Estimating prognostic relevant cutoff values for a multiplex PCR detecting BCR::ABL1 in chronic myeloid leukemia patients on tyrosine kinase inhibitor therapy in resource-limited settings.

The prognosis of chronic myeloid leukemia (CML) on tyrosine kinase inhibitor (TKI) treatment is based on the quantification of BCR::ABL1 fusion gene transcript copy number, harmonized by an international scale (IS) based on TaqMan-based real-time quantitative PCR (qRT-PCR). In Ethiopia, as in most low- and middle-income countries (LMICs), access to standard diagnostic, follow-up, and prognostic tools is very limited, and it has been challenging to strictly follow international guidelines. This seriously compromises clinical outcome, despite the availability of TKIs through the Glivec International Patient Assistance Program (GIPAP). Multiplex PCR (mpx-PCR), conventionally regarded as a "screening tool," offers a potential solution to this problem. A total of 219 samples from confirmed CML patients were assayed. In reference to qRT-PCR, the AUC of ROC curve for mpx-PCR was 0.983 (95% CI: 0.957 to 0.997). At the optimum cut-off value, equivalent to BCR::ABL1 (IS) transcript copy number of 0.6%, the specificity and sensitivity were 93% and 95%, respectively, with 94% accuracy. Albeit the sensitivity and accuracy of mpx-PCR decrease below the optimum cutoff of 0.6% (IS), the specificity at 0.1% (IS) was 100%, making it an attractive means to rule-out relapse and drug non-adherence at later stages of treatment, which is particularly an issue in a low income setting. We conclude that the relative simplicity and low cost of mpx-PCR and prognostic relevant cutoff values (0.1-0.6% IS) should allow its use in peripheral clinics and thus maximize the positive impact of TKIs made available through GIPAP in most LMICs.

Preservice laboratory education strengthening enhances sustainable laboratory workforce in Ethiopia.

BACKGROUND: There is a severe healthcare workforce shortage in sub Saharan Africa, which threatens achieving the Millennium Development Goals and attaining an AIDS-free generation. The strength of a healthcare system depends on the skills, competencies, values and availability of its workforce. A well-trained and competent laboratory technologist ensures accurate and reliable results for use in prevention, diagnosis, care and treatment of diseases. METHODS: An assessment of existing preservice education of five medical laboratory schools, followed by remedial intervention and monitoring was conducted. The remedial interventions included 1) standardizing curriculum and implementation; 2) training faculty staff on pedagogical methods and quality management systems; 3) providing teaching materials; and 4) procuring equipment for teaching laboratories to provide practical skills to complement didactic education. RESULTS: A total of 2,230 undergraduate students from the five universities benefitted from the standardized curriculum. University of Gondar accounted for 252 of 2,230 (11.3%) of the students, Addis Ababa University for 663 (29.7%), Jimma University for 649 (29.1%), Haramaya University for 429 (19.2%) and Hawassa University for 237 (10.6%) of the students. Together the universities graduated 388 and 312 laboratory technologists in 2010/2011 and 2011/2012 academic year, respectively. Practical hands-on training and experience with well-equipped laboratories enhanced and ensured skilled, confident and competent laboratory technologists upon graduation. CONCLUSIONS: Strengthening preservice laboratory education is feasible in resource-limited settings, and emphasizing its merits (ample local capacity, country ownership and sustainability) provides a valuable source of competent laboratory technologists to relieve an overstretched healthcare system.

Alteration of Endocrine Hormones and Antibody Responses in Different Spectrum of Tuberculosis Disease.

Effective control of Mycobacterium tuberculosis (Mtb) infection is mediated by multifaceted factors that involve both the endocrine and immune system. Profiling hormones and antibodies in different stages of TB provides insight in the pathogenesis of the disease. In this study, we profiled endocrine hormones (dehydroepiandrosterone (DHEA), cortisol, testosterone, estradiol, growth hormone and leptins) and Mtb strain H37RV lipoarabinomannan (LAM)-specific antibody levels in plasma samples, collected from pulmonary TB (PTB) patients, TB lymphadenitis (TBLN) patients and latently infected (QFT-positive) or uninfected (QFT-negative) apparently healthy individuals using ELISA. Plasma levels of leptin and DHEA were significantly low in PTB and TBLN patients compared to healthy controls (P<0.0001 and P=0.02, respectively), whereas these levels significantly increased following anti-TB treatment (P=0.002 and P=0.0001, respectively) among TB patients. The levels of estradiol and testosterone significantly improved following anti-TB treatment (P=0.03 and P=0.0003, respectively), whereas cortisol and growth hormones declined significantly (P <0.05). Similarly, LAM-specific IgG, IgM and IgA were significantly higher in PTB patients compared to other groups, whereas levels of IgG1 subtype were significantly higher among LTBI groups compared to both TB patients and QFT-negative individuals (P<0.0001). Overall, we observed significantly variable levels of endocrine hormones as well as immunoglobulins across the spectrum of TB illness and such profiling has a significant contribution in selection of effective biomarkers that have roles in TB treatment monitoring or diagnostics. Although this study did not show a functional association between hormones and antibodies, alterations in the levels of these biomarkers suggest the key roles these markers play in TB pathogenesis.

BCR-ABL Transcript Level as Compared to LDH and Uric Acid Among Chronic Myeloid Leukemic Patients.

BACKGROUND: Chronic myeloid leukemia is characterized by the presence of the Philadelphia chromosome, which is caused by the breakpoint cluster region-Abelson fusion or joined gene. A high concentration of BCR-ABL transcripts level can strongly forecast cytogenetic and hematologic reversion in CML patients. However, the molecular test for BCR-ABL is costly and hardly available in developing countries with low and middle-income. Owing to this, it is required to examine other cost-effective and best diagnostic (prognostic) biomarkers. OBJECTIVE: The present study aimed to estimate the total LDH and uric acid level as compared to BCR-ABL transcript level among treated and treatment-naive Chronic Myeloid Leukemia (CML) patients. METHODS: A comparative cross-sectional study design was used to include eighty-one (81) CML patients tested for BCR-ABL by GeneXpert RT-PCR transcript level at Tikur Anbessa Specialized Hospital. The current study correlates LDH with BCR-ABL and hematological parameters using the spearman correlation, Mann-Whitney U test, and roc curve data analysis tool. RESULTS: A total of 81 CML patients were assayed; 46(56.8%) of them were in the medically treated group, and the remaining 35 (43.2%) were treatment-naive patients. Significant positive correlations were observed between LDH and BCR-ABL (r=0.79, P<0.001).The correlation coefficient value of uric acid (r=0.295, p<0.008) with BCR-ABL showed a weak correlation between the two test parameters. There was a statistically noteworthy (p<0.05) difference in the median level of BCR-ABL and LDH among patients in the treatment group (median=21%, 350 U/L) and the treatment- naive group (median=57%, 1246 U/L), respectively. For uric acid, there was no statistically significant (p<0.542) difference between the study group. The AUC for LDH, Basophil, and WBC was 0.881, 0.889, and 0.748, respectively, which showed better performance for the follow-up of patients with CML than uric acid (0.695) and platelets (0.70). CONCLUSION: The CML LDH value strongly correlated with BCR-ABL transcript level, whereas uric acid was weakly correlated with BCR-ABL. Hence, in parallel with the BRC-ABL transcript level, these findings could be a patent for confirming the capability of LDH as an alternative cost-- effective diagnostic, prognostic biomarker, and a novel therapeutic target in CML disease.

Effect of Regular Khat Chewing on Serum Fasting Sugar Level in Diabetic patients versus Healthy Individuals; A comparative study.

BACKGROUND: Khat chewing is a long standing social-cultural habit in several countries. Even though many people chew khat simply for its pleasurable and stimulatory effect, evidence showed widely-held belief among khat chewers in Ethiopia and other part of the world that khat helps to lower blood glucose while some studies are contradicted on the effect of khat. There is limited data about khat's effect on blood glucose especially in our setting, Harar estern Ethiopia. OBJECTIVE: Primarily the present study aims to compare fasting blood sugar level among khat chewer diabetic and healthy individuals, and to asses risk factors associated with poor glycemic control in diabetic subjects. METHOD: A cross-sectional study included 200 confirmed diabetic and healthy subjects. Fasting blood sugar was determined by enzymatic method glucose oxidase and glucose hexokinase. Glycemic control was also determined for diabetic subjects based on the last 2-month diabetic clinic visits and current measurement. RESULT: (Median ± IQR [interquartile range]) fasting blood sugar difference among Khat chewer and non khat chewer were 159 ± 83 mg/dl and 202 ± 79 mg/dl respectively in diabetic subjects when tested by glucose oxidase. Similarly, in healthy non khat chewer and khat chewer, khat chewers has lower (Median ± IQR) fasting blood glucose level 82 ± 18 mg/dl than non khat chewers 94 ± 13 mg/dl when tested by glucose oxidase. Regarding risk factors associated with poor glycemic control in diabetic subjects, positive parental diabetes history, insulin medication, being overweight, obese were significantly associated with poor glycemic control. CONCLUSION: There was significant effect of khat on median FBS among khat chewers in diabetic and healthy individuals. And the proportion of glycemic control was high among diabetic subjects. RECOMMENDATION: Health care professional and patients should manage the risk factors to delay disease progression and restrain the damage. More studies should be conducted in randomized control trial manner to further elucidate khat effect on blood sugar level so that the actual effect of khat can be identified unlike in cross sectional where there may not be strong causal relationship.

External Quality Assessment on CD4+ T-Cell Count Using in-House Proficiency Testing Panels for CD4 Count Laboratories in Addis Ababa, Ethiopia.

BACKGROUND: CD4+ T-cell count External Quality Assessment program is important for the evaluation of performance of CD4 count laboratories. The aim of this study was to assess the quality of CD4count laboratory performance using in-house Proficiency testing panels that perform routine CD4 counts in Addis Ababa, Ethiopia, 2013/14. METHODS: Participating laboratories were 20, 23 and 25 in trials 1, 2 and 3, respectively. In-house prepared fresh whole blood samples both with "normal" and "low" CD4 values were sent to participating laboratories. Percentage and absolute counts of CD4+ T-lymphocytes were done using their routine procedures. Data were analyzed for each trial including trimmed mean, standard deviation (SD), percent coefficient of variation (%CV), residual, and standard deviation index (SDI) values for both absolute counts and percentages of CD4+ lymphocytes (%CD4). RESULTS: Most participating laboratories produced results that were within 2SD of the mean. Average inter-laboratory precision (trimmed %CV) was 10.87% and 5.14% for CD4 absolute counts and %CD4, respectively. For normal material, the trimmed mean %CV was 9.59% and3.23% for CD4 absolute counts and %CD4, respectively. For low material, the trimmed mean % CV was 12.15% and 7.05% for CD4 absolute counts and %CD4 respectively. BDFACSCount™ users showed the best accuracy and precision as evidenced by longitudinal analysis. CONCLUSION: This study was found to help facilities in early identifying their gaps with regard to their CD4 count performance and in avoiding the challenges encountered during participation in external EQA providers like the high cost, transportation problem, feedback delay and CD4laboratory coverage.

Gonadal and Cortisol Hormone Profile among Male Chronic Khat, Marijuana, and Heroin Abuses.

BACKGROUND: Drugs of abuse could interfere with the hypothalamic-pituitary-gonadal axis, causing impaired functions of the gland and associated functions of target organs. Drugs of abuse tend to cause changes in the endocrine system, and these changes could be physiological, molecular, biochemical, genetic, and cellular. METHOD: A case-control study design was conducted from a total of 171 male consented study participants (148 drug abusers and 23 controls). The serum gonadal and cortisol hormone levels were assayed using the electrochemiluminescence immunoassay method. Socio-demographic variables were collected using a semi-structured questionnaire by the interview method. Nonparametric statistical tools (Mann-Whitney test and median) were used to compare the groups. In all cases, P < 0.05 was considered statistically significant. RESULT: The median age for drug abusers and control groups was 27, and the age difference between drug abusers and control group was not statistically important. The median estradiol levels among chronic khat chewers (39.4 pg/ml), marijuana (44 pg/mL), and users of heroin (40.2 pg/mL) were significantly higher than control groups (23 pg/mL), P < 0.003. However, the median luteinizing hormone levels among chronic khat chewers (5 IU/L), marijuana users (5 IU/L), and heroin users (5.6 IU/L) were significantly lower than those of control groups (6.2 IU/L), P < 0.02. The median testosterone levels among chronic khat chewers (6.1 ng/mL), marijuana users (6.3 ng/mL), and heroin users (6.6 ng/mL) were significantly lower than control groups (8.0 ng/mL), P < 0.003. However, cortisol and follicle stimulating hormone did not show statistically significant difference between users of khat, marijuana, and heroin compared with the control group. CONCLUSION: The drug abuser group had significantly lower testosterone and luteinizing hormone levels compared with control groups. Drug abuse has been shown to affect gonadal hormones in an unusual physiological phenomenon. These findings reveal the need for intervention programs to solve these problems.

Errors in the Hematology Laboratory at St. Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia.

OBJECTIVE: The objective of this study was to determine the magnitude of pre-analytical, analytical and post-analytical laboratory errors in hematology tests. RESULTS: A total of 2606 hematology requests were studied. Out of the total, 562 (21.6%) pre-analytic, 14 (0.5%) analytical and 168 (6.4%) post-analytical errors were recorded which contribute a total frequency of 75.5, 1.9 and 22.6%, respectively. The name of the physician requesting the test was not provided on 2215 (85%) of request forms and 1827 (70.1%) of the request forms were unaccompanied with proper clinical details of the patient. Essential information required on the request forms was often missed. Close communication between clinicians and laboratory personnel is the key to improve laboratory quality in general.

Clinical chemistry laboratory errors at St. Paul's Hospital Millennium Medical College (SPHMMC), Addis Ababa, Ethiopia.

OBJECTIVE: This study was aimed to determine the magnitude of errors in clinical chemistry laboratory tests at different phases of the assay of clinical chemistry laboratory unit. RESULTS: From the total 1633 clinical chemistry laboratory tests done, overall, 541 (33.1%) errors occurred which accounts that 392 (72.3%), 45 (8.3%), and 104 (19.2%) were pre analytical, analytical and post analytical phases of errors, respectively. Incomplete clinical data of patient was observed on 1185 (72.6%) of CLL tests. Name, gender, and age of patients were missed on 8 (0.5%), 190 (11.6%), and 257 (15.7%) forms of the requests, respectively. The physician's name existed only on 248 (15.2%) and signature on 1137 (69.6%) of the request forms. An essential patient data were incomplete, which needs emphasis on awareness creation. Such practice improves laboratory data interpretation and thereby prevent misdiagnose and mistreatment of patients.

Antiretroviral treatment associated hyperglycemia and dyslipidemia among HIV infected patients at Burayu Health Center, Addis Ababa, Ethiopia: a cross-sectional comparative study.

BACKGROUND: The effects of highly active antiretroviral therapy (HAART) on glucose and lipid metabolism among sub-Saharan Africans, for whom access to antiretroviral therapy is expanding, remain largely unknown. Therefore, the aim of this study was to assess antiretroviral treatment associated hyperglycemia and dyslipidemia among HIV infected patients at Burayu health center, Addis Ababa, Ethiopia. METHODS: A cross-sectional comparative study was conducted among HIV infected adults at Burayu Health Center, Addis Ababa, Ethiopia from September, 2011 to May, 2012. Equal number of HAART naïve and HAART initiated patients (n = 126 each) were included in the study. Demographic data were collected using a well-structured questionnaire. Total cholesterol (TC), Triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and glucose were determined. The data were analyzed using SPSS version 20 software. RESULT: Of 252 study participants, 72.2% were females; mean age was 35.3 years; mean Body Mass Index (BMI) was 21.4(kg/m2); mean time living with the virus was 20.6 months and 15.5% were TB-HIV co-infected. The prevalence of hyperglycemia, increased LDL-C hypercholesterolemia, hypertriglyceridemia and decreased HDL-C were 7.9%, 23%, 42.1%, 46.8% and 50.8% in HAART and 5.6%, 7.1%, 11.1%, 31% and 73% in non-HAART groups, respectively. First line antiretrovirals were drugs containing 2 nucleoside backbones (from Zidovudine/Stavudine/Lamivudine/Tenofovir) with either Nevirapine or Efavirenz. There was statistically significant increase in serum lipid profile levels among HAART initiated patients than HAART naïve individuals (p =0.01 for TG and <0.001 for others). CONCLUSION: First-line HAART is associated with potentially atherogenic lipid profile levels in patients with HIV infection compared to untreated patients. This indicates glucose and lipid profile levels need to be monitored regularly in HIV infected patients taking antiretroviral treatment.

Burden of metabolic syndrome among HIV-infected patients in Southern Ethiopia.

BACKGROUND: HIV infection and highly active antiretroviral therapy (HAART) can induce metabolic disturbances including lipodystrophy, dyslipidemia, and insulin resistance, which are reminiscences of metabolic syndrome (MS). However, little is known regarding the magnitude of MS in Ethiopian HIV population. This study, aimed to estimate the prevalence of MS among HIV positive patients with and without HAART. METHODS: A cross-sectional study was conducted at Hawassa University Referral Hospital, southern Ethiopia between February 2012 and April 2013. Data on demographic and anthropometric characteristics were collected from a total of 374 HIV positive participants (188 on ART and 186 on Pre-ART) using WHO stepwise approach. Fasting blood glucose, total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol was measured. The International Diabetes Federation (IDF) and the National Cholesterol Education Program: Adult Treatment Panel III (ATP) Criteria were used to define MS. RESULT: Of the 374 study participants 68% were females, and 50.3% were receiving ART. Using the IDF criteria, metabolic syndrome was diagnosed in 25% of patients receiving ART compared to 22.5% of the ART naïve group (OR: 1.14 CI: 0.71-1.84). Using the ATP criteria, the prevalence of MS was 18.1% in the ART groups compared to 15.6% in ART naïve group (OR: 1.20, CI: 0.69-2.06). Patients receiving ART had significantly elevated Cholesterol, triglyceride, glucose and LDL-c levels but lower CD4(+) cell counts than the Pre-ART groups. Being a female, having BMI of at least 25, older age (i.e. age≥45 years) and having total cholesterol of at least 200mg/dl were significantly associated with the presence of MS. Using the ATP criteria to define MS, taking d4T-3TC-EFV regimen was significantly associated with higher odds of MS. CONCLUSION: Almost a quarter of HIV patients on ART developed metabolic syndrome. Furthermore patients on ART had elevated lipid profile and glucose metabolism disturbance than the ART naïve.

Establishing reference intervals for electrolytes in newborns and infants using direct ISE analyzer.

BACKGROUND: To generate clinically applicable reference intervals (RIs) for commonly requested electrolytes in Ethiopian newborns and infants that can help in early detection, close monitoring and correction of electrolyte abnormalities. Cord blood (from newborns, n = 60) and venous blood samples (from infants, n = 57) were collected and analyzed using direct ISE analyzer, AVL (9181). MedCalc® software was applied to determine the robust upper and lower end points covering 95% of the reference values of each electrolyte with respective 90% CIs. FINDINGS: This is an extension report of our recent study; and hence is resulted from the same data source. The level of Na+ and K+ showed difference in newborns and infants even though combined RIs were suggested by the Haris and Boyd rule as 126-143 mmol/l and 4.0-7.9 mmol/l respectively. However, Cl- values failed to show such a difference and thus a combined RI was determined to be 100-111 mmol/l. Almost all maternal, neonatal and infantile factors were not able to affect the values of the electrolytes. CONCLUSION: Combined RIs are suggested for the interpretation of electrolyte values in newborns and infants without taking the effect of maternal, neonatal and infantile factors into account. Since the RIs were different from previously reported values, it will be appropriate to apply such RIs for the interpretation of electrolyte values in Ethiopian pediatric population.

Is the difference in neonatal blood glucose concentration of caesarian and vaginally delivered term infants requiring separated reference intervals?

BACKGROUND: Mode of delivery has been reported to affect the cord blood glucose level of newborns. Vaginally delivered (VD) newborns were found to have relatively increased concentration of cord blood glucose than those delivered by cesarean section (CS). The aim of this study is thus to determine whether the difference in cord blood glucose concentration among VD and CS newborns is necessitating partitioned reference intervals (RIs) for the laboratory diagnosis of neonatal hypoglycemia. METHODS: A total of 60 newborns were included from Tikur Anbessa Specialized Hospital (TASH). Cord blood samples were collected and analyzed for glucose by Glucose-oxidase (GOD-PAP) method in TASH laboratory using HumaStar 300 from November 2010 to April 2011. All pre-analytical, analytical and post-analytical aspects were thoroughly controlled. A robust method was used for the determination of reference intervals using MedCalc® software Version 11.6.1. RESULTS: VD newborns accounted for 71.7% (n = 43) while the CS newborns accounted for 28.3% (n = 17). No statistically significant difference was noted in the studied demographic variables among VD and CS newborns except for blood glucose level. The RIs were then determined to be 2.46-6.85 mmol/l and 2.46-5.04 mmol/l for VD and CS newborns respectively. The combined RI was 2.24-6.48 mmol/l. CONCLUSION: Combined RI better be used for the interpretation of cord blood glucose values in VD and CS newborns. Cord blood glucose concentrations of 2.24 mmol/l can be used as statistical estimates of cut off points for neonatal hypoglycemia in newborns irrespective of their mode of deliveries.

Robust reference intervals for liver function test (LFT) analytes in newborns and infants.

BACKGROUND: Reference intervals (RIs) are ranges of upper and lower limits of a given analyte which are used for a laboratory test to determine whether a disease is present or absent or to know if the patient is at risk for future disease states. In Ethiopia, a country with highly diversified population groups and geographical sites, there are no established RIs to metabolic analytes including the liver function test (LFT) analytes for the pediatric population though it has been known that liver function assessment in this population is vital as a result of varied vulnerability to both endogenous and xenobiotic substances. METHODS: A cross sectional study was conducted in Tikur Anbessa Specialized Hospital (TASH) and Teklehaymanot Health Center (THC) from November 2010 to April 2011. 117 cord blood (from newborns) and venous blood samples (from infants) were collected and analyzed using HumaStar 300. All pre-analytical, analytical and post-analytical aspects were thoroughly controlled. A robust, CLSI/ IFCC recommended, method was used for the determination of upper and lower end points covering 95% of the reference values of each analyte with respective 90% CIs using MedCalc® software. RESULTS: Combined RIs for newborns and infants were established for albumin, AST, ALP, direct bilirubin and total bilirubin to be 3.88-5.82 g/dl, 16.1-55.4U/l, 130-831U/l, <0.41 mg/dl and <1.37 mg/dl respectively. But, separated RIs were indicated for ALT and GGT as 1.2-23.1U/l and 6.94-24.8U/l ALT; and 30.6-160.7U/L and 10-28.2U/l GGT for newborns and infants respectively. Some maternal and infantile factors were identified to affect the values of analytes. CONCLUSION: Almost all analytes were different from previously reported values for other target population of similar age group, kit insert values and adult values. So, interpretation of values of these analytes in newborns and infants of Ethiopian population sounds better to be performed by using such RIs taking the effect of some maternal and infantile factors in to account.

External quality assessment on CD4+ T-Cell Count using in-house proficiency testing panels for CD4 Count laboratories in Addis Ababa, Ethiopia

BACKGROUND: CD4+ T-cell count External Quality Assessment program is important for the evaluation of performance of CD4 count laboratories. The aim of this study was to assess the quality of CD4count laboratory performance using in-house Proficiency testing panels that perform routineCD4 counts in Addis Ababa, Ethiopia, 2013/14. METHODS: Participating laboratories were 20, 23 and 25 in trials 1, 2 and 3, respectively. In-house prepared fresh whole blood samples both with "normal" and "low" CD4 values were sent to participating laboratories. Percentage and absolute counts of CD4+ T-lymphocytes were done using their routine procedures. Data were analyzed for each trial including trimmed mean, standard deviation (SD), percent coefficient of variation (%CV), residual, and standard deviation index (SDI) values for both absolute counts and percentages of CD4+ lymphocytes (%CD4). RESULTS: Most participating laboratories produced results that were within 2SD of the mean. Average inter-laboratory precision (trimmed %CV) was 10.87% and 5.14% for CD4 absolute counts and %CD4, respectively. For normal material, the trimmed mean %CV was 9.59% and3.23% for CD4 absolute counts and %CD4, respectively. For low material, the trimmed mean % CV was 12.15% and 7.05% for CD4 absolute counts and %CD4 respectively. BDFACSCount™ users showed the best accuracy and precision as evidenced by longitudinal analysis. CONCLUSION: This study was found to help facilities in early identifying their gaps with regard to their CD4 count performance and in avoiding the challenges encountered during participation in external EQA providers like the high cost, transportation problem, feedback delay and CD4laboratory coverage