Managing costs in primary immunodeficiency: minimal immunophenotyping and three national references.

Our aim was to evaluate the cost-effectiveness of a minimal lymphocyte subset quantification (LSQ) by flow cytometry as the first screening in children with clinically suspected primary immunodeficiency (PID). Two hundred sixty-eight Brazilian patients (0-21 years old) were studied. They were divided by clinical and phenotypical features into those fulfilling criteria for PID (PID phenotype) according to the 2017 International Union of Immunological Societies (IUIS) classification and those not fulfilling these criteria (non-PID phenotype). We evaluated how many patients had values below the 10th percentile for five lymphocyte subsets in peripheral blood, (suggestive of PID) according to reference values for Brazil, Italy and USA. Three lymphocyte subsets (T CD3/CD4, B CD19 and NK CD16/CD56) had p-value < 0.05 and Odds Ratio (OR) indicating a risk at least two times higher for the diagnosis of a PID phenotype. The application of Kappa coefficient (k) on Brazilian vs Italian and Brazilian vs US data sets resulted in k compatible with strong or excellent level of agreement between the three classification systems. The authors conclude that a number of CD3+ /CD4+ , CD19+ and CD16+ /CD56+ (NK) cells in peripheral blood <10th percentile represented a significant risk for the diagnosis of PID in this cohort. Natural killer (NK) deficiency is quite rare and has a very specific clinical profile. So, the analysis of these cells could be requested only in some cases, saving even more costs. The minimal immunophenotyping, with quantification of T CD4+ , CD19+ and in some cases CD16+ /CD56+ cells, may be a useful tool for the first screening of PID, saving costs, especially in developing countries.

Post-transplant monitoring of NK cell counts as a simple approach to predict the occurrence of opportunistic infection in liver transplant recipients.

BACKGROUND: Monitoring of peripheral blood lymphocyte subpopulation (PBLS) counts might be useful for estimating the risk of infection after liver transplantation (LT). METHODS: We prospectively measured total lymphocyte and PBLS counts at baseline and post-transplant months 1 and 6 in 92 LT recipients. PBLS were enumerated by single-platform 6-color flow cytometry technology. Areas under receiver operating characteristic (ROC) curves were used to evaluate the accuracy of different PBLS for predicting cytomegalovirus (CMV) disease and overall opportunistic infection (OI). Adjusted hazard ratios (aHRs) for both outcomes were estimated by Cox regression. RESULTS: After a median follow-up of 730.0 days, 29 patients (31.5%) developed 38 episodes of OI (including 22 episodes of CMV disease). The counts of CD3(+) , CD4(+) , and CD8(+) T cells, and CD56(+) CD16(+) natural killer (NK) cells at month 1 were significantly lower in patients subsequently developing OI. The NK cell count was the best predictive parameter (area under ROC curve for predicting CMV disease: 0.78; P-value = 0.001). Patients with an NK cell count <0.050 × 10(3) cells/µL had higher cumulative incidences of CMV disease (P-value = 0.001) and overall OI (P-value <0.001). In the multivariate models, an NK cell count <0.050 × 10(3) cells/µL at month 1 post transplantation remained as an independent risk factor for CMV disease (aHR: 5.54; P-value = 0.003) and overall OI (aHR: 7.56; P-value <0.001). CONCLUSION: Post-transplant kinetics of NK cell counts may be used as a simple and affordable proxy to the cell-mediated immunity status in LT recipients and to their associated risk of OI.

Total lymphocyte count as a surrogate marker for CD4 count in resource-limited settings.

BACKGROUND: CD4 testing is the recognized gold standard used to stage HIV/AIDS, guide treatment decisions for HIV-infected persons and evaluate effectiveness of therapy. The need for a less expensive surrogate marker that can be used in resource-limited setting is however necessary. The study sought to assess the suitability of Total lymphocyte count (TLC) as a surrogate marker for CD4 count in resource-limited localities in Ghana. METHODS: This observational study was conducted at the Central Regional Hospital, which has one of the established antiretroviral therapy centres in Ghana. A total of one hundred and eighty-four (184) confirmed HIV I seropositive subjects were included in the study. Blood samples were taken from all the subjects for estimation of CD4 and total lymphocyte counts. The study subjects were further categorised into three (3) groups according to the Centers for Disease Control and Prevention (CDC) classification criteria as follows: CD4 counts (1) ≥ 500 cells/mm3 (2) 200-499 cells/mm3 and (3) <200 cells/mm3. Positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of various TLC cut-offs were computed for three groups. Correlation and Receiver Operator Characteristic analysis was performed for the various CD4 counts and their corresponding Total Lymphocyte count obtained. RESULTS: The sensitivity, specificity, positive and negative predictive values of TLC 1200 cells/ mm3 to predict CD4 count were <200 cells/mm3 72.2%, 100%, 100% and 95.7% respectively. A TLC of 1500 cells/ mm3 was found to have maximal sensitivity (96.67%), specificity (100%), PPV (100%) and NPV (75.0%) for predicting a CD4 cell count of 200-499 cell/mm3. A TLC of 1900 cells/mm3 was also found to have a maximal sensitivity (98.45%), specificity (100%), PPV (100%) and NPV (100%) for predicting CD4 count ≥500 cells/mm3. A positive correlation was noted between 184 paired CD4 and TLC counts (r = 0.5728). CONCLUSION: Total Lymphocyte count can therefore adequately serve as a surrogate marker for CD4 count in HIV patients who are naïve for antiretroviral therapy in resource-limited areas.

Absolute lymphocyte count: a cost-effective method of monitoring HIV-infected individuals.

AIM: Depletion of CD4 cell count is a hallmark of disease progression in AIDS. CD4 cell count is essential for physicians to decide about the timing of initiation of antiretroviral therapy (ART) and for prophylaxis of opportunistic infections. WHO has recommended that, absolute lymphocyte count (ALC) of ≤1200/µL can substitute CD4 cell count of ≤200/µL in resource-constrained countries throughout the world. MATERIALS AND METHODS: This study was undertaken to know whether there is a correlation between CD4 cell count and ALC in HIV-infected individuals. A single sample of blood was withdrawn for ALC and CD4 cell count. The samples received from December 1, 2004 to December 31, 2005 were analyzed. RESULTS: A total of 196 samples were collected from 185 patients. After exclusion, a total of 182 samples were analyzed. Results revealed that male:female ratio was 126:56 and their age ranged from 13 to 67 years. The median ALC was 1747 cells/µL, whereas the CD4 cell count ranged from 5 to 2848. The correlation coefficient between ALC and CD4 cell count was significant (0.714). There were 49 patients with an ALC of ≤1200/µL of whom 77.6% patients had CD4 cell count ≤ 200/µL (true positive) and 22.4% had CD4 cell count > 200/µL (false positive). There were 133 patients with an ALC of >1200/µL of whom 84.2% had CD4 cell count > 200/µL (true negative) and 15.8% had CD4 cell count ≤ 200/µL (false negative). Taking ALC of ≤1200/µL as a predictor of CD4 cell count ≤ 200/µL ,the sensitivity of the test was 64.4% and specificity was 91.1%. The positive predictive value was 77.6%, negative predictive value was 84.2%, and accuracy was 82.4%. CONCLUSION: We found that an ALC of ≤ 1520/µL has higher sensitivity (78%) for a CD4 cell count of ≤ 200/µL. The ALC was found to be significantly cost-effective in our setup but chances of missing out patients requiring ART was 1 in 5 using the WHO guidelines.

Comprehensive & cost effective laboratory monitoring of HIV/AIDS: an African role model.

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing > 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Rapid automated cell quantification on HIV microfluidic devices.

Lab-chip device analysis often requires high throughput quantification of fluorescent cell images, obtained under different conditions of fluorescent intensity, illumination, focal depth, and optical magnification. Many laboratories still use manual counting--a tedious, expensive process prone to inter-observer variability. The manual counting process can be automated for fast and precise data gathering and reduced manual bias. We present a method to segment and count cells in microfluidic chips that are labeled with a single stain, or multiple stains, using image analysis techniques in Matlab and discuss its advantages over manual counting. Microfluidic based cell capturing devices for HIV monitoring were used to validate our method. Captured CD4(+) CD3(+) T lymphocytes were stained with DAPI, AF488-anti CD4, and AF647-anti CD3 for cell identification. Altogether 4788 (76 x 3 x 21) gray color images were obtained from devices using discarded 10 HIV infected patient whole blood samples (21 devices). We observed that the automatic method performs similarly to manual counting for a small number of cells. However, automated counting is more accurate and more than 100 times faster than manual counting for multiple-color stained cells, especially when large numbers of cells need to be quantified (>500 cells). The algorithm is fully automatic for subsequent microscope images that cover the full device area. It accounts for problems that generally occur in fluorescent lab-chip cell images such as: uneven background, overlapping cell images and cell detection with multiple stains. This method can be used in laboratories to save time and effort, and to increase cell counting accuracy of lab-chip devices for various applications, such as circulating tumor cell detection, cell detection in biosensors, and HIV monitoring devices, i.e. CD4 counts.

Monoclonal antibody fluorescence for routine lymphocyte subpopulation analysis with the Abbott CELL-DYN Sapphire haematology analyser.

Using previously described procedures, this study quantified T-cell, T-cell subset, B-cell and NK-cell populations with the CD-Sapphire haematology analyser in a series of patients with mild to moderate lymphocytosis. Lymphocyte counts ranged from 6.0 to 14.9 x 10(9)/l, with 86/97 being <10.0 x 10(9)/l. Immunophenotyping (CD3/CD19/HLA-DR, CD4/CD8 and CD16/CD56 combinations) was performed using EDTA-anticoagulated blood, automated CD-Sapphire analysis and subsequent software processing. Of 35 samples from younger (<12 years) patients, 22 (63%) had nonspecific lymphocyte changes, 4 (11%) showed specific increases in nonreactive T-Helper or T-Suppressor cells, and five showed a reactive T-cell lymphocytosis. The remaining four were classified as 'Transient/Persistent NK-associated (NKa) Expansion' (n = 3) and specific B-cell lymphocytosis (n = 1). For older patients (n = 59), 15 (25%) had an increase (>1.5 x 10(9)/l) in B-cells, and seven investigated for surface immunoglobulin expression were all found to be clonal. The remaining samples were categorized as 'Transient/Persistent NK-associated (NKa) Expansion' (13/59), Reactive Lymphocytosis (5/59), 'Reactive Lymphocytosis or Transient/Persistent NKa Expansion' (8/59), specific T-Helper cell (n = 8) or T-Suppressor cell (n = 3) lymphocytosis, and 'Lymphocytosis of Undetermined Significance' (n = 7). This study has demonstrated the feasibility of applying limited immunophenotyping protocols to the investigation of patients with abnormal lymphocyte counts in routine haematology. By using commercially purchased liquid monoclonal reagents to determine lymphocyte subpopulation profiles, haematology laboratories can provide more definitive information of potential clinical importance.

Diagnostic accuracy and clinical utility of a simplified low cost method of counting CD4 cells with flow cytometry in Malawi: diagnostic accuracy study.

OBJECTIVES: To assess the diagnostic accuracy and clinical utility of a simplified low cost method for measuring absolute and percentage CD4 counts with flow cytometry. DESIGN: A CD4 counting method (Blantyre count) using a CD4 and CD45 antibody combination with reduced blood and reagent volumes. Diagnostic accuracy was assessed by measuring agreement of the index test with two other assays (TruCount and FACSCount). Clinical utility was investigated by comparing CD4 counts with the new assay with WHO clinical staging in patients with HIV. SETTING: Research laboratories and antiretroviral therapy clinic at a medical school and large government hospital in southern Malawi. PARTICIPANTS: Assay comparisons were performed on consecutive blood samples sent for CD4 counting from 129 patients with HIV. Comparison of CD4 count with staging was conducted on 253 consecutive new patients attending the antiretroviral therapy clinic. MAIN OUTCOME MEASURES: Limits of agreement with 95% confidence intervals between index test and reference standards. RESULTS: The limits of agreement for Blantyre count and TruCount were excellent (cell count -48.9 to 27.0 x10(9)/l for absolute counts in the CD4 range <400x10(9)/l and -2.42% to 2.37% for CD4 percentage). The assay was affordable with reagent costs per test of $0.44 ( pound0.22, euro0.33) for both absolute count and CD4 percentage, and $0.11 for CD4 percentage alone. Of 193 patients with clinical stage I or II disease, who were ineligible for antiretroviral therapy by clinical staging criteria, 73 (38%) had CD4 counts <200x10(9)/l. By contrast, 12 (20%) of 60 patients with stage III or IV disease had CD4 counts >350x10(9)/l. CONCLUSIONS: This simplified method of counting CD4 cells with flow cytometry has good agreement with established commercial assays, is affordable for routine clinical use in Africa, and could improve clinical decision making in patients with HIV.

Monitoring antiretroviral therapy in HIV/AIDS patients in resource-limited settings: CD4 counts or total lymphocyte counts?

OBJECTIVE: In order to improve the monitoring of disease progression and therapeutic effectiveness in the management of HIV/AIDS in a resource-limited setting, this study was carried out to establish a correlation between total lymphocyte counts (TLC) and CD4 lymphocyte counts in HIV-1 infected/AIDS adults in Yaoundé, Cameroon. METHODS: Full blood counts, differential white, and CD4 counts were measured in 149 patients using standard methods. The correlation coefficient established correlation between values. Sensitivity, specificity, and positive predictive values were calculated as required. RESULTS: The mean TLC, CD4 count, and CD4% as well as CD4/CD8 ratios were 1.932+/-0.895 x 10(9)/L, 268+/-183 cells/mm(3), 14.51+/-15.9%, and 0.34+/-0.25, respectively. Only a weak correlation was observed between TLC and CD4 counts (r=0.41, p=0.05). As a predictor of CD4 count, TLC cut-offs <2.0 and <1.0 x 10(9)/L were unable to predict these values reliably, but showed that at TLC cut-offs of <1.0 x 10(9)/L there was a high chance of CD4 counts being under 200 cells/mm(3). CONCLUSIONS: These data suggest that TLC are of limited value in predicting CD4 counts and should not be substituted for CD4 counts whenever possible. However, TLC may be reliably used in designing algorithms and programs for initiating patient management and follow-up in this setting.

Total lymphocyte count as a possible surrogate of CD4 cell count to prioritize eligibility for antiretroviral therapy among HIV-infected individuals in resource-limited settings.

OBJECTIVE: To characterize the value of total lymphocyte counts in predicting risk of death among patients initiating triple combination antiretroviral therapy. METHODS: Study subjects included antiretroviral-naive persons aged 18 years or older who initiated treatment with triple combination therapy between August 1 1996 and September 30 1999 in a population-based observational cohort of HIV-infected individuals. Total lymphocyte counts as well as CD4 count and plasma viral load were assessed at baseline. Separate Cox proportional hazards models were devised to evaluate the effect on survival of total lymphocyte count in lieu of or with CD4 count after adjustment for other prognostic factors including plasma viral load. RESULTS: A total of 733 antiretroviral-naive persons initiated triple drug combination antiretroviral therapy over the study period with a median follow-up of 29.5 months. In the first analysis, only baseline CD4 cell counts of 50-199 cells/microl or less than 50 microl were associated with an increased risk of mortality [adjusted relative risk (ARR) 2.90; 95% CI: 1.40, 5.98] and (ARR 6.30; 95% CI: 2.93, 13.54), respectively. When CD4 counts were excluded from the analysis as if unavailable, total lymphocyte count of between 0.8 and 1.4 G/I, and less than 0.8 G/I were both significantly associated with an increased risk of mortality (ARR 2.36; 95% CI: 1.16, 4.78) and (ARR 6.17; 95% CI: 2.93, 13.01), respectively. CONCLUSION: Total lymphocyte count may provide a simple and cost-effective alternative for prioritizing therapy initiation in resource-limited settings. Our results suggest that, if appropriately validated, judicious application of total lymphocyte counts could overcome one of the practical obstacles to more widespread provision of antiretroviral therapy in resource-poor settings.

Total lymphocyte count (TLC) is a useful tool for the timing of opportunistic infection prophylaxis in India and other resource-constrained countries.

BACKGROUND: In most resource-constrained countries, CD4 cell count testing is prohibitively expensive for routine clinical use and is not widely available. As a result, physicians are often required to make decisions about opportunistic infection (OI) chemoprophylaxis without a laboratory evaluation of HIV stage and level of immunosuppression. OBJECTIVES To evaluate the correlation of total lymphocyte count (TLC), an inexpensive and widely available parameter, to CD4 count. To determine a range of TLC cutoffs for the initiation of OI prophylaxis that is appropriate for resource-constrained settings. METHODS: Spearman correlation between CD4 count and TLC was assessed in patients attending an HIV/AIDS clinic in South India. Positive predictive value (PPV), negative predictive value (NPV), and sensitivity and specificity of various TLC cutoffs were computed for CD4 count <200 cells/mm3 and <350 cells/mm3. Correlation and statistical indices computed for all patients and for patients dually infected with HIV and active tuberculosis. RESULTS: High degree of correlation was noted between 650 paired CD4 and TLC counts (r = 0.744). TLC <1400 cells/mm3 had a 76% PPV, 86% NPV, and was 73% sensitive, 88% specific for CD4 count <200 cells/mm3. TLC <1700 cells/mm3 had a 86% PPV, 69% NPV, and was 70% sensitive, 86% specific for CD4 count <350 cells/mm3. The cost of a single CD4 count in India is approximately 30 US dollars, whereas the cost of a single TLC is 0.80 US dollars. CONCLUSION: TLC could serve as a low-cost tool for determining both a patient's risk of OI and when to initiate prophylaxis in resource-constrained settings. PPV, NPV, sensitivity, and specificity maximally aggregated at TLC <1400 cells/mm3 for CD4 <200 cell/mm3 and TLC <1700 cells/mm3 for CD4 <350 cells/mm3. Selection of appropriate TLC cutoffs for prophylaxis administration should be made on a regional basis depending on OI incidence, antimicrobial resistance patterns, and availability of the antimicrobials.

Effect of CD34+ peripheral blood progenitor cell dose on hematopoietic recovery.

The CD34+ cell surface antigen is expressed on progenitor cells required for blood stem cell transplantation. The number of cells expressing CD34+ can be used to assess the peripheral blood progenitor cell (PBPC) graft quality and predict hematopoietic recovery after engraftment. Because there is considerable variability among centers in the determination of CD34+ cell counts, standardizing flow cytometry methodology is essential. It is necessary to define a minimum safety threshold CD34+ cell dose for hematopoietic cell transplantation. This minimum dose would define a cell number in the graft, below which a proportion of patients would be expected to have delayed hematopoietic recovery or failure to engraft. We reviewed data from numerous studies. Although 1-2 x 10(6) CD34+ cells/kg can be considered an adequate graft, available data suggested that doses >5 x 10(6) CD34+ cells/kg were associated with more rapid engraftment and a lower probability of graft failure. The risk of delayed recovery was inversely related to CD34+ cell dose. Delayed recovery may result in greater transfusion requirements, longer hospitalization, increased antibiotic use and growth factor support, and higher health care costs. The extent of prior chemotherapy and radiation treatment are major risk factors for poor PBPC collection. To achieve an optimal CD34+ cell yield, PBPC collection should be initiated early during therapy. PBPC collection should be coordinated with the anticipated number of chemotherapy cycles, duration of chemotherapy, interval between chemotherapy and apheresis, need for radiotherapy, and exposure to the more progenitor cell-toxic drugs such as carmustine or busulfan.

Correlation between total and CD4 lymphocyte counts in HIV infection: not making the good an enemy of the not so perfect.

The aim of this study was to assess the correlation and average cost of total lymphocyte count compared with CD4 count as a broad estimate of immunosuppression in HIV-1 infected individuals. Spearman's partial rank correlation were calculated between total lymphocyte count, absolute CD4 count and CD4 per cent stratified by stage of HIV-1 infection for routinely collected samples. Data were collected prospectively from a T cell-subset register combined with clinical data obtained retrospectively from case notes of HIV-infected patients managed at St Mary's Hospital, London 1982-1991. Costing data were obtained through a survey of the departments of haematology and immunology (1989/90 prices). The correlation between 1534 paired absolute lymphocyte count and CD4 lymphocyte count was found to be high (R = 0.76). When analysed by stage of HIV infection, the correlation increased from R = 0.64 for asymptomatic patients, to R = 0.72 for patients with symptomatic non-AIDS HIV infection and R = 0.73 for AIDS patients. Correlations between absolute lymphocyte count and CD4 per cent were considerably weaker: R = 0.41 all paired counts; R = 0.32 for asymptomatic patients; R = 0.25 for symptomatic non-AIDS patients; R = 0.32 for AIDS patients. Average cost was pounds 8 per full blood count compared with pounds 38 per T-cell subset analysis. The high correlation between total and CD4 lymphocyte counts, especially for patients with symptomatic HIV disease, demonstrates the suitability of the use of total lymphocyte count in the absence of CD4 counts. Given the considerably lower prices of total lymphocyte counts compared with T-cell subset analysis, this is particularly relevant for developing countries.