelets in the concentrate. Since activation is generally lower in PC than PPP, it is unlikely that platelets provide the complement activating surface in PC but instead these data suggest that an enzyme activating surface is a result of storage container composition.

S35 AN ANIMAL MODEL TO STUDY REFRACTORINE'S TO ALLOGENEIC

LEUKODEPLETION. Morris A. Blaichmnn. Mindv Goldninn. Leslic I3nrdQqy and D.P. Sincal. Department of Pathology, McMastcr Univcrsity and tlic Canadian Rcd Cross Socicty BTS, Hamilton, and Montreal Ccntrcs.

D O N O R PLATELETS: T H E EFFECT O F PRE-STORAGE

ApprogmatclyM% of multitransfuscd individuals bcconic rcfractory to random donor platclets. Rcccnt clinical data indiwte that recipients of Icukoqc-dcpletcd (LD) blood produds are less likely to become rcfraaory to random donor platelets than recipients of non-LD produus. Lcukodeplction u n bc performed immcdiatcly after the collection of a unit of whole blood prior to its sloragc (prc-storagc LD) or just prior to transfusion after its storage (post-storage LD). The establishmcnt of an animal model could thus be extremely useful to study platelct rcfrauoriness due to alloimmunization and would allow the accurate quantitation of allogcneic platelet survival. This prcscnt study was undertaken 10 establish an animal model of allogcneic platelet refractoriness and to ascertain whcther pre-storage LD can reduce the frequency of refractoriness to allogeneic donor platelets compared to post-storage LD. In this model, two strains of rabbits were used; California Black rabbits were used as blood donors; while New Zealand White rabbits were the recipients. Eight weekly infusions of non-LD allqeneic fresh blood resulted in an allogeneic platelet refractory rate of 91.2% (31/34). If the allogeneic fresh blood was LD, this refractory rate was rcduwd to approximately 30%. When blood was stored for one week, both the rcfrauory rate and the platelet survival time of allogeneic platelets were signilicantly improved in animals that had received pre- storage LD blood compared to those that had received post-storage LD blood. Recipient animals that received pre-storage LD blood had-a refractory rate of 333% (5/15), whereas those that received post-storage LD blood had a refractory rate of 66.7% ( l O / f i ) . This model thus appears to be very useful to study the parameters causing refractoriness to allogeneic donor platelets. Moreover, these data provide evidence, for the first time, that the pre-storage lcukodepletion of allogcneic donor blood is associated with a lower frequency of refractoriness to allogeneic platelets than post-storage lcukodeplction.

Page 19: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Abstracts 89

S36 ALLOIMMUNIZATION TO PLATELET ANTIGEN HPA-1 a: INDIVIDUALS WITHOUT DRB30101 CAN ALSO PRODUCE ANTIBODIES. D. I 'AbbB. L, Tremblav, P. Chartrand, and F. DBcarv. Canadian Red Cross, Blood Services, MontrBal.

We and others have recently reported that 100% of the HPA-lb hornozygous women who developed an antibody response (responders) against the HPA-la platelets of their foetus carried the HLA- DRB3'0101 allele (DRw52a). However, this allele was also present in some of the women who did not produce antibodies (non- responders). We wanted to see if there was any difference at the sequence level in the DRB3.0101 alleles of responders vs non- responders. To do so, we sequenced the second exon of the DRB3 gene of 5 DRB3'0101 responders, 2 DRB3.0101 non-responders and 3 DRB3'0101 individuals picked from a group.of blood donors. All the DRB3'0101 sequences were found to be identical between themselves and to the published sequence. These results indicate that the responder state is not associated with a difference at the sequence level in the second exon of the DRB3.0101 allele. In order to extend the analysis of HLA-D association with alloirnmunization to HPA-la, we determined by oligotyping the DRB1, DRB3, DRB4, DRB5. DQA, DQB, DPA and DPB alleles present in 35 responders and 10 non-responders. Surprisingly, we found 2 responders carrying the DRB3'0301 (DRw52c) allele but not the DRB3'0101 allele. This was confirmed by sequencing. We also found the DQB'0201 allele to be present in 94% of the responders while its incidence in non-responders is of 30%. This data indicates that the DRB3'0101 allele is not necessary to develop an immune response to HPA-la and that in responders the DQB'0201 allele is present as frequently as the DRB3'0101 allele.

S37 ROLE OF THROMDOSPONDIK I N PUTELET ADtlESION: EFFECT OF DIVALENT CATIONS AND SHEAR RATE. F.R. Agbanvo, J.J. Sixma.* P.G. de G r o o t , * M.H. Ginsbe rg , and E .F . Plow. Committee on V a s c o l a r B i o l o g y , Research I n s t i t u c e of S c r i p p s C l i n i c , L a J o l l a , CA and Department of Hematology, U n i v e r s i t y H o s p i t a l U t r e c h t , Utrech t , The Nether l a n d s . *

I n s p i t e o f t h e s t r u c t u r a l s imi l a r i t i e s between throm- bospondin (TSP) and o t h e r p l a t e l e t a d h e s i v e p r o t e i n s , t h e e v i d e n c e p r e s e n t e d t o s u p p o r t i ts r o l e i n p l a t e l e t a d h e s i o n h a s been h i g h l y c o n t r a d i c t o r y . To f u r t h e r i n v e s t i g a t e t h e r o l e of TSP i n p l a t e l e t f u n c t i o n , a d h e s i o n s t u d i e s w e r e performed u n d e r b o t h s t a t i c and f low c o n d i t i o n s . It w a s d e m o n s t r a t e d i n e x p e r i m e n t s conducted i n t h e p r e s e n c e o r absence of d i v a l e n t c a t i o n s , t h a t t h e Ca-free c o n f o r m e r o f TSP f a i l e d to s u p p o r t p l a t e l e t adhes ion , and i n h i b i t e d t h e i n t e r a c t i o n of p l a t e l e t s w i t h o t h e r a d h e s i v e p r o t e i n s i n c l u d i n g f i b r i n o g e n , f i b r o n e c t i n (FN) and von W i l l e b r a n d f a c t o r . In c o n t r a s t , t h e Ca-conformer o f TSP s u p p o r t e d p l a t e l e t a d h e s i o n , and % s u r f a c e cove rage i n e r e a s e d from 5.4 5 0.3 'at 0 s-1, t o 41.5 2 6 .7 a t 1300 s-1. P l a t e l e t a d h e s i o n t o FN. o n t h e o t h e r hand, d e c r e a s e d w i t h increas- i n g s h e a r rates. While e x t e n s i v e p l a t e l e t s p r e a d i n g was c o n s i s t e n t l y o b s e r v e d on FN, p l a t e l e t s p r e a d i n g on TSP o c c u r r e d s p o r a d i c a l l y , and w a s o n l y s i g n i f i c a n t a t s h e a r rates above 800 s-l. These f i n d i n g s s u g g e s t t h a t a com- b i n a t i o n o f . T S P and FN may be n e c e s s a r y to mai ta in o p t i m a l p l a t e l e t a d h e s i o n a t v a r i o u s s h e a r rates I n Vivo. F u r t h e r - more, TSP may f u n c t i o n a s a modulator of c e l l u l a r a d h e s i v e f u n c t i o n s , by c h a n g i n g from an a d h e s i v e to a n o n a d h e s i v e conformer . unde r s p e c i f i c n i c r o e n v i r o n m e n t a l c o n d i t i o n s .

Page 20: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

90 CRCS Abstracts

S38 STAUROSPORINE ELJ3ATES BASAL LEVELS OF INOSITOL TRISPHOSPHATE IN PLATELETS. J. Turkson and K-Wonq. Canadian Red Cross Society, Calgary Centre, Calgary, Alberta.

Recent work in our laboratory shows Staurosporine, (STA), elevates intracellular Ca levels in neutrophils. For comparison, this effect of STA was investigated in platelets. Fura-2 studies showed a gradual but steady rise in intracellular Ca2+ upon stimulation with micromolar concentrations of STA. The time course of Inositol trisphosphate(IP3) isomers formation in platelets incubated with 3H myoinositol and later stimulated with STA was followed. IP3 formation has reached peak concentrations within 15s and stayed at this level for up to l5min. STA caused a dose-dependent increase in the levels of IP3; 2pM and IpM staurosporine elevated resting IP3 levels by 246.2 2 17.8% and 220.4 2 21.6% respectively. Pretreatment of platelet with Neonycin, a Phospholipase C(PLC) inhibitor, bloc ed the dose-dependent effect of STA. Results of 'H myoinositol studies sug est that the mobilization of intracellular Cay+ might be a consequence of the formation of IP3. Although the kinetics of elevation of intracellular Ca2+ appears to be different from that for the formation of the radiolabelled IP3, a cause and effect relationship between the two phenomena awaits further experimentation. On the basis of current results, we hypothesize that STA directly activates PLC-PIP2 pathway to elevate the level of Ins(1,4,5)P3 in platelets.

th8$

S39 Stem Cell Factor and the regulation of erythropoiesis. M.T. Aye and V. Fuller. Department of Medicine, University of Ottawa and the Canadian Red Cross Society, Ottawa Centre, Ottawa, Canada. It is gcnerally accepted that erythropoietin is thc spcCific long-range hormonal rcgolator of crythropoiesis. Other factors affccting clythropoicsis have been reported in two gcnetiwlly anemic strains o l mice. In one. known as thc W W mutant, the stem cell is defective. In thc other. Stcc l or SI/Sld, thcrc is a defect in the cellular microcnvironmcnt of the marrow. Thc genetic dcfcct in W W has bcen localised to mutations and/or delcrions in thc c-kit protouncogenc. Last year, the ligand (also known as Stcm Cell Factor or SCF) which binds to c-kit was cloned and mapped to the SI locus and the Stccl defect shown to be duc to mutations in this gene. Early repom showed that recombinant SCF enhanced colony formation sti.~nulatcd by IW, GM-CSF or epo. The studies described here was made possible by 'a generous gift of recombinant human SCF from Amgen I n c In these experiments marrow cells from 12 transplant donors were culturcd at 3 X I d cells/mUculture with 20% heparinked human plasma in methylcellulose. Celb were cultured 1) without epo or growth factors, 2) with 10% v/v PHA-LCM alone, 3) 1 unitfml epo alone, 4) PHA-LCM plus epo, S ) SCF 100 ngm/mI, 6) SCF plus PHA-LCM, 7) SCF plus epo and 8) SCF plus PHA-LCM and epo. Without epo or growth factors there was no colony formation. Niether were there any erythroid colonies in plates containing PHA-LCM alone. In the standard positive control cultures with PHA-LCM plus epo there was an average of 110 erythroid colonies. The most striking effect in plates containing SCF plus epo was the large number (mean 218) of macroscopic erythroid colonies with minimal growth of granulocytic colonies. We also detected a few small to medium sized and fully hemoglobinised erythroid colonies (mean 23) and megakaryayte colonies in plates with S C F alone. The number and size of such endogenous erythroid colonies were increased (mean 96) by the addition of PHA- LCM to the SCF-containing cultures. Lastly, cultures containing SCF plus PHA- LCM and epo gave a m a n of 265 colonies. These findings show that a) SCF can stimulate megakaryocyte colony formation, b) SCF by itself, in the absence of added epo, can stimulate erythroid colony formation and that c) PHA-LCM with SCF can promote erythropoiesis to leveb approaching cultures with 1 unit/ml of epo. We suggest that SCF, expressed by marrow mesenchymal cells. may be as important as epo in thc loc l l regulation of erythroid stem cell growth.

Page 21: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Abstracts 91

s40 INHIBITION OF HEMATOPOIESIS IN LONG-TERM MARROW CULTURES TREATED WITH RECOMBMANT HUMAN CSF-1 (M-CSq. Janowska-Wieczorck, Mavani, u. Guilbert. and S.C. Clark. C R C BTS, Edmonton Centre; Universify of Alberta and Genetics Institutc, Carobridge, h4A.

The effects of recombinant human macrophage colony-stimulating factor (rhCSF-1) in long-term marrow cultures (LTMC) established from normal bone marrow cells were examined. W h e n added during the first 3 weeks of culture (every second day, a t 15 ng/mL), rhCSF-1 strongly inhibited the growth of all hcmatopoietic progenitors analyzed (colony-forming unit-MIX [CRI-MIX), CFU- granulocyte macrophage [CFU-GM]. CFU-M, CFU-G, burst-forming unit- erythroid). Paralleling the inhibition of progenitors was the complete loss of adipocytes from the stromal layer of rhCSF-1-treated cultures. The inhjbitory effect of rhCSF-1 correlated in al l instances with the accumulation in the supernatants of these cultures of an activity (different from CSF-1) that inhibited colony formation in semisolid cultures. When addition of rhCSF-1 WY delayed 3 week, its inhibitory cffects were s i f l m t l y reduced, which correlated with rcduced inhibitory activity detected in the supernatants. Analysis of CSF-1 concentration by radioreceptor assay c o d m c d that added rhCSF-1 increased culturc CSF-1 levels and showed that the dccrcased inhibition observed when rhCSF-1 is added later in culture was not due to decreased CSF-1 levels at that point. In contrast, thc ability of rhCSF-1 to inhibit hematopoicsis and a m u l a t c inhibitory activity in LTMC correlated Glh it. rate of utilization, much higher in the first 2 weeks of culture, when the stromal layer was being established, than later. These obsenations documcnt the inhibitory effect of rhCSF-1 on all aspects of hcrnatopoiesis conducted in cultures that simulate the hcmatopoictic microenvironment, demonstrate the importance of accessory/stromal ceUs in mediating the cffects of rhCSF-1 in LTMC, and point to an inhibitory activity as the mediating agent.

s41 m a OF l ” E K W - 6 DEPENDENCY OF HUMW AND MLJfUNE M”A CELIS FOI.UXtNG FUSION WI’lIi NORMAL B -. D. Jobin, R. Ianieux and R. Bzin , Cinadian ~ e d ~ ~ Q S S Transfusion semi- and LaVal University, Quebec City.

Interleukin-6 (IL-6) has boen shown to stimulate the grawth of mrine plasma- and hunm myelcana ixnor d l s . pra3ression of primary turors to IL-6 irdependency has been shm to axur both in vivo ard in v i t ro ard it has been propcrsed that a genetic event leading to the cu&tutive a d v a t i o n of the IL-6 reaqtor might be responsible for this phenartlenon. In order to study the functioIlndL state of the Ik6 receptor on myelm cells, we have used the kram ability of newly-formed B-cell hybridarms to responCt to IL-6 and the fact that n a v k IL- 6 canrrot activate the hmm IL-6 receptor. Hunan-muse ( h m myel- ard m u r k B lymgi-~azytessj ard mxlse-hurrran ( m r h e myel- ard human B ly@ccytes) heterohyfridmas w e r e prepared ad tested in proliferation assays i n prey-ce of reccwbinant narrine and human IL-6. The results cbbmed shwd that h w ILd axld s t m a t e the growth of hth trpes of hybridcanas & d e the m u r k ILd a x l d OfiY - * the growth of the x m s e - h m hebmhybridcms derived frcw the wine myelma cells. These results mate t h a t the I L 6 ZeceptOIs active on B-cell hybridorrras are derived fm the myelam parimer and that this receptor is not c o n s t i w d y activated in IL-6 irdep=rdent myelcara cells s h its n o d function could be reactivakd by fusion with w d cells. lhus the y i o n of myelma d l s to IL-~ irdependency is llkely to involve a recessive wdification i n the gene(s) amtrolling the i n t r a ~ e l l u l a r siw transduction pathways.

Page 22: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

92 CRCS Abstracts

s42 ACTIVATION OF RAT COMPLEMENT BY LIPOSOMES: DIRECT EFFECTS OF CHOLESTEROL AND LIPOSOME SIZE. D.V. Devine and K. Serrano, CRCS BTS, Vancouver Centre and Department of Pathology, Univ. of British Columbia

The liposome is a basic structure upon which to construct an artificial platelet. Liposome compositions presently in use for targeted drug delivery have a relatively short circulation time before they are cleared by the RES. This clearance is in part due to opsonization by complement. In order to design a longer lived liposome, the parameters affectin complement activation were investigated. We have studie 8 the effects of the size and composition of small unilamellar vesicles (SUV) on the activation of com lement in the rat, a common model for in vivo li some

change in residual hemolytic activity after exposure to SUV. At lipid concentration > 1 mM, SUV bearing a net positive or negative charge activated C by the classical pathway; no alternative pathway activation was detected at any lipid concentration tested. The activation of complement by negatively charged liposomes was increased in a dose-dependent manner by the addition of cholesterol. To test the hypothesis that the cholesterol effect was due to alterations in physical properties of the bilayer, SUV were prepared using unsaturated lipids or lipids with increased fatty ac I chain length. SUV containing saturated lipids were more

unsaturated lipids of the same acyl chain length. However, complement activation was not increased by lengthening the fatty acyl chain. These observations suggest that increased membrane order per se does not account for the complement activating effect of cholesterol. Size also affected complement activation by Suv. At a iven concentration of exposed lipid, small SUV (50 or 100 nm? were less effective at complement activation than larger SUV (200 or 400 nm). This observation may reflect geometric constraints on the assembly of classical pathway components.

surviva P studies. Complement activation was detectegs the

ef Y ective complement activators than those containing

S43 MULTIMERIN: A SERIES OF DISULFIDE-LINKED, MULTIMERS CONTAINED WITHIN PLATELETS AND SYNTHESIZED BY A MEGAKARYOCYTIC CELL LWE AND BY ENDOTNELIAL CELLS. C.P. M. Havward. T. E. Warkentin, P. Horsewood. I.W. Smith, and=. Kelton, Department of Pathology McMaster University and the Canadian Red Cross, Hamilton Centre.

Multimerin is a unique, disulfide-linked multimeric, soluble platelet protein that is expressed on the surface of activated platelets. Investigation of the native protein by agarose/acrylamide gel electrophoresis demonstrated that this protein resembled von Willebrand factor in its complex multimeric composition with variability in multimer size. The native multimexs in platelets ranged in size from less than 450 kDa to many million Daltons. The platelet releasate contained mainly the smallest multimers, suggesting that the largest multimers bind preferentially to the platelet surface during activation. Using two-dimensional, non-reduced/reduced electrophoresis, we demonstrated that multirnerin and von Willebrand factor are the two largest proteins in platelets. As multimerin could not be detected in plasma, we looked for evidence of megakaxyocyte biosynthesis using Darni cells, a megakaxyocytic cell line. Metabolic labelling studies demonstrated that multimerin is synthesized and secreted by both Dami cells and by human umbilical vein endothelial cells. Only the smallest multimers were constiwively secreted by these cells. The unique structure of multimerin provides multiple functional sites for binding to its ligands. Multimerin's localization to both endotheliai cells and platelets and the activation-dependent nature of its expression on the platelet surface suggest a role in the cellular events assoaated with vascular injury.

Page 23: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

CRCS Abstracts 93

s44 A NOVEL APPROACH TO GENE THERAPY AIMED AT TREATING HEMOPHILIACS. J . C. Wallenburg, M. Richard, A. Belmaaza. and E Chamand. Canadian Red Cross Society, Blood Services, Montreal Centre.

The objective of gene therapy is to correct a genetic defect in a patient by introducing corrective genetic information. Effective therapy should produce a long-term cure, be advantageous to current therapies and be without risk to the patient. The method being currently evaluated in clinical trials is based on the use of retroviral vectors. Retroviral vectors are an extremely efficient vehicle to introduce genetic information in cells but have some drawbacks namely size constraint and mutagenic potential. Alternatives would be the random integration of plasmid vectors or gene targeting by homologous integration. However the former is also mutagenic while the latter is very inefficient and can only be used in the normal tissue of expression. Thus, we have begun to develop a novel approach to gene therapy aimed at treating hemophiIiacs based on the integration of corrective genetic material into the highly repetitive (LINES) sequences of the human genome via targeted integration. The possible advantages would be: 1. no size constraints; 2. autonomous expression; 3 . acceptable efficiency and 4 . greatly reduced mutagenic risk. These advantages would be particularly pertinent to the objective of introducing a large gene (ex. Factor VIII gene) in a tissue where it is not normally expressed (ex. bone marrow). Our results thus far indicate that LINE targeting is probably 100 fold more frequent than single gene targeting. This is based on the frequency of transformation when targeted events are enriched (the positive-negative selection of Mansour a [1988] Nature 336, 348) and the presence of a PCR amplified diagnostic fragment. We are currently confirming these results by cloning of the integration sites by plasmid rescue and also by using an innovative amplification approach adapted to the amplification of multiple targets.

S45 NEGATIVE CONTROL OF APOPTOSIS IN 8-CELL HYBRIDOMAS BY SHORT-LIVED PROTEIN(S). J. Perreault and R. Lemieux. Canadian Red Cross Transfusion Service and Lava1 University. Quebec City.

In the last two years, the programmed cell death (apoptosis) process has been shown to play an important role in the control of hematopoiesis and of the immune system. Apoptosis characterized by DNA fragmentation has been observed in several types of hematopoietic cells cultured in absence of various cytokines such as erythropoietin, CSF's and interleukins. The role of these cytokines in the prevention of apoptosis suggests that these molecules may represent maintenance factors for many types of cells. Our work with the 11A3 B-cell hybridoma cell line has also shown the induction of apoptosis in absence of IL-6. Further work using gene expression inhibitors (CHX, Act D) revealed the existence in several 8-cell hybridomas of a short-lived protein essential for preventing the final activation step of the apoptosis process. Unique characteristics of this experimental system are the rapid (90 rnin.) and synchronous induction of DNA fragmentation and RNA degradation and the fact that apoptosis could be activated by a limited inhibition (50%) of the normal gene expression level. Additional studies using this system should permit to better characterize the mechanism controlling the expression (cytokines?) and activity (inhibition of nuclease?) of the short-lived factor(s) and possibly to find ways by which apoptosis could be inhibited in cell culture systems.

Page 24: Abstracts of papers presented at the 7th Scientific Meeting of the Canadian Red Cross Society Blood Services, Quebec, Canada, 16–17 August 1991

94 CRCS Abstracfs

S ,46 HIGH YIELD PRODUCTION OF MONOCLONAL ANTIBODIES USING A

Morel, 3. Perron, B. Massie and R. Lemieux. Canadian Red Cross BTS, Quebec City and Biotechnology Research Institute-NRC, Montreal.

A current trend in the development of cell culture systems for mAb production is the use of small scale bioreactors in perfusion mode. We have developped and tested a tangential flow filtration (TFF) unit that can be used f o r the replacement of the culture medium and the retention of the hybridoma cells in a stirred-tank bioreactor. Coupled to a 3 li. Celligen bioreactor, the TFF unit has permitted to maintain long-term (> 60 days) steady-state culture conditions of the anti-A D5 hybridoma cell line. The steady- state viable cell concentration was directly proportionnal to the volume of medium perfused (up to 2 volumes per day). MAb productivity in the spent medium (150 pglml) was about two times higher that the one obtained in standard batch culture done in the same bioreactor. We are now in the process of determining the behaviour in this system of hybridoma cells cultured in serum-free medium. For this purpose, anti A- and anti B-secreting hybridomas have been adapted to growth in serum-free medium without loss of mAb productivity. In conclusion, the major advantages of the system developped are the low initial capital investment in equipment and the high mAb productivity which is comparable to the one of a 10 times larger bioreactor used in the batch mode.

PERFUSION CULTURE SYSTEM. a De I a Broisc, M. N o i w VL

s47 DOWNREGUIATION OF C ~ L SURFACE CLASS r M~IC ANTIGENS BY POXVIRUSIS. L. K. Boshkov, J . L. Macen and G. McFadden, Depts. of Medicine and Biochemistry, Univ. of Alberta and the Canadian Red Cross Blood Transfusion Service, Edmonton Centre, Edmonton AB.

Suppression of HLA gene products is of considerable theoretical and practical interest in transfusion medicine and transplantation. Downregulation of surface hlHC antigens is a recently reported strategy of adenoviruses and cytomegalovirus t o escape immuno recognition. Shope fibroma virus ( S N ) is a leporipoxvirus tha t causes self-limited localized fibromas in immunocompetent adult rabbits. Myxoma virus (MYX) and malignant rabbit fibroma virus (MRV) are related leporipoxviruses tha t induce rapidly lethal generalized infections accompanied by tumors and immunosuppression. Cell surface expression of MHC class I antigens was followed using FACS analysis of infected baby green monkey kidney cells stained with anti-HLA class I monoclonal antibodies (mAbs). Rapid decrease in cell surface expression of class I antigens is observed following infection with MYX and MRV, with surface class I reduction approaching background binding by 24 hours. Cell surface expression of the transferrin receptor is unaffected, suggesting a specific effect on class I. Minimai class I decrease is seen during infection with SFV or with vaccinia virus suggesting class I downregulation to be a specific feature of virulent feporipoxviral infection. Experiments using cycloheximide and cytosine arabinoside suggest the observed class I downregulation requires late viral gene expression. Preliminary immunofluorescence studies suggest class 1 accumulates intracellularly during the course of viral infection. W e a re currently characterizing the nature of class I downregulation in this novel viral system a t the genetic and molecular levels.